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Preparation of Silicon−Carbon-Based Dots@Dopamine and Its Application in Intracellular Ag+ Detection and Cell Imaging Yuliang Jiang, Zhaoyin Wang, and Zhihui Dai* Jiangsu Collaborative Innovation Center of Biomedical Functional Materials and Jiangsu Key Laboratory of Biofunctional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023, P. R. China S Supporting Information *
ABSTRACT: A novel nanocomposite, silicon−carbon-based dots@dopamine (Si-CDs@DA) was prepared using (3aminopropyl) triethoxysilane, glycerol, and dopamine as raw materials via a rapid microwave-assisted irradiation. This type of Si-CDs@DA exhibited ultrabright fluorescence emission (quantum yield of 12.4%) and could response to Ag+ selectively and sensitively. Moreover, the obtained Si-CDs@DA can be further applied in sensing intracellular Ag+ and cell imaging, because of its photostability, salt stability, and low cytotoxicity. This study provides a simple and efficient approach for preparing novel Ag+ fluorescent probes, which could expand the application of carbon nanomaterials in designing related biosensors. KEYWORDS: fluorescent probe, Si-CDs@DA, carbon-based dots, intracellular Ag+ sensing, cell imaging
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INTRODUCTION Silver ion (Ag+) is an important metal ion that is widely used in the electrical, photography/imaging, and pharmaceutical industries.1 However, it is also considered to be one of the most poisonous heavy metal ions because of its dangerous effects in humans and the environment.2−4 Therefore, the rapid, simple and sensitive detection of Ag+ is of great importance. Until now, methods such as atomic absorption spectroscopy, electrochemistry, and inductively coupled plasma mass spectroscopy have been used to detect metal ions with high sensitivity; however, these methods are usually complicated, time-consuming, and costly, whereas the fluorescence analysis method is currently considered to be a highly effective means for Ag+ detection. So far, there have been only a few reports on organic molecule fluorescence probes for the direct detection of Ag+, which impedes the wide application of this method.5−8 Therefore, it is of great significance to design and prepare a highly sensitive and selective fluorescent probe for Ag+ detection. In recent years, the emergence of different carbon nanomaterials, such as carbon nanotubes,9,10 fullerenes,11,12 graphene,13−16 and carbon dots17−21 have attracted intense attention because of their unusual properties. Among these carbon nanomaterials, carbon-based dots (CDs), including © XXXX American Chemical Society
carbon quantum dots and graphene quantum dots (GQDs) have been widely employed in various fields such as sensors,22 biomolecules,23 bioimaging,24 and oxygen reduction reaction.25 Compared with other fluorescent materials (conventional dyes, polymers, and semiconductor quantum dots), CDs could offer the advantageous features of bright fluorescence, high photostability, low toxicity, and resistance to metabolic degradation in bioapplications.26 However, problems such as the low yield and quantum efficiency still limit the applications of CDs. It has been demonstrated that the use of surface-passivated CDs is a highly effective approach for the improvement of the quantum yield (QY).27−32 Among these, functionalizing carbon nanomaterials with organosilicone can significantly enhance their optical properties and expand their novel applications.33 Recently, Chen’s group prepared a new type of CDs for sensitive and selective detection of quercetin by using N-(βaminoethyl)-γ-aminopropylmethyl-dimethoxysilane and citric acid as starting materials.34 However, so far there have been few reports on synthesizing novel silicon-doped photoSpecial Issue: Applied Materials and Interfaces in China Received: August 30, 2015 Accepted: October 16, 2015
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DOI: 10.1021/acsami.5b08089 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces Scheme 1. Schematic Illustration of the Procedure for Si-CDs@DA Synthesis
Figure 1. (a) UV spectrum, (b) IR spectrum, (c) Raman spectrum, (d) TEM image and HRTEM image (inset), (e) SEM image of Si-CDs@DA.
methods.35,36 In this work, we further prepared a novel silicon− carbon-based dots@dopamine (Si-CDs@DA) nanocomposite in a two-step procedure (Scheme 1). The obtained Si-CDs@ DA probe showed high QY (12.4%), photostability, salt
luminescent carbon nanomaterials with high QY. In our previous work, we successfully prepared two types of Ndoped CDs that exhibited highly selective and sensitive detection of Pb2+ and dopamine through different detection B
DOI: 10.1021/acsami.5b08089 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
Figure 2. (a) Emission spectrum of Si-CDs@DA at 400 nm excitation wavelength and excitation spectrum of Si-CDs@DA at the 478 nm emission wavelength (inset: Photographs of Si-CDs@DA taken under visible light and 365 nm), (b) PL intensity ratio (I/I0) of Si-CDs@DA at various pH values, (c) PL intensity ratio (I/I0) of Si-CDs@DA at different concentrations of KCl aqueous solution, (d) fluorescence intensity of Si-CDs@DA after different time periods. I0 and I are the fluorescence intensity at 400 nm in the absence and presence of ions, respectively. inverted fluorescence microscope (Nikon eclipse). Raman spectrum was measured using a Labram HR800 (Jobin Yvon). Synthesis of Si-CDs. The typical procedure for the microwaveassisted synthesis of Si-CDs was as follows: glycerol (5 mL) and APTES (20 μL) were mixed in a reaction bulb. The bottle was then placed inside the microwave reactor and irradiated for 5 min. After cooled to room temperature, the Si-CDs were obtained and stored in the refrigerator (4 °C) before use. Preparation of Si-CDs@DA. Si-CDs (10 mg) and dopamine (10 mg) were mixed in a pure water (15 mL). The mixture was stirred overnight at room temperature, and the reactant was filtered through a 0.22 μm microporous membrane to obtain the Si-CDs@DA. UV−Vis and Fluorescent Experiments. Si-CDs@DA was dissolved in phosphate buffer (pH 6.8) at an ambient temperature to obtain a stock solution (20.0 μM). The stock solution of metal ions was prepared in a pure water, and the concentration of metal ions was 20.0 μM. Test solutions were prepared by placing 3 mL of the stock solution into a cuvette. All experiments were performed at room temperature. MTT Assay. HeLa cells were harvested (the cell density was adjusted to 105 cells per mL) and seeded in a 96-well plate (90 μL well−1) overnight. Then different concentrations of Si-CDs@DA suspensions (20, 40, 60, 80, and 100 μg mL−1) were added. After that, the cells were cultivated for 24 h, and 20 μL of 1 mg/mL MTT solution was added to each cell well. After incubation for 4 h, the culture medium was discarded, and 150 μL of dimethyl sulfoxide was added. The obtained mixture was shaken for 15 min in the dark at room temperature, and its optical density was measured by using a microplate reader (Thermo). Cellular Imaging. Human Hela cells were cultured in a Dulbecco’s modified eagle medium (DMEM, Gibco, Grand Island, NY) containing 10% fetal bovine serum in a humidified incubator with 5% CO2 at 37 °C. The Si-CDs@DA suspension was injected into the well of a chamber slide with the final Si-CDs@DA concentration of 20
stability, and lower cytotoxicity, and thus these properties enabled its successful application in intracellular Ag+ sensing and cell imaging.
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EXPERIMENTAL SECTION
Materials and Reagents. Glycerol, (3-aminopropyl) triethoxysilane (APTES), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), and dopamine (DA) were purchased from Aladdin Chemistry Co. Ltd. (Shanghai, China) and were used without further purification. All other chemicals used in this work were obtained from commercial suppliers and used directly without further purification. Inorganic salts MnCl2, CaCl2, ZnCl2, FeCl2, NaNO3, KNO3, Co(NO3)2, Mg(NO3)2·6H2O, Fe(NO3)3·6H2O, Cu(NO3)2· 3H2O, Cd(NO3)2·2H2O, NiNO3·6H2O, AgNO3, and (CH3COO)2Pb were dissolved in doubly distilled water (100 mL) to obtain 20 μM aqueous solutions. All samples were prepared at room temperature and were shaken for 1 min before the test. Apparatus. The particle size distributions of Si-CDs@DA were observed using a transmission electron microscope (TEM, JEOL JEM2100, Japan). The microstructures of the Si-CDs@DA were examined using a JSM-7600F scanning electron microscope (SEM). The IR spectra of the product were measured by a Nicolet Nexus 670 Fourier transform infrared (FT-IR) spectrometer with a resolution of 4 cm−1 and scan times of 64 cm/s. IR samples were prepared by dropping the Si-CDs@DA ethanol solution onto the surface of a KBr wafer and then drying under an infrared lamp. Ultraviolet−visible (UV−vis) absorption spectra were measured using a Varian Cary 50 spectrophotometer with a 1 cm light path length. Fluorescence spectra were recorded using a Varian Cary Eclipse fluorescence spectrophotometer with an excitation wavelength of 400 nm. Si-CDs were prepared by using a microwave reactor (Yu Hua Instrument, China, 1 kW, 2.45 GHz). Live cell imaging was performed using an C
DOI: 10.1021/acsami.5b08089 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces μg mL−1. After incubation for 24 h, the cells were washed 3 times using phosphate buffered saline. The fluorescence images were characterized with an excitation wavelength of 488 nm.
fluorescent intensity (Figure 2d), suggesting that the stability of Si-CDs@DA is satisfactory. Fluorescent Response toward Metal Ions. We investigate the fluorescent response behaviors of Si-CDs@DA toward various metal ions (Cu2+, Ni2+, Mn2+, K+, Co2+, Cd2+, Ca2+, Na+, Fe3+, Ag+, Fe2+, Zn2+, Mg2+, Hg2+, and Pb2+). As shown in Figure 3, almost no fluorescent emission intensity
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RESULTS AND DISCUSSION Characterization of Si-CDs@DA. As shown in Figure 1a, two absorption bands at 280 and 350 nm appear in a typical UV−vis absorption spectrum. The absorption peak at approximately 280 nm is attributed to the π−π* transition corresponding to the carbon core CC of an aromatic π system. The shoulder peak at 350 nm can be regarded as the n−π* transition corresponding to the carbonyl/amine functional groups on the Si-CDs@DA surface.37 The functional groups on the Si-CDs@DA surface are further characterized by FT-IR. As is evident from Figure 1b, the several main absorption peaks can be assigned as the O−H stretching vibrations at approximately 3250−3500 cm−1, the C−H bond stretching vibrations at 2970 and 2860 cm−1, the characteristic CO stretching vibrations at 1730 cm−1, and the CC stretching vibrations at approximately 1400−1550 cm−1. The typical Si−O−Si asymmetric stretching peak is located at 1090 cm−1, and the C−Si stretching peak is located at 895 cm−1. Raman spectroscopy is also used to investigate the electronic properties of Si-CDs@DA as shown in Figure 1c. It is found that several typical peaks center at 1021.8 cm−1 (Si− O), 1266.4 cm−1 (C−O−C), 1580.6 cm−1 (CC, G peak), and 1790.1 cm−1 (CO), which are consistent with the IR spectrum data. Transmission electron microscopy (TEM) (Figure 1d) shows that the size of the as-prepared Si-CDs@DA is distributed in the 8−15 nm range with an average size of approximately 10 nm. In comparison with the Si-CDs TEM image (Figure S1), it can be found that the size of nanoparticles is not changed remarkably in the presence of DA. The lattice spacing in the HRTEM image is approximately 0.26 nm, indicating that the spacing array can be regarded as graphene layers (Figure 1d, inset) which is consistent with the previous report.38 Consistent with the TEM results, scanning electron microscopy (SEM) shows that most of the particles are small and spherical (Figure 1e). Fluorescence Properties. The excitation and emission spectra of Si-CDs@DA are presented in Figure 2a and show that the maximum excitation wavelength is 400 nm, possibly due to a transition from the σ and π highest occupied molecular orbital to the lowest unoccupied molecular orbital,39 and the maximum emission wavelength centers at 476 nm with a bright blue color that can be clearly observed under UV lamp illumination (λex = 365 nm, inset of Figure 2a). Using quinine sulfate as a reference, the QY of Si-CDs@DA in water at an excitation wavelength of 400 nm is found to be 12.4%. The effect of pH (in a range of 1−13) on fluorescence response of the Si-CDs@DA is examined. Experimental results depict that almost no fluorescence change is observed for Si-CDs@DA over a wide range of pH values from 4 to 8 as shown in Figure 2b. Moreover, the effect of salt concentration on fluorescent property is also investigated (Figure 2c). With the increase of KCl concentration from 0.2 to 2.5 M, the fluorescent emission intensity ratio (I/I0) of Si-CDs@DA does not change, demonstrating that the obtained Si-CDs@DA can preserve the strong photoluminescence under the extreme environmental condition of high salt concentration. The stability of SiCDs@DA is examined by keeping Si-CDs@DA in the air for two months. It is found that there is almost no change of the
Figure 3. Fluorescent intensity ratio (1 − F/F0) of the Si-CDs@DA (20 μM) in the presence of different individual metal ions (the concentration of metal ions was 20 μM). F0 and F are the fluorescent intensity at 400 nm in the absence and presence of ions, respectively.
change of the Si-CDs@DA can be observed after the addition of these metal ions except Ag+, indicating that Si-CDs@DA can be used to detect Ag+ with high selectivity. Sensing of Ag+. UV−vis assays of Si-CDs@DA toward Ag+ is further carried out. As depicted in Figure 4, with the increase
Figure 4. UV−vis spectra of Si-CDs@DA (10 μM) with different concentrations of Ag+ (from top to bottom, the concentrations of Ag+ are 0, 5, 10, 15, 20, 25, 30, 35, and 40 nM, respectively).
of Ag+ concentration from 0 to 40 nM, a new peak at 438 nm appear and increase gradually. To the best of our knowledge, Ag+ can be reduced to silver nanoparticles by using DA, which can be characterized by a typical UV absorption peak at about 430 nm.40 Therefore, we speculate that the novel nanocomposite, Si-CDs@DA, can also reduce Ag+. The relationship between Ag+ concentration and fluorescence intensity is further investigated. As shown in Figure 5a, the Ag+ efficiently quenches Si-CDs@DA fluorescence when the concentration of Ag+ reaches 80 nM, with the fluorescence intensity gradually D
DOI: 10.1021/acsami.5b08089 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
Figure 5. (a) Fluorescence emission spectrum of Si-CDs@DA (20 μM) in the presence of Ag+ (from top to bottom, the concentrations of Ag+ are 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, and 80 nM, respectively), (b) fluorescence intensity response of Si-CDs@DA to the concentration of Ag+ (λex = 400 nm).
decreasing to approximately 75% of its initial value. The relationship between the Si-CDs@DA fluorescence F/F0 and Ag+ ion concentration is linear in the 5−50 nM range of Ag+ concentration (Figure 5b). The fluorescence intensity is given by F/F0= −0.0145[Ag+] + 1.0018 (R2 = 0.997). On the basis of 3SD/k (where SD is the standard deviation of blank signal of the Si-CDs@DA and k is the slope of the calibration curve), the detection limit for Ag+ ions is estimated to be 2.5 nM, which is lower than some other fluorescent probes for Ag+ detection (Table 1). Table 1. Comparison of Analytical Performance of Different Ag+ probes probe
detection limit (μM)
ref
perylene derivatives phenanthro[9,10-d] imidazole derivative quinolinium/G-quadruplex naphthalimide derivatives metal−organic framework polytriazoles Si-CDs@DA
0.005 0.1 26.0 0.1 0.422 0.0025
41 42 43 44 45 46 This work
Figure 6. EDS analysis of Si-CDs@DA-Ag+.
out to evaluate the cytotoxicity of the Si-CDs@DA to Hela cells. As illustrated in Figure S4, the viability of Hela cells declines by only
