Ratiometric Fluorescent Biosensing of Hydrogen Peroxide and

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Ratiometric Fluorescent Biosensing of Hydrogen Peroxide and Hydroxyl Radical in Living Cells with Lysozyme−Silver Nanoclusters: Lysozyme as Stabilizing Ligand and Fluorescence Signal Unit Fang Liu,† Tao Bing,‡ Dihua Shangguan,‡ Meiping Zhao,§ and Na Shao*,† †

College of Chemistry, Beijing Normal University, Beijing 100875, P. R. China Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China § Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, P. R. China ‡

S Supporting Information *

ABSTRACT: Construction of facile ratiometric fluorescent probes which possess sensitive and selective sensing ability for bioactive small molecules is highly desirable and challenging. Herein, silver nanoclusters capped with denatured lysozyme (dLysAgNCs) were synthesized and proved to be dual emissive. The facility of the dLys-AgNCs ratiometric probe was attributed to the finding that the lysozyme acted not only as stabilizing ligand but also as fluorescence signal unit. In the presence of Fenton reagents, the emission of dLys-AgNCs at 640 nm was quenched by •OH, whereas the emission at 450 was enhanced due to • OH-induced oxidation of tyrosine in the lysozyme. This probe could be used for highly sensitive detection of H2O2. The fluorescence changes of F450/F640 had fantastic linearity to H2O2 concentrations in the range of 0.8−200 μmol/L (R2 = 0.9993), with a limit of detection (LOD) as low as 0.2 μmol/L. Additionally, this probe was also applied to H2O2-generated oxidase-based biosensing. As a proof-of-concept, glucose and acetylcholine chloride were detected with benefical LOD values of 0.6 μmol/L and 0.8 μmol/L, respectively. Furthermore, fluorescence confocal imaging demonstrated dLys-AgNCs had a sensitive response to fluctuation of •OH levels in living cells, which might have promising application in study of •OH-induced oxidative damage to proteins.

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The far-ranging impacts of H2O2 homeostasis have promoted the construction of highly accurate, sensitive, and selective sensors for detecting H2O2. To date, options for determination of H2O2 have been developed, such as electrochemistry,7,8 chemiluminescence,9 fluorometry,10−14 and chromatography.15 Among these approaches, fluorometric sensors are endowed with distinct merits in terms of convenience, sensitivity, and spatiotemporal resolution. To obtain more accurate readouts, ratiometric sensing has drawn considerable attention because of its self-calibration.16−18 Chang et al. reported a ratiometric

ydrogen peroxide (H2O2) is a crucial metabolite in living systems, and studies have proved its importance as a secondary messenger in cellular signal transduction.1,2 Numerous reactions can produce H2O2 as byproduct in the presence of O2-dependent oxidases (e.g., glucose oxidase, choline oxidase, cholesterol oxidase, uricase). If not scavenged in time, excessive H2O2 can react with low-oxidation-state metal ions like Fe2+ or Cu+ and produce hydroxyl radical (•OH), which is well-known as Fenton reaction.3,4 As one of the most aggressive reactive oxygen species (ROS), elevated levels of • OH can lead to oxidative stress such as oxidative damage of DNA and proteins, posing a substantial risk for incidence of cellular disorders.5,6 © 2016 American Chemical Society

Received: August 3, 2016 Accepted: October 7, 2016 Published: October 7, 2016 10631

DOI: 10.1021/acs.analchem.6b02995 Anal. Chem. 2016, 88, 10631−10638

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Analytical Chemistry fluorescent probe for H2O2 detection based on a twofluorophore cassette composed of a coumarin donor and a fluorescein acceptor.2 By employing H2O2 as intermediate, Wang et al. designed a fluorescent probe for ratiometric detection of choline and acetylcholine by conjugating poly(fluorene-co-phenylene) derivative with fluorescein.19 Although the synthetic procedures of the ratiometric probe are complicated, systematic errors can be eliminated greatly. Water-soluble metallic nanoclusters (MNCs), famous for their intrinsic values such as facile synthesis, ultrafine size, and excellent biocompatibility,20−22 have been well employed for fluorescence sensing platforms.23−25 In the procedures of MNCs synthesis, ligands are needed to protect MNCs against aggregation. Among various ligands, proteins are considered as ideal candidates because nanoclusters capped with proteins possesses excellent biocompatibility26 and very stable optical properties.27,28 Ratiometric fluorescence probes for H2O2 detection based on MNCs have been reported, wherein proteins were modified with organic fluorescent molecules and then served as ligands to synthesize MNCs.29,30 In such a strategy, MNCs acted as reference signal, whereas organic molecules which were susceptible to H2O2 served as response signal. Nonetheless, complicated modification of fluorophores and tedious synthetic procedures were inevitable in the construction of this kind of ratiometric sensors. Proteins which contain aromatic amino acids can emit intrinsic fluorescence.31 Biologically, the exposure of proteins to oxidative stress like oxygen radicals or other oxidants, may lead to tyrosine−tyrosine bonding, posing substantial effect on fluorescence of proteins.32,33 In the present work, the dual functions of proteins, intrinsic fluorescence and capping ligand, were simultaneously embodied in the synthesized lysozymestabilized AgNCs. The dLys-AgNCs had two emission peaks when excited at 365 nm: one strong emission at 640 nm from AgNCs and the other weak emission at 450 nm from aromatic amino acids in the ligand of lysozyme. In the presence of Fenton reagents, the red emission at 640 nm was decreased due to the corporate quenching effect of Fe3+ and •OH to AgNCs, whereas the blue emission at 450 nm was significantly enhanced in term of •OH-induced oxidative damage to the ligand of lysozyme. This probe could be utilized for ratiometric detection of H2O2 and further exploited to H2O2-generated oxidase-based biosensing, such as glucose and acetylcholine chloride. Also, dual channel fluorescence confocal images of • OH in living cells was realized using the dLys-AgNCs probe. Compared with previously reported ratiometric fluorescent sensors for H2O2 detection,29,30,34 our designed dLys-AgNCs possesses admirable highlights. First, the construction strategy of this ratiometric probe is facile. The denatured lysozyme acted not only as stabilized ligand for AgNCs but also as fluorescence response unit to Fenton reagents. No tedious fluorophore modification is involved. Second, the sensitivity is higher than common ratiometric probes in which an outside signal is changeable and a built-in reference signal is almost intact. The emission of our dLys-AgNCs at 450 nm is enhanced, whereas that at 640 nm is quenched by Fenton reagents. The divergent changes of the two emission greatly enlarge the ratio of F450/F640 (F450 and F640 are the fluorescence intensity of dLys-AgNCs at 450 and 640 nm, respectively).

Lysozyme, bovine serum albumin (BSA), Bull Immunoglobulin G (IgG), L-glutamic acid (Glu), glycine (Gly), and Lphenylalanine (Phe) was purchased from Beijing Dingguo Biotechnology Company. Sodium borohydride (NaBH4, 96%), H2O2 (30%), acetic acid, glucose, ferrous sulfate, ferric sulfate, sodium fluoride, potassium superoxide, sodium hypochlorite solution, thiourea, and dimethyl sulfoxide (DMSO) were purchased from Beijing Chemical Works. Glucose oxidase (GOx) was purchased from Amresco. Acetylcholine chloride (ACh), acetylcholinesterase (AChE), choline oxidase (ChOx), tert-butyl hydroperoxide solution (TBHP), ascorbic acid (Vc), reduced glutathione (GSH), phorbol-12-myristate-13-acetate (PMA), and N-acetylcysteine (NAC) were purchased from Sigma-Aldrich. •O2− was generated by ionization of KO2 in DMSO. ONOO− was synthesized as reported.35 •OtBu was generated by reaction of TBHP and Fe2+. 1O2 was generated by reaction of H2O2 and NaClO. Human prostatic carcinoma cells (PC-3) were obtained from Key Laboratory of Analytical Chemistry for Living Biosystems, Chinese Academy of Sciences (Beijing China). All other reagents were analytical reagent grade, and all solutions were prepared using ultrapure water (Millipore Milli-Q, 18.0 MΩ). Instruments and Characterization. Fluorescence spectra were measured on a Hitachi F-4600 fluorescence spectrophotometer. UV−visible absorption spectra were carried out using a TU-1901 diode-array spectrophotometer. Fluorescence images were performed on an Olympus FV1000-IX81 laser confocal microscope. Transmission electron microscopy (TEM) graphs were obtained from a TF20 (FEI). Timeresolved fluorescence measurements were measured on an OB920 single-photon counting fluorometer. Circular dichroism (CD) spectra were detected by Applied Photophysics Chirascan 250. The pH of buffer solution was adjusted by a HI 2211 pH/ORP meter. Reaction temperature was controlled precisely by a stirrer with temperature sensor and a thermometer. Synthesis of dLys-AgNCs. The dLys-AgNCs were synthesized according to the report by Chen et al. with modification.27 In brief, 1 mL of 5 mmol/L AgNO3 solution was added to 5 mL of 15 mg/mL lysozyme solution. The molar ratio of Ag+ and lysozyme was approximately 1:1. Subsequently, 0.3 mL of 1 mol/L NaOH was added to the above mixture drop by drop, under continuous stirring. Then 10 μL of 20 mmol/L NaBH4 was injected into the above-mentioned mixture solution. After reaction at room temperature for 15 min, 0.5 mL of 1 mol/L acetic acid was added to neutralize excess NaOH and make the dLys-AgNCs more stable. Consequently, the as-prepared dLys-AgNCs were dispersed in acetate buffer solution with a pH about 4.8. Fluorescent Detection of H2O2, Glucose, and ACh. For H2O2 sensing, 100 μL of dLys-AgNCs were diluted into a volume of 1 mL containing freshly prepared 0.5 mmol/L Fe2+ and reacted with different concentrations of H2O2 at room temperature for 10 min. For glucose sensing, 100 μL of dLys-AgNCs were diluted into a volume of 1 mL containing freshly prepared 0.5 mmol/L Fe2+, 1 U/mL GOx and then were reacted with different concentrations of glucose at 37 °C for 20 min. The procedure for ACh detection was the following: First, aliquots of 20 mmol/L Tris-HCl buffer (pH 7.5) containing 1 U/mL AChE, 0.5 U/mL ChOx, and different concentrations of ACh were equilibrated at 37 °C for 20 min. Then, 100 μL of dLys-AgNCs and freshly prepared 0.5 mmol/L Fe2+ were added

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EXPERIMENTAL SECTION Chemicals and Reagents. Silver nitrate (AgNO3) was purchased from Tianjin Heowns Biochem Technologies LLC. 10632

DOI: 10.1021/acs.analchem.6b02995 Anal. Chem. 2016, 88, 10631−10638

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Figure 1. (A) Fluorescence spectra of dLys-AgNCs (a) excitation spectra, (b) emission spectra excited at 490 nm, and (c) emission spectra excited at 365 nm. (B) Fluorescence spectra of denatured lysozyme (a) excitation spectra and (b) emission spectra.

to the aforementioned mixture for another 10 min, wherein the pH was adjusted to 4.8 with acetic acid. All of the fluorescence spectra were measured under excitation at 365 nm. Fluorescence Imaging of •OH in Live Cells. PC-3 cells were seeded in glass-bottom culture dishes and cultivated with 10% FBS at 37 °C in 5% CO2 atmosphere for 24 h. To monitor • OH, PC-3 cells was preprocessed with or without 1 μmol/L PMA or 1.5 mmol/L NAC for 30 min, followed by incubation with dLys-AgNCs for 30 min. To remove the unbound dLysAgNCs, the cells were washed three times with PBS and then observed under an Olympus FV1000-IX81 laser confocal microscope equipped with an oil immersion 60× objective. Under the excitation of 405 nm, the images of dLys-AgNCs were captured in blue channel (425−480 nm) and red channel (600−680 nm).

Figure 2. Fluorescence spectra of (a) dLys-AgNCs, (b) dLys-AgNCs + 0.5 mmol/L Fe2+ + 200 μmol/L H2O2, (c) dLys-AgNCs + 200 μmol/ L H2O2, (d) dLys-AgNCs + 0.5 mmol/L Fe2+, and (e) dLys-AgNCs + 0.5 mmol/L Fe3+.

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RESULT AND DISCUSSION Spectroscopic Properties of dLys-AgNCs. The asprepared dLys-AgNCs were first characterized by UV−visible absorption spectroscopy. As shown in Figure S1, a broad absorption band centered around 480 nm indicated the formation of AgNCs. No plasmon resonance peak around 425 nm was observed, which confirmed the absence of bigger nanoparticles in solution. The fluorescence spectra of dLys-AgNCs, as shown in Figure 1A, had a single red emission at 640 nm when excited at 490 nm. When excited at 365 nm, two emission bands emerged: one strong emission centered at 640 nm and the other weak emission centered at 450 nm. The emission at 640 nm was the intrinsic fluorescence of AgNCs, which have been verified in previous research.27,36,37 To elucidate the origin of emission at 450 nm, a control experiment was carried out wherein AgNO3free lysozyme was treated in the same conditions as that for synthesis of dLys-AgNCs. Compared with the fluorescence spectra of denatured lysozyme shown in Figure 1B, the emission at 450 nm of dLys-AgNCs should be assigned to the ligand of denatured lysozyme. Responses of dLys-AgNCs to Fenton Reagents. Fenton reaction occurs as eq 1. H 2O2 + Fe 2 + → ·OH + Fe3 + + OH−

reasons that caused the fluorescence changes, fluorescence characteristics of dLys-AgNCs in the presence of H2O2, Fe2+, and Fe3+ were investigated, respectively. As illustrated in Figure 2c,d, H2O2 or Fe2+ induced negligible fluorescence changes of dLys-AgNCs, while Fe3+ quenched the fluorescence of both 450 and 640 nm evidently (Figure 2e). It was speculated that •OH could enhance the blue emission at 450 nm. The apparent fluorescence enhancement of dLys-AgNCs at 450 nm should be the combined effect of Fe3+-induced quenching and •OHinduced enhancement. Fe3+ (as a quenching agent) and •OH (as an oxidizing agent) were corporately responsible for the fluorescence quenching of 640 nm. To further examine the above inferences, F− and thiourea, serving as masking reagents for Fe3+ and •OH scavenger,38 respectively, were added to dLys-AgNCs prior to Fenton reagents, respectively. When Fe3+ from Fenton reagents was masked by F−, fluorescence quenching of 640 nm were suppressed partly, about 60% recovering, and the fluorescence intensity of 450 nm was enhanced to a larger degree compared with that without masking (curves b and c, Figure 3A). When Fe3+ from outside introduction by Fe2(SO4)3 was masked by F−, the fluorescence quenching of 640 and 450 nm were suppressed completely, and the fluorescence spectra were superimposable on that of dLys-AgNCs (Figure S2). These results indicated that Fe3+ had a quenching effect on both AgNCs and the ligand of denatured lysozyme. The reasons were presumably related to complexation of Fe3+ with amino acids in ligand and simultaneous aggregation of AgNCs.39,40 The TEM image in Figure 3B revealed that Fenton reagents induced serious aggregation of dLys-AgNCs, which gave rise to

(1)

The effect of Fenton reagents on the fluorescence of dualemitting dLys-AgNCs was investigated. As shown in Figure 2b, the red emission of AgNCs was quenched evidently while the blue emission of the ligand of lysozyme was dramatically enhanced in the presence of Fenton reagents. To explore the 10633

DOI: 10.1021/acs.analchem.6b02995 Anal. Chem. 2016, 88, 10631−10638

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Figure 3. (A) Fluorescence spectra of (a) dLys-AgNCs, (b) dLys-AgNCs + 0.5 mmol/L Fe2+ + 200 μmol/L H2O2, (c) dLys-AgNCs + 0.5 mmol/L Fe2+ + 200 μmol/L H2O2 + 15 mmol/L NaF, (d) dLys-AgNCs + 0.5 mmol/L Fe2+ + 200 μmol/L H2O2 + 1.5 mmol/L thiourea. (B) HRTEM images of (a) dLys-AgNCs and (b) dLys-AgNCs + 0.5 mmol/L Fe2+ + 200 μmol/L H2O2. (C) Lifetime of (a) dLys-AgNCs and (b) dLys-AgNCs + 0.5 mmol/L Fe2+ + 200 μmol/L H2O2. (D) Fluorescence spectra of (a) tyrosine and (b−d) tyrosine with 0.5 mmol/L Fe2+ and 50, 100, 200 μmol/L H2O2.

the fluorescence quenching of 640 nm. When •OH produced from the Fenton reagents was scavenged by thiourea, the emission of denatured lysozyme at 450 nm remained almost unchanged compared with that of dLys-AgNCs only (curves a and d, Figure 3A). Furthermore, the emission of AgNCs at 640 nm experienced fluorescence quenching but had a conspicuously higher intensity than that in the absence of thiourea (curves b and d, Figure 3A). It was supposed that fluorescence quenching at 640 nm induced by •OH was ascribed to the aggressive power of •OH which oxidized Ag(0) in AgNCs to Ag(I).41,42 In control experiments, F− or thiourea alone had little influence on the fluorescence of dLys-AgNCs (Figure S3). The average lifetimes of dLys-AgNCs at 640 nm before and after adding Fenton reagents were 1.1 and 0.66 ns, respectively. When Fenton reagents were added, as depicted in Figure 3C, two of three lifetimes of dLys-AgNCs significantly decreased. The shortening of lifetimes might be attributed to the state change of AgNCs, which were induced by complexation of Fe3+ with ligand and •OH-induced oxidation of ligand as well as Ag in AgNCs. Oxidative damage from •OH might effectively induce conformational changes of denatured lysozyme. To verify such changes, circular dichroism (CD) measurement which gives information on protein conformational behaviors was employed. After addition of Fenton reagents, the peak at 208 nm was blue-shifted to 206 nm, accompanied by less-negative ellipticity (Figure S4), implying the loss of α-helix component and increase in β-sheet conformation.43,44 This suggested that the oxidation of ligand resulted in conformation changes and facilitated the emission at 450 nm.

Protein−protein cross-linkage via tyrosine−tyrosine bonding, viz., dityrosine or polytyrosine, has been widely used as a marker for oxidative stress.45 To investigate the tentative mechanism underlying the fluorescence enhancement at 450 nm, natural tyrosine was treated in the same conditions as that for synthesis of dLys-AgNCs and then mixed with Fenton reagents. As shown in Figure 3D, upon excitation at 365 nm, the fluorescence around 460 nm emerged and increased dramatically when an increasing amount of Fenton reagents were added into the treated tyrosine solution. Tyrosine tends to cross-link in the presence of highly oxidative •OH.46 The emerging fluorescence around 460 nm might originate from dityrosine or polytyrosine. Compared with the fluorescence induced by Fenton reagents for dLys-AgNCs at 450 nm, the maximum emission wavelength of tyrosine experienced a red shift about 10 nm, and this might stem from the environment change of oxidized tyrosine: the former was covalently linked with other amino acids located in lysozyme, and the latter was free in solution. In brief, the fluorescent responding mechanism of dLysAgNCs to Fenton Reagents was illustrated as Scheme 1. In acid medium, Fe3+ and •OH were produced from the reaction of H2O2 and Fe2+. The blue emission of dLys-AgNCs at 450 nm was enhanced dramatically in term of •OH-induced oxidative damage to the ligand of lysozyme, of which tyrosine was presumably oxidized to dityrosine or polytyrosine. On the other hand, the emission at 640 nm was decreased due to the corporate effect of Fe3+-induced quenching to AgNCs and • OH-induced oxidation of Ag(0). Ratiometric Fluorescent Detection of H2O2. Based on the above responding mechanism, the feasibility of dLys10634

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Ratiometric Fluorescent Detection of H2O2-Generated Substrates. By taking advantage of H2O2 as intermediate, the ratiometric fluorescent probe was configured into biosensors for H2O2-generated substances. Oxidase-triggered oxidation of substrates, with H2O2 as byproduct and Fenton reagentsinduced cascade reactions in the presence of Fe2+ could lead to fluorescence changes of the dual-emitting Lys-AgNCs. As a proof-of-concept, glucose was first detected. Glucose plays key roles in the human body, and the level of blood glucose is the most representative monitor of diabetes.50,51 Glucose can be catalyzed by GOx specifically in the presence of O2 to produce H2O2.52,53 In the presence of Fe2+, glucose, and GOx, the fluorescence spectra of dLys-AgNCs experienced similar changes (Figure S9d) as that containing Fe2+ and H2O2. GOx or glucose alone exerted little influence on the fluorescence of dLys-AgNCs (Figure S9b,c). As shown in Figure S10, the optimal concentration of GOx for glucose detection was 1U/mL. When the amount of GOx was less than 1U/mL, oxidation of glucose proceeded slowly, and the yield of H2O2 was low for a given amount of glucose. Time-dependent fluorescence changes as shown in Figure S11 demonstrated that the cascade reactions reached a plateau within 20 min at 37 °C. Under the optimal conditions, the fluorescence spectra of dLys-AgNCs in the presence of 0.5 mmol/L Fe2+ and 1U/mL GOx upon increasing the amount of glucose in acetate buffer solution (pH 4.8) are depicted in Figure 5A. In the wide range of 2−500 μmol/L, the calibration curves were established by plotting the fluorescence intensity ratio of F450/F640versus glucose concentrations. The linearity was excellent (R2 = 0.9936), and the LOD for glucose detection was down to 0.6 μmol/L. Ratiometric fluorescent sensing of H2O2-generated oxidasebased substrates was also exploited to ACh. ACh is a neurotransmitter in both peripheral nervous system and central nervous system.54 The level of Ach is highly related to cholinergic dysfunction, contributing factors underlying Alzheimer’s diseases and Parkinson’s.55 Ach can be hydrolyzed specifically by AChE to form choline that is in turn oxidized by ChOx to produce H2O2.56 An acid medium was indispensable for the Fenton reaction, whereas a weak alkaline medium at pH 8.0 to 9.0 and 7.0 to 8.0 were optimal for AChE and ChOx enzyme activities, respectively.12,57 Thus, H2O2 was first generated in Tris-HCl buffer solution (pH 7.5) from the enzyme reaction system containing ACh, AChE, and ChOx at 37 °C. Then in acid medium (pH 4.8), dLys-AgNCs and Fe2+ were separately introduced into above H2O2-containing solution. As expected, the fluorescence emission of 640 nm was quenched evidently while that of 450 nm was enhanced dramatically (Figure S12d). In addition, ACh, AChE, or ChOx exerted negligible fluorescence change of dLys-AgNCs (Figure S12). The AChE/ChOx concentrations and reaction time were also optimized. As shown in Figure S13, the maximum response of dLys-AgNCs to ACh could be obtained when the concentrations of AChE and ChOx were 1.0 U/mL and 0.5 U/mL, respectively. High concentrations of AChE and ChOx not only led to high background signal but also reduced reaction efficiency, and these may be explained by the fact that massive macromolecular enzymes gave rise to opaque solution and problematic adsorption of the enzymes on the surfaces of dLys-AgNCs.58 Time-dependent fluorescence changes shown

Scheme 1. Schematic of Fluorescent Responding Mechanism of dLys-AgNCs to Fenton Reagents

AgNCs probe for H2O2 detection was investigated. As expected, in the presence of Fe 2+ , by varying H 2 O 2 concentrations, ratiometric sensing platform for H2O2 detection was established. In order to obtain better sensing performance, probe and Fe2+ concentrations were optimized. As shown in Figure S5 and Figure S6, 10-fold diluted dLys-AgNCs and 0.5 mM Fe2+ were chosen in further experiments. The ratio of F450/ F640 reached a plateau within 10 min at room temperature (Figure S7). Since an acid medium was favorable to Fenton reaction,47 the effect of pH (from 3.0 to 5.5) on the fluorescence sensing of dLys-AgNCs was evaluated. As shown in Figure S8, the signal change decreased with increasing pH. Taking into account of practical sample analysis, pH 4.8 was chosen throughout further detection. With increasing amount of H2O2, the emission of dLysAgNCs at 640 nm was quenched gradually while that at 450 nm increased dramatically (Figure 4). The relationship between

Figure 4. Ratiometric fluorescent responses of dLys-AgNCs to different concentrations of H2O2. The arrows indicated the signal changes with increasing H2O2 concentrations (0, 0.8, 8, 20, 40, 80, 120, and 200 μmol/L). Inset: plot of fluorescence intensity ratio (F450/ F640) of dLys-AgNCs versus H2O2 concentrations.

fluorescence intensity ratio of F450/F640 and H2O2 concentrations is shown in the inset of Figure 4. The fluorescence intensity ratio of F450/F640 presented fantastic linearity (R2 = 0.9993) to H2O2 concentrations in the wide range of 0.8−200 μmol/L. Compared with some reported ratiometric fluorescent approaches for H2O2 detection,30,48,49 our dLys-AgNCs probe showed lower LOD (S/N = 3) of 0.2 μmol/L and 35-fold increase of F450/F640, suggesting that it was one of the most sensitive probes for H2O2 detection. 10635

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Figure 5. Ratiometric fluorescent responses of dLys-AgNCs to (A) glucose and (B) ACh. The arrows indicated the signal changes with increasing concentrations of glucose and ACh. Inset: plot of fluorescence ratio (F450/F640) of dLys-AgNCs versus glucose concentrations (0, 2, 20, 50, 100, 200, 300, and 500 μmol/L) and ACh concentrations (0, 2, 10, 20, 40, 70, and 100 μmol/L).

Fluorescence confocal imaging of •OH in living cells was performed. Compared with control experiments (Figure 7A,E), the fluorescence confocal images of PC-3 cells after being treated with dLys-AgNCs (Figure 7B,F) showed considerable fluorescence signal in blue and red channels. This confirmed that the dLys-AgNCs probe had good cell permeability and could be readily taken in by cancerous cells. To further prove that the fluorescence image signal could be regulated by •OH, PMA that can stimulate the generation of • OH60 and NAC that can scavenge free-radicals61,62 in cancer cells were added prior to dLys-AgNCs, respectively. For PMAincubated cells, the fluorescence of dLys-AgNCs in blue channel was dramatically enhanced while that in red channel was weakened (Figure 7C,G). On the other side, the images of PC-3 cells incubated with NAC presented faint fluorescence in blue emission and clear increase in red emission (Figure 7D,H). These results were in accordance with the fact that PMA and NAC can activate and inactivate the generation of •OH, respectively. It should be noted that the fluorescence images of cells with dLys-AgNCs and NAC was a bit weaker in blue channel and slightly stronger in red channel (Figure 7D,H) compared with that with dLys-AgNCs only (Figure 7B,F). This could be explained by the result demonstrating that endogenous •OH in PC-3 cells enhanced the fluorescence of dLys-AgNCs at 450 nm and weakened that at 640 nm to some extent. Taken together, the dLys-AgNCs probe had sufficient selectivity for •OH imaging in living cells, where it could sensitively response to fluctuation of •OH levels.

in Figure S14 demonstrated that the ACh-triggered enzyme reactions reached a plateau within 30 min. Under the optimal conditions, ACh detection was performed, and the fluorescence spectra are shown in Figure 5B. On plotting the F450/F640 increment against the Ach concentrations, a fine linear relationship (R2 = 0.9899) for ACh quantification was observed in the range of 2−100 μmol/L with a LOD as low as 0.8 μmol/L. Fluorescence Confocal Imaging of •OH in Living Cells. The complexity of the intracellular system challenges biosensors not only in sensitivity but more significantly in selectivity. To further verify the selectivity of our dLys-AgNCs to Fenton reagents, various reactive oxygen species (ROS), reactive nitrogen species (RNS), and biochemical antioxidants that exist in balance with ROS,59 were also examined. As shown in Figure 6, compared with Fenton reagents, other ROS, RNS,

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Figure 6. Ratiometric fluorescent response of dLys-AgNCs to Fenton reagents (200 μmol/L H2O2 + 0.5 mmol/L Fe2+), various ROS, RNS, biochemical antioxidants, proteins, and amino acids. The concentrations of ONOO−, •O2−, •OtBu, Vc, and GSH were 500 μmol/L. The concentrations of ClO−, 1O2, Glu, Gly, and Phe were 100 μmol/ L. The concentrations of BSA and IgG were 20 μmol/L.

CONCLUSIONS In summary, we have developed a facile ratiometric fluorescent probe for detection of bioactive small molecules. The sensing mechanism was the quenching effect of Fe3+ and •OH to AgNCs, and •OH-induced oxidative damage to ligand of lysozyme. The dual-emitting dLys-AgNCs probe achieved admirable results for H2O2 detection with high sensitivity, wide detection range, and fantastic linearity. Meanwhile, this probe was exploited to the detection of glucose and ACh, holding potential application for sensing other H2O2-generated oxidase systems. Furthermore, monitoring the fluctuation of • OH levels sensitively in living cells enabled the dLys-AgNCs to be a new candidate for fluorescence imaging of •OH, which might have promising application in study of •OH-induced oxidative damage to protein. It is anticipated that this research will enrich the prevalence of dual-emitting metal nanoclusters

and antioxidants (Vc and GSH) induced minor changes of F450/F640. No obvious responses were observed for other related biological species including proteins and amino acids. In addition, little effect on the fluorescence intensity ratio was obtained upon exposing the dLys-AgNCs to metal ions (Figure S15). The high selectivity of the dLys-AgNCs for Fenton reagents could be explained by •OH-induced cross-linkage of tyrosine in ligand of denatured lysozyme and effective quenching of Fe3+ and •OH to AgNCs. These results indicated that this dual-emitting probe should fulfill the demand for monitoring •OH in biological system. 10636

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Figure 7. Fluorescence confocal images of PC-3 cells alone (A,E,I), PC-3 cells treated with dLys-AgNCs probe (B,F,J), PC-3 cells incubated with PMA (C,G,K) and NAC (D,H,L) prior to treatment with dLys-AgNCs. Top images: fluorescence confocal images of blue channel. Middle images: fluorescence confocal images of red channel. Bottom images: overlay images of blue and red channels.

in which stabilizing ligands can emit fluorescence intrinsically and no tedious fluorophore modification is involved.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b02995. UV−visible absorption spectra of dLys-AgNCs and its fluorescent response to Fenton reagents; experiments of optimizing conditions for the detection of H2O2, glucose, and ACh; interference study of metal ions (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +86-10-58802146. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by the National Natural Science Foundation of China (No. 20905008) and the Fundamental Research Funds for the Central Universities.

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