Article pubs.acs.org/ac
“Light-on” Sensing of Antioxidants Using Gold Nanoclusters Lianzhe Hu, Lin Deng, Shahad Alsaiari, Dingyuan Zhang, and Niveen M. Khashab* Controlled Release and Delivery Lab, Advanced Membranes and Porous Materials Center, King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia S Supporting Information *
ABSTRACT: Depletion of intracellular antioxidants is linked to major cytotoxic events and cellular disorders, such as oxidative stress and multiple sclerosis. In addition to medical diagnosis, determining the concentration of antioxidants in foodstuffs, food preservatives, and cosmetics has proved to be very vital. Gold nanoclusters (Au-NCs) have a core size below 2 nm and contain several metal atoms. They have interesting photophysical properties, are readily functionalized, and are safe to use in various biomedical applications. Herein, a simple and quantitative spectroscopic method based on Au-NCs is developed to detect and image antioxidants such as ascorbic acid. The sensing mechanism is based on the fact that antioxidants can protect the fluorescence of Au-NCs against quenching by highly reactive oxygen species. Our method shows great accuracy when employed to detect the total antioxidant capacity in commercial fruit juice. Moreover, confocal fluorescence microscopy images of HeLa cells show that this approach can be successfully used to image antioxidant levels in living cells. Finally, the potential application of this “light-on” detection method in multiple logic gate fabrication was discussed using the fluorescence intensity of Au-NCs as output. fluorescent gold NCs (Au-NCs) have received considerable attention owing to their colloidal stability, facile synthesis, low cytotoxicity, and good photophysical properties.20−22 These features make them attractive for a wide variety of biochemical applications, such as sensing,23−26 in vitro and in vivo imaging,27−29 and cancer therapy.30,31 “Light-on” fluorescent sensors designed using Au-NCs are still relatively rare, as most reported sensors based on Au-NCs depend on the decrease in fluorescence intensity.32,33 Moreover, although Au-NCs have been demonstrated as attractive fluorescence agents in cellular imaging, the use of Au-NCs as specific probes for intracellular imaging of selected analytes also remains largely unexplored.34 Chen et al. have recently reported that the strong fluorescence of Au-NCs can be quenched by highly reactive oxygen species (hROS), including •OH, ClO−, and ONOO−.35 The fluorescent imaging of hROS in living cells has been developed only by using Au-NC-decorated silica nanoparticles as fluorescent probes. Herein, a simple approach to detect and image antioxidant based on protecting the strong fluorescence of Au-NCs against hROS quenching is presented (Scheme 1). Using ascorbic acid (AA) as representative antioxidant and •OH as hROS model, the quantitative fluorescence analysis of AA concentration is achieved. The proposed method shows good selectivity toward AA compared to nonantioxidants, and its potential applications in screening antioxidant capacity of different antioxidants and sensing the antioxidant activity of commercial fruit juice is demonstrated. In addition, the use of
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eactive oxygen species (ROS) represent an important class of radical species that are generated in metabolic processes and usually include superoxide (O2−), hydroxyl radical (•OH), hypochlorite (ClO−), and peroxynitrite (ONOO−). High concentration of ROS may cause oxidative stress, which can induce damage of proteins, DNA, and lipids, and thus lead to numerous human diseases, including cardiovascular disease, neurological disorders, and cancers.1 To protect the cells and organs against oxidative stress, living organisms developed sophisticated and complex antioxidant protection system.2 In normal aerobic cells, ROS exist in balance with biochemical antioxidants such as glutathione (GSH).3 Increasing intake of antioxidants through fresh fruits and vegetables also helps to maintain an adequate antioxidant status in human body.4 Uric and ascorbic acids are by far the antioxidants in highest concentration in human blood. Recent reports have linked uric acid concentration in blood to early symptoms of multiple sclerosis (MS), as it is difficult to diagnose such disease at early stages.5−9 Therefore, measuring the antioxidant capacity in biological fluids and foodstuffs is of high interest for various fields, including cell biology, food chemistry, pharmaceuticals screening, and diagnosis of oxidative stress-associated diseases in clinical chemistry.10−17 Metal nanoclusters (NCs) are a subclass of metal nanoparticles with a core size below 2 nm and typically contain several to tens of metal atoms.18,19 This size is comparable to the Fermi wavelength of conduction electrons. Owing to the strong quantum confinement of free electrons in this size regime, metal NCs possess discrete electronic states and exhibit interesting moleculelike properties, including quantized charging and size-dependent fluorescence. Among metal NCs, © 2014 American Chemical Society
Received: February 6, 2014 Accepted: April 30, 2014 Published: April 30, 2014 4989
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concentrations of AA in 10 mM PBS (pH 5.0), followed by the addition of 50 μL of 3 mM Fe2+ and 50 μL of 3 mM H2O2, respectively. After incubation at room temperature for 5 min, the solution fluorescence spectra were measured upon being excited at 375 nm. For commercial juice samples determination, the orange, apple, and mango juices were first diluted using 10 mM PBS (pH 5.0) by 1000-, 300-, and 250-fold, respectively. Then the diluted juice samples were tested following the procedure for AA detection. Cell Culture and Cell Viabilities. HeLa cells were cultured in EMEM medium containing 10% FBS and penicillin− streptomycin at 37 °C in a humidified 5% CO2 atmosphere. The cytotoxicity of Au-NCs was evaluated by using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. HeLa Cells were seeded at a density of 5 × 103 cells per well in 96-well flat bottom plates and incubated for 12 h. Cells were washed by DPBS buffer and incubated in the culture media with Au-NCs for 24 h at 37 °C. Cell viability was evaluated by the MTT colorimetric procedure. Intracellular Imaging. After cell attachment, 20 μg/mL Au-NCs was added and incubated with the EMEM solution at 37 °C for 2 h for cell uptake. In the assay of ClO−, the cells were washed three times, followed by the addition of 500 μM NaClO in PBS (pH 7.4) and incubation for 5 min before fluorescence imaging. In the assay of AA, the cells were washed three times, followed by the addition of 1 mM AA in PBS (pH 7.4) and incubation for 5 min. NaClO (500 μM) was then added, and the cells were incubated for another 5 min before fluorescence imaging.
Scheme 1. Sensing Principle of the Antioxidant Sensor Based on Au-NCs
fluorescent Au-NCs for intracellular imaging of antioxidant levels is also achieved. The ultrasmall fluorescent Au-NCs show excellent biocompatibility and are efficiently internalized by HeLa cells. Moreover, Au-NCs exhibited rapid response toward ClO− and AA/ClO−, thus showing great potential for imaging the ROS status and the antioxidant levels in living cells. Molecular logic gates have attracted much attention in the field of molecular-scale electronics, as well as chemical and biological computers.36−38 Since the fluorescence of Au-NCs can be tuned by the addition of Fe2+/H2O2 or •OH/AA, a multiple molecular logic gate system, including a NAND logic gate and an IMPLICATION logic gate, is developed. The NAND logic gate with Fe2+ and H2O2 as inputs allows the monitoring of Fe2+ and H2O2, while the IMPLICATION logic gate with •OH and AA as inputs can reflect the relative antioxidant levels in the solutions.
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RESULTS AND DISCUSSION Synthesis of Au-NCs. The Au-NCs are synthesized using a glutathione (GSH) template according to a previous report.39 The average size of the resulting Au-NCs is about 2 nm, as revealed by transmission electron microscopy (TEM) [Figure S1, Supporting Information (SI)]. The excitation and emission fluorescence spectra of as-prepared Au-NCs are shown in Figure S2 (SI). Upon excitation at 375 nm, the Au-NCs showed an emission band centered at 610 nm. The characteristics of the spectra are consistent with those reported in the literature, which confirmed the successful preparation of AuNCs. Responsiveness of Au NCs to Antioxidants. The fluorescence quenching of Au-NCs in the presence of •OH, ClO−, and ONOO− has been investigated by Chen et al.35 The fluorescence quenching was correlated linearly to the concentration of hROS, which was consistent with the quasifirst-order reaction mechanism. Among these hROS, •OH was found the most effective quencher. The fluorescence response of Au-NCs to •OH and •OH/AA is shown in Figure 1A. The •OH is generated by the Fenton reaction (Fe2+ + H2O2 → Fe3+ + •OH + OH−) through mixing ferrous ions with H2O2. In the presence of both Fe2+ and H2O2, the fluorescence of Au-NCs is quenched. This is consistent with previous reports, and the fluorescence quenching is attributed to the strong oxidative ability of •OH, which oxidizes the Au(0) in Au-NCs to Au(I).35 Because the amount of •OH generated can be adjusted by changing the amount of Fe2+ and H2O2, the fluorescence intensity of Au-NCs depends on the concentration of both Fe2+ and H2O2, which can be used for fluorescence sensing of Fe2+ and H2O2 as well (Figure S3, SI). To investigate the effect of antioxidants on the fluorescence of Au-NCs, we used ascorbic acid (AA) as a representative
EXPERIMENT SECTION Materials and Instruments. Chloroauric acid (HAuCl4· 4H2O), glutathione (GSH), hydrogen peroxide (H2O2), sodium hypochlorite (NaClO), ferrous chloride, and ascorbic acid (AA) were purchased from Sigma-Aldrich. All reagents were of analytical reagent grade and used without further purification. Commercial fruit juice was purchased from Abuljadeyel Beverages International Co. The human cer-vical tumor cell line (HeLa) was purchased from ATTC. Eagle’s minimal essential medium (EMEM) and fetal bovine serum (FBS) were purchased from Invitrogen. Fluorescence measurements were performed on a Cary Eclipse fluorescence spectrophotometer (Varian). The emission spectra were recorded upon excitation at 375 nm. The slits for excitation and emission were set at 10 nm. Absorbance spectra were measured using a Cary-5000 UV−vis−NIR spectrophotometer (Varian) with a 1 cm cell path length. Transmission electron microscopy (TEM) images were obtained with a Tecnai T12 transmission electron microscope. Confocal laser scan microscopy images were collected using a fluorescence microscope (CLSM, Zeiss) with an excitation wavelength of 405 nm. Synthesis of Au-NCs. Au-NCs were prepared as follows.39 Typically, 2.5 mL of aqueous HAuCl4 (4 mM) was added to 2.5 mL of GSH solution (8 mM) under vigorous stirring at room temperature. After 5 min of mixing, the solution was allowed to react at 70 °C for 24 h. The solution was then dialyzed using a 1000 Da MWCO dialysis bag in double distilled water for 48 h to remove unreacted compounds. The final solution was stored at 4 °C in refrigerator when not in use. Antioxidant Sensing. For AA detection, 100 μL of asprepared Au-NCs were mixed with 2.8 mL of different 4990
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Figure 2. TEM of Au-NCs after being treated with •OH (A) and with •OH and AA (B).
intensity of Au-NCs should definitely be ascribed to their oxidation by the generated •OH, which is consisted with previous report.35 Sensing of AA and Antioxidant Screening. As shown is Figure 3, the fluorescence intensity of Au-NCs increased as the
Figure 1. (A) The fluorescence intensity of (a) 50 μg/mL Au NCs, (b) 50 μg/mL Au NCs + 50 μM Fe2+ + 50 μM H2O2, and (c) 50 μg/ mL Au NCs + 50 μM Fe2+ + 50 μM H2O2 + 150 μM AA. (B) The fluorescence intensity of (a) 50 μg/mL Au NCs, (b) 50 μg/mL Au NCs + 50 μM Fe2+, (c) 50 μg/mL Au NCs + 50 μM H2O2, and (d) 50 μg/mL Au NCs + 150 μM AA. Figure 3. Fluorescence intensity of Au NCs/Fe2+/H2O2 system in the presence of AA from 0, 5, 10, 20, 30, 40, 60, 80, 100, to 120 μM. Inset: the linear relationship between the fluorescence intensity and the concentration of AA.
analyte. As shown in Figure 1A, the fluorescence of Au-NCs was more than 95% of its original intensity in the presence of AA and •OH. AA is a well-known •OH scavenger; thus, the removal of •OH by AA is the reason behind the protection of fluorescence against quenching. In a control experiment, using only Fe2+, H2O2, or AA showed negligible effect on the fluorescence intensity (Figure 1B). This further demonstrates that the generation of •OH and the antioxidant capacity of AA result in the fluorescence intensity change. We also evaluated the reversibility of the fluorescence quenching. However, the fluorescence of •OH-quenched Au-NCs failed to recover, even in the presence of a 10-fold concentration of AA, which suggests the oxidation of Au-NCs is irreversible in this case. As shown in Figure 1A, a small peak centered at 510 nm appears after the fluorescence of Au-NCs is quenched by •OH. This may be due to light scattering of the oxidized product of Au-NCs. The UV−vis absorption spectra of Au-NCs are presented in Figure S4 (SI). In the presence of •OH, the spectrum of Au-NCs shows a broad range adsorption increase because of the oxidation. The absorption spectrum shows no surface plasmon resonance (SPR) peak, suggesting that larger Au nanoparticles were not generated. In the presence of AA, the UV−vis spectra are similar to the original absorption of AuNCs. Figure 2 shows the TEM images of Au-NCs after being treated with •OH and •OH/AA, respectively. No obvious morphology changes of Au-NCs are observed after treatment with •OH. This indicates that the decrease in fluorescence
concentration of AA increased in the presence of a fixed amount of •OH. The fluorescence intensity versus the AA concentration is linear over a concentration range of 5−100 μM (R = 0.979). The relative standard deviation (RSD) was 2.1% for six repeated measurements of 20 μM AA. The detection limit of AA is 5 μM at a signal-to-noise ratio of 3. The selectivity of Au-NCs toward AA is also studied. The fluorescent responses of Au-NCs solutions to some common biological molecules are monitored and compared to that of AA. Figure 4 shows that the addition of 100 μM AA results in about 6-fold increase of the fluorescence intensity of Au-NCs. In contrast, no obvious increase in fluorescent intensity is observed by adding 100 μM of biological ions, amino acids, or proteins. This data demonstrates that the antioxidant capacity of AA caused the increase in fluorescence intensity; thus, AuNCs can be used as a simple and reliable fluorescent probe of the light-on detection of AA with minor interference from other nonantioxidant molecules. The proposed method is further used to screen antioxidants with respect to their ROS-scavenging capacities. At present, on the basis of the chemical reaction involved, major antioxidant capacity assays can be divided into two categories, including assays based on electron transfer and hydrogen atom transfer.40 4991
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acid, gallic acid, and tartaric acid are tested as model scavengers of •OH. All of these antioxidants can induce the increase in fluorescence intensity of Au-NCs. Citric acid and AA show higher •OH removing ability, while tartaric acid and gallic acid perform less well. Compared with previous optical methods for antioxidant capacity assays,13,41,42 our approach employing AuNCs as fluorescent probes has more benefits, including rapid response, large Stokes shift, low toxicity, and ease of use. Antioxidants Sensing in Fruit Juices. Foodstuffs are a good exogenous source of natural antioxidants. The total antioxidant capacity (TAC) of food samples is usually used to evaluate their health beneficial effects. In order to demonstrate the potential application of the proposed method, the TAC of commercial fruit juice is tested. The results of TAC in three types of fruit juice are expressed in AA concentration and shown in Table S1 (SI). The TAC values of orange, apple, and mango juices were 49.69, 17.61, and 11.44 mM, respectively. The relative standard deviation ranged from 2.3% to 3.5% for three replicate measurements of fruit juices, indicating the good repeatability of this method. To further demonstrate the reliability of this method, the concentration of AA in each sample was tested three times by adding fixed amounts of AA to commercial fruit juices. The analytical recoveries of added AA ranged from 91.3% to 94.8%. These results confirm that the proposed method has great potential application in TAC determination in commercial fruit juices. Intracellular Imaging. The antioxidant levels in living cells are essential to control cellular function. The ability of Au-NCs to image antioxidant levels is explored using HeLa cells as a model system. Upon incubation with Au-NCs (20 μg/mL) in a serum-supplemented cell culture medium for 2 h, appreciable amounts of Au-NCs are observed inside the HeLa cells (Figure 6a). This result indicates that the Au-NCs are rapidly uptaken into cells and maintain their fluorescence emission characteristic. The Au-NC-loaded cells were then treated with ClO− as a typical hROS because it is difficult to generate •OH at physiological pH using the Fenton reaction. The fluorescence of Au-NCs can be quenched by ClO− at physiological pH, and AA can also protect the fluorescence against quenching. The fluorescence spectra of Au-NCs in the presence of different
Figure 4. Fluorescence response of Au-NCs/Fe2+/H2O2 system in the presence of Zn2+, Mg2+, Fe3+, Ca2+, Cu2+, proline (Pro), serine (Ser), phenylalanine (Phe), tyrosine (Tyr), bovine serum albumin (BSA), glucose oxidase (GOx), lysozyme (Lys), and AA.
In the hydrogen atom transfer based assays, the capability of an antioxidant to quench free radicals is measured. •OH is a universal radical in organisms and can be used to evaluate antioxidant capacity in vitro. As shown in Figure 5, AA, citric
Figure 5. Fluorescence intensity of 50 μg/mL Au-NCs with different concentrations of (a) citric acid, (b) AA, (c) tartaric acid, and (d) gallic acid.
Figure 6. Confocal fluorescence microscopy images of HeLa cells treated with (a) PBS buffer, (b) 500 μM ClO−, and (c) 1 mM AA with 500 μM ClO− and corresponding bright-field images of (d) PBS buffer, (e) 500 μM ClO−, and (f) 1 mM AA with 500 μM ClO−. Scale bar: 20 μm. 4992
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concentrations of ClO− and AA/ClO− in PBS buffer (pH 7.4) are shown in Figure S5 (SI). When the Au-NC-loaded cells are incubated with 500 μM ClO− for 10 min, the fluorescence signal observed is much weaker (Figure 6b). This implies that the fluorescent Au-NCs are also oxidized by ClO− in living cells. To validate the response of this nanosensor toward antioxidants, the cells were incubated with 1 mM AA before the addition of ClO−. In this case, the fluorescence signal increases notably, confirming that AA can also protect the fluorescence against quenching by ClO− in living cells (Figure 6c). The corresponding bright-field images are also given in Figure 6d−f, which also showed good response toward the ClO− and AA/ClO−. The biocompatibility of Au-NCs is then tested by using the MTT assay (Figure S6, SI). After 24 h incubation with Au-NCs, the cell viability is estimated to be greater than 85% over a wide concentration range of 10−5−102 μg/mL, which confirms the excellent biocompatibility and the great potential of Au-NCs for ROS status and antioxidant level imaging in living cells. Molecular Logic Gates Based on Au-NCs. On the basis of the observation that the fluorescence of Au-NCs is quenched by •OH, Boolean NAND logic gate are developed to show the potential of this method for multiplexed detection of analytes. As shown in Figure 7A, Fe2+ and H2O2 are used as inputs and
A similar NAND logic gate composed of Au-NCs using nitrite and H2O2 as inputs was recently developed.43 Compared with previous reports, the advantage of this study is that more complex logic gates can be created. By controlling the concentration of •OH and AA, the first IMPLICATION logic gate based on Au NCs is designed (Figure 7B). The •OH is generated through mixing ferrous ions with H2O2 beforehand. Only with the input of •OH and without AA (1/0) is the fluorescence of Au-NCs quenched, and the output is “0”. In all other cases, the outputs are “1”. This IMPLICATION logic gate can be used to reflect the relative concentrations of antioxidants in solutions. The multifunctional system developed in this study allows the monitoring of Fe2+/H2O2 and •OH/AA levels by using only one fluorescence probe. The fluorescence spectra of the NAND and the IMPLICATION logic gates are shown in Figure S7 (SI).
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CONCLUSIONS A simple and selective spectroscopic method for antioxidant sensing and imaging is successfully demonstrated. The sensing mechanism is based on the ability of antioxidants to protect the fluorescence of Au-NCs against quenched by hROS. This method is used for evaluating the antioxidant content in commercial fruit juices and proves to be superior to existing spectroscopic sensing methods in terms of rapid response, ease of use, and good biocompatibility. Intracellular studies showed the great potential of Au-NCs for probing antioxidant levels in living cells. Through integrating the fluorescence feature of nanoclusters with the Boolean logic gate concept, a multiple logic gate system is reported using Au-NCs, which demonstrates the potential of this system to be employed in complex logic devices at the molecular level for multiplex analysis. Thus, Au-NCs are foreseen as promising fluorescent probes in antioxidant sensing, imaging, drug antioxidant capacity screening, and logic gate applications in molecular devices.
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ASSOCIATED CONTENT
S Supporting Information *
TEM, fluorescence, and UV−vis spectra, MTT assay and juices determination results (Figures S1−S7 and Table S1). This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +966-128021172. Fax: +966-128082410. E-mail: Niveen.
[email protected]. Author Contributions
Figure 7. (A) The truth table, symbol, and algebraic expression of the NAND logic gate based on Au-NCs. (B) The truth table, symbol, and algebraic expression of the IMPLICATION logic gate based on AuNCs.
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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the fluorescence intensity of Au-NCs as output. For input, the presence of Fe2+ or H2O2 is defined as 1 and their absence as 0. The fluorescence intensity at 610 nm serves as the output (1 or 0). The absence of both inputs (0/0) or the presence of either input (1/0, 0/1) gives an output signal of “1”. Only the presence of both inputs (1/1) causes a significant fluorescence quenching, giving an output signal of “0”. The NAND logic operation could be realized by controlling the concentration of Fe2+ and H2O2.
ACKNOWLEDGMENTS The authors gratefully acknowledge King Abdullah University of Science and Technology (KAUST) for the support of this work.
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