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silver alloy nanoclusters in metal-enhanced singlet oxygen generation and their correlation with photoluminescence
Materials Science & Engineering C 109 (2020) 110525
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Protein-protected gold/silver alloy nanoclusters in metal-enhanced singlet oxygen generation and their correlation with photoluminescence Yong Yua, Wen Di Leeb, Yen Nee Tana,c,
T
⁎
a
Institute of Materials Research and Engineering, The Agency for Science, Technology and Research (A*STAR), 2 Fusionopolis Way, #08-03, Innovis, 138634, Singapore School of Materials Science & Engineering, Nanyang Technological University, Nanyang Avenue, 639798, Singapore c Faculty of Science, Agriculture & Engineering, Newcastle University, Newcastle Upon Tyne NE1 7RU, United Kingdom b
A R T I C LE I N FO
A B S T R A C T
Keywords: Gold-silver alloy Metal nanoclusters Galvanic replacement Photosensitizer Metal enhanced singlet oxygen generation
Photoluminescent noble metal nanoclusters (NCs, core size < 2 nm) have recently emerged as a new type of photosensitizers advantageous over conventional photosensitizers due to their high singlet oxygen (1O2) generation efficiency, excellent photostability and water solubility, as well as good biocompatibility for photodynamic therapy and bioimaging. However, no correlation has been established between the intrinsic 1O2 generation and photoluminescence properties of metal NCs with their size, composition, and concentration, which is important to customize the molecule-like properties of NCs for different applications. Herein, we report a systematic study to uncover the rational design of bimetallic NCs with controllable 1O2 generation efficiency by tuning their compositions through spontaneous galvanic displacement reaction. A series of ultrasmall gold/silver alloy nanoclusters (AuAgNCs) were synthesized by reacting bovine serum albumin (BSA) protein-protected Ag13NCs (13 Ag atoms/cluster) with varying concentrations of gold precursor at room temperature. It was found that the 1O2 generation efficiency of the resultant BSA-protected AuAgNCs were inversely correlated to their photoluminescence intensity. Interestingly, plasmonic gold nanoparticles (> 10 nm) were also formed simultaneously by photobleaching of the BSA-AuAgNCs, leading to significant metal enhancement effect to the 1 O2 generation rate much higher (~45 times) than that of the monometallic BSA-Ag13NC. This versatile two-forone strategy to develop next generation metal-enhanced bimetallic NC photosensitizers in one pot opens up new opportunities in designing advanced hybrid nanomaterials with complementary and/or enhanced functionalities.
1. Introduction Reactive oxygen species (ROS) such as singlet oxygen (1O2) have attracted enormous research interests due to its significance in diverse fields such as photodynamic therapy (PDT) for cancer treatment, fine chemicals synthesis, waste water decontamination, and blood sterilization etc. [1–3]. There are three essential components to generate ROS, which include a photosensitizer, oxygen containing medium (e.g., molecular oxygen and/or water), and incident light of appropriate wavelength. Among the three, photosensitizer is of most interest as it is the medium to pass the energy of incident light to the nearby molecules in generating ROS. In particular, paramount of efforts have been made to develop effective and biocompatible photosensitizers for biomedical applications due to the efficacy and non-invasive nature of the light therapy. For example, hematoporphyrin and its derivatives (the first
generation of photosensitizers) and porphyrin-based photosensitizers (the second generation photosensitizer, e.g., chlorines) have been accepted for the treatment of non-oncological diseases as well as various cancers at clinical level [4–7]. However, these photosensitizers are still suffering from intrinsic issues such as poor photostability and aqueous solubility [5]. More recently, nanomaterials-based photosensitizers have been developed to overcome the problems faced by current commercially available photosensitizers for biomedical applications [8,9]. In recent years, ligand-protected gold (Au) or silver (Ag) nanoclusters (NCs), which are ultrasmall nanoparticles with a core size of < 2 nm and protected by various organic molecules (e.g., thiolates [10–17], proteins [18–22], peptides [23–29], DNA [30–33] and synthetic polymers [34–39]), have emerged as a new type of promising photosensitizers due to their excellent water solubility, better
⁎ Corresponding author at: Institute of Materials Research and Engineering, the Agency for Science, Technology and Research (A*STAR), 2 Fusionopolis Way, #0803, Innovis 138634, Singapore, and Faculty of Science, Agriculture & Engineering, Newcastle University, Newcastle Upon Tyne NE1 7RU, United Kingdom. E-mail address: [email protected] (Y.N. Tan).
https://doi.org/10.1016/j.msec.2019.110525 Received 29 August 2019; Received in revised form 12 November 2019; Accepted 5 December 2019 Available online 05 December 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.
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2.2. Galvanic replacement synthesis of gold/silver alloy nanoclusters
photostability and good biocompatibility. Kawasaki et al. discovered that the atomically precise Au25(SR)18 cluster (RS- denotes the thiolate ligand 2-phenylethanethiol) was able to generate 1O2 effectively as confirmed by both fluorescence spectroscopy and chemical trapping methods using 1O2 selective probes [40]. Yamamoto et al. have studied the ligand effect on singlet oxygen generation (SOG) property of both protein (bovine serum albumin or BSA) and peptide (glutathione or GSH) protected Au25 and found that protein-protected Au25 cluster (i.e., BSA-Au25) could generate 1O2 with a rate of 6 times faster than that using peptide-protected Au25(SG)18 [41]. Most recently, our study have shown that the silver-based nanoclusters formed within the BSA protein template, which consist of 13 Ag atoms per cluster (i.e., BSA-Ag13NC) is capable of achieving 10 times higher 1O2 quantum efficiency than its gold analogue BSA-Au25 [8]. In addition, MTT assay with MCF-7 cells showed no dark toxicity of the BSA-Ag13NC which validated its application as a biocompatible theranostic agent. In view of the synergistic effects that are commonly observed in the metal nanocomposite [42–44], there is a possibility that bimetallic or alloy NCs could achieve even higher SOG efficiencies than its monometallic counterpart. However, no rational design rules or synthesis strategy have been established to better correlate the intrinsic 1O2 generation and photoluminescence properties of bimetallic NCs with their size, composition, and concentration, which is important to customize the molecular-like properties of NCs for different applications. Furthermore, some of the biotemplated metal NC-based photosensitizers which also exhibit bright photoluminescence have been successfully applied for bioimaging and photodynamic cancer therapy in in vitro and in vivo studies [8,40,45], showing their great promise as effective biocompatible theranostic agents for nanomedicine [46]. In this study, we report a new synthesis strategy to systematically tune the singlet oxygen generation property of bimetallic NCs by alloying different amount of Au atoms into the BSA-protected Ag13NC via a thermodynamically favorable galvanic displacement reaction at room temperature. The resultant bimetallic nanomaterials were comprehensively characterized using various techniques, including UV–vis and photoluminenscence spectroscopy, transmission electron microscopy, X-ray photoelectron spectroscopy (XPS), energy-dispersive X-ray (EDX) spectroscopy, and matrix assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry. A chemical trapping method using 9,10-Anthracenediyl-bis(methylene) dimalonic acid (ABDA) as the 1O2 detection probe were employed to measure the SOG efficiency of the resultant BSA-protected alloy AuAgNCs, which was correlated to the amount of Au ions used in the replacement reaction and their photoluminescence properties. Interestingly, it was found that plasmonic gold nanoparticles with an estimated size of 60 nm were also formed during the photobleaching process to enhance the SOG efficiency of BSA-AuAg NCs on site, achieving an unprecedented rate of single oxygen generation (i.e., 45 times) higher than that of the monometallic BSA-Ag13NC photosensitizers. Presented below are the details of this study.
The protein-protected gold/silver alloy nanoclusters (AuAgNCs) were synthesized in a two-step procedure. BSA-protected Ag13NC was first prepared following our previously reported protocol [8]. In a typical synthesis, aqueous solutions of silver nitrate (AgNO3, 4 mL, 15 × 10−3 M) and bovine serum albumin (BSA, 4 mL, 74 mg mL−1) were mixed in a 20 mL glass vial containing 4 mL of ultrapure water under vigorous, constant stirring. After 2 min, a solution of sodium hydroxide (NaOH, 400 μL, 1 M) was added to the glass vial. Another 30 min later, aliquots of freshly prepared sodium borohydride solution (NaBH4, 300 μL, 11.2 × 10−3 M in 0.1 M NaOH) was added to the glass vial. The reaction proceeded for 1 h and the final product was collected and desalted with a cellulose dialysis tubing (MWCO of 6000). The purified sample was then used as precursor for the synthesis of alloy AuAgNCs via a galvanic replacement route. In a typical synthesis, a solution of purified BSA-Ag13NC (400 μL, 4 × 10−3 M) was mixed with varying volumes of HAuCl4 (15 × 10−3 M). The reaction solution was topped up to 1.2 mL with calculated amount of ultrapure water. After 3 h, the product was centrifuged (15,000 ×g, 45 min, 4 °C) in a Hettich Mikro 220R Centrifuge. The supernatant was transferred to an Amicon Ultra-0.5 Centrifugal Filter and centrifuged at 14,000 ×g for 30 min at 25 °C. The concentrated solute was recovered by reverse-spinning and topped up with ultrapure water to reach the initial concentration. The purified samples were stored in a 4 °C fridge for later use.
2.3. Characterization of BSA protein-protected AuAgNCs Photoabsorption UV–vis and photoemission spectra were recorded on a Shimadzu UV-2450 UV–visible spectrophotometer and a Tecan Infinite M200 Microplate Reader, respectively. Transmission Electron Microscopy (TEM) images and Energy-Dispersive X-ray (EDX) Spectroscopy results were obtained on a Philips CM300 Transmission Electron Microscope (High Resolution). X-ray Photoelectron Spectroscopy (XPS) study was done on a Theta Probe Angle-Resolved Xray Photoelectron Spectrometer. Matrix Assisted Laser Desorption Ionization (MALDI) Time-of-Flight (TOF) mass spectrometry study was done on a Bruker Daltonics Autoflex II TOF/TOF system. The MALDITOF samples were prepared by mixing 2 μL sample with 2 μL matrix solution. The matrix solution was prepared by dissolving 2,5-dihydroxybenzoic acid (DHB) solution to saturation in 50% acetonitrile.
2.4. Measurement of singlet oxygen generation by BSA-AuAgNCs Singlet oxygen generation property of the BSA-protected alloy nanoclusters (BSA-AuAgNCs) was studied by an optical method using 9,10-Anthracenediyl-bis(methylene) dimalonic acid (ABDA) as 1O2 probe. In a typical experiment, 200 × 10−6 M of the photosensitizer and 50 × 10−6 M of ABDA were mixed in a cuvette and buffered to 1 mL using ultrapure water. The absorption spectra of the mixture solution was measured prior to irradiation (denoted as 0 min) and after every 2–10 min up to 60 min of irradiation time. A halogen bulb (output: 100 W, 3200 K) was used as the white light source. Irradiation was stopped when the absorption peaks of ABDA are almost flat (A/ A0 < 5% twice consecutively) or when there is no longer any change in the absorption peak (A/A0 < 1% twice consecutively), whichever occurs first. To acquire a better representative of the 1O2 production rates, the overhead contribution of the BSA-protected AuAgNCs to the ABDA peak intensities was subtracted from each raw spectra reading. The overhead contribution was measured by replacing the 50 × 10−6 M ABDA with an equal volume of ultrapure water. The percentage change in peak intensity at 370 nm was then calculated using the subtracted values.
2. Experimental 2.1. Materials Gold (III) chloride solution (HAuCl4, 30% in dilute HCl), silver nitrate (AgNO3, 99.9999%), bovine serum albumin (BSA, 96%), sodium hydroxide (NaOH, 97%), sodium borohydride (NaBH4, 99.99%), 9,10Anthracenediyl-bis(methylene)dimalonic acid (ABDA, 90%), and 2,5dihydroxybenzoic acid (DHB, 98%) were purchased from SigmaAldrich. All reagents were used as received and without further purification. All glassware were washed with Aqua Regia (HCl:HNO3 volume ratio = 3:1) and rinsed with ethanol and ultrapure water. (Caution: Aqua Regia is a very corrosive oxidizing agent, which should be handled with great care.) Ultrapure water with a specific resistance of 18.2 MΩ was used throughout the experiment. 2
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(A) Galvanic displacement
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Fig. 1. (A) Schematic illustration of the BSA-RAuxAg(13−x)NCs formed by galvanic replacement reaction. (B) UV–vis and (C) photoemission spectra (λex = 480 nm) of BSA-protected Ag13NC (dashed lines) and BSA-RAuxAg(13−x)NCs (solid lines) where R indicates the molar ratio of Au ions to AgNC. (Inset of B) TEM image of BSA24AuxAg(13−x)NCs. The red circles highlight the individual alloy nanoclusters. (Inset of C) Enlarged photoemission spectra of BSA-RAuxAg(13−x)NCs for R = 12, 15, 18, 21 and 24 respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3. Results and discussion
absorption spectrum depending on their composition and morphology. Due to the ultrasmall size of metal NCs (< 2 nm), the atoms per cluster are too few to support the characteristic LSPR peaks that are commonly observed for the gold nanoparticle (AuNP @ 520 nm [48,49]) and silver nanoparticle (AgNP@ 400 nm [50]). Thus, the absence of LSPR peaks of AuNP or AgNPs in Fig. 1B provides an indirect evidence to indicate the formation of bimetallic NCs. Transmission electron microscopy (TEM) was also used to further confirm the formation of BSA-protected alloy AuAgNCs. Inset of Fig. 1A shows the particle size of < 3 nm in diameter, which were in line with the UV–vis spectra that ruled out the formation of plasmonic Au or AgNPs with size larger than 5 nm. Furthermore, the as-synthesized BSA-AuAgNCs showed weak red emission under UV irradiation, which is the intrinsic property of metal NCs due to the quantum confinement effect. However, the photoluminescent (PL) intensity of the BSA-AuAgNCs decreased rapidly as the concentration of Au ions (or R ratio) increased (Fig. 1C). To better understand the composition of BSA-RAuxAg(13−x)NC (i.e., to determine the value of x), elemental analysis using X-ray Photoelectron Spectroscopy (XPS) and Energy-Dispersive X-ray (EDX) Spectroscopy have been conducted. Surprisingly, only Au4f signals were observed in the XPS spectra (Fig. S1, Supporting Information). Considering that XPS is a surface analysis technique with a typical depth of < 5 nm, there is a high chance that no Ag3d signal could be detected if most of the Ag were replaced by the Au ions on the surface of NCs. It is also possible that the BSA-RAuxAg(13−x)NC were piled up when they were dried on the substrate during the sample preparation process, leading to the inaccuracy of detection for Ag elements located in the core structure of alloy AuAgNCs. As such, we have proceeded to characterize the as-synthesized metallic nanoclusters using EDX, which is a technique with a typical analysis depth of 1–3 μm. While EDX results give an Au-to-Ag ratio of 1.35, 0.99 and 2.32 for BSARAuxAg(13−x)NCs with R = 15, 18 and 21, respectively; no Au and Ag
Fig. 1A shows the synthesis of BSA-protected gold/silver alloy nanoclusters through the galvanic replacement reaction of BSA-Ag13NC with gold precursor (Au ions) at room temperature. As the standard redox potential of AuCl4−/Au (0.99 V vs standard hydrogen electrode, SHE) is higher than that of Ag+/Ag (0.80 V vs SHE), this reaction is thermodynamically favorable and proceeds spontaneously [47]. The BSA-Ag13NC which contained 13 Ag atoms in a single nanocluster was first synthesized according to a previous reported method [8]. It was red brown in solution (under day light) and exhibited a shoulder peak at around 435 nm on the UV–vis spectrum (Fig. 1B, dashed line). Upon UV irradiation, the BSA-Ag13 NC showed red fluorescent with an emission peak located at 670 nm wavelength (Fig. 1C, dashed line). The protein-protected alloy AuAgNCs were then synthesized by adding a solution of gold precursor (HAuCl4) to the BSA-Ag13NC in various mole ratios of Au ions to AgNC. Upon addition of HAuCl4, the solutions turned murky but cleared up slightly after 3 h of reaction time. The final products (denoted as BSA-RAuxAg(13−x)NC where R is the molecular ratio of Au ions to 1 mol of BSA-Ag13NC initially used in the synthesis) were clear and colorless. For example, BSA-12AuxAg(13−x)NC represents the product formed by reacting 1 mol of AgNC with 12 times of Au ions (i.e., R = 12). Totally five samples were collected for further investigations, namely BSA-12AuxAg(13−x)NC, BSA-15AuxAg(13−x)NC, BSA-18AuxAg(13−x)NC, BSA-21AuxAg(13−x)NC and BSA24AuxAg(13−x)NC, respectively. As shown in Fig. 1B, UV–vis absorption spectra of all bimetallic NCs are featureless (solid lines). Disappearance of the shoulder peak at 435 nm as observed on the spectrum of Ag13NCs, suggesting successful oxidation of Ag atoms and incorporation of Au atoms into the alloy NCs for all samples. Typically, noble metal nanoparticles in the size range of 5 to 100 nm exhibit unique localized surface plasmon resonance (LSPR) peaks in the UV–visible 3
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Fig. 2. Time course absorption spectra of ABDA solution (50 × 10−6 M) in the presence of 200 μM of (A) BSA-Ag13NC, (B) BSA-12AuxAg(13−x)NC, (C) BSA15AuxAg(13−x)NC, (D) BSA-18AuxAg(13−x)NC, (E) BSA-21AuxAg(13−x)NC, and (F) BSA-24AuxAg(13−x)NC.
of using current techniques to determine the exact molecular formula of the NC samples tested in this study, the initial composition of each NC based on the R ratio is used throughout the article for the convenience of discussion. Next, the SOG properties of the as-synthesized BSARAuxAg(13−x)NCs were studied by a chemical trapping method using 9,10-Anthracenediyl-bis(methylene) dimalonic acid (ABDA) as the singlet oxygen (1O2) detection probe. As shown in Fig. 2, ABDA has four characteristic peaks at 342, 359, 378 and 400 nm in the absorption spectrum. When 1O2 is generated, ABDA will react with it to yield a steady state endoperoxide, leading to the disappearance of the aforementioned characteristic absorption peaks. This decrease in the absorption peak (ΔO.D.) is directly correlated to the amount of ABDA degraded and corresponded to the amount of 1O2 reacted. Therefore, 1O2 generation property of BSA-RAuxAg(13−x)NCs can be characterized by the decrease in the absorption peak of ABDA. Fig. 2(B–F) show that all
signals were detected for R = 12 and 24 (Figs. S2 to S4, Supporting Information). This could be due to the fact that the BSARAuxAg(13−x)NCs synthesized in this study are very small (< 3 nm) and protected by the bulky protein templates that are hard to be observed even under HR-TEM due to poor contrast, making it hard to determine the exact composition when selecting an area of interest in TEM for EDX analysis. Although mass spectrometry such as matrix assisted laser desorption ionization (MALDI) time-of-flight (TOF) is a powerful technique to identify the atomic composition of single metallic (Au or Ag) nanoclusters stabilised by thiol ligands, it is relatively difficult to assign the atomic composition for the bimetallic nanoclusters protected by the large protein template with unknown molecular formula. Furthermore, the molecular weight of two Ag atoms is very close to that of one Au atom, which compounds the difficulty in assigning the exact formula of the resulted alloy AuAgNCs based on the mass difference between BSA-RAuxAg(13−x)NCs and BSA-Ag13NC. Due to the limitations 4
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Fig. 3. Decomposition rate constants of ABDA in the presence of 200 μM of (A) BSA-Ag13NC, (B) BSA-12AuxAg(13−x)NC, (C) BSA-15AuxAg(13−x)NC, (D) BSA18AuxAg(13−x)NC, (E) BSA-21AuxAg(13−x)NC, and (F) BSA-24AuxAg(13−x)NC under white light irradiation.
reported herein possess unusually high 1O2 generation property. Several control experiments have also been conducted to confirm that the degradation of ADBA was due to the photo-induced generation of 1O2 by the BSA protein-protected alloy AuAgNCs. Firstly, it was observed that the BSA template itself could not degrade ABDA under white light irradiation for up to 30 min (Fig. S5A, Supporting Information). Secondly, ABDA would not degrade under dark condition even in the presence of BSA-RAuxAg(13−x)NCs (e.g., BSA-24AuxAg(13−x)), for a prolonged period of time (Fig. S5B, Supporting Information). This is in contrast to the rapid ABDA degradation (i.e., 4 min) in the presence of the same alloy NCs under white light treatment (Fig. 2F), showing the intrinsic SOG property of the as-synthesized BSA-RAuxAg(13−x)NCs. The degradation rate of ABDA in the presence of BSA-RAuxAg13NCs were calculated and compared. The natural logarithm of the inverse of
the characteristic absorbance peaks of ABDA eventually disappeared in a certain period of time in the presence of BSA-RAgxAu(13−x)NCs (R = 12, 15, 18, 21 and 24) under white light irradiation. This result indicates that the all the as-synthesized BSA-RAuxAg(13−x)NC are capable of generating 1O2 under white light irradiation. Additionally, it was discovered that the more Au ions were introduced to replace BSAAg13NC as precursor in forming alloy AuAgNCs, the faster the degradation rate of ABDA. For example, it took 50 min for the ABDA peaks to disappear completely for BSA-RAuxAg(13−x)NC with R = 12, followed by 45 min for R = 15, 10 min for R = 18, and only 4 min for R = 21 and 24, respectively. As a comparison with the original BSAAg13NC, the rate of ABDA degradation was much slower and about half of the ABDA was still remained intact after 1 h (Fig. 2A), suggesting that BSA-RAuxAg(13−x)NCs synthesized through galvanic replacement 5
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Fig. 4. (A) The 1O2 generation efficiency (square) of BSA-RAuxAg(13−x)NCs (R is molar ratio of Au ions to Ag13NC) is inversely correlated to their photoluminescence (PL) intensities (circle). (Insets) Digital photos of the BSA-RAuxAg(13−x)NC (R = 0, 12, 15, 18, 21 and 24, respectively; R = 0, i.e., BSA-Ag13NC) under UV light irradiation. (B) A simplified Jablonski diagram of BSA-RAuxAg(13−x)NCs upon photoexcitation in water.
Fig. 5. Time course study showing the in-situ formation of plasmonic AuNPs to enhance the SOG property of BSA-RAuxAg(13−x) alloy NCs. (A) UV–vis spectra of the mixture solution of BSA-24AuxAg(13−x)NC and ABDA under white light irradiation for 10 min (2 min of time interval). (B) UV–vis spectra of the mixture solution of BSA-24AuxAg(13−x)NC and ABDA under dark condition for 10 min. (C) UV–vis spectra of BSA-24AuxAg(13−x)NC alone (no ABDA) under white light irradiation for 10 min. (D) Schematic illustration of metal enhanced 1O2 generation by the photobleaching degradation of BSA-AuAgNCs to form large plasmonic AuNPs.
the original BSA-Ag13NC was only 0.0116 min−1 (Fig. 3A). The rate of SOG was as high as ~6 to 45 times for the BSA-protected alloy AuAgNCs as compared to the monometallic BSA-Ag13NC, suggesting the excellent SOG properties of the BSA-RAuxAg(13−x)NCs and their potency as effective water soluble biotemplated photosensitizers. Ultrasmall metallic NCs possess molecular-like properties such as photoluminescence due to the quantum confinement effect. Therefore, we also investigated the correlation of the intrinsic photoluminescence and SOG properties of the resulted BSA-RAuxAg(13−x) alloy NCs. It was discovered for the first that time that the SOG rate of the as-synthesized BSA-protected alloy AuAgNCs is inversely correlated to their photoluminescence (PL) intensities. As shown in Fig. 4A, the PL intensities of
relative absorbance (ln(A0/A)) was plotted against the reaction time (t), and the slope of linear fitted line (k) represented the degradation rate constants. As shown in Fig. 3(B–F), the rate constants of ABDA degradation are 0.0705 (R = 12), 0.0797 (R = 15), 0.254 (R = 18), 0.529 (R = 21), and 0.465 (R = 24) min−1 in the presence of BSARAuxAg(13−x) photosensitizer with different molar ratio R. It should be noted that ABDA was completely degraded in < 10 min for using BSA18AuxAg(13−x) as photosensitizer and 4 min for using both BSA21AuxAg(13−x) and BSA-24AuxAg(13−x)NCs, which curves have plateaued out at each respective time. These results show that the degradation rate of ABDA increases as the amount of Au in the precursor increases. In comparison, the degradation rate constant of ABDA with 6
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metallic molar ratios (R) were systematically studied with a chemical trapping method using ABDA as the 1O2 detection probe and UV–visible/fluorescent spectroscopy. It was found that the singlet oxygen generation rate of the bimetallic nanoclusters increased as their photoluminescence intensity decreased. The inverse correlation between the intrinsic photoluminescence and SOG property of the BSARAuxAg(13−x)NCs gives a clear evidence of the competition between the pathways of photosensitization of nearby medium to form reactive oxygen species (e.g., 1O2) and radiative decay of the excited photosensitizer to generate photoluminescence. The photobleaching degradation of BSA-RAuxAg(13−x)NCs to form large gold nanoparticles (AuNPs > 10 nm) has been identified and accounted for the very high rate of 1O2 generation of BSA-RAuxAg(13−x)NCs (~45 times of the monometallic BSA-Ag13NCs) due to plasmonic enhancement effect. This study not only reports a new methodology of making photosensitizers with excellent 1O2 generation property via a simple metal replacement approach, but also exemplifies the metal-enhanced ROS generation of a photosensitizer by the in situ formation of plasmonic gold nanoparticles, opening up new opportunities in designing advanced hybrid nanomaterials with complementary and/or enhanced functionalities in one pot.
all the BSA-RAuxAg13NCs were much lower than that of BSA-Ag13NC (< 20% that of the BSA-Ag13NC) despite of their higher SOG rates. Furthermore, when the PL intensity of these BSA-RAuxAg(13−x)NCs decreased by ~80% from R = 12 to 24, the corresponding ABDA degradation rate increased by > 5 times. According to the classic Jablonski diagram as shown in Fig. 4B, it is intuitive that when the more excited states of a photosensitizer are used to sensitize triplet oxygen, the fewer will remain to return to the ground states in the pathway of radiative decay since the total amount of excited states is a constant given that the light condition is fixed. Photoluminescence is used instead of fluorescence in this diagram because the lifetime of metal NCs could be at the order of a few nanoseconds to a few microseconds [8,30]. It has been observed previously that the degradation rate constant (k) of BSA-Au25NC with a higher PL quantum yield (10.5%) is only 3% of that BSA-Ag13NC with a much lower PL quantum yield (0.4%). However, the difference in metal species of the two NCs makes the aforementioned relation less direct. The tendency of delayed photoluminescence [51] has been suggested for the poor 1O2 generation efficiency despite the seemingly large population of triplet states in the BSA-Au25NC. Hence, it is more straightforward to establish correlation of the portion of excited states used for both photosensitization of triplet oxygen and photoluminescence, because the BSA-RAuxAg(13−x)NCs reported herein have a similar composition and were synthesized using the same methodology. Although full understanding of the unique 1O2 generation property of BSA-RAuxAg(13−x)NCs is yet to be elucidated, investigation of the optical property of BSA-RAuxAg(13−x)NC in the course of ABDA degradation in this study may shed some light on the enhanced 1O2 generation. As shown in Fig. 5A, UV–vis spectrum of the reaction mixture with BSA-24AuxAg(13−x) and ABDA under white light irradiation shows an absorption peak at ~550 nm. This peak which spans in the wavelengths of 400–800 nm increases gradually along with the irradiation time within 10 min, indicating the formation of plasmonic AuNPs [52]. TEM image of the reaction mixture further confirms the size of as-formed AuNPs is ~60 nm (Fig. S6). It has been reported that plasmonic nanoparticles such as AuNPs and AgNPs are able to enhance the 1O2 generation efficiency of their nearby photosensitizer, analogous to the metal-enhanced photoemission phenomenon [53–55]. It is thus hypothesized that the photobleaching of BSA-RAuxAg(13−x)NCs leads to their gradual decomposition to form large AuNPs in the proximity of BSA-RAuxAg(13−x)NCs. These AuNPs serve as antennas to absorb more incident light due to the strong LSPR. The enhanced localized electric field of AuNPs then amplifies the 1O2 generation property of BSARAuxAg(13−x)NCs (Fig. 5D). As such, UV–vis spectrum of the BSA24AuxAg(13−x)NC and ABDA mixture in the dark conditions were also examined. As shown in Fig. 5B, the characteristic LSPR peaks of AuNPs were not observed for the same period of time, suggesting that ABDA alone does not react with BSA-24AuxAg(13−x)NC to form large AuNPs wherein the major contribution of ABDA is to trap the generated 1O2. As a comparison, a small peak at 540 nm was observed in the UV–vis spectrum of BSA-24AuxAg(13−x)NC under white light irradiation for 10 min (Fig. 5C), which was not unexpected as photobleaching is deemed as an intrinsic property of a photosensitizer while prolonged irradiation could destabilize the alloy AuAgNCs to form large AuNPs. In addition, this process will be greatly accelerated in the presence of ABDA, as trapping of the generated 1O2 by ABDA could further drove the destabilization and conversion of BSA-24AuxAg(13−x)NC as shown in Fig. 5A.
Declaration of competing interest None. Acknowledgement The authors acknowledge funding and support from the Institute of Materials Research and Engineering, Agency of Science, Technology and Research (A*STAR), under Bioinspired Approaches to Biomimetic Materials program (IMRE/17-1P1404), in collaboration with Newcatle University (RSA/CCEAMD5010) and Nanyang Technological University, Singapore. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.msec.2019.110525. References [1] M.C. DeRosa, R.J. Crutchley, Photosensitized singlet oxygen and its applications, Coord. Chem. Rev. 233-234 (2002) 351–371. [2] H. Wu, W. Chen, H. Yang, S. Wang, S.T. Lim, W. Yao, J. Guo, T. Li, M.W. Wong, D. Huang, Improved synthesis dimethylhomoecoerdianthrone (HOCD) and its functionalization through facile amination reactions, Dyes Pigments 130 (2016) 154–161. [3] S. Wang, R. Gao, F. Zhou, M. Selke, Nanomaterials and singlet oxygen photosensitizers: potential applications in photodynamic therapy, J. Mater. Chem. 14 (2004) 487–493. [4] A.E. O’Connor, W.M. Gallagher, A.T. Byrne, Porphyrin and nonporphyrin photosensitizers in oncology: preclinical and clinical advances in photodynamic therapy, Photochem. Photobiol. 85 (2009) 1053–1074. [5] R. Bonnett, Photosensitizers of the porphyrin and phthalocyanine series for photodynamic therapy, Chem. Soc. Rev. 24 (1995) 19–33. [6] P. Zhang, W. Steelant, M. Kumar, M. Scholfield, Versatile photosensitizers for photodynamic therapy at infrared excitation, J. Am. Chem. Soc. 129 (2007) 4526–4527. [7] S. Brown, Clinical antimicrobial photodynamic therapy: phase II studies in chronic wounds, J. Natl. Compr. Cancer Netw. 10 (2012) S–80-S-83. [8] Y. Yu, J. Geng, E.Y.X. Ong, V. Chellappan, Y.N. Tan, Bovine serum albumin proteintemplated silver nanocluster (BSA-Ag13): an effective singlet oxygen generator for photodynamic cancer therapy, Adv. Healthcare Mater. 5 (2016) 2528–2535. [9] X.T. Zheng, Y.C. Lai, Y.N. Tan, Nucleotide-derived theranostic nanodots with intrinsic fluorescence and singlet oxygen generation for bioimaging and photodynamic therapy, Nanoscale Adv 1 (2019) 2250–2257. [10] Y. Yu, Q. Yao, Z. Luo, X. Yuan, J.Y. Lee, J. Xie, Precursor engineering and controlled conversion for the synthesis of monodisperse thiolate-protected metal nanoclusters, Nanoscale 5 (2013) 4606–4620. [11] P. Maity, S. Xie, M. Yamauchi, T. Tsukuda, Stabilized gold clusters: from isolation toward controlled synthesis, Nanoscale 4 (2012) 4027–4037. [12] R. Jin, Quantum sized, thiolate-protected gold nanoclusters, Nanoscale 2 (2010) 343–362.
4. Conclusion In conclusion, a series of ultrasmall BSA-protected gold/silver alloy (core size < 2 nm) with unique photoluminescence and singlet oxygen generation (SOG) properties have been successfully synthesized by galvanic replacement between BSA-Ag13NCs and gold ions precursor (HAuCl4). The as-synthesized BSA-RAuxAg(13−x)NCs of different 7
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Protein-protected gold/silver alloy nanoclusters in metal-enhanced singlet oxygen generation and their correlation with photoluminescence Yong Yua, Wen Di Leeb, Yen Nee Tana,c,
T
⁎
a
Institute of Materials Research and Engineering, The Agency for Science, Technology and Research (A*STAR), 2 Fusionopolis Way, #08-03, Innovis, 138634, Singapore School of Materials Science & Engineering, Nanyang Technological University, Nanyang Avenue, 639798, Singapore c Faculty of Science, Agriculture & Engineering, Newcastle University, Newcastle Upon Tyne NE1 7RU, United Kingdom b
A R T I C LE I N FO
A B S T R A C T
Keywords: Gold-silver alloy Metal nanoclusters Galvanic replacement Photosensitizer Metal enhanced singlet oxygen generation
Photoluminescent noble metal nanoclusters (NCs, core size < 2 nm) have recently emerged as a new type of photosensitizers advantageous over conventional photosensitizers due to their high singlet oxygen (1O2) generation efficiency, excellent photostability and water solubility, as well as good biocompatibility for photodynamic therapy and bioimaging. However, no correlation has been established between the intrinsic 1O2 generation and photoluminescence properties of metal NCs with their size, composition, and concentration, which is important to customize the molecule-like properties of NCs for different applications. Herein, we report a systematic study to uncover the rational design of bimetallic NCs with controllable 1O2 generation efficiency by tuning their compositions through spontaneous galvanic displacement reaction. A series of ultrasmall gold/silver alloy nanoclusters (AuAgNCs) were synthesized by reacting bovine serum albumin (BSA) protein-protected Ag13NCs (13 Ag atoms/cluster) with varying concentrations of gold precursor at room temperature. It was found that the 1O2 generation efficiency of the resultant BSA-protected AuAgNCs were inversely correlated to their photoluminescence intensity. Interestingly, plasmonic gold nanoparticles (> 10 nm) were also formed simultaneously by photobleaching of the BSA-AuAgNCs, leading to significant metal enhancement effect to the 1 O2 generation rate much higher (~45 times) than that of the monometallic BSA-Ag13NC. This versatile two-forone strategy to develop next generation metal-enhanced bimetallic NC photosensitizers in one pot opens up new opportunities in designing advanced hybrid nanomaterials with complementary and/or enhanced functionalities.
1. Introduction Reactive oxygen species (ROS) such as singlet oxygen (1O2) have attracted enormous research interests due to its significance in diverse fields such as photodynamic therapy (PDT) for cancer treatment, fine chemicals synthesis, waste water decontamination, and blood sterilization etc. [1–3]. There are three essential components to generate ROS, which include a photosensitizer, oxygen containing medium (e.g., molecular oxygen and/or water), and incident light of appropriate wavelength. Among the three, photosensitizer is of most interest as it is the medium to pass the energy of incident light to the nearby molecules in generating ROS. In particular, paramount of efforts have been made to develop effective and biocompatible photosensitizers for biomedical applications due to the efficacy and non-invasive nature of the light therapy. For example, hematoporphyrin and its derivatives (the first
generation of photosensitizers) and porphyrin-based photosensitizers (the second generation photosensitizer, e.g., chlorines) have been accepted for the treatment of non-oncological diseases as well as various cancers at clinical level [4–7]. However, these photosensitizers are still suffering from intrinsic issues such as poor photostability and aqueous solubility [5]. More recently, nanomaterials-based photosensitizers have been developed to overcome the problems faced by current commercially available photosensitizers for biomedical applications [8,9]. In recent years, ligand-protected gold (Au) or silver (Ag) nanoclusters (NCs), which are ultrasmall nanoparticles with a core size of < 2 nm and protected by various organic molecules (e.g., thiolates [10–17], proteins [18–22], peptides [23–29], DNA [30–33] and synthetic polymers [34–39]), have emerged as a new type of promising photosensitizers due to their excellent water solubility, better
⁎ Corresponding author at: Institute of Materials Research and Engineering, the Agency for Science, Technology and Research (A*STAR), 2 Fusionopolis Way, #0803, Innovis 138634, Singapore, and Faculty of Science, Agriculture & Engineering, Newcastle University, Newcastle Upon Tyne NE1 7RU, United Kingdom. E-mail address: [email protected] (Y.N. Tan).
https://doi.org/10.1016/j.msec.2019.110525 Received 29 August 2019; Received in revised form 12 November 2019; Accepted 5 December 2019 Available online 05 December 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.
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2.2. Galvanic replacement synthesis of gold/silver alloy nanoclusters
photostability and good biocompatibility. Kawasaki et al. discovered that the atomically precise Au25(SR)18 cluster (RS- denotes the thiolate ligand 2-phenylethanethiol) was able to generate 1O2 effectively as confirmed by both fluorescence spectroscopy and chemical trapping methods using 1O2 selective probes [40]. Yamamoto et al. have studied the ligand effect on singlet oxygen generation (SOG) property of both protein (bovine serum albumin or BSA) and peptide (glutathione or GSH) protected Au25 and found that protein-protected Au25 cluster (i.e., BSA-Au25) could generate 1O2 with a rate of 6 times faster than that using peptide-protected Au25(SG)18 [41]. Most recently, our study have shown that the silver-based nanoclusters formed within the BSA protein template, which consist of 13 Ag atoms per cluster (i.e., BSA-Ag13NC) is capable of achieving 10 times higher 1O2 quantum efficiency than its gold analogue BSA-Au25 [8]. In addition, MTT assay with MCF-7 cells showed no dark toxicity of the BSA-Ag13NC which validated its application as a biocompatible theranostic agent. In view of the synergistic effects that are commonly observed in the metal nanocomposite [42–44], there is a possibility that bimetallic or alloy NCs could achieve even higher SOG efficiencies than its monometallic counterpart. However, no rational design rules or synthesis strategy have been established to better correlate the intrinsic 1O2 generation and photoluminescence properties of bimetallic NCs with their size, composition, and concentration, which is important to customize the molecular-like properties of NCs for different applications. Furthermore, some of the biotemplated metal NC-based photosensitizers which also exhibit bright photoluminescence have been successfully applied for bioimaging and photodynamic cancer therapy in in vitro and in vivo studies [8,40,45], showing their great promise as effective biocompatible theranostic agents for nanomedicine [46]. In this study, we report a new synthesis strategy to systematically tune the singlet oxygen generation property of bimetallic NCs by alloying different amount of Au atoms into the BSA-protected Ag13NC via a thermodynamically favorable galvanic displacement reaction at room temperature. The resultant bimetallic nanomaterials were comprehensively characterized using various techniques, including UV–vis and photoluminenscence spectroscopy, transmission electron microscopy, X-ray photoelectron spectroscopy (XPS), energy-dispersive X-ray (EDX) spectroscopy, and matrix assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry. A chemical trapping method using 9,10-Anthracenediyl-bis(methylene) dimalonic acid (ABDA) as the 1O2 detection probe were employed to measure the SOG efficiency of the resultant BSA-protected alloy AuAgNCs, which was correlated to the amount of Au ions used in the replacement reaction and their photoluminescence properties. Interestingly, it was found that plasmonic gold nanoparticles with an estimated size of 60 nm were also formed during the photobleaching process to enhance the SOG efficiency of BSA-AuAg NCs on site, achieving an unprecedented rate of single oxygen generation (i.e., 45 times) higher than that of the monometallic BSA-Ag13NC photosensitizers. Presented below are the details of this study.
The protein-protected gold/silver alloy nanoclusters (AuAgNCs) were synthesized in a two-step procedure. BSA-protected Ag13NC was first prepared following our previously reported protocol [8]. In a typical synthesis, aqueous solutions of silver nitrate (AgNO3, 4 mL, 15 × 10−3 M) and bovine serum albumin (BSA, 4 mL, 74 mg mL−1) were mixed in a 20 mL glass vial containing 4 mL of ultrapure water under vigorous, constant stirring. After 2 min, a solution of sodium hydroxide (NaOH, 400 μL, 1 M) was added to the glass vial. Another 30 min later, aliquots of freshly prepared sodium borohydride solution (NaBH4, 300 μL, 11.2 × 10−3 M in 0.1 M NaOH) was added to the glass vial. The reaction proceeded for 1 h and the final product was collected and desalted with a cellulose dialysis tubing (MWCO of 6000). The purified sample was then used as precursor for the synthesis of alloy AuAgNCs via a galvanic replacement route. In a typical synthesis, a solution of purified BSA-Ag13NC (400 μL, 4 × 10−3 M) was mixed with varying volumes of HAuCl4 (15 × 10−3 M). The reaction solution was topped up to 1.2 mL with calculated amount of ultrapure water. After 3 h, the product was centrifuged (15,000 ×g, 45 min, 4 °C) in a Hettich Mikro 220R Centrifuge. The supernatant was transferred to an Amicon Ultra-0.5 Centrifugal Filter and centrifuged at 14,000 ×g for 30 min at 25 °C. The concentrated solute was recovered by reverse-spinning and topped up with ultrapure water to reach the initial concentration. The purified samples were stored in a 4 °C fridge for later use.
2.3. Characterization of BSA protein-protected AuAgNCs Photoabsorption UV–vis and photoemission spectra were recorded on a Shimadzu UV-2450 UV–visible spectrophotometer and a Tecan Infinite M200 Microplate Reader, respectively. Transmission Electron Microscopy (TEM) images and Energy-Dispersive X-ray (EDX) Spectroscopy results were obtained on a Philips CM300 Transmission Electron Microscope (High Resolution). X-ray Photoelectron Spectroscopy (XPS) study was done on a Theta Probe Angle-Resolved Xray Photoelectron Spectrometer. Matrix Assisted Laser Desorption Ionization (MALDI) Time-of-Flight (TOF) mass spectrometry study was done on a Bruker Daltonics Autoflex II TOF/TOF system. The MALDITOF samples were prepared by mixing 2 μL sample with 2 μL matrix solution. The matrix solution was prepared by dissolving 2,5-dihydroxybenzoic acid (DHB) solution to saturation in 50% acetonitrile.
2.4. Measurement of singlet oxygen generation by BSA-AuAgNCs Singlet oxygen generation property of the BSA-protected alloy nanoclusters (BSA-AuAgNCs) was studied by an optical method using 9,10-Anthracenediyl-bis(methylene) dimalonic acid (ABDA) as 1O2 probe. In a typical experiment, 200 × 10−6 M of the photosensitizer and 50 × 10−6 M of ABDA were mixed in a cuvette and buffered to 1 mL using ultrapure water. The absorption spectra of the mixture solution was measured prior to irradiation (denoted as 0 min) and after every 2–10 min up to 60 min of irradiation time. A halogen bulb (output: 100 W, 3200 K) was used as the white light source. Irradiation was stopped when the absorption peaks of ABDA are almost flat (A/ A0 < 5% twice consecutively) or when there is no longer any change in the absorption peak (A/A0 < 1% twice consecutively), whichever occurs first. To acquire a better representative of the 1O2 production rates, the overhead contribution of the BSA-protected AuAgNCs to the ABDA peak intensities was subtracted from each raw spectra reading. The overhead contribution was measured by replacing the 50 × 10−6 M ABDA with an equal volume of ultrapure water. The percentage change in peak intensity at 370 nm was then calculated using the subtracted values.
2. Experimental 2.1. Materials Gold (III) chloride solution (HAuCl4, 30% in dilute HCl), silver nitrate (AgNO3, 99.9999%), bovine serum albumin (BSA, 96%), sodium hydroxide (NaOH, 97%), sodium borohydride (NaBH4, 99.99%), 9,10Anthracenediyl-bis(methylene)dimalonic acid (ABDA, 90%), and 2,5dihydroxybenzoic acid (DHB, 98%) were purchased from SigmaAldrich. All reagents were used as received and without further purification. All glassware were washed with Aqua Regia (HCl:HNO3 volume ratio = 3:1) and rinsed with ethanol and ultrapure water. (Caution: Aqua Regia is a very corrosive oxidizing agent, which should be handled with great care.) Ultrapure water with a specific resistance of 18.2 MΩ was used throughout the experiment. 2
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(A) Galvanic displacement
Ag+
Au3+
BSA-RAuxAg(13-x)NC
BSA-Ag13NC 5000
10 nm
Absorbance
1.0 0.8
BSA-Ag13NC R = 12 R = 15 R = 18 R = 21 R = 24
(C) 4000
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Fig. 1. (A) Schematic illustration of the BSA-RAuxAg(13−x)NCs formed by galvanic replacement reaction. (B) UV–vis and (C) photoemission spectra (λex = 480 nm) of BSA-protected Ag13NC (dashed lines) and BSA-RAuxAg(13−x)NCs (solid lines) where R indicates the molar ratio of Au ions to AgNC. (Inset of B) TEM image of BSA24AuxAg(13−x)NCs. The red circles highlight the individual alloy nanoclusters. (Inset of C) Enlarged photoemission spectra of BSA-RAuxAg(13−x)NCs for R = 12, 15, 18, 21 and 24 respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3. Results and discussion
absorption spectrum depending on their composition and morphology. Due to the ultrasmall size of metal NCs (< 2 nm), the atoms per cluster are too few to support the characteristic LSPR peaks that are commonly observed for the gold nanoparticle (AuNP @ 520 nm [48,49]) and silver nanoparticle (AgNP@ 400 nm [50]). Thus, the absence of LSPR peaks of AuNP or AgNPs in Fig. 1B provides an indirect evidence to indicate the formation of bimetallic NCs. Transmission electron microscopy (TEM) was also used to further confirm the formation of BSA-protected alloy AuAgNCs. Inset of Fig. 1A shows the particle size of < 3 nm in diameter, which were in line with the UV–vis spectra that ruled out the formation of plasmonic Au or AgNPs with size larger than 5 nm. Furthermore, the as-synthesized BSA-AuAgNCs showed weak red emission under UV irradiation, which is the intrinsic property of metal NCs due to the quantum confinement effect. However, the photoluminescent (PL) intensity of the BSA-AuAgNCs decreased rapidly as the concentration of Au ions (or R ratio) increased (Fig. 1C). To better understand the composition of BSA-RAuxAg(13−x)NC (i.e., to determine the value of x), elemental analysis using X-ray Photoelectron Spectroscopy (XPS) and Energy-Dispersive X-ray (EDX) Spectroscopy have been conducted. Surprisingly, only Au4f signals were observed in the XPS spectra (Fig. S1, Supporting Information). Considering that XPS is a surface analysis technique with a typical depth of < 5 nm, there is a high chance that no Ag3d signal could be detected if most of the Ag were replaced by the Au ions on the surface of NCs. It is also possible that the BSA-RAuxAg(13−x)NC were piled up when they were dried on the substrate during the sample preparation process, leading to the inaccuracy of detection for Ag elements located in the core structure of alloy AuAgNCs. As such, we have proceeded to characterize the as-synthesized metallic nanoclusters using EDX, which is a technique with a typical analysis depth of 1–3 μm. While EDX results give an Au-to-Ag ratio of 1.35, 0.99 and 2.32 for BSARAuxAg(13−x)NCs with R = 15, 18 and 21, respectively; no Au and Ag
Fig. 1A shows the synthesis of BSA-protected gold/silver alloy nanoclusters through the galvanic replacement reaction of BSA-Ag13NC with gold precursor (Au ions) at room temperature. As the standard redox potential of AuCl4−/Au (0.99 V vs standard hydrogen electrode, SHE) is higher than that of Ag+/Ag (0.80 V vs SHE), this reaction is thermodynamically favorable and proceeds spontaneously [47]. The BSA-Ag13NC which contained 13 Ag atoms in a single nanocluster was first synthesized according to a previous reported method [8]. It was red brown in solution (under day light) and exhibited a shoulder peak at around 435 nm on the UV–vis spectrum (Fig. 1B, dashed line). Upon UV irradiation, the BSA-Ag13 NC showed red fluorescent with an emission peak located at 670 nm wavelength (Fig. 1C, dashed line). The protein-protected alloy AuAgNCs were then synthesized by adding a solution of gold precursor (HAuCl4) to the BSA-Ag13NC in various mole ratios of Au ions to AgNC. Upon addition of HAuCl4, the solutions turned murky but cleared up slightly after 3 h of reaction time. The final products (denoted as BSA-RAuxAg(13−x)NC where R is the molecular ratio of Au ions to 1 mol of BSA-Ag13NC initially used in the synthesis) were clear and colorless. For example, BSA-12AuxAg(13−x)NC represents the product formed by reacting 1 mol of AgNC with 12 times of Au ions (i.e., R = 12). Totally five samples were collected for further investigations, namely BSA-12AuxAg(13−x)NC, BSA-15AuxAg(13−x)NC, BSA-18AuxAg(13−x)NC, BSA-21AuxAg(13−x)NC and BSA24AuxAg(13−x)NC, respectively. As shown in Fig. 1B, UV–vis absorption spectra of all bimetallic NCs are featureless (solid lines). Disappearance of the shoulder peak at 435 nm as observed on the spectrum of Ag13NCs, suggesting successful oxidation of Ag atoms and incorporation of Au atoms into the alloy NCs for all samples. Typically, noble metal nanoparticles in the size range of 5 to 100 nm exhibit unique localized surface plasmon resonance (LSPR) peaks in the UV–visible 3
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Fig. 2. Time course absorption spectra of ABDA solution (50 × 10−6 M) in the presence of 200 μM of (A) BSA-Ag13NC, (B) BSA-12AuxAg(13−x)NC, (C) BSA15AuxAg(13−x)NC, (D) BSA-18AuxAg(13−x)NC, (E) BSA-21AuxAg(13−x)NC, and (F) BSA-24AuxAg(13−x)NC.
of using current techniques to determine the exact molecular formula of the NC samples tested in this study, the initial composition of each NC based on the R ratio is used throughout the article for the convenience of discussion. Next, the SOG properties of the as-synthesized BSARAuxAg(13−x)NCs were studied by a chemical trapping method using 9,10-Anthracenediyl-bis(methylene) dimalonic acid (ABDA) as the singlet oxygen (1O2) detection probe. As shown in Fig. 2, ABDA has four characteristic peaks at 342, 359, 378 and 400 nm in the absorption spectrum. When 1O2 is generated, ABDA will react with it to yield a steady state endoperoxide, leading to the disappearance of the aforementioned characteristic absorption peaks. This decrease in the absorption peak (ΔO.D.) is directly correlated to the amount of ABDA degraded and corresponded to the amount of 1O2 reacted. Therefore, 1O2 generation property of BSA-RAuxAg(13−x)NCs can be characterized by the decrease in the absorption peak of ABDA. Fig. 2(B–F) show that all
signals were detected for R = 12 and 24 (Figs. S2 to S4, Supporting Information). This could be due to the fact that the BSARAuxAg(13−x)NCs synthesized in this study are very small (< 3 nm) and protected by the bulky protein templates that are hard to be observed even under HR-TEM due to poor contrast, making it hard to determine the exact composition when selecting an area of interest in TEM for EDX analysis. Although mass spectrometry such as matrix assisted laser desorption ionization (MALDI) time-of-flight (TOF) is a powerful technique to identify the atomic composition of single metallic (Au or Ag) nanoclusters stabilised by thiol ligands, it is relatively difficult to assign the atomic composition for the bimetallic nanoclusters protected by the large protein template with unknown molecular formula. Furthermore, the molecular weight of two Ag atoms is very close to that of one Au atom, which compounds the difficulty in assigning the exact formula of the resulted alloy AuAgNCs based on the mass difference between BSA-RAuxAg(13−x)NCs and BSA-Ag13NC. Due to the limitations 4
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Fig. 3. Decomposition rate constants of ABDA in the presence of 200 μM of (A) BSA-Ag13NC, (B) BSA-12AuxAg(13−x)NC, (C) BSA-15AuxAg(13−x)NC, (D) BSA18AuxAg(13−x)NC, (E) BSA-21AuxAg(13−x)NC, and (F) BSA-24AuxAg(13−x)NC under white light irradiation.
reported herein possess unusually high 1O2 generation property. Several control experiments have also been conducted to confirm that the degradation of ADBA was due to the photo-induced generation of 1O2 by the BSA protein-protected alloy AuAgNCs. Firstly, it was observed that the BSA template itself could not degrade ABDA under white light irradiation for up to 30 min (Fig. S5A, Supporting Information). Secondly, ABDA would not degrade under dark condition even in the presence of BSA-RAuxAg(13−x)NCs (e.g., BSA-24AuxAg(13−x)), for a prolonged period of time (Fig. S5B, Supporting Information). This is in contrast to the rapid ABDA degradation (i.e., 4 min) in the presence of the same alloy NCs under white light treatment (Fig. 2F), showing the intrinsic SOG property of the as-synthesized BSA-RAuxAg(13−x)NCs. The degradation rate of ABDA in the presence of BSA-RAuxAg13NCs were calculated and compared. The natural logarithm of the inverse of
the characteristic absorbance peaks of ABDA eventually disappeared in a certain period of time in the presence of BSA-RAgxAu(13−x)NCs (R = 12, 15, 18, 21 and 24) under white light irradiation. This result indicates that the all the as-synthesized BSA-RAuxAg(13−x)NC are capable of generating 1O2 under white light irradiation. Additionally, it was discovered that the more Au ions were introduced to replace BSAAg13NC as precursor in forming alloy AuAgNCs, the faster the degradation rate of ABDA. For example, it took 50 min for the ABDA peaks to disappear completely for BSA-RAuxAg(13−x)NC with R = 12, followed by 45 min for R = 15, 10 min for R = 18, and only 4 min for R = 21 and 24, respectively. As a comparison with the original BSAAg13NC, the rate of ABDA degradation was much slower and about half of the ABDA was still remained intact after 1 h (Fig. 2A), suggesting that BSA-RAuxAg(13−x)NCs synthesized through galvanic replacement 5
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Fig. 4. (A) The 1O2 generation efficiency (square) of BSA-RAuxAg(13−x)NCs (R is molar ratio of Au ions to Ag13NC) is inversely correlated to their photoluminescence (PL) intensities (circle). (Insets) Digital photos of the BSA-RAuxAg(13−x)NC (R = 0, 12, 15, 18, 21 and 24, respectively; R = 0, i.e., BSA-Ag13NC) under UV light irradiation. (B) A simplified Jablonski diagram of BSA-RAuxAg(13−x)NCs upon photoexcitation in water.
Fig. 5. Time course study showing the in-situ formation of plasmonic AuNPs to enhance the SOG property of BSA-RAuxAg(13−x) alloy NCs. (A) UV–vis spectra of the mixture solution of BSA-24AuxAg(13−x)NC and ABDA under white light irradiation for 10 min (2 min of time interval). (B) UV–vis spectra of the mixture solution of BSA-24AuxAg(13−x)NC and ABDA under dark condition for 10 min. (C) UV–vis spectra of BSA-24AuxAg(13−x)NC alone (no ABDA) under white light irradiation for 10 min. (D) Schematic illustration of metal enhanced 1O2 generation by the photobleaching degradation of BSA-AuAgNCs to form large plasmonic AuNPs.
the original BSA-Ag13NC was only 0.0116 min−1 (Fig. 3A). The rate of SOG was as high as ~6 to 45 times for the BSA-protected alloy AuAgNCs as compared to the monometallic BSA-Ag13NC, suggesting the excellent SOG properties of the BSA-RAuxAg(13−x)NCs and their potency as effective water soluble biotemplated photosensitizers. Ultrasmall metallic NCs possess molecular-like properties such as photoluminescence due to the quantum confinement effect. Therefore, we also investigated the correlation of the intrinsic photoluminescence and SOG properties of the resulted BSA-RAuxAg(13−x) alloy NCs. It was discovered for the first that time that the SOG rate of the as-synthesized BSA-protected alloy AuAgNCs is inversely correlated to their photoluminescence (PL) intensities. As shown in Fig. 4A, the PL intensities of
relative absorbance (ln(A0/A)) was plotted against the reaction time (t), and the slope of linear fitted line (k) represented the degradation rate constants. As shown in Fig. 3(B–F), the rate constants of ABDA degradation are 0.0705 (R = 12), 0.0797 (R = 15), 0.254 (R = 18), 0.529 (R = 21), and 0.465 (R = 24) min−1 in the presence of BSARAuxAg(13−x) photosensitizer with different molar ratio R. It should be noted that ABDA was completely degraded in < 10 min for using BSA18AuxAg(13−x) as photosensitizer and 4 min for using both BSA21AuxAg(13−x) and BSA-24AuxAg(13−x)NCs, which curves have plateaued out at each respective time. These results show that the degradation rate of ABDA increases as the amount of Au in the precursor increases. In comparison, the degradation rate constant of ABDA with 6
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metallic molar ratios (R) were systematically studied with a chemical trapping method using ABDA as the 1O2 detection probe and UV–visible/fluorescent spectroscopy. It was found that the singlet oxygen generation rate of the bimetallic nanoclusters increased as their photoluminescence intensity decreased. The inverse correlation between the intrinsic photoluminescence and SOG property of the BSARAuxAg(13−x)NCs gives a clear evidence of the competition between the pathways of photosensitization of nearby medium to form reactive oxygen species (e.g., 1O2) and radiative decay of the excited photosensitizer to generate photoluminescence. The photobleaching degradation of BSA-RAuxAg(13−x)NCs to form large gold nanoparticles (AuNPs > 10 nm) has been identified and accounted for the very high rate of 1O2 generation of BSA-RAuxAg(13−x)NCs (~45 times of the monometallic BSA-Ag13NCs) due to plasmonic enhancement effect. This study not only reports a new methodology of making photosensitizers with excellent 1O2 generation property via a simple metal replacement approach, but also exemplifies the metal-enhanced ROS generation of a photosensitizer by the in situ formation of plasmonic gold nanoparticles, opening up new opportunities in designing advanced hybrid nanomaterials with complementary and/or enhanced functionalities in one pot.
all the BSA-RAuxAg13NCs were much lower than that of BSA-Ag13NC (< 20% that of the BSA-Ag13NC) despite of their higher SOG rates. Furthermore, when the PL intensity of these BSA-RAuxAg(13−x)NCs decreased by ~80% from R = 12 to 24, the corresponding ABDA degradation rate increased by > 5 times. According to the classic Jablonski diagram as shown in Fig. 4B, it is intuitive that when the more excited states of a photosensitizer are used to sensitize triplet oxygen, the fewer will remain to return to the ground states in the pathway of radiative decay since the total amount of excited states is a constant given that the light condition is fixed. Photoluminescence is used instead of fluorescence in this diagram because the lifetime of metal NCs could be at the order of a few nanoseconds to a few microseconds [8,30]. It has been observed previously that the degradation rate constant (k) of BSA-Au25NC with a higher PL quantum yield (10.5%) is only 3% of that BSA-Ag13NC with a much lower PL quantum yield (0.4%). However, the difference in metal species of the two NCs makes the aforementioned relation less direct. The tendency of delayed photoluminescence [51] has been suggested for the poor 1O2 generation efficiency despite the seemingly large population of triplet states in the BSA-Au25NC. Hence, it is more straightforward to establish correlation of the portion of excited states used for both photosensitization of triplet oxygen and photoluminescence, because the BSA-RAuxAg(13−x)NCs reported herein have a similar composition and were synthesized using the same methodology. Although full understanding of the unique 1O2 generation property of BSA-RAuxAg(13−x)NCs is yet to be elucidated, investigation of the optical property of BSA-RAuxAg(13−x)NC in the course of ABDA degradation in this study may shed some light on the enhanced 1O2 generation. As shown in Fig. 5A, UV–vis spectrum of the reaction mixture with BSA-24AuxAg(13−x) and ABDA under white light irradiation shows an absorption peak at ~550 nm. This peak which spans in the wavelengths of 400–800 nm increases gradually along with the irradiation time within 10 min, indicating the formation of plasmonic AuNPs [52]. TEM image of the reaction mixture further confirms the size of as-formed AuNPs is ~60 nm (Fig. S6). It has been reported that plasmonic nanoparticles such as AuNPs and AgNPs are able to enhance the 1O2 generation efficiency of their nearby photosensitizer, analogous to the metal-enhanced photoemission phenomenon [53–55]. It is thus hypothesized that the photobleaching of BSA-RAuxAg(13−x)NCs leads to their gradual decomposition to form large AuNPs in the proximity of BSA-RAuxAg(13−x)NCs. These AuNPs serve as antennas to absorb more incident light due to the strong LSPR. The enhanced localized electric field of AuNPs then amplifies the 1O2 generation property of BSARAuxAg(13−x)NCs (Fig. 5D). As such, UV–vis spectrum of the BSA24AuxAg(13−x)NC and ABDA mixture in the dark conditions were also examined. As shown in Fig. 5B, the characteristic LSPR peaks of AuNPs were not observed for the same period of time, suggesting that ABDA alone does not react with BSA-24AuxAg(13−x)NC to form large AuNPs wherein the major contribution of ABDA is to trap the generated 1O2. As a comparison, a small peak at 540 nm was observed in the UV–vis spectrum of BSA-24AuxAg(13−x)NC under white light irradiation for 10 min (Fig. 5C), which was not unexpected as photobleaching is deemed as an intrinsic property of a photosensitizer while prolonged irradiation could destabilize the alloy AuAgNCs to form large AuNPs. In addition, this process will be greatly accelerated in the presence of ABDA, as trapping of the generated 1O2 by ABDA could further drove the destabilization and conversion of BSA-24AuxAg(13−x)NC as shown in Fig. 5A.
Declaration of competing interest None. Acknowledgement The authors acknowledge funding and support from the Institute of Materials Research and Engineering, Agency of Science, Technology and Research (A*STAR), under Bioinspired Approaches to Biomimetic Materials program (IMRE/17-1P1404), in collaboration with Newcatle University (RSA/CCEAMD5010) and Nanyang Technological University, Singapore. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.msec.2019.110525. References [1] M.C. DeRosa, R.J. Crutchley, Photosensitized singlet oxygen and its applications, Coord. Chem. Rev. 233-234 (2002) 351–371. [2] H. Wu, W. Chen, H. Yang, S. Wang, S.T. Lim, W. Yao, J. Guo, T. Li, M.W. Wong, D. Huang, Improved synthesis dimethylhomoecoerdianthrone (HOCD) and its functionalization through facile amination reactions, Dyes Pigments 130 (2016) 154–161. [3] S. Wang, R. Gao, F. Zhou, M. Selke, Nanomaterials and singlet oxygen photosensitizers: potential applications in photodynamic therapy, J. Mater. Chem. 14 (2004) 487–493. [4] A.E. O’Connor, W.M. Gallagher, A.T. Byrne, Porphyrin and nonporphyrin photosensitizers in oncology: preclinical and clinical advances in photodynamic therapy, Photochem. Photobiol. 85 (2009) 1053–1074. [5] R. Bonnett, Photosensitizers of the porphyrin and phthalocyanine series for photodynamic therapy, Chem. Soc. Rev. 24 (1995) 19–33. [6] P. Zhang, W. Steelant, M. Kumar, M. Scholfield, Versatile photosensitizers for photodynamic therapy at infrared excitation, J. Am. Chem. Soc. 129 (2007) 4526–4527. [7] S. Brown, Clinical antimicrobial photodynamic therapy: phase II studies in chronic wounds, J. Natl. Compr. Cancer Netw. 10 (2012) S–80-S-83. [8] Y. Yu, J. Geng, E.Y.X. Ong, V. Chellappan, Y.N. Tan, Bovine serum albumin proteintemplated silver nanocluster (BSA-Ag13): an effective singlet oxygen generator for photodynamic cancer therapy, Adv. Healthcare Mater. 5 (2016) 2528–2535. [9] X.T. Zheng, Y.C. Lai, Y.N. Tan, Nucleotide-derived theranostic nanodots with intrinsic fluorescence and singlet oxygen generation for bioimaging and photodynamic therapy, Nanoscale Adv 1 (2019) 2250–2257. [10] Y. Yu, Q. Yao, Z. Luo, X. Yuan, J.Y. Lee, J. Xie, Precursor engineering and controlled conversion for the synthesis of monodisperse thiolate-protected metal nanoclusters, Nanoscale 5 (2013) 4606–4620. [11] P. Maity, S. Xie, M. Yamauchi, T. Tsukuda, Stabilized gold clusters: from isolation toward controlled synthesis, Nanoscale 4 (2012) 4027–4037. [12] R. Jin, Quantum sized, thiolate-protected gold nanoclusters, Nanoscale 2 (2010) 343–362.
4. Conclusion In conclusion, a series of ultrasmall BSA-protected gold/silver alloy (core size < 2 nm) with unique photoluminescence and singlet oxygen generation (SOG) properties have been successfully synthesized by galvanic replacement between BSA-Ag13NCs and gold ions precursor (HAuCl4). The as-synthesized BSA-RAuxAg(13−x)NCs of different 7
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