DOI: 10.1002/chem.201404160
Full Paper
& Ratiometric Probes
A Single-Wavelength-Emitting Ratiometric Probe Based on Phototriggered Fluorescence Switching of Graphene Quantum Dots Zhi-bei Qu,[a] Min Zhang,[a] Tianshu Zhou,[b] and Guoyue Shi*[a]
Abstract: Ratiometric fluorescent probes are of great importance in research, because a built-in correction for environmental effects can be provided to reduce background interference. However, the traditional ratiometric fluorescent probes require two luminescent materials with different emission bands. Herein a novel ratiometric probe based on a single-wavelength-emitting material is reported. The probe works by regulating the luminescent property of graphene
quantum dots with UV illumination as activator. The ratiometric sensor shows high sensitivity and specificity for iron ions. Moreover, the ratiometric sensor was successfully employed to monitor ferritin levels in Sprague Dawley rats with chemical-induced acute liver damage. The proposed singlewavelength ratiometric fluorescent probe may greatly broaden the applicability of ratiometric sensors in diagnostic devices, medical applications, and analytical chemistry.
Introduction
Ratiometric fluorescent probes have recently attracted much research interest,[11] on account of their avoiding the influences of excitation intensity, concentration, and solution environments. Conventional ratiometric fluorescent probes require two luminescent materials with different emission colors, which limits their construction. It is highly desirable to expand the ratiometric strategy, especially to search for new ratiometric methods using monoprobes with single emission wavelengths. The construction of a single-wavelength ratiometric fluorescent probe would greatly broaden the application of ratiometric sensors. Herein, the photoregulated fluorescent property of GQDs was discovered and a novel ratiometric probe for iron ions with a single emission band was realized with the aid of UV irradiation. The working mechanism of the probe was well elucidated. The sensing system was further applied to monitor ferritin levels in Sprague Dawley (SD) rats.
Graphene quantum dots (GQDs),[1] which serve as versatile fluorescent probes[2] and general platforms for labeling,[3] have been at the center of research efforts in a wide range of fields including material science, bioimaging,[4] and analytical chemistry.[5] This is largely attributed to their excellent photostability and biocompatibility. Prepared from graphene oxide, GQDs mostly have oxygen-rich surface functional groups such as hydroxyl and carboxyl groups, which provide an avenue for further modification. In addition to their extraordinary phototoluminescence properties, GQDs also show great potential in photocatalysis[6] and enzyme mimetics,[7] which have been successfully applied in electrochemical[8] and colorimetric sensors.[9] The greatest virtue of GQDs is their tunable optical properties.[10] In other word, the fluorescence of GQDs is largely affected by their characteristics and solution conditions, such as the size of the nanodots, edge modification, functional groups, surface potential and passivation, pH, and the solvent. GQDs are the most promising candidates for the development of ratiometric fluorescent probes, which have great potential in chemical sensors and bioimaging.
Results and Discussion The GQDs employed in this work were fabricated by using a previously reported simple hydrothermal method.[12] The average size of the GQDs was 2.6 nm and the average height was 1.2 nm (about 1–2 layers of graphene sheets), characterized by TEM and AFM (see Figure S1 in the Supporting Information). GQDs can be quenched by various metal ions, such as Hg2 + , Ag + , Cu2 + , and Fe3 + , at high concentration.[11a, 13] Fe3 + can be detected simply by use of quenching mechanism, albeit with an obvious lack of selectivity. In addition, the simple quenching method for iron ions does not show good sensitivity. The fluorescence of GQDs can be efficiently quenched only when the concentration of Fe3 + is higher than 10 mm in aqueous solution. As shown in Figure S2 in the Supporting Information, the fluorescence of GQDs decreases
[a] Z.-b. Qu, Dr. M. Zhang, Prof. Dr. G. Y. Shi Department of Chemistry East China Normal University Dongchuan RD 500, Shanghai, 200241 (P. R. China) Fax: (+ 86) 21-54340043 E-mail: [email protected] [b] Prof. Dr. T. S. Zhou Department of Environmental Science East China Normal University Dongchuan RD 500, Shanghai, 200241 (P. R. China) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201404160. Chem. Eur. J. 2014, 20, 1 – 7
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Full Paper under UV irradiation due to a photobleaching process. However, in the presence of Fe2 + /Fe3 + ions, the GQDs were either quenched or turned on before and after UV irradiation. In other word, GQDs show contrasting response to iron ions before and after UV irradiation. Thus, a ratiometric probe for iron ions was constructed (Scheme 1). As Fe2 + and Fe3 + ions showed exactly the same response in the sensing system, only the data for Fe3 + ions are shown in this work.
To compare the photocatalytic ability of GQDs to produce ROS with other carbon materials including GO,[16] reduced GO (rGO),[17] nitrogen-doped graphene sheets (N-GSs)[18] and carboxylated carbon nanotubes (CNTs), 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) was used to monitor the presence of ROS. Oxidation of ABTS by ROS to produces a green product.[19] The production of ROS can be quantified by the absorbance of ABTS at 405 nm. In the experiments, 0.05 mg mL1 of GQDs, GO, rGO, N-GSs, and CNTs was employed. The GQDs showed much better photocatalytic ROS production than other carbon materials at the same mass concentration under UV irradiation (Figure 1). ROS production can be induced by UV light (low-pressure Hg lamp, 254 nm, 8 W; Hg-vapor lamp, 400 W), sunlight, a green laser (532 nm, 20 mW), and incandescence (40 W). The outstanding photocatalytic production of ROS by GQDs is attributed to their large Scheme 1. Schematic representation of the working mechanism of the GQD-based sensor for iron ions. specific surface area, versatile The fluorescent intensity of GQDs decreases by 43.6 % in the presence of 200 mm Fe3 + ions in solution without illumination (control group), which serves as the turn-off channel. However, the UV-treated GQDs without Fe3 + ions suffer strong photobleaching, whereas those in the presence of Fe3 + ions still show strong fluorescence. The fluorescence of UV-treated GQDs in the presence of Fe3 + ions is about ten times (981 %) more intense than that of GQDs after UV irradiation in the absence of iron ions. The fluorescence peak of GQDs also exhibited a small redshift from 428 to 445 nm after UV treatment, which implies a change in the surface functional groups of GQDs. In the turn-off channel, luminescence of GQDs is quenched by Fe3 + ions. UV/Vis spectroscopy was employed to elucidate the quenching mechanism (see Figure S3 in the Supporting Information). It was found that the absorption band of Fe3 + ions overlaps with the emission band of GQDs. Therefore, fluorescence resonance energy transfer (FRET) can be invoked to explain the quenching effect of GQDs by Fe3 + . On the other hand, transition metal ions such as Fe3 + have empty d orbitals that may accept electrons donated from GQDs, so a photoinduced electron-transfer mechanism may also contribute to quenching of the GQDs. The turn-on effect of Fe3 + ions is proposed to be related to generation of reactive oxygen species (ROS) catalyzed by GQDs. Because they can act as both electron donors and acceptors, GQDs are efficient photosensitizers for production of ROS under visible light under in vitro and in vivo conditions.[14] ROS generation is proposed to be highly related to the defects and oxygen-rich functional groups at the edge of GQDs.[15] Experimental research coupled with theoretical calculations has proved that OH-functionalized Stone– Wales defects can efficiently lower the energy barrier for diffusion of oxygen radicals through graphene oxide (GO). &
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Figure 1. UV/Vis spectra (a) and photograph (b) of colorimetric responses of ABTS after UV (low-pressure Hg lamp, 254 nm, 8 W, 5 min) treatment in the the absence and presence of GQDs, GO, rGO, N-GSs, and CNTs. The concentration of each carbon material was 0.05 mg mL1 with 0.5 mm ABTS.
oxygen-rich functional groups serving as active centers, and unique quantum confinement property. It was further shown that the presence of Fe3 + can efficiently eliminate ROS and hinder the color change of ABTS (see Figure S5 in the Supporting Information). According to previous research, singlet oxygen was verified in the ROS produced by GQDs.[14] A reaction mechanism was proposed as follows. First, singlet oxygen (1O2) is produced by GQDs under UV irradiation. 1O2 is a highly oxidative ROS that would react with the C=C bonds of the GQDs. This process could destroy the ar2
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Full Paper omatic conjugation of GQDs and further result in photobleaching [Eq. (1)]. 3
UV=GQD
O2 ! 1 O2
ð1Þ
That is why the fluorescence of GQDs is turned off after UV treatment. In the presence of iron ions, however, the highly reactive singlet oxygen can react with Fe2 + and generate another ROS, namely, the superoxide
Figure 2. TEM images of GQDs (a) and GQDs after UV treatment in the absence (b) and presence (c) of Fe3 + ions.
anion [Eq. (2)]. 1
O2 þ Fe2þ ! O2 C þ Fe2þ þ
ð2Þ
It is known that disproportionation can be catalyzed by iron-containing compounds. Superoxide anions are possibly eliminated in the following reaction [Eq. (3)] and decomposed into less reactive compounds. Fe2þ =Fe3þ
O2 C þ 2 H2 O !H2 O2 þ 2 OH þ O2
ð3Þ
Through the mechanism assumed above, singlet oxygen was quenched in the presence of iron ions, and GQDs were protected from photobleaching. This provides a good explanation of why the fluorescence of GQDs was turned on by UV treatment in the presence of Fe2 + /Fe3 + . TEM was employed to study the morphological change of GQDs after UV irradiation (Figure 2). It was Figure 3. Fluorescent spectra of GQDs in the presence of different concentrations of observed that GQDs did not exhibit remarkable dif- Fe3 + ions before (a) and after (b) UV illumination (Hg-vapor lamp, 400 W, 30 min). The 3+ ferences in size after UV irradiation in the absence spectra were collected with 320 nm excitation. The concentrations of Fe ions were 0, 1, 5, 10, 15, 20, 25, 30, 35, 40, and 50 mm. c) Linear plot of the ratiometric fluorescent reand presence of iron ions. The average diameter of sponse as a function of Fe3 + concentration. GQDs of 2–3 nm was maintained and showed no statistical difference before and after UV treatment. It can be inferred that the difference in luminescent intensity of out UV treatment, the majority of the oxygen (58.8 %) was in GQDs before and after UV irradiation is not attributable to varthe form of CO single bonds of hydroxyl groups. The rest of iations in size. Notably, the UV-treated GQDs in the absence of oxygen was in the form of COOH (29.4 %) and COO (11.8 %), which corresponded to carboxyl groups in the GQDs. For Fe3 + showed relatively low crystallinity (Figure 3 b), which indicated that more structural defects were induced in GQDs GQDs after UV illumination, however, a larger fraction of the during UV irradiation. oxygen existed in carboxyl form (32.3 % COOH and 15.3 % X-ray photoelectron spectroscopy (XPS) and FTIR spectroscoCOO) in addition to an increase in the atomic fraction of py were used to illustrate the elemental and structural changes oxygen. In the presence of Fe3 + ions, UV-induced formation of of the functional groups of GQDs in the absence and presence carboxyl groups was inhibited. The amount of carboxyl oxygen of Fe3 + ions after UV treatment. XPS characterization revealed (30.0 % COOH and 14.7 % COO) was lower than in the abthat UV-illuminated GQDs have a lower C/O ratio (see Table S1 sence of Fe3 + ions but still higher than before UV treatment. in the Supporting Information), which implies that GQDs themFTIR spectra (see Figure S8 in the Supporting Information) selves suffered oxidation simultaneously with production of showed similar evidence of transformation of oxygen-rich funcROS. The C/O ratios were 10.3 and 9.7 before and after UV tional groups for GQDs treated with UV. The UV-illuminated treatment, respectively. Interestingly, in the presence of Fe3 + GQDs showed a higher carboxyl CO deformation peak at 1385 cm1, which proved a larger amount of carboxyl groups ions, however, the C/O ratio of GQDs stayed almost unchanged (C/O ratio: 10.2) after UV treatment. High-resolution XP spectra at the surface of GQDs. The carboxyl peak at 1385 cm1 of the revealed that three types of oxygen atoms emerged in GQDs UV-illuminated GQDs in the presence of Fe3 + ions showed no (see Figure S6 in the Supporting Information). For GQDs withobvious change after UV treatment. However, the alkoxyl CO Chem. Eur. J. 2014, 20, 1 – 7
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Full Paper peak at 1093 cm1 and C-O-C peak at 1093 cm1 stayed unchanged, which implies that carboxyl CO plays an essential role in the oxidation process of GQDs. As shown by Zhu and coworkers,[20] the fluorescence property of GQDs was highly affected by the oxygen-rich functional groups at their surface. A higher oxygen fraction and addiFigure 4. Relative ratiometric response of iron ions over various competing metal ions. The concentration of Fe3 + tional carboxyl groups would and Fe2 + ions was 40 mm, and the concentrations of other metal ions were 200 mm. lead to a lower quantum yield and a redshift of fluorescence, which are coincident with our results. these metal ions cannot induce quenching of GQDs. The rest As shown by colorimetric experiments with ABTS and fluoof metal ions including Hg2 + , Cd2 + , Cu2 + , and Pb2 + can be disrescence characterization of GQDs, iron ions behave as exceltinguished by the turn-on channel under UV irradiation. None lent reagents to eliminate ROS and can be further used to of the above metal ions can inhibit ROS generation and promodulate the fluorescence of GQDs. Thus, a ratiometric fluotect GQDs from photobleaching like iron ions. Overall, the rarescent probe for iron ions was developed. The response was tiometric probe showed improved selectivity for iron ions. Ag + calculated as follows [Eq. (4)] ions were not considered as interference, because Ag + ions would be photodecomposed by UV illumination. The ratiometric sensor could not distinguish Fe2 + and Fe3 + ions and UV Con I I showed exactly the same response to both ions, because Fe2 + ð4Þ R2 ¼ iUV 1 1 iCon I0 I0 and Fe3 + ions interconvert rapidly in the process of ROS generation/elimination. The high selectivity of the ratiometric fluowhere Ii is the fluorescent intensity of sample i, I0 the fluoresrescent probe indicates that it could be applied in complex cent intensity of the blank sample with zero concentration of systems. Fe3 + ions, superscript UV refers to the UV-treated group, and As the most abundant transition metal ions in the human Con to the control group stored in the dark. body, Fe3 + ions play an essential role in many biological proThe fluorescence of GQDs in the control group decreased cess. In many enzymes, such as hemoglobin and cytochrome c, when Fe3 + ions were added, whereas that of GQDs in the UViron ions serve as active centers in biological catalysis, which is often related to metabolism, diseases, cancers, cellular apoptotreated group increased on addition of Fe3 + ions (Figure 3). sis, and so forth. Lack of iron can lead to serious illnesses inThe sensitivity of the probe is remarkably higher than that of cluding iron-deficiency anemia and nerve deafness. Therefore, the simple quenching method. The fluorescent responses of the intake and storage of iron are very important to human both the control and UV-treated groups show good linearity as a function of Fe3 + concentration (see Figure S9 in the Supporthealth. Ferritin, an iron-containing protein, is the major form of iron storage in the liver, spleen, muscles, marrow, and blood. ing Information). Overall, the ratiometric response of the probe The ferritin concentration can reflect anemia, inflammation, for Fe3 + ions exhibits excellent linearity and a wide linear and hepatic illness.[21] In particular, a rise in ferritin level is range from 1 to 50 mm with a correlation coefficient of 0.996. The calculated limit of detection is 0.3 mm. The ratiometric restrongly related to hepatitis, hepatic fibrosis, leukemia, and sponse of the probe reveals a better linear fit than either the hepatic cancer. Monitoring of ferritin levels is of great imporquenching channel of the control group or the turn-on chantance in medical research. Here, we used SD rats as models to nel of the UV-treated group. This further evidences that the inverify the practical use of our proposed ratiometric probe. The troduction of a ratiometric strategy could efficiently improve ferritin levels of healthy rats and rats suffering from chemically the stability and accuracy of the sensor. induced acute liver damage[22] were estimated by measuring 3+ To examine the selectivity of the ratiometric probe for Fe serum iron concentrations. Damage to the liver would release a large amount of ferritin from hepatic tissues to the blood, /Fe2 + ions, the fluorescent responses of GQDs towards various which is reflected in a remarkable increase of iron concentracompeting metal ions (Al3 + , Ca2 + , Cd2 + , Co2 + , Cr3 + , Cu2 + , tion. The GQD-based ratiometric probe shows good accuracy Hg2 + , K + , Li + , Mg2 + , Mn2 + , Na + , NH4 + , Ni2 + , Pb2 + and Zn2 + ) and recovery for Fe3 + ions in rats. The fluorescent responses of was examined under the same conditions. The final concentration of ions was 200 mm, which was five times of that of iron acutely liver damaged rats were remarkably higher than those ions. Figure 4 shows that all the competing metal ions had no of health ones (see Table S4 in the Supporting Information). effect on the fluorescent responses of the sensing system. ReThe successful application of the GQD-based ratiometric probe garding the turn-off channel, all of the non-transition metal proves its suitability for complex systems and it may show ions (Al3 + , Ca2 + , K + , Li + , Mg2 + , Na + , NH4 + ) and Co2 + , Cr3 + , great potential in therapeutic diagnostics of acute hepatitis and hepatic cancer. ROS is generated in the sensing system Mn2 + , Ni2 + and Zn2 + ions can be easily excluded, because &
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Full Paper water was then slowly added by dropping funnel. The solution was heated to reflux for 30 min at 95 8C. 100 mL of water was added. Subsequently, 3 mL of H2O2 (30 %) was added dropwise to change the color of the solution from dark brown to yellow. The resulting suspension was filtered through a 0.22 mm microporous membrane, and further washed twice with 200 mL of HCl (1 m) and 200 mL of deionized water. The brown solid was vacuum-dried at 50 8C for 24 h to obtain GO.
and the ratiometric probe is a potential agent for use in photodynamic therapy to kill cancer cells.[21] Iron ions are able to eliminate ROS produced by GQDs. Therefore, iron-containing compounds are possible protectors that could reduce damage to healthy cells in photodynamic therapy. Moreover, the photoregulated fluorescence property was not only applied to GQDs top-down fabricated from GO, but also could be expanded to bottom-up synthesized carbon quantum dots (CQDs).[23] The ROS-related mechanism of UVand Fe2 + /Fe3 + -triggered reactions seem to be general to all carbon-based fluorescent materials (see Figure S10 in the Supporting Information).
Preparation of graphene GQDs GO (0.05 g) was added to a mixture of concentrated H2SO4 (10 mL) and HNO3 (30 mL). The solution was treated with mild ultrasonication for 12 h (500 W, 40 kHz). The mixture was then diluted with distilled water (100 mL) and filtered through a 0.22 mm microporous membrane to remove the acids. Purified GO was redispersed in distilled water (40 mL) and the pH was adjusted to 8.0 with NaOH. The suspension was transferred to a Teflon-lined autoclave and heated at 200 8C for 12 h. After cooling to room temperature, the solution was further dialyzed in a dialysis bag (retained molecular weight: 3500 Da) for 48 h to obtain the pristine GQDs.
Conclusion We have reported a new type of ratiometric probe based on single-wavelength-emitting GQDs for the sensitive and selective determination of iron ions. The probe is easily fabricated and suitable for complex systems. The ratiometric sensor was successfully employed to monitor ferritin levels in SD rats with chemically induced acute liver damage and shows potential for further applications in diagnosing acute hepatitis and hepatic cancer. The ROS-related process also has potential for photodynamic therapy. The development of this ratiometric probe not only introduces a novel method for detection of iron ions, but also gives a new view of the ratiometric strategy and could largely broaden the applicability of ratiometric sensors beyond analytical chemistry.
Animal experiments All procedures involving animals were conducted with the approval of the Animal Ethics Committee in East China Normal University (ECNU), China. Male SD rats (weight 200–250 g) were purchased from Shanghai SLAC Laboratory animal Co. Ltd and acclimatized for 4 d. The rats were divided into two groups at random, that is, the control group and the acute-liver- damage group. The acuteliver-damage groupwas intraperitoneally injected with 1.0 mL kg1 body weight CCl4 (dissolved in olive oil, 1/1), whereas rats in control group were injected with the same volume of olive oil. Rats of each group were sacrificed 24 h after CCl4/oil injection. The serum of each rat was collected for Fe3 + determination.
Experimental Section Reagents and materials
Acknowledgements
CNTs were obtained from Shenzhen Carbon Nanotechnologies Co. Ltd. Graphite powder (spectral grade) and other chemicals (analytical grade) were provided by Sinopharm Group Chemical Regent Co., Ltd. (Shanghai, China). All solvents and chemicals in this work were used without further purification unless stated. Double-distilled water was used throughout the experiments.
This work was supported by the National Natural Science Foundation of China (No. 21275055, 21277048) and Project funded by China Postdoctoral Science Foundation (2014M550224).
Instrumentation Keywords: analytical methods graphene · iron · quantum dots
The fluorescence and absorption spectra were recorded with a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan) and a Shimadzu UV-1800 spectrophotometer (Tokyo, Japan), respectively. A JEM-2010 transmission electron microscope (JEOL Ltd. Japan) and BioScope atomic force microscope (NanoScope IIIa SPM System, Digital Instruments, Inc., U.S.A) were used to study the morphology of the prepared GQDs. FTIR spectra were obtained with a Nicolet Nexus 670 spectrometer. XPS experiments were carried out on an AXIS UltraDLD system with AlKa radiation (hn = 1486.6 eV).
Graphene oxide was synthesized from graphite powder by a modified Hummers method. Typically, 0.5 g of graphite, 0.5 g of NaNO3, and 23 mL of H2SO4 were mixed in an ice bath. 3 g of KMnO4 was carefully added into the mixture over 10 min. The suspension was transferred to a 35 8C water bath and stirred for 2 h. 40 mL of www.chemeurj.org
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FULL PAPER & Ratiometric Probes Z.-b. Qu, M. Zhang, T. S. Zhou, G. Y. Shi* && – && A Single-Wavelength-Emitting Ratiometric Probe Based on Phototriggered Fluorescence Switching of Graphene Quantum Dots
A new ratiometric strategy: A new type of ratiometric probe based on graphene quantum dots (GQDs) emitting single-wavelength fluorescence was developed for the sensitive and selective
Chem. Eur. J. 2014, 20, 1 – 7
determination of iron ions. The analytical method is based on the different responses of the GQDs to iron ions before and after UV irradiation (see figure; ROS: reactive oxygen species).
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& Ratiometric Probes
A Single-Wavelength-Emitting Ratiometric Probe Based on Phototriggered Fluorescence Switching of Graphene Quantum Dots Zhi-bei Qu,[a] Min Zhang,[a] Tianshu Zhou,[b] and Guoyue Shi*[a]
Abstract: Ratiometric fluorescent probes are of great importance in research, because a built-in correction for environmental effects can be provided to reduce background interference. However, the traditional ratiometric fluorescent probes require two luminescent materials with different emission bands. Herein a novel ratiometric probe based on a single-wavelength-emitting material is reported. The probe works by regulating the luminescent property of graphene
quantum dots with UV illumination as activator. The ratiometric sensor shows high sensitivity and specificity for iron ions. Moreover, the ratiometric sensor was successfully employed to monitor ferritin levels in Sprague Dawley rats with chemical-induced acute liver damage. The proposed singlewavelength ratiometric fluorescent probe may greatly broaden the applicability of ratiometric sensors in diagnostic devices, medical applications, and analytical chemistry.
Introduction
Ratiometric fluorescent probes have recently attracted much research interest,[11] on account of their avoiding the influences of excitation intensity, concentration, and solution environments. Conventional ratiometric fluorescent probes require two luminescent materials with different emission colors, which limits their construction. It is highly desirable to expand the ratiometric strategy, especially to search for new ratiometric methods using monoprobes with single emission wavelengths. The construction of a single-wavelength ratiometric fluorescent probe would greatly broaden the application of ratiometric sensors. Herein, the photoregulated fluorescent property of GQDs was discovered and a novel ratiometric probe for iron ions with a single emission band was realized with the aid of UV irradiation. The working mechanism of the probe was well elucidated. The sensing system was further applied to monitor ferritin levels in Sprague Dawley (SD) rats.
Graphene quantum dots (GQDs),[1] which serve as versatile fluorescent probes[2] and general platforms for labeling,[3] have been at the center of research efforts in a wide range of fields including material science, bioimaging,[4] and analytical chemistry.[5] This is largely attributed to their excellent photostability and biocompatibility. Prepared from graphene oxide, GQDs mostly have oxygen-rich surface functional groups such as hydroxyl and carboxyl groups, which provide an avenue for further modification. In addition to their extraordinary phototoluminescence properties, GQDs also show great potential in photocatalysis[6] and enzyme mimetics,[7] which have been successfully applied in electrochemical[8] and colorimetric sensors.[9] The greatest virtue of GQDs is their tunable optical properties.[10] In other word, the fluorescence of GQDs is largely affected by their characteristics and solution conditions, such as the size of the nanodots, edge modification, functional groups, surface potential and passivation, pH, and the solvent. GQDs are the most promising candidates for the development of ratiometric fluorescent probes, which have great potential in chemical sensors and bioimaging.
Results and Discussion The GQDs employed in this work were fabricated by using a previously reported simple hydrothermal method.[12] The average size of the GQDs was 2.6 nm and the average height was 1.2 nm (about 1–2 layers of graphene sheets), characterized by TEM and AFM (see Figure S1 in the Supporting Information). GQDs can be quenched by various metal ions, such as Hg2 + , Ag + , Cu2 + , and Fe3 + , at high concentration.[11a, 13] Fe3 + can be detected simply by use of quenching mechanism, albeit with an obvious lack of selectivity. In addition, the simple quenching method for iron ions does not show good sensitivity. The fluorescence of GQDs can be efficiently quenched only when the concentration of Fe3 + is higher than 10 mm in aqueous solution. As shown in Figure S2 in the Supporting Information, the fluorescence of GQDs decreases
[a] Z.-b. Qu, Dr. M. Zhang, Prof. Dr. G. Y. Shi Department of Chemistry East China Normal University Dongchuan RD 500, Shanghai, 200241 (P. R. China) Fax: (+ 86) 21-54340043 E-mail: [email protected] [b] Prof. Dr. T. S. Zhou Department of Environmental Science East China Normal University Dongchuan RD 500, Shanghai, 200241 (P. R. China) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201404160. Chem. Eur. J. 2014, 20, 1 – 7
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Full Paper under UV irradiation due to a photobleaching process. However, in the presence of Fe2 + /Fe3 + ions, the GQDs were either quenched or turned on before and after UV irradiation. In other word, GQDs show contrasting response to iron ions before and after UV irradiation. Thus, a ratiometric probe for iron ions was constructed (Scheme 1). As Fe2 + and Fe3 + ions showed exactly the same response in the sensing system, only the data for Fe3 + ions are shown in this work.
To compare the photocatalytic ability of GQDs to produce ROS with other carbon materials including GO,[16] reduced GO (rGO),[17] nitrogen-doped graphene sheets (N-GSs)[18] and carboxylated carbon nanotubes (CNTs), 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) was used to monitor the presence of ROS. Oxidation of ABTS by ROS to produces a green product.[19] The production of ROS can be quantified by the absorbance of ABTS at 405 nm. In the experiments, 0.05 mg mL1 of GQDs, GO, rGO, N-GSs, and CNTs was employed. The GQDs showed much better photocatalytic ROS production than other carbon materials at the same mass concentration under UV irradiation (Figure 1). ROS production can be induced by UV light (low-pressure Hg lamp, 254 nm, 8 W; Hg-vapor lamp, 400 W), sunlight, a green laser (532 nm, 20 mW), and incandescence (40 W). The outstanding photocatalytic production of ROS by GQDs is attributed to their large Scheme 1. Schematic representation of the working mechanism of the GQD-based sensor for iron ions. specific surface area, versatile The fluorescent intensity of GQDs decreases by 43.6 % in the presence of 200 mm Fe3 + ions in solution without illumination (control group), which serves as the turn-off channel. However, the UV-treated GQDs without Fe3 + ions suffer strong photobleaching, whereas those in the presence of Fe3 + ions still show strong fluorescence. The fluorescence of UV-treated GQDs in the presence of Fe3 + ions is about ten times (981 %) more intense than that of GQDs after UV irradiation in the absence of iron ions. The fluorescence peak of GQDs also exhibited a small redshift from 428 to 445 nm after UV treatment, which implies a change in the surface functional groups of GQDs. In the turn-off channel, luminescence of GQDs is quenched by Fe3 + ions. UV/Vis spectroscopy was employed to elucidate the quenching mechanism (see Figure S3 in the Supporting Information). It was found that the absorption band of Fe3 + ions overlaps with the emission band of GQDs. Therefore, fluorescence resonance energy transfer (FRET) can be invoked to explain the quenching effect of GQDs by Fe3 + . On the other hand, transition metal ions such as Fe3 + have empty d orbitals that may accept electrons donated from GQDs, so a photoinduced electron-transfer mechanism may also contribute to quenching of the GQDs. The turn-on effect of Fe3 + ions is proposed to be related to generation of reactive oxygen species (ROS) catalyzed by GQDs. Because they can act as both electron donors and acceptors, GQDs are efficient photosensitizers for production of ROS under visible light under in vitro and in vivo conditions.[14] ROS generation is proposed to be highly related to the defects and oxygen-rich functional groups at the edge of GQDs.[15] Experimental research coupled with theoretical calculations has proved that OH-functionalized Stone– Wales defects can efficiently lower the energy barrier for diffusion of oxygen radicals through graphene oxide (GO). &
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Figure 1. UV/Vis spectra (a) and photograph (b) of colorimetric responses of ABTS after UV (low-pressure Hg lamp, 254 nm, 8 W, 5 min) treatment in the the absence and presence of GQDs, GO, rGO, N-GSs, and CNTs. The concentration of each carbon material was 0.05 mg mL1 with 0.5 mm ABTS.
oxygen-rich functional groups serving as active centers, and unique quantum confinement property. It was further shown that the presence of Fe3 + can efficiently eliminate ROS and hinder the color change of ABTS (see Figure S5 in the Supporting Information). According to previous research, singlet oxygen was verified in the ROS produced by GQDs.[14] A reaction mechanism was proposed as follows. First, singlet oxygen (1O2) is produced by GQDs under UV irradiation. 1O2 is a highly oxidative ROS that would react with the C=C bonds of the GQDs. This process could destroy the ar2
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Full Paper omatic conjugation of GQDs and further result in photobleaching [Eq. (1)]. 3
UV=GQD
O2 ! 1 O2
ð1Þ
That is why the fluorescence of GQDs is turned off after UV treatment. In the presence of iron ions, however, the highly reactive singlet oxygen can react with Fe2 + and generate another ROS, namely, the superoxide
Figure 2. TEM images of GQDs (a) and GQDs after UV treatment in the absence (b) and presence (c) of Fe3 + ions.
anion [Eq. (2)]. 1
O2 þ Fe2þ ! O2 C þ Fe2þ þ
ð2Þ
It is known that disproportionation can be catalyzed by iron-containing compounds. Superoxide anions are possibly eliminated in the following reaction [Eq. (3)] and decomposed into less reactive compounds. Fe2þ =Fe3þ
O2 C þ 2 H2 O !H2 O2 þ 2 OH þ O2
ð3Þ
Through the mechanism assumed above, singlet oxygen was quenched in the presence of iron ions, and GQDs were protected from photobleaching. This provides a good explanation of why the fluorescence of GQDs was turned on by UV treatment in the presence of Fe2 + /Fe3 + . TEM was employed to study the morphological change of GQDs after UV irradiation (Figure 2). It was Figure 3. Fluorescent spectra of GQDs in the presence of different concentrations of observed that GQDs did not exhibit remarkable dif- Fe3 + ions before (a) and after (b) UV illumination (Hg-vapor lamp, 400 W, 30 min). The 3+ ferences in size after UV irradiation in the absence spectra were collected with 320 nm excitation. The concentrations of Fe ions were 0, 1, 5, 10, 15, 20, 25, 30, 35, 40, and 50 mm. c) Linear plot of the ratiometric fluorescent reand presence of iron ions. The average diameter of sponse as a function of Fe3 + concentration. GQDs of 2–3 nm was maintained and showed no statistical difference before and after UV treatment. It can be inferred that the difference in luminescent intensity of out UV treatment, the majority of the oxygen (58.8 %) was in GQDs before and after UV irradiation is not attributable to varthe form of CO single bonds of hydroxyl groups. The rest of iations in size. Notably, the UV-treated GQDs in the absence of oxygen was in the form of COOH (29.4 %) and COO (11.8 %), which corresponded to carboxyl groups in the GQDs. For Fe3 + showed relatively low crystallinity (Figure 3 b), which indicated that more structural defects were induced in GQDs GQDs after UV illumination, however, a larger fraction of the during UV irradiation. oxygen existed in carboxyl form (32.3 % COOH and 15.3 % X-ray photoelectron spectroscopy (XPS) and FTIR spectroscoCOO) in addition to an increase in the atomic fraction of py were used to illustrate the elemental and structural changes oxygen. In the presence of Fe3 + ions, UV-induced formation of of the functional groups of GQDs in the absence and presence carboxyl groups was inhibited. The amount of carboxyl oxygen of Fe3 + ions after UV treatment. XPS characterization revealed (30.0 % COOH and 14.7 % COO) was lower than in the abthat UV-illuminated GQDs have a lower C/O ratio (see Table S1 sence of Fe3 + ions but still higher than before UV treatment. in the Supporting Information), which implies that GQDs themFTIR spectra (see Figure S8 in the Supporting Information) selves suffered oxidation simultaneously with production of showed similar evidence of transformation of oxygen-rich funcROS. The C/O ratios were 10.3 and 9.7 before and after UV tional groups for GQDs treated with UV. The UV-illuminated treatment, respectively. Interestingly, in the presence of Fe3 + GQDs showed a higher carboxyl CO deformation peak at 1385 cm1, which proved a larger amount of carboxyl groups ions, however, the C/O ratio of GQDs stayed almost unchanged (C/O ratio: 10.2) after UV treatment. High-resolution XP spectra at the surface of GQDs. The carboxyl peak at 1385 cm1 of the revealed that three types of oxygen atoms emerged in GQDs UV-illuminated GQDs in the presence of Fe3 + ions showed no (see Figure S6 in the Supporting Information). For GQDs withobvious change after UV treatment. However, the alkoxyl CO Chem. Eur. J. 2014, 20, 1 – 7
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Full Paper peak at 1093 cm1 and C-O-C peak at 1093 cm1 stayed unchanged, which implies that carboxyl CO plays an essential role in the oxidation process of GQDs. As shown by Zhu and coworkers,[20] the fluorescence property of GQDs was highly affected by the oxygen-rich functional groups at their surface. A higher oxygen fraction and addiFigure 4. Relative ratiometric response of iron ions over various competing metal ions. The concentration of Fe3 + tional carboxyl groups would and Fe2 + ions was 40 mm, and the concentrations of other metal ions were 200 mm. lead to a lower quantum yield and a redshift of fluorescence, which are coincident with our results. these metal ions cannot induce quenching of GQDs. The rest As shown by colorimetric experiments with ABTS and fluoof metal ions including Hg2 + , Cd2 + , Cu2 + , and Pb2 + can be disrescence characterization of GQDs, iron ions behave as exceltinguished by the turn-on channel under UV irradiation. None lent reagents to eliminate ROS and can be further used to of the above metal ions can inhibit ROS generation and promodulate the fluorescence of GQDs. Thus, a ratiometric fluotect GQDs from photobleaching like iron ions. Overall, the rarescent probe for iron ions was developed. The response was tiometric probe showed improved selectivity for iron ions. Ag + calculated as follows [Eq. (4)] ions were not considered as interference, because Ag + ions would be photodecomposed by UV illumination. The ratiometric sensor could not distinguish Fe2 + and Fe3 + ions and UV Con I I showed exactly the same response to both ions, because Fe2 + ð4Þ R2 ¼ iUV 1 1 iCon I0 I0 and Fe3 + ions interconvert rapidly in the process of ROS generation/elimination. The high selectivity of the ratiometric fluowhere Ii is the fluorescent intensity of sample i, I0 the fluoresrescent probe indicates that it could be applied in complex cent intensity of the blank sample with zero concentration of systems. Fe3 + ions, superscript UV refers to the UV-treated group, and As the most abundant transition metal ions in the human Con to the control group stored in the dark. body, Fe3 + ions play an essential role in many biological proThe fluorescence of GQDs in the control group decreased cess. In many enzymes, such as hemoglobin and cytochrome c, when Fe3 + ions were added, whereas that of GQDs in the UViron ions serve as active centers in biological catalysis, which is often related to metabolism, diseases, cancers, cellular apoptotreated group increased on addition of Fe3 + ions (Figure 3). sis, and so forth. Lack of iron can lead to serious illnesses inThe sensitivity of the probe is remarkably higher than that of cluding iron-deficiency anemia and nerve deafness. Therefore, the simple quenching method. The fluorescent responses of the intake and storage of iron are very important to human both the control and UV-treated groups show good linearity as a function of Fe3 + concentration (see Figure S9 in the Supporthealth. Ferritin, an iron-containing protein, is the major form of iron storage in the liver, spleen, muscles, marrow, and blood. ing Information). Overall, the ratiometric response of the probe The ferritin concentration can reflect anemia, inflammation, for Fe3 + ions exhibits excellent linearity and a wide linear and hepatic illness.[21] In particular, a rise in ferritin level is range from 1 to 50 mm with a correlation coefficient of 0.996. The calculated limit of detection is 0.3 mm. The ratiometric restrongly related to hepatitis, hepatic fibrosis, leukemia, and sponse of the probe reveals a better linear fit than either the hepatic cancer. Monitoring of ferritin levels is of great imporquenching channel of the control group or the turn-on chantance in medical research. Here, we used SD rats as models to nel of the UV-treated group. This further evidences that the inverify the practical use of our proposed ratiometric probe. The troduction of a ratiometric strategy could efficiently improve ferritin levels of healthy rats and rats suffering from chemically the stability and accuracy of the sensor. induced acute liver damage[22] were estimated by measuring 3+ To examine the selectivity of the ratiometric probe for Fe serum iron concentrations. Damage to the liver would release a large amount of ferritin from hepatic tissues to the blood, /Fe2 + ions, the fluorescent responses of GQDs towards various which is reflected in a remarkable increase of iron concentracompeting metal ions (Al3 + , Ca2 + , Cd2 + , Co2 + , Cr3 + , Cu2 + , tion. The GQD-based ratiometric probe shows good accuracy Hg2 + , K + , Li + , Mg2 + , Mn2 + , Na + , NH4 + , Ni2 + , Pb2 + and Zn2 + ) and recovery for Fe3 + ions in rats. The fluorescent responses of was examined under the same conditions. The final concentration of ions was 200 mm, which was five times of that of iron acutely liver damaged rats were remarkably higher than those ions. Figure 4 shows that all the competing metal ions had no of health ones (see Table S4 in the Supporting Information). effect on the fluorescent responses of the sensing system. ReThe successful application of the GQD-based ratiometric probe garding the turn-off channel, all of the non-transition metal proves its suitability for complex systems and it may show ions (Al3 + , Ca2 + , K + , Li + , Mg2 + , Na + , NH4 + ) and Co2 + , Cr3 + , great potential in therapeutic diagnostics of acute hepatitis and hepatic cancer. ROS is generated in the sensing system Mn2 + , Ni2 + and Zn2 + ions can be easily excluded, because &
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Full Paper water was then slowly added by dropping funnel. The solution was heated to reflux for 30 min at 95 8C. 100 mL of water was added. Subsequently, 3 mL of H2O2 (30 %) was added dropwise to change the color of the solution from dark brown to yellow. The resulting suspension was filtered through a 0.22 mm microporous membrane, and further washed twice with 200 mL of HCl (1 m) and 200 mL of deionized water. The brown solid was vacuum-dried at 50 8C for 24 h to obtain GO.
and the ratiometric probe is a potential agent for use in photodynamic therapy to kill cancer cells.[21] Iron ions are able to eliminate ROS produced by GQDs. Therefore, iron-containing compounds are possible protectors that could reduce damage to healthy cells in photodynamic therapy. Moreover, the photoregulated fluorescence property was not only applied to GQDs top-down fabricated from GO, but also could be expanded to bottom-up synthesized carbon quantum dots (CQDs).[23] The ROS-related mechanism of UVand Fe2 + /Fe3 + -triggered reactions seem to be general to all carbon-based fluorescent materials (see Figure S10 in the Supporting Information).
Preparation of graphene GQDs GO (0.05 g) was added to a mixture of concentrated H2SO4 (10 mL) and HNO3 (30 mL). The solution was treated with mild ultrasonication for 12 h (500 W, 40 kHz). The mixture was then diluted with distilled water (100 mL) and filtered through a 0.22 mm microporous membrane to remove the acids. Purified GO was redispersed in distilled water (40 mL) and the pH was adjusted to 8.0 with NaOH. The suspension was transferred to a Teflon-lined autoclave and heated at 200 8C for 12 h. After cooling to room temperature, the solution was further dialyzed in a dialysis bag (retained molecular weight: 3500 Da) for 48 h to obtain the pristine GQDs.
Conclusion We have reported a new type of ratiometric probe based on single-wavelength-emitting GQDs for the sensitive and selective determination of iron ions. The probe is easily fabricated and suitable for complex systems. The ratiometric sensor was successfully employed to monitor ferritin levels in SD rats with chemically induced acute liver damage and shows potential for further applications in diagnosing acute hepatitis and hepatic cancer. The ROS-related process also has potential for photodynamic therapy. The development of this ratiometric probe not only introduces a novel method for detection of iron ions, but also gives a new view of the ratiometric strategy and could largely broaden the applicability of ratiometric sensors beyond analytical chemistry.
Animal experiments All procedures involving animals were conducted with the approval of the Animal Ethics Committee in East China Normal University (ECNU), China. Male SD rats (weight 200–250 g) were purchased from Shanghai SLAC Laboratory animal Co. Ltd and acclimatized for 4 d. The rats were divided into two groups at random, that is, the control group and the acute-liver- damage group. The acuteliver-damage groupwas intraperitoneally injected with 1.0 mL kg1 body weight CCl4 (dissolved in olive oil, 1/1), whereas rats in control group were injected with the same volume of olive oil. Rats of each group were sacrificed 24 h after CCl4/oil injection. The serum of each rat was collected for Fe3 + determination.
Experimental Section Reagents and materials
Acknowledgements
CNTs were obtained from Shenzhen Carbon Nanotechnologies Co. Ltd. Graphite powder (spectral grade) and other chemicals (analytical grade) were provided by Sinopharm Group Chemical Regent Co., Ltd. (Shanghai, China). All solvents and chemicals in this work were used without further purification unless stated. Double-distilled water was used throughout the experiments.
This work was supported by the National Natural Science Foundation of China (No. 21275055, 21277048) and Project funded by China Postdoctoral Science Foundation (2014M550224).
Instrumentation Keywords: analytical methods graphene · iron · quantum dots
The fluorescence and absorption spectra were recorded with a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan) and a Shimadzu UV-1800 spectrophotometer (Tokyo, Japan), respectively. A JEM-2010 transmission electron microscope (JEOL Ltd. Japan) and BioScope atomic force microscope (NanoScope IIIa SPM System, Digital Instruments, Inc., U.S.A) were used to study the morphology of the prepared GQDs. FTIR spectra were obtained with a Nicolet Nexus 670 spectrometer. XPS experiments were carried out on an AXIS UltraDLD system with AlKa radiation (hn = 1486.6 eV).
Graphene oxide was synthesized from graphite powder by a modified Hummers method. Typically, 0.5 g of graphite, 0.5 g of NaNO3, and 23 mL of H2SO4 were mixed in an ice bath. 3 g of KMnO4 was carefully added into the mixture over 10 min. The suspension was transferred to a 35 8C water bath and stirred for 2 h. 40 mL of www.chemeurj.org
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FULL PAPER & Ratiometric Probes Z.-b. Qu, M. Zhang, T. S. Zhou, G. Y. Shi* && – && A Single-Wavelength-Emitting Ratiometric Probe Based on Phototriggered Fluorescence Switching of Graphene Quantum Dots
A new ratiometric strategy: A new type of ratiometric probe based on graphene quantum dots (GQDs) emitting single-wavelength fluorescence was developed for the sensitive and selective
Chem. Eur. J. 2014, 20, 1 – 7
determination of iron ions. The analytical method is based on the different responses of the GQDs to iron ions before and after UV irradiation (see figure; ROS: reactive oxygen species).
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