Article pubs.acs.org/ac
Sensitive and Selective Detection of Copper Ions with Highly Stable Polyethyleneimine-Protected Silver Nanoclusters Zhiqin Yuan,† Na Cai,† Yi Du, Yan He,* and Edward S. Yeung College of Chemistry and Chemical Engineering, College of Biology, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, Hunan, People’s Republic of China. S Supporting Information *
ABSTRACT: Copper is a highly toxic environmental pollutant with bioaccumulative properties. Therefore, sensitive Cu2+ detection is very important to prevent over-ingestion, and visual detection using unaugmented vision is preferred for practical applications. In this study, hyperbranched polyethyleneimine-protected silver nanoclusters (hPEI-AgNCs) were successfully synthesized using a facile, one-pot reaction under mild conditions. The hPEI-AgNCs were very stable against extreme pH, ionic strength, temperature, and photoillumination and could act as sensitive and selective Cu2+ sensing nanoprobes in aqueous solutions with a 10 nM limit of detection. In addition, hPEI-AgNCs-doped agarose hydrogels were developed as an instrument-free and regenerable platform for visual Cu2+ and water quality monitoring.
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materials for sensitive and selective Cu2+ detection need to be developed. Recently, fluorescent noble metal (e.g., Au, Ag, and Pt) nanoclusters that are less than 2 nm in size and consist of several to hundreds of atoms have attracted attention due to their unique physical and optical properties.23−28 These noble metal nanoclusters exhibit excellent photostability and large Stokes shifts, making them a powerful alternative to fluorescent dye molecules. Among them, silver nanoclusters (AgNCs) displaying bright fluorescence and good biocompatibility have gathered wide research interest in the past decade.29−32 Considerable efforts have been dedicated to the syntheses of water-soluble AgNCs using various templates/additives (e.g., polyelectrolytes and thiolates). Red-emitting AgNCs were synthesized using poly(methacrylic acid) as a template under UV irradiation.33 Seven-atom AgNCs were prepared using 2,3dimercaptosuccinic acid or mercaptosuccinic acid as stabilizing ligand.34,35 In addition to polyelectrolytes and thiolates, biomolecules, such as DNA, were also exploited for the preparation of AgNCs.36−38 These DNA-encapsulated AgNCs (DNA-AgNCs) are highly fluorescent with tunable emission wavelengths; they have been utilized to sense metal ions,39 DNA,40 and protein.41 However, some of these AgNCs are not stable toward high ionic strength or extreme pH, which restricts their practical applications. Because they are amine-rich, polyamines may serve as effective stabilizing agents during the synthesis and surface
opper is an essential transition metal element for human health and serves as a critical cofactor for many enzymes because it can undergo a redox-active Cu(I)/Cu(II) conversion.1,2 However, copper is also highly toxic toward organisms when presented in excess because it may promote the generation of reactive oxygen species (ROS).3 ROS interfere with cellular signaling, cause damage to cell structures, or induce apoptosis.4−7 Gastrointestinal disturbance, as well as liver or kidney damage, may also be induced with high concentration of Cu2+.8 Additionally, copper accumulation in the neuronal cytoplasm might cause Alzheimer’s, Parkinson’s, and amyotrophic lateral sclerosis diseases.9,10 The U.S. Environmental Protection Agency (EPA) limits the Cu2+ levels in drinking water to 1.3 mg/L or ∼20 μM. Because Cu2+containing materials are widely used in electronic device production and industrial and agricultural processes, Cu2+ contamination is a continuous problem. Therefore, sensitive and selective detection of Cu2+ in environmental and biological samples is required. Toward this goal, many techniques have been developed for Cu2+ detection, such as atomic absorption spectrometry,11 resonance scattering spectrometry,12 inductively coupled plasma mass spectroscopy,13 and fluorimetric spectrometry.14,15 In particular, fluorescence-based detection techniques have attracted wide interest because they exhibit high sensitivity and strong interference rejection. Many chemosensors using organic fluorophores have been developed by modulating photoinduced electron transfer, internal charge transfer, or fluorescence energy transfer processes based on the paramagnetic properties of Cu2+.16−22 These organic probes often provide admirable selectivity; however, their preparation usually requires specialized synthetic skills and complicated purification procedures. Therefore, accessible fluorescent © XXXX American Chemical Society
Received: July 15, 2013 Accepted: November 4, 2013
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and Fe3+ were prepared from KCl, NaCl, CaCl2, MgCl2, CrCl3, CuCl2, Zn(Ac)2, NiCl2, Co(Ac)2, AgNO3, CdCl2, Mn(Ac)2, Hg(NO3)2, Pb(Ac)2, and FeCl3, respectively. Aqueous solution of F−, Cl−, Br−, I−, Ac−, citrate3−, CN−, EDTA2−, N3−, NO3−, H2PO4−, S2−, S2O32−, SCN−, SDS−, and SO42− were prepared from NaF, NaCl, NaBr, KI, NaAc, trisodium citrate, disodium EDTA, NaN3, NaNO3, NaH2PO4, Na2S, NaS2O3, KSCN, SDS, and Na2SO4, respectively. Buffer solutions from pH 2 to 11 were prepared according to standard protocols, and the pH values were measured with a benchtop pH meter (Orion plus, Thermo-Fisher, U.S.A.). The concentrations of all buffer solutions were 0.1 M, including glycine−HCl (pH 2−3), HAc−NaAc (pH 4−6), NaH2PO4−Na2HPO4 (pH 7−8), and NaHCO3−Na2CO3 buffers (pH 9−11). All glassware was cleaned with fresh aqua regia (HCl/HNO3 = 3:1, v/v) before use. Synthesis of hPEI-AgNCs. In a typical assay, 2.5 μmol of hPEI and 2.5 μmol of AgNO3 were dissolved in 10 mL of ultrapure water and stirred for 2 h to facilitate complexation between Ag+ and the amine ligands. Subsequently, the pH was adjusted to 5 with HAc, and 30 μmol of AA was added to the solution before it was stirred continuously for 2 days. All reactions were carried out at room temperature. Characterization. The fluorescence spectra of the AgNCs were obtained with a F-7000 fluorescence spectrophotometer (Hitachi, Japan). The UV absorption spectra of the AgNCs were obtained with a UV-1800 spectrophotometer (Shimadzu, Japan). High-resolution transmission electron microscopy (HRTEM) images were collected with a Tecnai F20 highresolution transmission electron microscope (FEI, U.S.A.). Fluorescence lifetime decays were measured using an Edinburgh FL 900 photocounting system (Livingston, U.K.). The X-ray photoelectron spectroscopy (XPS) measurements were preformed using a K-Alpha 1063 instrument (Thermo Fisher, U.K.), and all binding energies were calibrated by C1s (284.6 eV) as the reference energy. Selectivity and Sensitivity Measurements. Copper dichloride (CuCl2) was used as the Cu2+ source during sensitivity studies. A CuCl2 stock solution (0.1 M) was prepared, and various concentrations were obtained by serial dilution of the stock solution. To detect the Cu2+, solutions with different Cu2+ concentrations were added to 1 mL of 10 mM Ac buffer of pH 4 diluted hPEI-protected AgNCs solution. The fluorescence spectra were collected by using a F-7000 spectrophotometer. The following metal ions were used to evaluate the selectivity of the AgNCs: K+, Na+, Ca2+, Mg2+, Cr3+, Cu2+, Zn2+, Ni2+, Co2+, Ag+, Cd2+, Mn2+, Hg2+, Pb2+, and Fe3+. Various anions were also used to study the selectivity of AgNCs: F−, Cl−, Br−, I−, Ac−, citrate3−, CN−, EDTA2−, N3−, NO3−, H2PO4−, S2−, S2O32−, SCN−, SDS−, and SO42−. Thermostability and Reversibility Test. The solution temperature was controlled with an Eppendorf thermal cycler; after a 10 min equilibration, the fluorescence spectra were collected with a F-7000 spectrophotometer. Preparation of hPEI-AgNCs-Doped Agarose Hydrogel. The 1% hPEI-AgNC-doped agarose hydrogel was prepared as follows: 20 mg of agarose was completely dissolved in 2 mL of water under microwave heating. When the solution cooled to 70 °C after removal from the heater, 20 μL of AgNC solution was added with rapid stirring for 2 min. Subsequently, the mixture was kept static for 1 h to form the hydrogel. Visual Cu2+ Detection with hPEI-AgNCs-Doped Agarose Hydrogel. The hPEI-AgNC-doped agarose hydrogel was
modification of nanomaterials. In particular, hyperbranched polyethyleneimine (hPEI) is a positively charged polyamine that contains primary, secondary, and tertiary amine groups and has been used as polyvalence ligand to modify and stabilize metallic or metallic oxide/sulfide nanocrystals because the amine groups interact strongly with metal atoms. ZnO nanorods,42 Au nanoparticles,43 Ag nanoparticles,44 and CdS quantum dots45 have been successfully synthesized with hPEI in the reactant mixture. Similarly, hPEI can also provide scaffolds that interact with the Ag atoms during the formation of AgNCs. For instance, Qu et al. synthesized blue emissive hPEI (MW 10K) templated AgNCs by using formaldehyde as the reducing agent.46−48 However, these AgNCs are not stable under low pH and highly ionic conditions. Hydrogels are networks of hydrophilic polymers cross-linked through physical, ionic, or covalent interactions; these materials possess many attractive physical properties. Hydrogels are particularly useful when constructing visual detection platforms because they have negligible background colors and fluorescence emissions, as well as large loading capacities and controllable shapes. Because instrument-free and operationally simple tools are advantageous during practical analysis, using hydrogels in visual sensing applications has been extensively explored recently. For example, DNA-functionalized hydrogels were developed as platforms for visual detection of heavy metal ions (e.g., Hg2+ and Pb2+) and adenosine.49−51 Recently, fluorescent AgNCs-doped DNA hydrogels were fabricated by directly reducing silver ions dispersed in DNA hydrogels52 and have been utilized for visual Hg2+ sensing.53 In this study, we synthesized highly stable hPEI-AgNCs with bluish-green emission using a facile, one-pot method. We demonstrated that the AgNCs might be viable, highly selective, and sensitive fluorescent probes for Cu2+ detection with a 10 nM limit of detection. In addition, the as-prepared hPEIAgNCs were completely water-soluble and remarkably stable under extreme pH, ionic strengths, and temperature conditions, facilitating the development of a regenerable visual sensing platform for Cu2+ detection based on hPEI-AgNCs-doped agarose hydrogels. The strongly fluorescent hPEI-AgNCsdoped hydrogels almost completely stopped emitting when immersed in 5 μM Cu2+ solution; this concentration is lower than the EPA’s upper limit of ∼20 μM. Therefore, this simple sensing strategy might be used to monitor the Cu2+ levels in water samples, such as river, tap, lake, and spring water.
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EXPERIMENTAL SECTION Chemicals. Branched polyethyleneimine (MW 25K) and agarose were purchased from Sigma-Aldrich (Milwaukee, U.S.A.). Branched polyethyleneimine (MW 10K, 1.8K, and 0.6K) were obtained from Alfa Aesar (Heysham, U.K.). Silver nitrate (AgNO3), ascorbic acid (AA), sodium hydroxide (NaOH), acetic acid (HAc), sodium borohydride (NaBH4), concentrated nitric acid (HNO3), hydrochloric acid (HCl), and other chemicals were obtained from Sinopharm Chemical Reagent Corporation (Shanghai, China). Glutathione (GSH), cysteine (Cys), mercaptoethyl amine (MEN), mercaptoacetic acid (MAA), and D-penicillamine (DPA) were obtained from Beijing Dingguo Changsheng Biotechnology Corporation (Beijing, China). All chemicals were used without further purification. Ultrapure water was obtained using a Milli-Q integral water purification system purchased from Millipore Corporation (Billerica, U.S.A.). Solutions of K+, Na+, Ca2+, Mg2+, Cr3+, Cu2+, Zn2+, Ni2+, Co2+, Ag+, Cd2+, Mn2+, Hg2+, Pb2+, B
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Scheme 1. Synthetic Strategy of hPEI-AgNCs
Figure 1. (a) UV−vis absorption spectrum, (b) fluorescence excitation (black) and emission (blue) spectra, and (c) HRTEM image of the asprepared hPEI-AgNCs.
spectrometry, steady-state and time-resolved fluorescence spectrometry, HRTEM, and XPS. The pH was adjusted to become weakly acidic or neutral before adding the reducing agent because the original pH was approximately 9 due to the abundant amine motifs in the hPEI molecules. Under basic conditions, hPEI may interact with Ag+ to form stable hPEI− Ag+ complexes that hinder the reduction of Ag+. Even with a strong reducing agent, such as NaBH4, only large nonfluorescent silver nanoparticles formed at pH 9. The AgNC formation reaction was successfully carried out at pH 5. After AA reduction, the solution turned from colorless to bright yellow, and strong bluish-green emission was easily observed under UV illumination. None of the reactants, including the hPEI-Ag+ complex, had a similar fluorescence profile, suggesting that fluorescent hPEI-AgNCs were formed. From the absorption spectra of the solution, a visible absorption band centered at 361 nm was observed and assigned to the hPEIAgNCs; a surface plasmon resonance absorption peak
immersed in 100 mL water samples containing varied concentrations of Cu2+; and the fluorescence was visually observed under a hand-held UV lamp. To regenerate the hydrogel, the Cu2+-adsorbed AgNC-doped agarose hydrogels were immersed in 100 mL of 1 mM EDTA. All experiments were conducted in 10 mM Ac buffer of pH 4.
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RESULTS AND DISCUSSION Synthesis and Characterization of hPEI-AgNCs. It is well-known that the hPEI is synthesized by ring-opening polymerization of aziridine. Although the degree of branching can hardly be controlled and the purchased 25K hPEI has a large molecular weight distribution ranging from 20 000 to 30 000, the polymer contains ample amine motifs and can interact and stabilize many metal ions, making it a good template and protection agent for synthesis of metal nanocomplexes. The hPEI-protected AgNCs (hPEI-AgNCs) were synthesized in one pot, as illustrated in Scheme 1, and characterized by UV−vis C
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Figure 2. (a) Relative fluorescence intensity (F/F0) of hPEI-AgNCs at different pH values. (b) F/F0 of hPEI-AgNCs after adding various concentrations of NaCl in both pH 7.4 and pH 4 solutions. (c) Reversible temperature dependence of F/F0 of hPEI-AgNCs during thermocycling processes. (d) Fluorescence emission spectra of hPEI-AgNCs upon addition of 1 mM thiolates. (e) Relative fluorescence intensity variation of hPEIAgNCs as a function of time under 361 nm light illumination.
hPEI-AgNCs were mainly composed of Ag0.55 The percentage of Ag+ was calculated to be ∼11% by fitting the Ag 3d XPS spectrum with two components. Because the NaBH4 treated AgNCs had a much higher quantum yield (2.8%), all of the subsequent experiments were performed with NaBH4-treated AgNCs. When utilizing the hPEI-AgNCs for practical sensing applications, the material must be water-soluble and stable toward ambient environments. The hPEI-AgNCs are inherently water-soluble because they include abundant amine groups in the hPEI structures. Due to the strong interaction between amine groups, the Ag atoms, and the hyperbranched structure of hPEI molecules, we believe that the as-prepared hPEIAgNCs might possess enhanced stability toward extreme pH or high ionic strengths in solution. As presented in Figure 2a, the fluorescence intensities at 490 nm demonstrated little change from pH 2 to 11, indicating the as-prepared hPEI-AgNCs were quite stable toward different pH values. Meanwhile, the fluorescence intensities of the hPEI-AgNCs in solutions with different NaCl concentrations were almost the same as the solutions without NaCl (pH 4 or pH 7.4); the emission remained greater than 97%, even in 1 M NaCl solution (Figure 2b), suggesting that the hPEI-AgNCs were highly stable toward highly ionic conditions. Interestingly, when the temperature increased from 20 to 75 °C, the fluorescence intensity of the hPEI-AgNCs decreased by ∼one-third, but completely recovered after returning to 20 °C; this process was repeatable (Figure 2c), suggesting that the hPEI-AgNCs were thermally stable. The above fluorescence intensity measurement results were further supported by the fluorescence decay measurements. As shown in Supporting Information Table S1, the fluorescence lifetime values had no obvious change at different
appearing at between 400 and 500 nm was not observed, indicating the absence of any large silver nanoparticles (Figure 1a).54 The fluorescence spectra revealed an excitation peak centered at 361 nm and an emission peak centered at 490 nm (Figure 1b); the hPEI-AgNC fluorescence quantum yield was calculated to be ∼1.5% by using quinine sulfate as the reference. Time-resolved fluorescence lifetime measurements revealed two components at 2.3 ns (63.6%) and 8.7 ns (36.4%). To further verify the formation of hPEI-AgNC, HRTEM images were obtained. As displayed in Figure 1c, the asprepared hPEI-AgNCs had a spherical shape and uniform size distribution; the hPEI-AgNC diameters were 1.8 ± 0.2 nm. Therefore, the hPEI-AgNCs were successfully synthesized using a one-pot reaction. The large Stokes shift (∼130 nm) exhibited by the hPEI-AgNCs implied that they might be excellent fluorescent nanoprobes for chemo/biosensing and imaging. Further experiments indicated that the formation of the fluorescent hPEI-AgNCs might be affected by several factors, such as pH, Ag+/hPEI molar ratio, and AA/Ag+ molar ratio. A systematic survey was performed to optimize these experimental conditions. The strongest hPEI-AgNC emission was achieved when the solution pH was 5, the molar ratio between Ag+ ions and the hPEI polymers was 1:1, and the AA/Ag+ molar ratio was 12:1 (Supporting Information Figure S1). To determine the oxidation state of the hPEI-AgNCs, excess NaBH4 was added and the fluorescence intensity doubled, indicating that some Ag+ remained on the as-prepared AgNC surface (Supporting Information Figure S2). To further investigate the oxidation state of the silver atoms, XPS measurements were performed. As can be seen in Supporting Information Figure S3, two binding energy peaks of Ag 3d were centered at 368.1 and 374.1 eV, respectively, suggesting the D
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For the sensitivity evaluation, emission spectra of the hPEIAgNCs solution were recorded after adding different Cu2+ concentrations. Figure 4 indicates that increasing the Cu2+
pH or NaCl concentrations. They decreased a little at elevated temperature but recovered after cooling. Moreover, the addition of thiolates (1 mM) had almost no effect on the fluorescence of hPEI-AgNCs (Figure 2d), though too high concentration of Cys (50 mM) did lead to some intensity decrease (Supporting Information Figure S4). Finally, their fluorescence intensities have no obvious decrease after 1 h of light illumination at 361 nm (Figure 2e), suggesting excellent photostability of hPEI-AgNCs. The high stability of the asprepared hPEI-AgNCs under various conditions made them remarkably different from the 10K-hPEI-templated AgNCs reported by Qu et al. (Supporting Information Figure S5), which were synthesized by using formaldehyde as the reducing agent and were sensitive to pH47 and halidic ions.46 Such extraordinary stability might arise from the shielding effect of the protective hPEI layer, whose amine groups are preserved after AA reduction of Ag+ at pH 5. To confirm that, we synthesized AgNCs with hPEIs of different molecular weights (MW = 0.6K, 1.8K, 10K, and 25K). As shown in Supporting Information Figure S6, the fluorescence intensity of AgNCs increased with the MW of hPEI, implying that high molecular weight hPEI provided good protection to AgNCs and improved their stability. Taken together, our data suggest that these highly stable hPEI-AgNCs could be utilized for practical Cu2+ sensing, even under extreme conditions. Cu2+ Sensing in Aqueous Media. After adding Cu2+ to the hPEI-AgNC solution, the strong bluish-green emission was noticeably diminished. To examine the specificity of the copper(II)’s fluorescence quenching behavior, the fluorescence response of the hPEI-AgNCs toward various metal ions including K+, Na+, Ca2+, Mg2+, Cr3+, Cu2+, Zn2+, Ni2+, Co2+, Ag+, Cd2+, Mn2+, Hg2+, Pb2+, and Fe3+ was examined under the same conditions; the final concentration of the ions was 20 μM. It can be seen that only Cu2+ caused dramatic fluorescence quenching (Figure 3), and the best selectivity occurred at pH 4
Figure 4. Fluorescence emission spectra of hPEI-AgNCs after adding various concentrations of Cu2+ (from top to bottom: 0, 0.01, 0.05, 0.17, 0.27, 0.52, 0.77, 1.7, 2.7, 7.7, 17.7, and 27.7 μM). Inset: plots of intensity ratio (F0/F) vs the concentrations of Cu2+.
concentration gradually quenches the hPEI-AgNCs fluorescence emission (361 nm excitation), but no obvious changes in the maximum emission wavelength or spectral shape were observed. The intensity ratio F0/F displays a good linear relationship (R2 = 0.993) versus Cu2+ concentration ranging from 10 nM to 7.7 μM (Figure 4 inset) and was easily described by the Stern−Volmer equation, F0/F = 1 + Ksv[Q], where F0 and F are the fluorescence intensity of the hPEIAgNCs at 490 nm in the absence and presence of Cu2+, Ksv is the Stern−Volmer fluorescence quenching constant, and [Q] is the concentration of Cu2+. Ksv was calculated to be 3.1 × 105 M−1 by linear regression of the plot. The quantized limit of detection (LOD) was 10 nM. It has been reported that the fluorescence of hPEI-capped carbon quantum dots could be quenched by Cu2+ due to the inner filter effect of cupric amine complexes,56 as the cupric amine coordinates may quench the fluorescence of dye molecules.57 Therefore, we speculate that the Cu2+-mediated fluorescence quenching of the hPEI-AgNCs was induced by Cu2+ binding to the hPEI protection layer and subsequent energy transfer from the AgNCs to the cupric amine coordinates. This assumption was supported by the following facts: after adding Cu2+ to the AgNCs solution, the solution turned blue with the appearance of a broad absorption band in the 500−1000 nm range and a small shoulder at 300 nm (Supporting Information Figure S10a), indicating the formation of cupric amine coordinates.56 Except for its original emission peak centered at 490 nm, no extra emission peaks appeared in the red and near-infrared wavelength ranges (Supporting Information Figure S10b), implying no formation of fluorescent complexes or coordinates. Furthermore, fluorescein-quenching tests with Cu2+ were conducted in both the absence and presence of hPEI, suggesting that the cupric amine coordinates have a strong quenching efficiency (Supporting Information Figure S11). In addition, fluorescence quenching experiments with hPEI-AgNCs were performed with the addition of excess free hPEI. As illustrated in Supporting Information Figure S12, free hPEI reduced the fluorescence quenching. Some Cu2+ may have interacted with the amine motifs in the free hPEI molecules, causing lower quenching efficiency and suggesting
Figure 3. Relative fluorescence intensity (F/F0) of hPEI-AgNCs after adding various metal ions in the absence or presence of Cu2+ in pH 4 solution (from 1 to 16: blank, K+, Na+, Ca2+, Ag+, Mg2+, Co2+, Ni2+, Mn2+, Zn2+, Cd2+, Pb2+, Cr3+, Hg2+, Al3+, and Fe3+).
(Supporting Information Figure S7). At this pH, anions could not affect the fluorescence (Supporting Information Figure S8), and the efficient fluorescence response toward Cu2+ was fundamentally preserved in the presence of 150 mM of NaCl (Supporting Information Figure S9). The high selectivity of the hPEI-AgNCs indicates that they could be used to sense Cu2+ in complex systems. All subsequent experiments were performed at pH 4. E
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Scheme 2. Schematic Illustration of hPEI-AgNCs-Based Nanoprobes for the Detection of Cu2+
that the distance between the AgNC and cupric amine coordinates determines the quenching capabilities. Moreover, Cu2+-mediated hPEI-AgNC fluorescence quenching was reversible. After adding EDTA, the fluorescence of the hPEIAgNCs was effectively recovered (Supporting Information Figure S13), implying that the binding capacity of amine groups for Cu2+ caused the quenching activity. Therefore, the energy transfer between the hPEI-AgNCs and the cupric amine complexes led to the observed fluorescence quenching (Scheme 2). Because the assay had high sensitivity and selectivity, we can conclude that the hPEI protective layer on the AgNC surface played at least two significant roles: (1) to form coordinates with Cu2+ and quenching the AgNC fluorescence, and (2) to prevent other metal ions, anions, and small molecules from approaching the AgNC surface. Visual Cu2+ Detection Using hPEI-AgNCs-Doped Agarose Hydrogels. Because of the remarkable stability of the hPEI-AgNCs under complicated and extreme conditions, we exploited them for visual detection of Cu2+ using naked eyes, which is preferable for practical applications. Immobilizing the hPEI-AgNCs into hydrogels is a logical solution since these materials have high loading capacity and low background fluorescence. The hyperbranched structure and large molecular weight of hPEI could restrict the diffusion and prevent the release of the 1.8 nm hPEI-AgNCs from the hydrogels. And the accumulative properties of functional hydrogels allow them to be adapted to visual detection of Cu2+. For this purpose, we prepared hPEI-AgNCs-doped agarose hydrogels. Capitalizing on the excellent stability of hPEI-AgNCs toward high temperatures, the heating process during the hydrogel preparation has no effect on the optical properties of the AgNCs and the transparent agarose hydrogels emitted strong fluorescence under UV irradiation, suggesting that the hPEIAgNCs and hydrogels were successfully combined. To evaluate the performance of the hPEI-AgNCs-doped agarose hydrogels for Cu2+ detection, the hydrogels were immersed into Cu2+ solutions of various concentrations at pH 4. When the Cu2+ concentration increased from 0 to 5 μM, the optically transparent hydrogels gradually turned blue, indicating the adsorption of Cu2+ and the formation of cupric amine coordinates (Figure 5a); the fluorescence of the hydrogels under a hand-held UV lamp completely disappeared after 5 μM Cu2+ treatment (Figure 5b). Because the EPA maximum Cu2+ allowance is ∼20 μM, these hPEI-AgNCs-doped agarose hydrogels are applicable for instrument-free visual Cu 2+ detection and water quality control under practical conditions.
Figure 5. (a) Visual detection of Cu2+ using hPEI-AgNCs-doped agarose hydrogels under visible light (from I to IV: 0, 0.05, 0.5, 5 μM). (b) Corresponding photographs under 365 nm UV light. (c) Fluorescence regeneration results for quenching with Cu2+ after adding EDTA.
As far as we know, this is the first example of visual Cu2+ detection utilizing hPEI-AgNCs-doped hydrogels. To serve as an environmentally friendly platform for visual Cu2+ detection, the functional hydrogels must be regenerable. Otherwise, the spent materials have to be disposed and fresh functional hydrogels must be constructed. The regeneration capability of the Cu2+-accumulative hydrogels was evaluated using EDTA as an effective extraction agent. The fluorescence of the Cu2+-accumulative hydrogels recovered within a few hours (Figure 5c), suggesting desorption of Cu2+ and regeneration of the hPEI-AgNCs-doped agarose hydrogels. Subsequently, the fluorescence was fully quenched upon readdition of Cu2+, indicating that the hPEI-AgNCs-doped hydrogels were recyclable. Therefore, the hPEI-AgNCs-doped agarose hydrogels could serve as an instrument-free and regenerable fluorescent platform for visual Cu2+ detection. To demonstrate the practical applicability of this platform, several environmental water samples were tested, including river, lake, tap, and spring water. These water samples were successfully cleaned, and the remaining Cu2+ was less than 5 μM (Supporting Information Figure S14), suggesting that the F
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(12) Guo, Y.; Wang, Z.; Qu, W.; Shao, H.; Jiang, X. Biosens. Bioelectron. 2011, 26, 4064−4069. (13) Becker, J. S.; Matusch, A.; Depboylu, C.; Dobrowolska, J.; Zoriy, M. V. Anal. Chem. 2007, 79, 6074−6080. (14) Luan, C. L.; Li, Y. Q.; Yang, J.; Yang, J. G.; Zhong, X. Q. Chin. J. Anal. Chem. 2001, 29, 1083−1085. (15) Chan, Y. H.; Chen, J. X.; Liu, Q. S.; Wark, S. E.; Son, D. H.; Batteas, J. D. Anal. Chem. 2010, 82, 3671−3678. (16) Que, E. L.; Chang, C. J. J. Am. Chem. Soc. 2006, 128, 15942− 15943. (17) Mei, L.; Xiang, Y.; Li, N.; Tong, A. J. Talanta 2007, 72, 1717− 1722. (18) White, B. R.; Holcombe, J. A. Talanta 2007, 71, 2015−2020. (19) Turel, M.; Duerkop, A.; Yegorova, A.; Scripinets, Y.; Lobnik, A.; Samec, N. Anal. Chim. Acta 2009, 644, 53−60. (20) Ciesienski, K. L.; Hyman, L. M.; Derisavifard, S.; Franz, K. J. Inorg. Chem. 2010, 49, 6808−6810. (21) Elanchezhian, V. S.; Kandaswamy, M. Inorg. Chem. Commun. 2010, 13, 1109−1113. (22) Lin, W. Y.; Long, L. L.; Chen, B. B.; Tan, W.; Gao, W. S. Chem. Commun. 2010, 46, 1311−1313. (23) Zheng, J.; Nicovich, P. R.; Dickson, R. M. Annu. Rev. Phys. Chem. 2007, 58, 409−431. (24) Shang, L.; Dong, S.; Nienhaus, G. U. Nano Today 2011, 6, 401− 418. (25) Lu, Y. Z.; Chen, W. Chem. Soc. Rev. 2012, 41, 3594−3623. (26) Yuan, Z.; Peng, M.; He, Y.; Yeung, E. S. Chem. Commun. 2011, 47, 11981−11983. (27) Su, Y. T.; Lan, G. Y.; Chen, W. Y.; Chang, H. T. Anal. Chem. 2010, 82, 8566−8572. (28) Tanaka, S.-I.; Miyazaki, J.; Tiwari, D. K.; Jin, T.; Inouye, Y. Angew. Chem., Int. Ed. 2011, 50, 431−435. (29) Guo, C.; Irudayaraj, J. Anal. Chem. 2011, 83, 2883−2889. (30) Xu, H.; Suslick, K. S. Adv. Mater. 2010, 22, 1078−1082. (31) Diez, I.; Ras, R. H. A. Nanoscale 2011, 3, 1963−1970. (32) Choi, S.; Dickson, R. M.; Yu, J. Chem. Soc. Rev. 2012, 41, 1867− 1891. (33) Shang, L.; Dong, S. Chem. Commun. 2008, 44, 1088−1090. (34) Wu, Z.; Lanni, E.; Chen, W.; Bier, M. E.; Ly, D.; Jin, R. J. Am. Chem. Soc. 2009, 131, 16672−16674. (35) Udaya Bhaskara Rao, T.; Pradeep, T. Angew. Chem., Int. Ed. 2010, 49, 3925−3929. (36) Petty, J. T.; Zheng, J.; Hud, N. V.; Dickson, R. M. J. Am. Chem. Soc. 2004, 126, 5207−5212. (37) Richards, C. I.; Choi, S.; Hsiang, J.-C.; Antoku, Y.; Vosch, T.; Bongiorno, A.; Tzeng, Y.-L.; Dickson, R. M. J. Am. Chem. Soc. 2008, 130, 5038−5039. (38) Morishita, K.; MacLean, J. L.; Liu, B.; Jiang, H.; Liu, J. Nanoscale 2013, 5, 2840−2849. (39) Lan, G. Y.; Huang, C. C.; Chang, H. T. Chem. Commun. 2010, 46, 1257−1259. (40) Yeh, H.-C.; Sharma, J.; Han, J. J.; Martinez, J. S.; Werner, J. H. Nano Lett. 2010, 10, 3106−3110. (41) Sharma, J.; Yeh, H.-C.; Yoo, H.; Werner, J. H.; Martinez, J. S. Chem. Commun. 2011, 47, 2294−2296. (42) Zhou, Y.; Wu, W. B.; Hu, G. D.; Wu, H. T.; Cui, S. G. Mater. Res. Bull. 2008, 43, 2113−2118. (43) Wen, S. H.; Zheng, F. Y.; Shen, M. W.; Shi, X. Y. Colloids Surf., A 2013, 419, 80−86. (44) Tan, S.; Erol, M.; Attygalle, A.; Du, H.; Sukhishvili, S. Langmuir 2007, 23, 9836−9843. (45) Hassan, M. L.; Ali, A. F. J. Cryst. Growth 2008, 310, 5252−5258. (46) Qu, F.; Li, N. B.; Luo, H. Q. Anal. Chem. 2012, 84, 10373− 10379. (47) Qu, F.; Li, N. B.; Luo, H. Q. Langmuir 2013, 29, 1199−1205. (48) Zhang, N.; Qu, F.; Luo, H. Q.; Li, N. B. Anal. Chim. Acta 2013, 791, 46−50. (49) Dave, N.; Chan, M. Y.; Huang, P.-J. J.; Smith, B. D.; Liu, J. J. Am. Chem. Soc. 2010, 132, 12668−12673.
hPEI-AgNCs-doped agarose hydrogels may be applied to practical environmental analysis.
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CONCLUSIONS In summery, fluorescent hPEI-AgNCs were successfully synthesized using a simple, one-pot method under mild conditions. The hPEI-AgNCs demonstrated high stability toward extreme pH, ionic strength, and temperature conditions. Due to the energy transfer between the hPEI-AgNCs and the cupric amine complexes, these hPEI-AgNCs displayed selective response toward Cu2+ over other metal ions and anions at pH 4 and provided rapid, sensitive, and reliable nanoprobes for fluorimetric Cu2+ sensing in aqueous media with a 10 nM limit of detection. By combining hPEI-AgNCs with hydrogels, hPEIAgNCs-doped agarose hydrogels were constructed as a visual Cu2+ detection platform. A dramatic fluorescence quenching was observed with 5 μM Cu2+, suggesting this system is a competent instrument-free water quality monitoring tool. Considering the recyclability of the hPEI-AgNCs-doped agarose hydrogels, this platform may be used for Cu2+ removal. Because of the strong fluorescence, large Stokes shift, and unusual stability, we expect that the hPEI-protected AgNCs have great potential as chemo/biosensors and cell imaging agents.
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ASSOCIATED CONTENT
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AUTHOR INFORMATION
S Supporting Information *
Additional figures as indicated in the main text. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author
*E-mail: [email protected]. Phone: +86 731 88823074. Fax: +86 731 88821904. Author Contributions †
Z.Y. and N.C. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China with Grant Nos. 20975036 and 91027037. REFERENCES
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Sensitive and Selective Detection of Copper Ions with Highly Stable Polyethyleneimine-Protected Silver Nanoclusters Zhiqin Yuan,† Na Cai,† Yi Du, Yan He,* and Edward S. Yeung College of Chemistry and Chemical Engineering, College of Biology, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, Hunan, People’s Republic of China. S Supporting Information *
ABSTRACT: Copper is a highly toxic environmental pollutant with bioaccumulative properties. Therefore, sensitive Cu2+ detection is very important to prevent over-ingestion, and visual detection using unaugmented vision is preferred for practical applications. In this study, hyperbranched polyethyleneimine-protected silver nanoclusters (hPEI-AgNCs) were successfully synthesized using a facile, one-pot reaction under mild conditions. The hPEI-AgNCs were very stable against extreme pH, ionic strength, temperature, and photoillumination and could act as sensitive and selective Cu2+ sensing nanoprobes in aqueous solutions with a 10 nM limit of detection. In addition, hPEI-AgNCs-doped agarose hydrogels were developed as an instrument-free and regenerable platform for visual Cu2+ and water quality monitoring.
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materials for sensitive and selective Cu2+ detection need to be developed. Recently, fluorescent noble metal (e.g., Au, Ag, and Pt) nanoclusters that are less than 2 nm in size and consist of several to hundreds of atoms have attracted attention due to their unique physical and optical properties.23−28 These noble metal nanoclusters exhibit excellent photostability and large Stokes shifts, making them a powerful alternative to fluorescent dye molecules. Among them, silver nanoclusters (AgNCs) displaying bright fluorescence and good biocompatibility have gathered wide research interest in the past decade.29−32 Considerable efforts have been dedicated to the syntheses of water-soluble AgNCs using various templates/additives (e.g., polyelectrolytes and thiolates). Red-emitting AgNCs were synthesized using poly(methacrylic acid) as a template under UV irradiation.33 Seven-atom AgNCs were prepared using 2,3dimercaptosuccinic acid or mercaptosuccinic acid as stabilizing ligand.34,35 In addition to polyelectrolytes and thiolates, biomolecules, such as DNA, were also exploited for the preparation of AgNCs.36−38 These DNA-encapsulated AgNCs (DNA-AgNCs) are highly fluorescent with tunable emission wavelengths; they have been utilized to sense metal ions,39 DNA,40 and protein.41 However, some of these AgNCs are not stable toward high ionic strength or extreme pH, which restricts their practical applications. Because they are amine-rich, polyamines may serve as effective stabilizing agents during the synthesis and surface
opper is an essential transition metal element for human health and serves as a critical cofactor for many enzymes because it can undergo a redox-active Cu(I)/Cu(II) conversion.1,2 However, copper is also highly toxic toward organisms when presented in excess because it may promote the generation of reactive oxygen species (ROS).3 ROS interfere with cellular signaling, cause damage to cell structures, or induce apoptosis.4−7 Gastrointestinal disturbance, as well as liver or kidney damage, may also be induced with high concentration of Cu2+.8 Additionally, copper accumulation in the neuronal cytoplasm might cause Alzheimer’s, Parkinson’s, and amyotrophic lateral sclerosis diseases.9,10 The U.S. Environmental Protection Agency (EPA) limits the Cu2+ levels in drinking water to 1.3 mg/L or ∼20 μM. Because Cu2+containing materials are widely used in electronic device production and industrial and agricultural processes, Cu2+ contamination is a continuous problem. Therefore, sensitive and selective detection of Cu2+ in environmental and biological samples is required. Toward this goal, many techniques have been developed for Cu2+ detection, such as atomic absorption spectrometry,11 resonance scattering spectrometry,12 inductively coupled plasma mass spectroscopy,13 and fluorimetric spectrometry.14,15 In particular, fluorescence-based detection techniques have attracted wide interest because they exhibit high sensitivity and strong interference rejection. Many chemosensors using organic fluorophores have been developed by modulating photoinduced electron transfer, internal charge transfer, or fluorescence energy transfer processes based on the paramagnetic properties of Cu2+.16−22 These organic probes often provide admirable selectivity; however, their preparation usually requires specialized synthetic skills and complicated purification procedures. Therefore, accessible fluorescent © XXXX American Chemical Society
Received: July 15, 2013 Accepted: November 4, 2013
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and Fe3+ were prepared from KCl, NaCl, CaCl2, MgCl2, CrCl3, CuCl2, Zn(Ac)2, NiCl2, Co(Ac)2, AgNO3, CdCl2, Mn(Ac)2, Hg(NO3)2, Pb(Ac)2, and FeCl3, respectively. Aqueous solution of F−, Cl−, Br−, I−, Ac−, citrate3−, CN−, EDTA2−, N3−, NO3−, H2PO4−, S2−, S2O32−, SCN−, SDS−, and SO42− were prepared from NaF, NaCl, NaBr, KI, NaAc, trisodium citrate, disodium EDTA, NaN3, NaNO3, NaH2PO4, Na2S, NaS2O3, KSCN, SDS, and Na2SO4, respectively. Buffer solutions from pH 2 to 11 were prepared according to standard protocols, and the pH values were measured with a benchtop pH meter (Orion plus, Thermo-Fisher, U.S.A.). The concentrations of all buffer solutions were 0.1 M, including glycine−HCl (pH 2−3), HAc−NaAc (pH 4−6), NaH2PO4−Na2HPO4 (pH 7−8), and NaHCO3−Na2CO3 buffers (pH 9−11). All glassware was cleaned with fresh aqua regia (HCl/HNO3 = 3:1, v/v) before use. Synthesis of hPEI-AgNCs. In a typical assay, 2.5 μmol of hPEI and 2.5 μmol of AgNO3 were dissolved in 10 mL of ultrapure water and stirred for 2 h to facilitate complexation between Ag+ and the amine ligands. Subsequently, the pH was adjusted to 5 with HAc, and 30 μmol of AA was added to the solution before it was stirred continuously for 2 days. All reactions were carried out at room temperature. Characterization. The fluorescence spectra of the AgNCs were obtained with a F-7000 fluorescence spectrophotometer (Hitachi, Japan). The UV absorption spectra of the AgNCs were obtained with a UV-1800 spectrophotometer (Shimadzu, Japan). High-resolution transmission electron microscopy (HRTEM) images were collected with a Tecnai F20 highresolution transmission electron microscope (FEI, U.S.A.). Fluorescence lifetime decays were measured using an Edinburgh FL 900 photocounting system (Livingston, U.K.). The X-ray photoelectron spectroscopy (XPS) measurements were preformed using a K-Alpha 1063 instrument (Thermo Fisher, U.K.), and all binding energies were calibrated by C1s (284.6 eV) as the reference energy. Selectivity and Sensitivity Measurements. Copper dichloride (CuCl2) was used as the Cu2+ source during sensitivity studies. A CuCl2 stock solution (0.1 M) was prepared, and various concentrations were obtained by serial dilution of the stock solution. To detect the Cu2+, solutions with different Cu2+ concentrations were added to 1 mL of 10 mM Ac buffer of pH 4 diluted hPEI-protected AgNCs solution. The fluorescence spectra were collected by using a F-7000 spectrophotometer. The following metal ions were used to evaluate the selectivity of the AgNCs: K+, Na+, Ca2+, Mg2+, Cr3+, Cu2+, Zn2+, Ni2+, Co2+, Ag+, Cd2+, Mn2+, Hg2+, Pb2+, and Fe3+. Various anions were also used to study the selectivity of AgNCs: F−, Cl−, Br−, I−, Ac−, citrate3−, CN−, EDTA2−, N3−, NO3−, H2PO4−, S2−, S2O32−, SCN−, SDS−, and SO42−. Thermostability and Reversibility Test. The solution temperature was controlled with an Eppendorf thermal cycler; after a 10 min equilibration, the fluorescence spectra were collected with a F-7000 spectrophotometer. Preparation of hPEI-AgNCs-Doped Agarose Hydrogel. The 1% hPEI-AgNC-doped agarose hydrogel was prepared as follows: 20 mg of agarose was completely dissolved in 2 mL of water under microwave heating. When the solution cooled to 70 °C after removal from the heater, 20 μL of AgNC solution was added with rapid stirring for 2 min. Subsequently, the mixture was kept static for 1 h to form the hydrogel. Visual Cu2+ Detection with hPEI-AgNCs-Doped Agarose Hydrogel. The hPEI-AgNC-doped agarose hydrogel was
modification of nanomaterials. In particular, hyperbranched polyethyleneimine (hPEI) is a positively charged polyamine that contains primary, secondary, and tertiary amine groups and has been used as polyvalence ligand to modify and stabilize metallic or metallic oxide/sulfide nanocrystals because the amine groups interact strongly with metal atoms. ZnO nanorods,42 Au nanoparticles,43 Ag nanoparticles,44 and CdS quantum dots45 have been successfully synthesized with hPEI in the reactant mixture. Similarly, hPEI can also provide scaffolds that interact with the Ag atoms during the formation of AgNCs. For instance, Qu et al. synthesized blue emissive hPEI (MW 10K) templated AgNCs by using formaldehyde as the reducing agent.46−48 However, these AgNCs are not stable under low pH and highly ionic conditions. Hydrogels are networks of hydrophilic polymers cross-linked through physical, ionic, or covalent interactions; these materials possess many attractive physical properties. Hydrogels are particularly useful when constructing visual detection platforms because they have negligible background colors and fluorescence emissions, as well as large loading capacities and controllable shapes. Because instrument-free and operationally simple tools are advantageous during practical analysis, using hydrogels in visual sensing applications has been extensively explored recently. For example, DNA-functionalized hydrogels were developed as platforms for visual detection of heavy metal ions (e.g., Hg2+ and Pb2+) and adenosine.49−51 Recently, fluorescent AgNCs-doped DNA hydrogels were fabricated by directly reducing silver ions dispersed in DNA hydrogels52 and have been utilized for visual Hg2+ sensing.53 In this study, we synthesized highly stable hPEI-AgNCs with bluish-green emission using a facile, one-pot method. We demonstrated that the AgNCs might be viable, highly selective, and sensitive fluorescent probes for Cu2+ detection with a 10 nM limit of detection. In addition, the as-prepared hPEIAgNCs were completely water-soluble and remarkably stable under extreme pH, ionic strengths, and temperature conditions, facilitating the development of a regenerable visual sensing platform for Cu2+ detection based on hPEI-AgNCs-doped agarose hydrogels. The strongly fluorescent hPEI-AgNCsdoped hydrogels almost completely stopped emitting when immersed in 5 μM Cu2+ solution; this concentration is lower than the EPA’s upper limit of ∼20 μM. Therefore, this simple sensing strategy might be used to monitor the Cu2+ levels in water samples, such as river, tap, lake, and spring water.
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EXPERIMENTAL SECTION Chemicals. Branched polyethyleneimine (MW 25K) and agarose were purchased from Sigma-Aldrich (Milwaukee, U.S.A.). Branched polyethyleneimine (MW 10K, 1.8K, and 0.6K) were obtained from Alfa Aesar (Heysham, U.K.). Silver nitrate (AgNO3), ascorbic acid (AA), sodium hydroxide (NaOH), acetic acid (HAc), sodium borohydride (NaBH4), concentrated nitric acid (HNO3), hydrochloric acid (HCl), and other chemicals were obtained from Sinopharm Chemical Reagent Corporation (Shanghai, China). Glutathione (GSH), cysteine (Cys), mercaptoethyl amine (MEN), mercaptoacetic acid (MAA), and D-penicillamine (DPA) were obtained from Beijing Dingguo Changsheng Biotechnology Corporation (Beijing, China). All chemicals were used without further purification. Ultrapure water was obtained using a Milli-Q integral water purification system purchased from Millipore Corporation (Billerica, U.S.A.). Solutions of K+, Na+, Ca2+, Mg2+, Cr3+, Cu2+, Zn2+, Ni2+, Co2+, Ag+, Cd2+, Mn2+, Hg2+, Pb2+, B
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Scheme 1. Synthetic Strategy of hPEI-AgNCs
Figure 1. (a) UV−vis absorption spectrum, (b) fluorescence excitation (black) and emission (blue) spectra, and (c) HRTEM image of the asprepared hPEI-AgNCs.
spectrometry, steady-state and time-resolved fluorescence spectrometry, HRTEM, and XPS. The pH was adjusted to become weakly acidic or neutral before adding the reducing agent because the original pH was approximately 9 due to the abundant amine motifs in the hPEI molecules. Under basic conditions, hPEI may interact with Ag+ to form stable hPEI− Ag+ complexes that hinder the reduction of Ag+. Even with a strong reducing agent, such as NaBH4, only large nonfluorescent silver nanoparticles formed at pH 9. The AgNC formation reaction was successfully carried out at pH 5. After AA reduction, the solution turned from colorless to bright yellow, and strong bluish-green emission was easily observed under UV illumination. None of the reactants, including the hPEI-Ag+ complex, had a similar fluorescence profile, suggesting that fluorescent hPEI-AgNCs were formed. From the absorption spectra of the solution, a visible absorption band centered at 361 nm was observed and assigned to the hPEIAgNCs; a surface plasmon resonance absorption peak
immersed in 100 mL water samples containing varied concentrations of Cu2+; and the fluorescence was visually observed under a hand-held UV lamp. To regenerate the hydrogel, the Cu2+-adsorbed AgNC-doped agarose hydrogels were immersed in 100 mL of 1 mM EDTA. All experiments were conducted in 10 mM Ac buffer of pH 4.
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RESULTS AND DISCUSSION Synthesis and Characterization of hPEI-AgNCs. It is well-known that the hPEI is synthesized by ring-opening polymerization of aziridine. Although the degree of branching can hardly be controlled and the purchased 25K hPEI has a large molecular weight distribution ranging from 20 000 to 30 000, the polymer contains ample amine motifs and can interact and stabilize many metal ions, making it a good template and protection agent for synthesis of metal nanocomplexes. The hPEI-protected AgNCs (hPEI-AgNCs) were synthesized in one pot, as illustrated in Scheme 1, and characterized by UV−vis C
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Figure 2. (a) Relative fluorescence intensity (F/F0) of hPEI-AgNCs at different pH values. (b) F/F0 of hPEI-AgNCs after adding various concentrations of NaCl in both pH 7.4 and pH 4 solutions. (c) Reversible temperature dependence of F/F0 of hPEI-AgNCs during thermocycling processes. (d) Fluorescence emission spectra of hPEI-AgNCs upon addition of 1 mM thiolates. (e) Relative fluorescence intensity variation of hPEIAgNCs as a function of time under 361 nm light illumination.
hPEI-AgNCs were mainly composed of Ag0.55 The percentage of Ag+ was calculated to be ∼11% by fitting the Ag 3d XPS spectrum with two components. Because the NaBH4 treated AgNCs had a much higher quantum yield (2.8%), all of the subsequent experiments were performed with NaBH4-treated AgNCs. When utilizing the hPEI-AgNCs for practical sensing applications, the material must be water-soluble and stable toward ambient environments. The hPEI-AgNCs are inherently water-soluble because they include abundant amine groups in the hPEI structures. Due to the strong interaction between amine groups, the Ag atoms, and the hyperbranched structure of hPEI molecules, we believe that the as-prepared hPEIAgNCs might possess enhanced stability toward extreme pH or high ionic strengths in solution. As presented in Figure 2a, the fluorescence intensities at 490 nm demonstrated little change from pH 2 to 11, indicating the as-prepared hPEI-AgNCs were quite stable toward different pH values. Meanwhile, the fluorescence intensities of the hPEI-AgNCs in solutions with different NaCl concentrations were almost the same as the solutions without NaCl (pH 4 or pH 7.4); the emission remained greater than 97%, even in 1 M NaCl solution (Figure 2b), suggesting that the hPEI-AgNCs were highly stable toward highly ionic conditions. Interestingly, when the temperature increased from 20 to 75 °C, the fluorescence intensity of the hPEI-AgNCs decreased by ∼one-third, but completely recovered after returning to 20 °C; this process was repeatable (Figure 2c), suggesting that the hPEI-AgNCs were thermally stable. The above fluorescence intensity measurement results were further supported by the fluorescence decay measurements. As shown in Supporting Information Table S1, the fluorescence lifetime values had no obvious change at different
appearing at between 400 and 500 nm was not observed, indicating the absence of any large silver nanoparticles (Figure 1a).54 The fluorescence spectra revealed an excitation peak centered at 361 nm and an emission peak centered at 490 nm (Figure 1b); the hPEI-AgNC fluorescence quantum yield was calculated to be ∼1.5% by using quinine sulfate as the reference. Time-resolved fluorescence lifetime measurements revealed two components at 2.3 ns (63.6%) and 8.7 ns (36.4%). To further verify the formation of hPEI-AgNC, HRTEM images were obtained. As displayed in Figure 1c, the asprepared hPEI-AgNCs had a spherical shape and uniform size distribution; the hPEI-AgNC diameters were 1.8 ± 0.2 nm. Therefore, the hPEI-AgNCs were successfully synthesized using a one-pot reaction. The large Stokes shift (∼130 nm) exhibited by the hPEI-AgNCs implied that they might be excellent fluorescent nanoprobes for chemo/biosensing and imaging. Further experiments indicated that the formation of the fluorescent hPEI-AgNCs might be affected by several factors, such as pH, Ag+/hPEI molar ratio, and AA/Ag+ molar ratio. A systematic survey was performed to optimize these experimental conditions. The strongest hPEI-AgNC emission was achieved when the solution pH was 5, the molar ratio between Ag+ ions and the hPEI polymers was 1:1, and the AA/Ag+ molar ratio was 12:1 (Supporting Information Figure S1). To determine the oxidation state of the hPEI-AgNCs, excess NaBH4 was added and the fluorescence intensity doubled, indicating that some Ag+ remained on the as-prepared AgNC surface (Supporting Information Figure S2). To further investigate the oxidation state of the silver atoms, XPS measurements were performed. As can be seen in Supporting Information Figure S3, two binding energy peaks of Ag 3d were centered at 368.1 and 374.1 eV, respectively, suggesting the D
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For the sensitivity evaluation, emission spectra of the hPEIAgNCs solution were recorded after adding different Cu2+ concentrations. Figure 4 indicates that increasing the Cu2+
pH or NaCl concentrations. They decreased a little at elevated temperature but recovered after cooling. Moreover, the addition of thiolates (1 mM) had almost no effect on the fluorescence of hPEI-AgNCs (Figure 2d), though too high concentration of Cys (50 mM) did lead to some intensity decrease (Supporting Information Figure S4). Finally, their fluorescence intensities have no obvious decrease after 1 h of light illumination at 361 nm (Figure 2e), suggesting excellent photostability of hPEI-AgNCs. The high stability of the asprepared hPEI-AgNCs under various conditions made them remarkably different from the 10K-hPEI-templated AgNCs reported by Qu et al. (Supporting Information Figure S5), which were synthesized by using formaldehyde as the reducing agent and were sensitive to pH47 and halidic ions.46 Such extraordinary stability might arise from the shielding effect of the protective hPEI layer, whose amine groups are preserved after AA reduction of Ag+ at pH 5. To confirm that, we synthesized AgNCs with hPEIs of different molecular weights (MW = 0.6K, 1.8K, 10K, and 25K). As shown in Supporting Information Figure S6, the fluorescence intensity of AgNCs increased with the MW of hPEI, implying that high molecular weight hPEI provided good protection to AgNCs and improved their stability. Taken together, our data suggest that these highly stable hPEI-AgNCs could be utilized for practical Cu2+ sensing, even under extreme conditions. Cu2+ Sensing in Aqueous Media. After adding Cu2+ to the hPEI-AgNC solution, the strong bluish-green emission was noticeably diminished. To examine the specificity of the copper(II)’s fluorescence quenching behavior, the fluorescence response of the hPEI-AgNCs toward various metal ions including K+, Na+, Ca2+, Mg2+, Cr3+, Cu2+, Zn2+, Ni2+, Co2+, Ag+, Cd2+, Mn2+, Hg2+, Pb2+, and Fe3+ was examined under the same conditions; the final concentration of the ions was 20 μM. It can be seen that only Cu2+ caused dramatic fluorescence quenching (Figure 3), and the best selectivity occurred at pH 4
Figure 4. Fluorescence emission spectra of hPEI-AgNCs after adding various concentrations of Cu2+ (from top to bottom: 0, 0.01, 0.05, 0.17, 0.27, 0.52, 0.77, 1.7, 2.7, 7.7, 17.7, and 27.7 μM). Inset: plots of intensity ratio (F0/F) vs the concentrations of Cu2+.
concentration gradually quenches the hPEI-AgNCs fluorescence emission (361 nm excitation), but no obvious changes in the maximum emission wavelength or spectral shape were observed. The intensity ratio F0/F displays a good linear relationship (R2 = 0.993) versus Cu2+ concentration ranging from 10 nM to 7.7 μM (Figure 4 inset) and was easily described by the Stern−Volmer equation, F0/F = 1 + Ksv[Q], where F0 and F are the fluorescence intensity of the hPEIAgNCs at 490 nm in the absence and presence of Cu2+, Ksv is the Stern−Volmer fluorescence quenching constant, and [Q] is the concentration of Cu2+. Ksv was calculated to be 3.1 × 105 M−1 by linear regression of the plot. The quantized limit of detection (LOD) was 10 nM. It has been reported that the fluorescence of hPEI-capped carbon quantum dots could be quenched by Cu2+ due to the inner filter effect of cupric amine complexes,56 as the cupric amine coordinates may quench the fluorescence of dye molecules.57 Therefore, we speculate that the Cu2+-mediated fluorescence quenching of the hPEI-AgNCs was induced by Cu2+ binding to the hPEI protection layer and subsequent energy transfer from the AgNCs to the cupric amine coordinates. This assumption was supported by the following facts: after adding Cu2+ to the AgNCs solution, the solution turned blue with the appearance of a broad absorption band in the 500−1000 nm range and a small shoulder at 300 nm (Supporting Information Figure S10a), indicating the formation of cupric amine coordinates.56 Except for its original emission peak centered at 490 nm, no extra emission peaks appeared in the red and near-infrared wavelength ranges (Supporting Information Figure S10b), implying no formation of fluorescent complexes or coordinates. Furthermore, fluorescein-quenching tests with Cu2+ were conducted in both the absence and presence of hPEI, suggesting that the cupric amine coordinates have a strong quenching efficiency (Supporting Information Figure S11). In addition, fluorescence quenching experiments with hPEI-AgNCs were performed with the addition of excess free hPEI. As illustrated in Supporting Information Figure S12, free hPEI reduced the fluorescence quenching. Some Cu2+ may have interacted with the amine motifs in the free hPEI molecules, causing lower quenching efficiency and suggesting
Figure 3. Relative fluorescence intensity (F/F0) of hPEI-AgNCs after adding various metal ions in the absence or presence of Cu2+ in pH 4 solution (from 1 to 16: blank, K+, Na+, Ca2+, Ag+, Mg2+, Co2+, Ni2+, Mn2+, Zn2+, Cd2+, Pb2+, Cr3+, Hg2+, Al3+, and Fe3+).
(Supporting Information Figure S7). At this pH, anions could not affect the fluorescence (Supporting Information Figure S8), and the efficient fluorescence response toward Cu2+ was fundamentally preserved in the presence of 150 mM of NaCl (Supporting Information Figure S9). The high selectivity of the hPEI-AgNCs indicates that they could be used to sense Cu2+ in complex systems. All subsequent experiments were performed at pH 4. E
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Scheme 2. Schematic Illustration of hPEI-AgNCs-Based Nanoprobes for the Detection of Cu2+
that the distance between the AgNC and cupric amine coordinates determines the quenching capabilities. Moreover, Cu2+-mediated hPEI-AgNC fluorescence quenching was reversible. After adding EDTA, the fluorescence of the hPEIAgNCs was effectively recovered (Supporting Information Figure S13), implying that the binding capacity of amine groups for Cu2+ caused the quenching activity. Therefore, the energy transfer between the hPEI-AgNCs and the cupric amine complexes led to the observed fluorescence quenching (Scheme 2). Because the assay had high sensitivity and selectivity, we can conclude that the hPEI protective layer on the AgNC surface played at least two significant roles: (1) to form coordinates with Cu2+ and quenching the AgNC fluorescence, and (2) to prevent other metal ions, anions, and small molecules from approaching the AgNC surface. Visual Cu2+ Detection Using hPEI-AgNCs-Doped Agarose Hydrogels. Because of the remarkable stability of the hPEI-AgNCs under complicated and extreme conditions, we exploited them for visual detection of Cu2+ using naked eyes, which is preferable for practical applications. Immobilizing the hPEI-AgNCs into hydrogels is a logical solution since these materials have high loading capacity and low background fluorescence. The hyperbranched structure and large molecular weight of hPEI could restrict the diffusion and prevent the release of the 1.8 nm hPEI-AgNCs from the hydrogels. And the accumulative properties of functional hydrogels allow them to be adapted to visual detection of Cu2+. For this purpose, we prepared hPEI-AgNCs-doped agarose hydrogels. Capitalizing on the excellent stability of hPEI-AgNCs toward high temperatures, the heating process during the hydrogel preparation has no effect on the optical properties of the AgNCs and the transparent agarose hydrogels emitted strong fluorescence under UV irradiation, suggesting that the hPEIAgNCs and hydrogels were successfully combined. To evaluate the performance of the hPEI-AgNCs-doped agarose hydrogels for Cu2+ detection, the hydrogels were immersed into Cu2+ solutions of various concentrations at pH 4. When the Cu2+ concentration increased from 0 to 5 μM, the optically transparent hydrogels gradually turned blue, indicating the adsorption of Cu2+ and the formation of cupric amine coordinates (Figure 5a); the fluorescence of the hydrogels under a hand-held UV lamp completely disappeared after 5 μM Cu2+ treatment (Figure 5b). Because the EPA maximum Cu2+ allowance is ∼20 μM, these hPEI-AgNCs-doped agarose hydrogels are applicable for instrument-free visual Cu 2+ detection and water quality control under practical conditions.
Figure 5. (a) Visual detection of Cu2+ using hPEI-AgNCs-doped agarose hydrogels under visible light (from I to IV: 0, 0.05, 0.5, 5 μM). (b) Corresponding photographs under 365 nm UV light. (c) Fluorescence regeneration results for quenching with Cu2+ after adding EDTA.
As far as we know, this is the first example of visual Cu2+ detection utilizing hPEI-AgNCs-doped hydrogels. To serve as an environmentally friendly platform for visual Cu2+ detection, the functional hydrogels must be regenerable. Otherwise, the spent materials have to be disposed and fresh functional hydrogels must be constructed. The regeneration capability of the Cu2+-accumulative hydrogels was evaluated using EDTA as an effective extraction agent. The fluorescence of the Cu2+-accumulative hydrogels recovered within a few hours (Figure 5c), suggesting desorption of Cu2+ and regeneration of the hPEI-AgNCs-doped agarose hydrogels. Subsequently, the fluorescence was fully quenched upon readdition of Cu2+, indicating that the hPEI-AgNCs-doped hydrogels were recyclable. Therefore, the hPEI-AgNCs-doped agarose hydrogels could serve as an instrument-free and regenerable fluorescent platform for visual Cu2+ detection. To demonstrate the practical applicability of this platform, several environmental water samples were tested, including river, lake, tap, and spring water. These water samples were successfully cleaned, and the remaining Cu2+ was less than 5 μM (Supporting Information Figure S14), suggesting that the F
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hPEI-AgNCs-doped agarose hydrogels may be applied to practical environmental analysis.
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CONCLUSIONS In summery, fluorescent hPEI-AgNCs were successfully synthesized using a simple, one-pot method under mild conditions. The hPEI-AgNCs demonstrated high stability toward extreme pH, ionic strength, and temperature conditions. Due to the energy transfer between the hPEI-AgNCs and the cupric amine complexes, these hPEI-AgNCs displayed selective response toward Cu2+ over other metal ions and anions at pH 4 and provided rapid, sensitive, and reliable nanoprobes for fluorimetric Cu2+ sensing in aqueous media with a 10 nM limit of detection. By combining hPEI-AgNCs with hydrogels, hPEIAgNCs-doped agarose hydrogels were constructed as a visual Cu2+ detection platform. A dramatic fluorescence quenching was observed with 5 μM Cu2+, suggesting this system is a competent instrument-free water quality monitoring tool. Considering the recyclability of the hPEI-AgNCs-doped agarose hydrogels, this platform may be used for Cu2+ removal. Because of the strong fluorescence, large Stokes shift, and unusual stability, we expect that the hPEI-protected AgNCs have great potential as chemo/biosensors and cell imaging agents.
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ASSOCIATED CONTENT
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AUTHOR INFORMATION
S Supporting Information *
Additional figures as indicated in the main text. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author
*E-mail: [email protected]. Phone: +86 731 88823074. Fax: +86 731 88821904. Author Contributions †
Z.Y. and N.C. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China with Grant Nos. 20975036 and 91027037. REFERENCES
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