Carbon Dots Based Dual-Emission Silica Nanoparticles as a

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Carbon Dots Based Dual-Emission Silica Nanoparticles as a Ratiometric Nanosensor for Cu2+ Xiangjun Liu, Nan Zhang, Tao Bing, and Dihua Shangguan* Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China S Supporting Information *

ABSTRACT: A simple and effective strategy for designing ratiometric fluorescent nanosensor has been described in this work. A carbon dots (CDs) based dual-emission nanosensor for Cu2+ detection was prepared by coating CDs on the surface of Rhodamine B-doped silica nanoparticles. The fluorescent CDs were synthesized using N-(β-aminoethyl)-γ-aminopropyl methyldimethoxysilane (AEAPMS) as the main raw material, so that the residual ethylenediamine groups and methoxysilane groups on the surface of CDs can serve as the Cu2+ recognition sites and the silylation reaction groups. The obtained nanosensor showed characteristic fluorescence emissions of Rhodamine B (red) and CDs (blue) under a single excitation wavelength. Upon binding to Cu2+, only the fluorescence of CDs was quenched, resulting in the ratiometric fluorescence response of the dual-emission silica nanoparticles. This ratiometric nanosensor exhibited good selectivity to Cu2+ over other substances, such as metal ions, amino acids, proteins, and vitamin C. The ratio of F467/F585 linearly decreased with the increasing of Cu2+ concentration in the range of 0 to 3 × 10−6 M, a detection limit as low as 35.2 nM was achieved. Additionally, this nanosensor was successfully applied for the ratiometric fluorescence imaging of Cu2+ in cells and determination of Cu2+ in real tap water.

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luorescent carbon dots (CDs), a fascinating class of quantum dots (QDs) based on carbon materials, have attracted increasing attention in recent years. Compared with conventional semiconductor QDs that usually contain heavy metal, for example, Cd2+, the good photostability, high biocompatibility, and low cytotoxicity make CDs good alternatives to QDs for biosensing. Up until now, many methods have been developed for preparation of CDs and the applications of CDs are mainly focusing on the optoelectronic devices and bioimaging.1−9 Recently, through surface functionalization, CDs have been endowed with molecular recognition ability and applied in chemical sensing. Dong et al. have developed polyamine-functionalized CDs and used them for selective detection of Cu2+.10 Qu et al. have developed N-(2aminoethyl)-N,N,N′-tris(pyridine-2-ylmethyl) ethane-1,2-diamine modified CDs and used them for specific determination and cellular imaging of Cu2+.11 Additionally, the CDs prepared using pomelo peel as a carbon source have been demonstrated for sensitive and selective detection of Hg2+.12 Zhu et al. have developed a simple method for preparation of CDs using citric acid and ethylenediamine, which shows high quantum yield and good selectivity for Fe3+.13 Copper plays important roles in many areas, such as chemical, electronic, environment, and biological field. Especially, copper is a cofactor for numerous enzymes and plays an important role in some physiological and pathological events, such as Wilson disease and Alzheimer’s disease.14,15 © 2014 American Chemical Society

However, copper is highly toxic to human health in high concentration, such as serious infant liver damage and childhood cirrhosis.16 Along with the widespread use of copper in industry, the copper contamination of the environment has attracted more and more attention. Many methods, such as atomic absorption spectrometry, inductively coupled plasma atomic emission spectrometry, and inductively coupled plasma mass spectroscopy, have been developed for the detection of trace copper ions.17−20 Recently, fluorescent chemosensors for copper ions detection have attracted increasing attention due to the high sensitivity, good selectivity, and easy operation.21−29 However, most of the chemosensors respond to copper ions by increasing or decreasing the fluorescence intensity, this singleintensity-based sensing is usually compromised by some other factors, such as the concentration change of sensors, drift of light source or detector, or environmental effects in complex samples. Ratiometric fluorescent sensors can avoid these problems and have attracted much attention recently.26,30,31 However, this strategy is limited by the molecular design and synthesis of new dual-emission fluorophores that are sensitive and selective to Cu2+. Besides, many chemosensors are suffering from poor water solubility. Received: October 14, 2013 Accepted: January 29, 2014 Published: January 29, 2014 2289

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Technical Note

Dual-emission fluorescent nanoparticles used for ratiometric fluorescence detection have attracted significant attention in recent years. They are simply obtained by combining two different fluorophores in one nanoparticle, one fluorophore as reference and another as a signal report unit.32−38 Compared with the organic fluorophores, dual-emission fluorescent nanoparticles do not need elaborate molecular design and complicated synthesis. Recently, CDs-based dual-emission nanohybrid materials are prepared and used as a ratiometric fluorescent probe for Cu2+; because the CDs itself do not have the ability of Cu2+ recognition, an organic molecule specific for Cu2+ was first immobilized on the surface of CDs, which increased the complexity of preparation.39 Herein, we developed a simple method to prepare dualemission nanoparticles for ratiometric detection of Cu2+. The CDs with Cu2+ recognition sites were simply prepared using N(β-aminoethyl)-γ-aminopropyl methyldimethoxysilane (AEAPMS) as the main raw material and then covalently linked to Rhodamine B-doped silica nanoparticles through silylation reaction without any other surface functionalization. The selectivity and sensitivity of this ratiometric fluorescence nanosensor to Cu2+ were carefully investigated. The feasibility of the dual-emission silica nanoparticles for Cu2+ determination in real water samples and Cu2+ imaging in cells were also investigated.

at room temperature to obtain RBITC-APTES. A volume of 100 μL of the above RBITC-APTES reaction solution and 50 μL of TEOS was added into 4.0 mL of ethanol; after stirring for 30 min, 200 μL of ammonia was injected and the mixture was stirred at room temperature for 8 h. Then another 80 μL of TEOS was added and stirred for 24 h at room temperature. Subsequently, the reaction mixture was centrifuged and the silica nanoparticles were collected and washed 2 times with ethanol. The obtained nanoparticles were ultrasonically dispersed in 4.0 mL of ethanol for 20 min, then 10 μL of TEOS and 100 μL of ammonia was injected to the dispersion solution and stirred at room temperature for 24 h. The resultant dye-doped silica nanoparticles were collected by centrifugation and washed 3 times with ethanol and redispersed into 4.0 mL of ethanol. Preparation of CDs Coated Dual-Emission Silica Nanoparticles. A volume of 3.0 mL of the above dye-doped silica nanoparticles was ultrasonically dispersed for 20 min and was added with 100 μL of ammonia. After stirring for 30 min, 10 μL of CDs was added and stirred for about 15 h at room temperature. Then, the reaction mixture was centrifuged and the obtained dual-emission silica nanoparticles were washed 4 times with ethanol and then redispersed in ethanol at a concentration of 10 mg/mL for use. After suspension in ethanol for 24 h, the dual-emission silica nanoparticles were spun down and resuspended in ethanol again. No fluorescence was found in the supernatant and the fluorescence of the resuspended silica nanoparticles did not decrease (Supporting Information, Figure S1), indicating that the free CDs adsorbed on the obtained dual-emission silica nanoparticles were washed off thoroughly. Fluorescence Assay of Cu2+. The fluorescence detection was all performed in phosphate buffer (PB, 10 mM, pH 7.4) solution. Typically, 0.5 mL of dual-emission silica nanoparticles suspension was centrifuged and redispersed into 5.0 mL of PB solution, which was used as the working solution of ratiometric nanosensor. Cu2+ was directly added to nanosensor solution, the fluorescence spectra were collected on a Hitachi F-4600 fluorescence spectrophotometer with excitation at 360 nm. Selectivity and Interference Tests. The stock solutions of other metal ions (Ca2+, Mg2+, Hg2+, Co2+, Cd2+, Ni2+, Pb2+, Ba2+, Mn2+, Zn2+, Ag+, K+, Fe3+, Al3+) were prepared in ultrapure water at the concentration of 1.0 mM. Additionally, considering that amino acids and proteins in the biological system may interact with dual-emission silica nanoparticles, and interfere with the detection of Cu2+, the common amino acids (Gly, Ala, Phe, Cys, His, Trp, Glu, Lys, Pro, Arg, Ser, Ile, Ser, Cystine), two proteins (HSA, BSA), and vitamin C (VC, a common and highly abundant vitamin) were chosen for the interference test. For the selectivity and interference study, a small aliquot of the stock solution of interference substances (other metal ions, amino acids, proteins, or VC) were mixed with dual-emission silica nanoparticles solution; and then the fluorescence spectra were recorded. Then, a small aliquot of Cu2+ stock solution was further added to the mixtures, and the fluorescence spectra were collected again. Cell Culture and Fluorescence Imaging. MCF-7 cells (5 × 104 per dish) were seeded in glass bottom culture dishes and grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in 5% CO2 atmosphere for 24 h. After removal of the culture medium, cells were incubated with dual-emission silica nanoparticles in 1.0 mL of fresh culture medium (0.2 mg/mL) for 1.5 h. Then the medium was

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EXPERIMENTAL SECTION Reagents. AEAPMS and anhydrous citric acid were purchased from Alfa Aesar (Tianjin, China). Aminopropyltriethoxysilane (APTES) was purchased from J&K (Beijing, China). Rhodamine B isothiocyanate (RBITC) was purchased from SERVA. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Beijing, China). Penicillin and streptomycin were purchased from Hyclone (Beijing, China). Tetraethoxysilane was purchased from Xilong Chemical (Shantou, China). Ethanol, NH4OH, and other reagents were purchased from Beijing Chemical. Ultrapure water was used throughout all the experiments. Apparatus. Fluorescence measurements were performed on a Hitachi F-4600 fluorescence spectrophotometer. Fluorescence images were performed on an Olympus FV1000-IX81 laser confocal microscope. Dynamic light scattering (DLS) was performed on a Zetasizer nano ZS (ZEN3600) instrument. Transmission electron microscopy (TEM) measurements were obtained on a JEM-2011 electron microscope. Cu2+ in real tap water samples was detected on an Agilent 7700 inductively coupled plasma mass spectrometer (ICPMS). The element contents on the surface of the nanoparticles were determined on an ESCALab22I-XL X-ray photoelectron spectrometer (XPS). Synthesis of CDs. CDs were synthesized according to a reported method.7 Briefly, 2.0 mL of AEAPMS was heated to 230 °C, then 100 mg of anhydrous citric acid was added quickly under vigorous stirring. After 3 min, the reaction stopped and cooled to room temperature. Then the products were purified by precipitation with petroleum ether three times. A volume of 0.4 mL of final product was taken into 1.6 mL of ethanol for the next use. Preparation of Dye-Doped Silica Nanoparticles. For preparation of dye-doped silica nanoparticles, RBITC was precoupled to APTES. A total of 4.7 mg of RBITC and 50 μL of APTES were added into 0.47 mL of ethanol, stirred for 24 h 2290

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Scheme 1. Schematic Illustration of the Preparation of Dual-Emission Silica Nanoparticles and Its Sensing Mechanism to Cu2+

removed, and the cells were washed two times with PBS to remove the residual nanoparticles. Subsequently, 0.5 mL of 4% paraformaldehyde was added to fix cells for 15 min. Then cells were washed three times with PBS and permeabilized by 0.5 mL of 0.5% triton X-100 for 5 min. After washing three times with PBS, cells were suspended in 0.5 mL of PBS and observed under an Olympus FV1000-IX81 laser confocal microscope. Blue and red emissions from CDs and Rhodamine B were excited at 405 nm. Analysis of Cu2+ in Real Tap Water Samples. Tap water samples were collected from our lab and filtered through a 0.22 membrane prior to use. Different concentrations of Cu2+ were spiked in the water samples and analyzed by ratiometric nanosensor.

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RESULTS AND DISCUSSION The preparation and sensing mechanism of the ratiometric nanosensor are shown in Scheme 1. First, dye-doped silica nanoparticles (red) were prepared using RBITC-APTES and TEOS by a stöber method. Then a thin silica shell was deposited on the red silica core. Lastly, CDs were coupled on the surface of the dye-doped silica nanoparticles through the silylation reaction. The obtained hybrid nanomaterials showed two emission peaks at 467 and 585 nm, which can be assigned to the CDs emission and Rhodamine B emission. Because of the presence of large amount of ethylenediamine groups on the surface of CDs, Cu2+ can selectively bind to CDs resulting in quenching of the blue fluorescence, whereas the red fluorescence of the dye-doped silica core still remained, thereby realizing the ratiometric fluorescence response to Cu2+. To avoid leakage of free dye, RBITC was covalently linked to APTES. A thin silica shell was further deposited on the dyedoped nanoparticle core to avoid the direct contact of Rhodamine B with components in the sample, which may interfere in the detection. The CDs used in this study were synthesized according to the reported method.7 Because N-(βaminoethyl)-γ-aminopropyl methyldimethoxysilane (AEAPMS) was used as the main raw material, the obtained CDs bore a large amount of ethylenediamine groups and methoxysilane groups. The ethylenediamine groups could recognize Cu2+, and the methoxysilane groups could couple on the surface of silica nanoparticle through silylation reaction. The fluorescence spectra of CDs at different excitation wavelength were shown in Figure 1a. When excited at wavelengths in the range of 300−400 nm, especially in 340− 380 nm, the CDs showed strong blue emission. However, along

Figure 1. (a) Fluorescence spectra of CDs excited at different excitation wavelength and (b) size distribution of the CDs in ethanol.

with further increasing of the excitation wavelength, the emission red-shifted and the intensity decreased greatly. Under the excitation of 360 nm, the CDs exhibited the strongest emission with a fluorescence quantum yield of 0.25 (quinine sulfate (ΦF = 0.546 in 0.1 M H2SO4) as the standard). The fluorescence intensity of CDs did not change significantly with pH in the range 3.0−11.0 but decreased at pH lower than 3.0 and increased at pH higher than 11.0 (Supporting Information Figure S2), which suggests that the CDs can be used in biological samples and in many environmental samples. Additionally, the results of dynamic light scattering measurements indicated that the average diameter of CDs in ethanol is about 2.3 nm (Figure 1b). The TEM images showed that the average diameter of the dye-doped silica nanoparticles was about 145 nm (Figure 2a). 2291

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Figure 2. TEM images of the dye-doped silica nanoparticles (a) and dual-emission silica nanoparticles (b). (c) XPS of C, Si, and O element of dyedoped silica nanoparticles (red) and dual-emission silica nanoparticles (black). (d) Fluorescence spectra of CDs (blue), dye-doped silica nanoparticles (red), and dual-emission silica nanoparticles (black).

The response of the resultant dual-emission fluorescent nanoparticles toward different concentrations of Cu2+ was investigated. The fluorescence spectra were shown in Figure 3. In the absence of Cu2+, two well-resolved emission peaks at 467 and 585 nm were observed with a single excitation at 360 nm, which were attributed to the emission of the CDs on the surface and the Rhodamine B in the core of the silica nanoparticles, respectively. The blue fluorescence at 467 nm decreased significantly along with the increasing of the Cu2+ concentration in the range of 0 to 10.0 μM, which indicated that the fluorescence of CDs was quenched by Cu2+. When the concentration of Cu2+ was higher than 20 μM, the fluorescence of CDs was almost completely quenched and the fluorescence spectra of the dual-emission nanoparticles no longer changed with the increase of Cu2+. The red fluorescence at 585 nm was also found decreased with the increasing of the Cu 2+ concentration, but the extent of decrease was much less than that of the blue fluorescence and the emission peak become constant when the concentration of Cu2+ was higher than 20 μM. As shown in Figure 3b, there is a spectral overlap between

After coated with CDs, the average diameter of the obtained nanoparticles increased to about 160 nm, suggesting the successful coating of CDs on the surface of silica nanoparticles (Figure 2b). The coating of CDs on the surface of dye-doped silica nanoparticles was further confirmed by XPS. The XPS results showed that the carbon content increased from 59.21% to 74.74%, the silicon content decreased from 7.97% to 1.82%, and the oxygen content decreased from 24.65% to 16.01% after the modification of CDs on the dye-doped silica nanoparticles (Figure 2c). The fluorescence spectra of the dual-emission silica nanoparticles, CDs, and dye-doped silica nanoparticles are shown in Figure 2d. The dye-doped silica nanoparticles showed Rhodamine B emission with a maximum at 585 nm (Figure 2d, red line). When the CDs were coated on the surface of the dyedoped silica nanoparticles, a well-resolved dual emission spectrum at 467 and 585 nm was displayed at a single excitation wavelength (360 nm) (Figure 2d, black line), which demonstrated that the CDs was successfully coupled onto the dye-doped silica nanoparticles. 2292

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The fluorescence intensity ratio at 467−585 nm (F467/F585) against the Cu2+ concentration was shown in Figure 3c. The ratio of F467/F585 gradually decreased with the increasing of Cu 2+ concentration and reached a plateau when the concentration of Cu2+ was higher than 20 μM, a good linearity was obtained in the range of 0 to 3 × 10−6 M with a correlation coefficient of 0.9933 (Figure 3, inset). And the detection limit was calculated to be 35.2 nM based on the ratio of signal-tonoise of 3, which is much lower than the maximum concentration (1.3 ppm, about 20 μM) of Cu2+ in drinking water permitted by the U.S. Environment Protection Agency. The fluorescence of the dual-emission fluorescent nanoparticles at different pH was also tested. The ratio of F467/F585 in the absence of Cu2+ was not affected by pH changes in the range of 3.0−11.0 (Supporting Information Figure S3), which is consistent with the fluorescence change of CDs only. The fluorescence ratio in the presence of Cu2+ was almost not changed with pH in the range of 5.0−10.0. The higher ratios in the pH range 2.0−4.0 may be due to the protonation of the amino groups, resulting in the reduction of the chelation with Cu2+; and the increased ratios at pH higher than 10.0 may be partially due to the interaction of Cu2+ with a high concentration of OH−. Since the dual-emission silica nanoparticles can be well dispersed in PB buffer, the Cu2+ detection was performed in buffer solution without any organic solvent, suggesting the potential application of this nanosensor in environmental or biological samples. The response of this nanosensor to Cu2+ is fast, and the whole detection process is very simple. To evaluate the selectivity of the dual-emission nanoparticles to Cu2+, the fluorescence intensity ratio (F467/F585) of the nanosensor in the presence of other metal ions, such as Ca2+, Mg2+, Hg2+, Co2+, Cd2+, Ni2+, Pb2+, Ba2+, Mn2+, Zn2+, Ag+, K+, Fe3+, and Al3+, were studied under the same conditions. As shown in Figure 4a, in the presence of other ions, the ratio of F467/F585 did not show significant change compared with that of the dual-emission nanoparticles, which suggests the good selectivity of the dual-emission nanoparticles to Cu2+ over other metal ions. Further addition of Cu2+ to the nanoparticles solution containing other metal ions, the ratios of F467/F585 decreased greatly, which indicates that the coexistence of these metal ions have negligible interference on the detection of Cu2+ and further demonstrated the high specificity of the ratiometric nanosensor for Cu2+. Furthermore, taking into account that amino acids and other bioactive substances may interact with dual-emission nanoparticles and interfere with Cu2+ sensing, some amino acids (Gly, Ala, Phe, Cystine, His, Trp, Glu, Lys, Pro, Arg, Ser, Ile, Cys, Thr), proteins (HSA, BSA), and vitamin C sodium (VC Na) were also chosen for investigation. As shown in Figure 4b, only very little changes of the intensity ratios (F467/F585) were observed upon addition of the above substances. However, after addition of Cu2+ to the nanoparticle solutions containing the above substances, the ratios of F467/F585 decreased immediately. Compared with that in the presence of only Cu2+, no significant changes of the ratio (F467/F585) were observed in the copresence of the above substances and Cu2+, which indicates that the resultant dual-emission silica nanoparticles showed high selectivity to Cu2+ against not only metal ions but also amino acids and other bioactive substances (HSA, BSA, and VC Na). The negligible interference from other substances suggests the potential utility of the resultant dual-emission silica nanoparticles for detection of Cu2+ in biological samples.

Figure 3. (a) Fluorescence spectra change of dual-emission nanoparticles upon addition of different concentrations of Cu2+. The concentration of Cu2+ from the top to the bottom: 0, 0.5, 1.0, 2.0, 3.0, 5.0, 10.0, 20.0, and 30.0 μM, respectively. (b) Absorption spectrum of RBITC (blue line) and emission spectrum of CDs (black line), excited at 360 nm. (c) Plot of fluorescence intensity ratio of F467/F585 versus the concentration of Cu2+. Inset: Linear fitting curve of F467/F585 versus a different concentration of Cu2+.

the emission of CDs and the absorption of RBITC, which suggests that a slight fluorescence resonance energy transfer (FRET) from CDs to Rhodamine B might occur. Therefore, the decrease of the emission at 585 nm might mainly attribute to the decrease of the FRET, which resulted from the reduction of the fluorescence intensity of CDs upon addition of Cu2+. 2293

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by pH in the range 3.0−9.0 (Supporting Information Figure S5). The fluorescence responses of CDs-APTES and CDsAEAMPS to Cu2+ were shown in Figure 5a,b. Cu2+ greatly quenched the fluorescence of CDs-AEAMPS but could not quench the fluorescence of CDs-APTES. Additionally, the fluorescence spectra of the CDs-APTES based dual-emission nanoparticles in the presence of Cu2+ and other metal ions were collected and shown in Figure 5c. Clearly different from the dual-emission nanoparticles based on CDs-AEAPMS (Figure 3a), the fluorescence spectra of CDs-APTES based dualemission nanoparticles did not show a significant change upon the addition of Cu2+ as well as other metal ions. Above results confirm that the ethylenediamine group in AEAPMS is essential for specific recognition of Cu2+. The practical application of this nanosensor was evaluated through the determination of Cu2+ spiked in real tap water samples. The water samples were simply filtered through a membrane (0.22 μm) prior to use without any other treatment. The results obtained from this nanosensor were compared to that from ICPMS as shown in Table 1. Good recoveries were obtained in the spiked samples by this nanosensor, and the obtained results were comparable with that obtained by ICPMS. Table 1. Determination of Cu2+ Spiked in Real Tap Water Samples

Figure 4. (a) Fluorescence response of dual-emission nanoparticles to various metal ions (10 μM except for K+ 1.0 mM, black bars) and the subsequent addition of Cu2+ (10 μM, gray bars). (b) Fluorescence response of dual-emission nanoparticles to 10 μM of amino acids, proteins (HSA, 30 μM; BSA, 50 μM) and 10 μM of VC Na (black bars) and the subsequent addition of Cu2+ (10 μM, gray bars).

a

spiked Cu2+ (μM)

nanosensor meana ± SDb

0 0.5 1.0 2.0

N/A 0.55 ± 0.06 1.24 ± 0.03 2.12 ± 0.13

ICPMS meana ± SDb 0.07 0.43 0.92 2.14

± ± ± ±

0.01 0.05 0.09 0.26

Mean value of three determinations. bSD: standard deviation.

To test the feasibility of dual-emission nanoparticles for intracellular Cu2+ imaging, MCF-7 cells were chosen to incubate with dual-emission nanoparticles. The fluorescence images were captured under a laser-scanning confocal microscope in blue (425−500 nm) and red (530−630 nm) channels with excitation by a 405 nm laser. As shown in Figure 6a,b, strong fluorescence from both channels could be observed in cells. After addition of Cu2+ to cells, the fluorescence intensity of both channels decreased (Figure 6e,f), but the blue fluorescence decreased more greatly than the red fluorescence, which agreed well with the fluorescence response of dualemission nanoparticles to Cu2+ in PB buffer. These results suggest that the dual-emission nanoparticles also respond to

Since AEAPMS was the main raw material to prepare the CDs, the ethylenediamine group in AEAPMS was speculated as the key factor that determines the selectivity of this nanosensor to Cu2+. In order to verify this supposition, APTES that only contains one amino group was selected to substitute AEAPMS for preparing CDs (CDs-APTES) and dual-emission nanoparticles. The fluorescence properties of CDs-APTES were almost the same with CDs-AEAPMS; the strongest emission was excited at 360−380 nm (Supporting Information Figure S4). The fluorescence intensity of CDs-APTES was not affected

Figure 5. (a) Fluorescence response of CDs-AEAPMS to Cu2+ (10 μM), (b) fluorescence response of CDs-APTES to Cu2+ (20 μM), and (c) fluorescence response of the CDs-APTES based dual-emission nanoparticles to 10 μM of different metal ions. 2294

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Figure 6. Confocal fluorescence images of MCF-7 cells before (top) and after (bottom) addition of Cu2+ (50 μM). (a,e) Fluorescence images from blue channel, (b,f) fluorescence images from red channel, (c,g) overlay of fluorescence images and bright field image, and (d,h) fluorescence ratio (Fred/Fblue) images.

Cu2+ when located in cells. The fluorescence ratio (Fred/Fblue) images clearly showed significant difference before and after addition of Cu2+ (Figure 6d,h); the addition of Cu2+ caused a great increase of the fluorescence ratio. These results confirm that the dual-emission nanoparticles can be used for ratiometric imaging of Cu2+ in cells.

CONCLUSIONS In summary, a ratiometric fluorescence nanosensor for Cu2+ has been designed and prepared through coating CDs on dyedoped silica nanoparticles. The CDs were simply synthesized by using AEAPMS as the main raw material, so that the obtained CDs could recognize Cu2+ directly because of the surface ethylenediamine group and could be coated on silica nanoparticles by the silylation reaction because of the surface methoxysilane group. The resultant nanoparticles exhibit the characteristic emissions of Rhodamine B and CDs under a single excitation wavelength, and the CDs emission can be quenched upon the binding of Cu2+, which results in the ratiometric fluorescence response to Cu2+ in aqueous solution. The selectivity assay reveals that this ratiometric fluorescence nanosensor has good selectivity to Cu2+ over other metal ions, amino acids, and other bioactive substances. The measurement of Cu2+ is simple and sensitive; the limit of detection down to 35.2 nM has been achieved. Additionally, this ratiometric nanosensor has been successfully applied for the determination of spiked Cu2+ in water samples and ratiometric imaging of Cu2+ in cells. This CDs-based dual-emission silica nanoparticle strategy could be extended to other analytes by changing the CDs with other specific recognition sites.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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ACKNOWLEDGMENTS

We gratefully acknowledge the financial support from Grant 973 Program (Grants 2011CB935800 and 2013CB933700) and NSF of China (Grants 21275149, 21205124, and 21321003).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone/fax: 86-10-62528509. Notes

The authors declare no competing financial interest. 2295

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Analytical Chemistry

Technical Note

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dx.doi.org/10.1021/ac404236y | Anal. Chem. 2014, 86, 2289−2296