Article pubs.acs.org/ac
Boron-Doped Graphene Quantum Dots for Selective Glucose Sensing Based on the “Abnormal” Aggregation-Induced Photoluminescence Enhancement Li Zhang,§ Zhi-Yi Zhang,§ Ru-Ping Liang, Ya-Hua Li, and Jian-Ding Qiu* Department of Chemistry, Nanchang University, Nanchang, Jiangxi 330031, P.R. China S Supporting Information *
ABSTRACT: A hydrothermal approach for the cutting of boron-doped graphene (BG) into boron-doped graphene quantum dots (BGQDs) has been proposed. Various characterizations reveal that the boron atoms have been successfully doped into graphene structures with the atomic percentage of 3.45%. The generation of boronic acid groups on the BGQDs surfaces facilitates their application as a new photoluminescence (PL) probe for label free glucose sensing. It is postulated that the reaction of the two cis-diol units in glucose with the two boronic acid groups on the BGQDs surfaces creates structurally rigid BGQDs−glucose aggregates, restricting the intramolecular rotations and thus resulting in a great boost in the PL intensity. The present unusual “aggregation-induced PL increasing” sensing process excludes any saccharide with only one cis-diol unit, as manifested by the high specificity of BGQDs for glucose over its close isomeric cousins fructose, galactose, and mannose. It is believed that the doping of boron can introduce the GQDs to a new kind of surface state and offer great scientific insights to the PL enhancement mechanism with treatment of glucose.
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imaging.15 Recent progress involving doping GQDs with electron-rich nitrogen has motivated our curiosity to examine the corresponding performance of its counterpart by doping GQDs with electron-deficient boron. Intuitively, doping GQDs with chemically bonded boron atoms should alter their optical characteristics and offer more active sites, thus producing new phenomena and unexpected properties. As far as we are aware, however, no attempt has been made to synthesize boron-doped GQDs (BGQDs) and the application development of heteroatom-doped GQDs remains inchoate, which might be attributed to the limitation in synthesis methods and the difficulties in construction of an effective GQD-based sensing platform. Much research has indicated that diabetes and some cancers are correlated with the breakdown of glucose transport in the human body. One of the major challenges in the management of these diseases is the monitoring of glucose concentrations. Thus, far various methods for detecting glucose have been presented, including nanoparticle aggregation-induced colorimetric detection17−19 and electrochemical and fluorescence detection.20−23 Among the fluorescent sensing devices, boronic acid composites are the most widely used fluorescent probes for glucose, serving as either fluorophore or quencher.24−27 In our
raphene, containing a single layer of sp2-hybridized carbon atoms, has fuelled intensive research interest because of its unique two-dimensional (2D) crystalline structure, fascinating physical and chemical properties, and potential applications in electronic devices and molecule sensors.1,2 Both theoretical and experimental studies have shown that not only the size and shape but also the geometry and chemical nature determine the properties of graphene nanostructures.3,4 Graphene nanosheets of less than 100 nm in size, which are known as graphene quantum dots (GQDs),3 are particularly encouraging owing to their outstanding photoluminescence (PL) properties. Various methods have been demonstrated in preparation of GQDs, such as electrochemical oxidation processes,5,6 oxidation cutting methods,7−9 and carbonizing organics routes.10−13 Doping GQDs with heteroatoms can effectively tune their intrinsic properties, including optical characteristics and surface and local chemical features. For instance, nitrogen-doped GQDs (NGQDs) are attracting increasing interest in the field of fuel cells and optoelectronics for their effective electrocatalytic activity and strong electronwithdrawing effect.14−16 Li et al. proposed a simple electrochemical approach to synthesize luminescent NGQDs and investigated their use as metal-free oxygen reduction reaction (ORR) catalysts in fuel cells.14 Liu et al. prepared NGQDs by a facile solvothermal method using dimethylformamide as a solvent and nitrogen source and demonstrated their application as two-photon fluorescent probes for cellular and deep-tissue © 2014 American Chemical Society
Received: January 22, 2014 Accepted: April 5, 2014 Published: April 7, 2014 4423
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Scheme 1. (a) Schematic Representation of the Boron-Doped Graphene Quantum Dots (BGQDs). (b) Proposed “AggregationInduced PL Increasing” Mechanism for the Glucose-Specific Sensing by BGQDs
reagents were of analytical grade and were used as received without further purification. All solutions were prepared and diluted using ultrapure water having a resistivity of 18.2 MΩ from the Millipore Milli-Q system. Characterization. The morphologies of BGQDs samples were characterized by transmission electron microscopy (TEM) using a JEM-2010 TEM facility (JEOL, Japan) with a 200 kV accelerating voltage. The TEM samples were prepared by drying a droplet of the BGQDs solution on a Cu grid. Atomic force microscopy (AFM) images were collected on a Bruker Multimode 8 AFM/SPM (Bruker, Germany) system with NanoScope Analysis Version 1.40 software. Imaging was performed in ScanAsyst mode under ambient conditions. X-ray photoelectron spectroscopy (XPS) characterizations were conducted by using a VG Multilab 2000X instrument (Thermal Electron, USA). Raman spectra were collected using a Renishaw InVia micro-Raman (Renishaw, UK) system with the excitation wavelength at 514 nm. X-ray diffraction (XRD) patterns of samples were obtained on a D/MAX2200PC Rigaku powder diffractometer (Rigaku, Japan) equipped with CuKa1 radiation (λ = 1.54 Å). Isoelectric point measurement was used to monitor the surface potential of BGQDs with a Zetasizer Nano ZS90 (Malvern, UK). UV−vis absorption spectra were recorded on an UV-2450 spectrophotometer (Shimadzu, Japan). Fourier transform infrared (FT-IR) spectra of the B-free GQDs and BGQDs were obtained using a Tenson 27 Fourier Transform Infrared spectrometer (Bruker, Germany) at resolution of 4 cm−1 in the range of 600−4000 cm−1. The photoluminescence (PL) spectra and the light scattering (LS) spectra of the resulting solutions were recorded on a F7000 spectrophotometer (Hitachi, Japan). All the experiments were performed at room temperature, and the pH value was calibrated with a pH meter. Synthesis of Boron-Doped Graphene. Boron-doped graphene (BG) was synthesized through graphene sheets (GSs) in the presence of boron oxide (B2O3), in which boron atoms coming from B2O3 vapor can replace carbon atoms within graphene structures at high temperature in a tubular
recent research, a cationic boronic acid-substituted bipyridinium salt (BBV) has been utilized to quench the PL of GQDs, and the interaction between boronic acid group and glucose neutralizes the net charge of the BBV, thus recovering the PL of GQDs.28 The sensing approach is simple but lacks selectivity among saccharides, and other monosaccharides, e.g., fructose, mannose, and galactose, can largely affect the accuracy of glucose determination. Most recently, Qu and co-workers employed phenylboronic acid functionalized GQDs as a fluorescent probe for the selective sensing of glucose,29 where a functionalization process was necessary, and the “turn off” sensing principle might be interfered with a variety of ligands or solvents, leading to “false positive”. To develop new PL sensors with improved glucose selectivity, new approaches based on new concepts need to be devised to exclude the interferences from participating in the PL responsive processes. Herein, we for the first time report a hydrothermal approach for the cutting of boron-doped graphene (BG) into BGQDs. The generation of boronic acid groups on the BGQDs surfaces facilitates their application as a novel PL probe for label free glucose sensing. It is postulated that the reaction of the two cisdiol units in glucose with the two boronic acid groups on the BGQDs surfaces creates structurally rigid BGQDs−glucose aggregates, restricting the intramolecular rotations and thus resulting in a great boost in the PL intensity (Scheme 1). In contrast to the conventional “aggregation-induced quenching” mode,30−33 an interesting “aggregation-induced PL increasing” phenomena is first observed for the GQDs. The present sensing process excludes any saccharide with only one cis-diol unit, as manifested by the high specificity of BGQDs for glucose over its close isomeric cousins fructose, galactose, and mannose.
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EXPERIMENTAL SECTION Chemicals and Reagents. Graphite flakes (99.8%, 325 mesh) were provided by Alfa Aesar. Other chemicals such as boron oxide (B2O3), sulfuric acid (H2SO4), sodium hydroxide (NaOH), and nitric acid (HNO 3) were bought from Sinopharm Chemical Reagent Co. Ltd. (China). All the 4424
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Figure 1. Morphology and optical properties of BGQDs. (a) TEM image of the dispersive BGQDs and the inset shows the diameter distribution of the BGQDs. (b) AFM image of the dispersive BGQDs deposited on freshly cleaved mica. size: 3 × 3 μm; the inset shows the height distribution of the BGQDs. (c) UV−vis absorption and excitation-dependent PL behavior of BGQDs; Inset: photographs of the BGQDs taken under visible light and 365 nm UV light (from left to right), respectively. (d) FT-IR spectra of BGQDs and B-free GQDs; Inset: the magnified FT-IR spectra of BGQDs and B-free GQDs from 1350 to 1440 cm−1.
through a 0.22 μm microporous membrane to remove the acids. The filter cake was collected and dried and then redispersed in 20 mL of ultrapure water. The pH was adjusted to 8 with NaOH. The solution was then put into a poly(tetrafluoroethylene) (Teflon)-lined autoclave and heated at 200 °C for 11.5 h. The resulting solution was filtered through a 0.22 μm microporous membrane to remove the large tracts. The pale yellow filtrate was dialyzed in a dialysis bag (retained molecular weight: 3500 Da) for 12 h, and the resultant BGQDs showed blue photoluminescence under UV light. Detection Procedure of Glucose. Twenty μL of BGQDs stock solution (50 μg/mL) was added to 340 μL of phosphate buffer solution (PB, 100 mM, pH 7.4), followed by the addition of 40 μL different concentrations of glucose, and then, the mixture solution was sonicated with moderate intensity at room temperature for 10 min to accelerate the aggregation reaction. Finally, the fluorescence spectra were recorded under excitation at 310 nm.
furnace (TOL 1200, Nanjing NanDa Instrument Plant).34 GSs were produced by heating the dried graphene oxide (GO, synthesized by the modified Hummers method35) at 300 °C for 2 h with a continuous flow of nitrogen at a heating rate of 5 °C/min in a tube furnace. 1.0 g of GSs was put onto the surface of 1.25 g of B2O3, which was then placed in the center of a corundum tube with a continuous flow of argon to guarantee an inert atmosphere in the tube furnace. The center temperature of the tube furnace was heated to 1100 °C at an increasing rate of 5 °C/min. After maintaining 1100 °C for 4 h, the sample was cooled to room temperature slowly under an argon atmosphere. The obtained product was then refluxed in 3 M NaOH aqueous solution for 2 h to remove any of the unreacted boron oxide. After filtration and water washing to neutral, the product was dried in a vacuum at 60 °C. Synthesis of Boron-Doped Graphene Quantum Dots. Boron-doped graphene quantum dots (BGQDs) were prepared from oxidized BG (BGO) by a hydrothermal approach similar to the B-free GQDs.8 BG was first refluxed in 40% HNO3 for 24 h; after filtration and water washing to neutral, the product (BGO) was dried in a vacuum at 60 °C. Then, the BGO was heated to 300 °C with a heating rate of 5 °C/min and then maintained at 300 °C for 2 h in a tube furnace under an argon atmosphere. 0.05 g of the product was oxidized with concentrated H2SO4 and HNO3 (volume ratio 1:3) for 17 h under mild ultrasonication without any pausing. The solution was diluted with 250 mL of ultrapure water and then filtered
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RESULTS AND DISCUSSION Characterization of the BGQDs. The BGQDs were synthesized from BG by the hydrothermal approach described in the Experimental Section. The as-prepared BGQDs solution remains homogeneous even after 3 months at room temperature without any perceptible changes (e.g., aggregation or color change), which could be further characterized by the
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Figure 2. XPS spectra. (a) The wide scan spectra of GO, BG, and the as-produced BGQDs. (b) The magnification of (a) from 100 to 250 eV. (c) The high-resolution C 1s XPS spectrum of BG. (d) The high-resolution C 1s XPS spectrum of BGQDs. (e) The high-resolution B 1s XPS spectrum of BG. (f) The high resolution B 1s XPS spectrum of BGQDs.
almost unchanged absorption and PL spectra (Figure S1 in the Supporting Information). The transmission electron microscopy (TEM) image shows fairly uniform BGQDs with diameters of ca. 2−4 nm (Figure 1a), which are much smaller than those of the B-free counterparts synthesized hydrothermally (∼10 nm) but well-consistent with those of NGQDs prepared solvothermally.8,15 The corresponding atomic force microscopy (AFM) image (Figure 1b) reveals a typical topographic height of 0.5−0.8 nm (inset of Figure 1b), suggesting that most of the BGQDs consist of a single graphene layer. To explore the optical properties of the BGQDs, UV−vis absorption and excitation-dependent PL behavior of BGQDs were investigated. The BGQDs show a broad UV−vis absorption band with a weak shoulder at 295 nm (Figure 1c), which is blue-shifted by ca. 15 nm with respect to that of previous B-free GQDs.33 The photograph shows that BGQDs solution is pale-yellow, transparent, and clear under daylight and exhibits blue PL under irradiation by a 365 nm UV light (inset of Figure 1c). It has been reported that isolated sp2hybridized clusters with a size of ca. 3 nm within the carbon− oxygen matrix could yield band gaps consistent with blue emission due to the localization of electron−hole pairs,36
suggesting that both the size and surface effects make an important contribution to the observed blue PL emission from BGQDs. Upon excitation with a 310 nm beam, the PL spectrum shows a strong peak at 440 nm with a Stokes shift of 130 nm. Like other luminescent carbon nanoparticles, the BGQDs also exhibit an excitation-dependent PL behavior (Figure 1c). As the excitation wavelength increases, the emission peak shifts to longer wavelength and its intensity decreases rapidly. It is suspected that the giant red-edge effect is responsible for the strong dependence of the PL peak position upon the wavelength of the excitation source.37 The chemically synthesized BGQDs are readily water-dispersible due to the presence of hydroxyl and carboxylic groups at the surface and edges, which is confirmed by Fourier transform infrared (FTIR) measurement. The peaks at about 1636 and 1386 cm−1 indicate the existence of COO− (Figure 1d), while the peak at 3442 cm−1 corresponds to the OH stretching mode. Compared with B-free GQDs, the magnified FT-IR spectrum of BGQDs (inset of Figure 1d) displays the additional absorption peak from asymmetric B−O stretching at 1410 cm−1,38 confirming the successful preparation of the BGQDs. X-ray photoelectron spectroscopy (XPS) characterization was then carried out to explore the content and configuration 4426
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Figure 3. (a) XRD patterns and (b) Raman spectra of the pristine graphene, BG, B-free GQDs, and BGQDs.
compound. The above results indicate that B atoms are mainly in the three different bonding characters inserted into the graphene network of GQDs (Scheme 1). Figure 3a shows the typical X-ray diffraction (XRD) profiles for BG, B-free GQDs, and BGQDs. The XRD peak for BG shifts to a higher degree (ca. 25.8°) compared with the pristine graphene obtained from thermally annealing GO under the same conditions (ca. 24.3°), suggesting the smaller interlayer spacing as a result of the more thorough reduction in the presence of boron oxide. As in the case of B-free GQDs, the BGQDs show a broader diffraction peak at ca. 25°, indicating that the oxidation reaction during the BGQDs preparation process has introduced more active sites on the surfaces of BGQDs, as confirmed by XPS (Figure 2) and FT-IR spectroscopy investigations (Figure 1d). The XRD peak for as-prepared BGQDs shifts to a lower degree compared with the BG sample (ca. 25.8°), indicating that the BGQDs have a larger interlayer spacing than the BG does. The increased interlayer spacing in BGQDs (ca. 0.356 nm) could be attributed to the weak π−π stacking of graphenes with more O-containing functional groups surrounding the edges of the graphene layers in BGQDs. It is also noteworthy that the prepared BGQDs do not show any diffractions in the 2θ ≈ 10° region characteristic of GO, evidently indicating that the BGQDs are different from GO, though both contain oxygen-enriched functional groups. Raman spectra offer clear evidence of B-doping in the graphene lattice. Figure 3b shows the Raman spectra of the BG, B-free GQDs, and BGQDs. For comparison, the spectrum of pristine graphene obtained from thermally annealing GO under the same conditions is also displayed. The spectra exhibit two remarkable peaks at around 1365 and 1596 cm−1 corresponding to the well-defined D band and G band, respectively. As is known, the G band related to the E2g vibration mode of sp2 carbon domains can be used to explain the degree of graphitization, while the D band is associated with structural defects and partially disordered structures of the sp2 domains.41 The ID/IG ratio in the Raman spectra is generally used to evaluate the disorder in the graphene materials. Although there is no significant change in the position of D and G bands, the intensity ratio of ID/IG increases from pristine graphene (0.79) to BG (0.87), clearly suggesting a broken hexagonal symmetry of the graphene, most presumably by the heterogeneous Bdopants.42 Interestingly, it is observed that both the BGQDs and B-free GQDs have an ID/IG ratio of ca. 0.7, which is much lower than that of the BG (0.87) (Figure 3b), indicating the efficient structural restoration of graphitic framework during the hydrothermal process to yield high-quality GQDs.14 In comparison with B-free GQDs, however, the BGQDs exhibit
of doped boron in the BG and BGQDs. Compared with graphene oxide (GO) involving only the core levels of C 1s and O 1s (Figure 2a), the full range XPS analysis of the resultant BGQDs sample clearly shows the presence of boron (B), carbon (C), and oxygen (O) with atomic percentages of 3.45%, 70.16%, and 26.39%, and the corresponding B 1s, C 1s, and O 1s peaks center at 190, 284, and 532 eV, respectively (Figure 2a,b), while for the BG sample, the corresponding atomic percentage of B 1s, C 1s, and O 1s is 4.25%, 80.4%, and 15.35%, respectively. The O/C atomic ratio for the BGQDs is ca. 37.6%, higher than that of the BG (ca. 19.1%), suggesting that oxidation reaction takes place during the HNO3 refluxing for the BGQDs preparation (see the Experimental Section). In addition, the B 1s peak appears at a higher binding energy (B 1s peak: 190 eV as compared to 187 eV for pure boron), which suggests that boron atoms coming from B2O3 vapor have been partly bonded to the carbon atoms in the sp2 carbon network.39,40 On the basis of the Shirley algorithm, the C 1s peak of BG can be fitted into three components (Figure 2c). The most intense peak located at 283.9 eV is assigned to sp2 hybrid carbon atoms, and small signals at higher binding energies indicate some C−O (285.5 eV) and CO (286.5 eV) species remain in the sample after thermal annealing.5 It should be noticed that the signal at 281.8 eV, corresponding to carbon atoms neighboring with boron atoms, is not observed due to the relatively low boron content.34 In comparison with BG, Figure 2d clearly reveals the wider C 1s peak of BGQDs to be associated with the more oxygen-containing groups. A highresolution spectrum of C 1s confirmed the presence of CC (284.4 eV), C−O (285.2 eV), CO (286.5 eV), and O−C O (288 eV) bonds, indicating the as-prepared BGQDs are rich in hydroxyl, carbonyl, and carboxylic acid groups on the surfaces,5,14 which is consistent with the corresponding FT-IR spectra (Figure 1d). In the high-resolution B 1s spectrum of BG (Figure 2e), the peak at 189.0 eV is attributed to the BC3 structures, while the minor peak centered at 188.0 eV may be assigned to the B4C species, which are not shown in the scheme due to its extremely low content in the sample. The most intense peak at 189.2 eV corresponds to the structure of boron atoms bonding to carbon and oxygen atoms (BC2O), suggesting a large fraction of boron exists in the form of epoxy. The signal at 191.9 eV reveals that boron atoms are surrounded by carbon and oxygen atoms (BCO2), indicating the presence of boronic acid group in this sample.34 The highresolution B 1s spectrum of BGQDs is similar to that of BG except for the increased percentage of BCO2 species (Figure 2f), which is favorable for the subsequent glucose sensing application as they act as anchoring sites for polyhydroxy4427
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Figure 4. (a) Characteristic PL response of the BGQDs with different concentrations of glucose (from 1 to 13:0, 0.1, 0.5, 1, 2, 4, 6, 8, 10, 20, 50, 100, 200 mM). The inset picture shows the PL increasing in the BGQDs solution illuminated with an UV lamp, and the homologous concentrations of glucose were 0, 2, 8, 20, and 50 mM (from left to right). (b) Plot of the PL intensity vs glucose concentration in pH 7.4 phosphate buffer. (c) PL spectra of the BGQDs in the presence of 10 mM of different saccharides. (d) PL response of the BGQDs to different saccharides (10 mM). The inset picture shows the saccharides treated BGQDs solution illuminated with an UV lamp.
a broader D band, suggesting that the intercalation of B atoms into the conjugated carbon backbone has led to somewhat disordered structures.14,41 Selective Sensing of Glucose by BGQDs. Since the asprepared BGQDs involve boronic acid structure (BCO2 species) through the B-doping, which has been proven by the XPS analysis, it is expected that the BGQDs could tightly but reversibly recognize diols, thus giving rapid and sensitive PL response to glucose. To explore this attractive possibility, we investigated the PL evolution of BGQDs in the absence and presence of different amounts of glucose. Since the pKa of BGQDs is about 3.73 (Figure S2 in the Supporting Information), BGQDs with negative charge when pH > 3.73 can reversibly react with diols of glucose, and the reaction extent could be enhanced with increasing pH in alkaline aqueous solutions (Figure S3 in the Supporting Information). When glucose is added to the BGQDs solution in pH 7.4 phosphate buffer, the PL intensity of the BGQDs linearly increases with the glucose concentration in the range of 0.1−10 mM with a detection limit of 0.03 mM (Figure 4a,b), which is comparable to or better than that of previous boronic acidbased glucose sensors.23,26,30,43−45 At a glucose concentration of 10 mM, the BGQDs become very emissive, with an intensity ∼6-fold higher than that in the absence of glucose. As expected, the sensor demonstrates a relatively wider linear range (0.05− 10 mM) and a lower detection limit (about 0.01 mM) at pH 10 phosphate buffer (Figure S4 in the Supporting Information) compared with that in pH 7.4 phosphate buffer. Considering the potential application of the proposed method in real samples (such as whole blood), we assess the sensor response in neutral pH due to its physiological relevance. In comparison with the significant changes caused by glucose, the emission spectrum of BGQDs is only slightly intensified even when a
large amount (10 mM) of fructose is added, and similar results are observed in the cases of galactose and mannose (Figure 4c,d). It should be noted that the affinity constants (Ka) for binding of the other three monosaccharides (fructose, galactose, and mannose) to the boronic acid group are all larger than that of glucose.46 This suggests that the binding affinity strength is not the decisive parameter in the selective PL recognition of BGQDs to glucose. Working Mechanism of the Unusual PL Response of BGQDs to Glucose. On the basis of the above experimental data and enlightened by previous studies of the chemical reactions of tetraphenylethene (TPE)-cored diboronic acid with glucose,45 we propose a working mechanism for the specific PL response of BGQDs to glucose as shown in Scheme 1. At a concentration of glucose higher than 0.1 mM, thanks to the accessibility of the cis-5,6-diol units of glucose by the boronate group of the negatively charged BGQDs, BGQDs− glucose aggregates may form, where two BGQDs are fastened by a glucose linker. The intramolecular rotation of the luminescence center in the aggregates involves simultaneous movements of the glucose linker and another luminescence center in the neighboring unit. The high energy barrier to such motions stiffens the aggregate structure, blocks the nonradiative relaxation channels, and populates the radiative decay, thus making the luminescence center highly emissive.45,47−50 The possibility of aggregate formation increases with increasing glucose concentration, with the PL intensity reaching its maximum at glucose of about 50 mM. Compared with glucose, fructose has no additional cis conformational diol unit to further bind with BGQDs to form aggregates though it is easier to covalently combine with BGQDs than glucose.24 This is why no obvious change in PL is observed at high concentration of fructose. Similar to the case for fructose, galactose and mannose 4428
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Figure 5. (a) LS spectra of the BGQDs with different concentrations of glucose. The concentrations of glucose were 0, 0.1, 0.5, 1, 2, 4, 6, 8, 10, 20, 50, 100, and 200 mM (from 1 to 13). (b) Plot of LS intensity vs glucose concentration in pH 7.4 phosphate buffer. (c) TEM image of the aggregation of BGQDs with 10 mM glucose. (d) AFM image of the aggregation of BGQDs with 10 mM glucose, size: 3 × 3 μm. The inset is the height distribution of the BGQDs aggregates.
have no more cis-diol units to enhance the PL of BGQDs by cross-linking aggregation. Clearly, the existence of a pair of cisdiol units in the 1,2- and 5,6-positions in glucose is the key structural feature that confers a glucose-specific response on BGQDs. In other words, it is the aggregates of BGQDs with glucose that enhances the emission of the luminescence center. Light scattering (LS) spectra coupled TEM and AFM characterization give reliable evidence of the unusual aggregation-induced PL increasing mechanism. The LS signals of aggregated BGQDs were measured by scanning synchronously both the excitation and emission monochromators of a common spectrofluorometer. As shown in Figure 5a, the LS intensity of the solution containing BGQDs is very weak. Notably, the LS intensities at 200−500 nm for BGQDs treated with various amounts of glucose are enhanced, and the LS spectra maintain almost the same pattern as the concentration of glucose increases. It is found that the enhanced LS signals linearly increase with the increasing glucose concentration over the range of 0.1 to 10 mM with a limit of detection of around 0.06 mM (3σ) (Figure 5b). To further confirm the aggregationinduced PL increasing mechanism, we have also monitored the LS response to various amounts of glucose in pH 10 phosphate buffer. As expected, the LS intensity was enhanced with increasing glucose concentrations, demonstrating a better performance (linear range over 0.05−10 mM with a detection limit of 0.03 mM, Figure S5 in the Supporting Information) compared with that in pH 7.4 phosphate buffer, consistent with the PL results in different pH buffer solutions. TEM and AFM images gave more direct information on the aggregation of the
BGQDs. Upon interaction with 10 mM glucose, the aggregation of BGQDs was monitored, as indicated by the formation of wirelike chains (Figure 5c,d). The topographic heights of the cross-linked BGQDs are mostly between 1.0 and 1.6 nm, suggesting that most of the BGQDs aggregates are of bilayered structure. These observations support our suggestion that the PL increase should be ascribed to the formation of BGQDs aggregates as a result of the intense and exclusive combination of boronate group on the BGQDs surfaces with glucose, offering strong support to the proposed working mechanism shown in Scheme 1.
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CONCLUSION In summary, we report on a novel hydrothermal approach for the cutting of BG into BGQDs; the boronic acid groups on the BGQDs surfaces could provide important application in glucose selective sensing. It works on the basis of a conceptually new mechanism: the PL of BGQDs is enhanced by the restriction of the intramolecular rotations activated by the peculiar BGQDs−glucose interactions. In contrast to the conventional “aggregation-induced quenching” mode, which is not preferred in practice because there are a variety of ligands or solvents that may interfere with quenching, leading to “false positive”, the reaction of the two cis-diol units in glucose with the two boronic acid groups on the BGQDs surfaces creates structurally rigid BGQDs−glucose aggregates, resulting in a great boost in the PL intensity. The present sensing process excludes any saccharide with only one cis-diol unit, as manifested by the high specificity of BGQDs for glucose over 4429
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its close isomeric cousins fructose, galactose, and mannose. It is reasonable to believe that the doping of boron can introduce the GQDs to a new kind of surface state, which offers great scientific insights to the PL enhancement mechanism after treatment with glucose.
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ASSOCIATED CONTENT
S Supporting Information *
The stability of BGQDs (Figure S1); the isoelectric point of BGQDs (Figure S2); the effects of pH values on the PL response to the glucose (Figure S3); the PL as well as the LS response of the BGQDs to different concentrations of glucose in pH 10 phosphate buffer (Figures S4 and S5). This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Author Contributions §
L.Z. and Z.-Y.Z. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (21163014, 21105044, and 21265017) and the Program for New Century Excellent Talents in University (NCET-11-1002, NCET-13-0848).
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REFERENCES
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Boron-Doped Graphene Quantum Dots for Selective Glucose Sensing Based on the “Abnormal” Aggregation-Induced Photoluminescence Enhancement Li Zhang,§ Zhi-Yi Zhang,§ Ru-Ping Liang, Ya-Hua Li, and Jian-Ding Qiu* Department of Chemistry, Nanchang University, Nanchang, Jiangxi 330031, P.R. China S Supporting Information *
ABSTRACT: A hydrothermal approach for the cutting of boron-doped graphene (BG) into boron-doped graphene quantum dots (BGQDs) has been proposed. Various characterizations reveal that the boron atoms have been successfully doped into graphene structures with the atomic percentage of 3.45%. The generation of boronic acid groups on the BGQDs surfaces facilitates their application as a new photoluminescence (PL) probe for label free glucose sensing. It is postulated that the reaction of the two cis-diol units in glucose with the two boronic acid groups on the BGQDs surfaces creates structurally rigid BGQDs−glucose aggregates, restricting the intramolecular rotations and thus resulting in a great boost in the PL intensity. The present unusual “aggregation-induced PL increasing” sensing process excludes any saccharide with only one cis-diol unit, as manifested by the high specificity of BGQDs for glucose over its close isomeric cousins fructose, galactose, and mannose. It is believed that the doping of boron can introduce the GQDs to a new kind of surface state and offer great scientific insights to the PL enhancement mechanism with treatment of glucose.
G
imaging.15 Recent progress involving doping GQDs with electron-rich nitrogen has motivated our curiosity to examine the corresponding performance of its counterpart by doping GQDs with electron-deficient boron. Intuitively, doping GQDs with chemically bonded boron atoms should alter their optical characteristics and offer more active sites, thus producing new phenomena and unexpected properties. As far as we are aware, however, no attempt has been made to synthesize boron-doped GQDs (BGQDs) and the application development of heteroatom-doped GQDs remains inchoate, which might be attributed to the limitation in synthesis methods and the difficulties in construction of an effective GQD-based sensing platform. Much research has indicated that diabetes and some cancers are correlated with the breakdown of glucose transport in the human body. One of the major challenges in the management of these diseases is the monitoring of glucose concentrations. Thus, far various methods for detecting glucose have been presented, including nanoparticle aggregation-induced colorimetric detection17−19 and electrochemical and fluorescence detection.20−23 Among the fluorescent sensing devices, boronic acid composites are the most widely used fluorescent probes for glucose, serving as either fluorophore or quencher.24−27 In our
raphene, containing a single layer of sp2-hybridized carbon atoms, has fuelled intensive research interest because of its unique two-dimensional (2D) crystalline structure, fascinating physical and chemical properties, and potential applications in electronic devices and molecule sensors.1,2 Both theoretical and experimental studies have shown that not only the size and shape but also the geometry and chemical nature determine the properties of graphene nanostructures.3,4 Graphene nanosheets of less than 100 nm in size, which are known as graphene quantum dots (GQDs),3 are particularly encouraging owing to their outstanding photoluminescence (PL) properties. Various methods have been demonstrated in preparation of GQDs, such as electrochemical oxidation processes,5,6 oxidation cutting methods,7−9 and carbonizing organics routes.10−13 Doping GQDs with heteroatoms can effectively tune their intrinsic properties, including optical characteristics and surface and local chemical features. For instance, nitrogen-doped GQDs (NGQDs) are attracting increasing interest in the field of fuel cells and optoelectronics for their effective electrocatalytic activity and strong electronwithdrawing effect.14−16 Li et al. proposed a simple electrochemical approach to synthesize luminescent NGQDs and investigated their use as metal-free oxygen reduction reaction (ORR) catalysts in fuel cells.14 Liu et al. prepared NGQDs by a facile solvothermal method using dimethylformamide as a solvent and nitrogen source and demonstrated their application as two-photon fluorescent probes for cellular and deep-tissue © 2014 American Chemical Society
Received: January 22, 2014 Accepted: April 5, 2014 Published: April 7, 2014 4423
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Scheme 1. (a) Schematic Representation of the Boron-Doped Graphene Quantum Dots (BGQDs). (b) Proposed “AggregationInduced PL Increasing” Mechanism for the Glucose-Specific Sensing by BGQDs
reagents were of analytical grade and were used as received without further purification. All solutions were prepared and diluted using ultrapure water having a resistivity of 18.2 MΩ from the Millipore Milli-Q system. Characterization. The morphologies of BGQDs samples were characterized by transmission electron microscopy (TEM) using a JEM-2010 TEM facility (JEOL, Japan) with a 200 kV accelerating voltage. The TEM samples were prepared by drying a droplet of the BGQDs solution on a Cu grid. Atomic force microscopy (AFM) images were collected on a Bruker Multimode 8 AFM/SPM (Bruker, Germany) system with NanoScope Analysis Version 1.40 software. Imaging was performed in ScanAsyst mode under ambient conditions. X-ray photoelectron spectroscopy (XPS) characterizations were conducted by using a VG Multilab 2000X instrument (Thermal Electron, USA). Raman spectra were collected using a Renishaw InVia micro-Raman (Renishaw, UK) system with the excitation wavelength at 514 nm. X-ray diffraction (XRD) patterns of samples were obtained on a D/MAX2200PC Rigaku powder diffractometer (Rigaku, Japan) equipped with CuKa1 radiation (λ = 1.54 Å). Isoelectric point measurement was used to monitor the surface potential of BGQDs with a Zetasizer Nano ZS90 (Malvern, UK). UV−vis absorption spectra were recorded on an UV-2450 spectrophotometer (Shimadzu, Japan). Fourier transform infrared (FT-IR) spectra of the B-free GQDs and BGQDs were obtained using a Tenson 27 Fourier Transform Infrared spectrometer (Bruker, Germany) at resolution of 4 cm−1 in the range of 600−4000 cm−1. The photoluminescence (PL) spectra and the light scattering (LS) spectra of the resulting solutions were recorded on a F7000 spectrophotometer (Hitachi, Japan). All the experiments were performed at room temperature, and the pH value was calibrated with a pH meter. Synthesis of Boron-Doped Graphene. Boron-doped graphene (BG) was synthesized through graphene sheets (GSs) in the presence of boron oxide (B2O3), in which boron atoms coming from B2O3 vapor can replace carbon atoms within graphene structures at high temperature in a tubular
recent research, a cationic boronic acid-substituted bipyridinium salt (BBV) has been utilized to quench the PL of GQDs, and the interaction between boronic acid group and glucose neutralizes the net charge of the BBV, thus recovering the PL of GQDs.28 The sensing approach is simple but lacks selectivity among saccharides, and other monosaccharides, e.g., fructose, mannose, and galactose, can largely affect the accuracy of glucose determination. Most recently, Qu and co-workers employed phenylboronic acid functionalized GQDs as a fluorescent probe for the selective sensing of glucose,29 where a functionalization process was necessary, and the “turn off” sensing principle might be interfered with a variety of ligands or solvents, leading to “false positive”. To develop new PL sensors with improved glucose selectivity, new approaches based on new concepts need to be devised to exclude the interferences from participating in the PL responsive processes. Herein, we for the first time report a hydrothermal approach for the cutting of boron-doped graphene (BG) into BGQDs. The generation of boronic acid groups on the BGQDs surfaces facilitates their application as a novel PL probe for label free glucose sensing. It is postulated that the reaction of the two cisdiol units in glucose with the two boronic acid groups on the BGQDs surfaces creates structurally rigid BGQDs−glucose aggregates, restricting the intramolecular rotations and thus resulting in a great boost in the PL intensity (Scheme 1). In contrast to the conventional “aggregation-induced quenching” mode,30−33 an interesting “aggregation-induced PL increasing” phenomena is first observed for the GQDs. The present sensing process excludes any saccharide with only one cis-diol unit, as manifested by the high specificity of BGQDs for glucose over its close isomeric cousins fructose, galactose, and mannose.
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EXPERIMENTAL SECTION Chemicals and Reagents. Graphite flakes (99.8%, 325 mesh) were provided by Alfa Aesar. Other chemicals such as boron oxide (B2O3), sulfuric acid (H2SO4), sodium hydroxide (NaOH), and nitric acid (HNO 3) were bought from Sinopharm Chemical Reagent Co. Ltd. (China). All the 4424
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Figure 1. Morphology and optical properties of BGQDs. (a) TEM image of the dispersive BGQDs and the inset shows the diameter distribution of the BGQDs. (b) AFM image of the dispersive BGQDs deposited on freshly cleaved mica. size: 3 × 3 μm; the inset shows the height distribution of the BGQDs. (c) UV−vis absorption and excitation-dependent PL behavior of BGQDs; Inset: photographs of the BGQDs taken under visible light and 365 nm UV light (from left to right), respectively. (d) FT-IR spectra of BGQDs and B-free GQDs; Inset: the magnified FT-IR spectra of BGQDs and B-free GQDs from 1350 to 1440 cm−1.
through a 0.22 μm microporous membrane to remove the acids. The filter cake was collected and dried and then redispersed in 20 mL of ultrapure water. The pH was adjusted to 8 with NaOH. The solution was then put into a poly(tetrafluoroethylene) (Teflon)-lined autoclave and heated at 200 °C for 11.5 h. The resulting solution was filtered through a 0.22 μm microporous membrane to remove the large tracts. The pale yellow filtrate was dialyzed in a dialysis bag (retained molecular weight: 3500 Da) for 12 h, and the resultant BGQDs showed blue photoluminescence under UV light. Detection Procedure of Glucose. Twenty μL of BGQDs stock solution (50 μg/mL) was added to 340 μL of phosphate buffer solution (PB, 100 mM, pH 7.4), followed by the addition of 40 μL different concentrations of glucose, and then, the mixture solution was sonicated with moderate intensity at room temperature for 10 min to accelerate the aggregation reaction. Finally, the fluorescence spectra were recorded under excitation at 310 nm.
furnace (TOL 1200, Nanjing NanDa Instrument Plant).34 GSs were produced by heating the dried graphene oxide (GO, synthesized by the modified Hummers method35) at 300 °C for 2 h with a continuous flow of nitrogen at a heating rate of 5 °C/min in a tube furnace. 1.0 g of GSs was put onto the surface of 1.25 g of B2O3, which was then placed in the center of a corundum tube with a continuous flow of argon to guarantee an inert atmosphere in the tube furnace. The center temperature of the tube furnace was heated to 1100 °C at an increasing rate of 5 °C/min. After maintaining 1100 °C for 4 h, the sample was cooled to room temperature slowly under an argon atmosphere. The obtained product was then refluxed in 3 M NaOH aqueous solution for 2 h to remove any of the unreacted boron oxide. After filtration and water washing to neutral, the product was dried in a vacuum at 60 °C. Synthesis of Boron-Doped Graphene Quantum Dots. Boron-doped graphene quantum dots (BGQDs) were prepared from oxidized BG (BGO) by a hydrothermal approach similar to the B-free GQDs.8 BG was first refluxed in 40% HNO3 for 24 h; after filtration and water washing to neutral, the product (BGO) was dried in a vacuum at 60 °C. Then, the BGO was heated to 300 °C with a heating rate of 5 °C/min and then maintained at 300 °C for 2 h in a tube furnace under an argon atmosphere. 0.05 g of the product was oxidized with concentrated H2SO4 and HNO3 (volume ratio 1:3) for 17 h under mild ultrasonication without any pausing. The solution was diluted with 250 mL of ultrapure water and then filtered
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RESULTS AND DISCUSSION Characterization of the BGQDs. The BGQDs were synthesized from BG by the hydrothermal approach described in the Experimental Section. The as-prepared BGQDs solution remains homogeneous even after 3 months at room temperature without any perceptible changes (e.g., aggregation or color change), which could be further characterized by the
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Figure 2. XPS spectra. (a) The wide scan spectra of GO, BG, and the as-produced BGQDs. (b) The magnification of (a) from 100 to 250 eV. (c) The high-resolution C 1s XPS spectrum of BG. (d) The high-resolution C 1s XPS spectrum of BGQDs. (e) The high-resolution B 1s XPS spectrum of BG. (f) The high resolution B 1s XPS spectrum of BGQDs.
almost unchanged absorption and PL spectra (Figure S1 in the Supporting Information). The transmission electron microscopy (TEM) image shows fairly uniform BGQDs with diameters of ca. 2−4 nm (Figure 1a), which are much smaller than those of the B-free counterparts synthesized hydrothermally (∼10 nm) but well-consistent with those of NGQDs prepared solvothermally.8,15 The corresponding atomic force microscopy (AFM) image (Figure 1b) reveals a typical topographic height of 0.5−0.8 nm (inset of Figure 1b), suggesting that most of the BGQDs consist of a single graphene layer. To explore the optical properties of the BGQDs, UV−vis absorption and excitation-dependent PL behavior of BGQDs were investigated. The BGQDs show a broad UV−vis absorption band with a weak shoulder at 295 nm (Figure 1c), which is blue-shifted by ca. 15 nm with respect to that of previous B-free GQDs.33 The photograph shows that BGQDs solution is pale-yellow, transparent, and clear under daylight and exhibits blue PL under irradiation by a 365 nm UV light (inset of Figure 1c). It has been reported that isolated sp2hybridized clusters with a size of ca. 3 nm within the carbon− oxygen matrix could yield band gaps consistent with blue emission due to the localization of electron−hole pairs,36
suggesting that both the size and surface effects make an important contribution to the observed blue PL emission from BGQDs. Upon excitation with a 310 nm beam, the PL spectrum shows a strong peak at 440 nm with a Stokes shift of 130 nm. Like other luminescent carbon nanoparticles, the BGQDs also exhibit an excitation-dependent PL behavior (Figure 1c). As the excitation wavelength increases, the emission peak shifts to longer wavelength and its intensity decreases rapidly. It is suspected that the giant red-edge effect is responsible for the strong dependence of the PL peak position upon the wavelength of the excitation source.37 The chemically synthesized BGQDs are readily water-dispersible due to the presence of hydroxyl and carboxylic groups at the surface and edges, which is confirmed by Fourier transform infrared (FTIR) measurement. The peaks at about 1636 and 1386 cm−1 indicate the existence of COO− (Figure 1d), while the peak at 3442 cm−1 corresponds to the OH stretching mode. Compared with B-free GQDs, the magnified FT-IR spectrum of BGQDs (inset of Figure 1d) displays the additional absorption peak from asymmetric B−O stretching at 1410 cm−1,38 confirming the successful preparation of the BGQDs. X-ray photoelectron spectroscopy (XPS) characterization was then carried out to explore the content and configuration 4426
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Figure 3. (a) XRD patterns and (b) Raman spectra of the pristine graphene, BG, B-free GQDs, and BGQDs.
compound. The above results indicate that B atoms are mainly in the three different bonding characters inserted into the graphene network of GQDs (Scheme 1). Figure 3a shows the typical X-ray diffraction (XRD) profiles for BG, B-free GQDs, and BGQDs. The XRD peak for BG shifts to a higher degree (ca. 25.8°) compared with the pristine graphene obtained from thermally annealing GO under the same conditions (ca. 24.3°), suggesting the smaller interlayer spacing as a result of the more thorough reduction in the presence of boron oxide. As in the case of B-free GQDs, the BGQDs show a broader diffraction peak at ca. 25°, indicating that the oxidation reaction during the BGQDs preparation process has introduced more active sites on the surfaces of BGQDs, as confirmed by XPS (Figure 2) and FT-IR spectroscopy investigations (Figure 1d). The XRD peak for as-prepared BGQDs shifts to a lower degree compared with the BG sample (ca. 25.8°), indicating that the BGQDs have a larger interlayer spacing than the BG does. The increased interlayer spacing in BGQDs (ca. 0.356 nm) could be attributed to the weak π−π stacking of graphenes with more O-containing functional groups surrounding the edges of the graphene layers in BGQDs. It is also noteworthy that the prepared BGQDs do not show any diffractions in the 2θ ≈ 10° region characteristic of GO, evidently indicating that the BGQDs are different from GO, though both contain oxygen-enriched functional groups. Raman spectra offer clear evidence of B-doping in the graphene lattice. Figure 3b shows the Raman spectra of the BG, B-free GQDs, and BGQDs. For comparison, the spectrum of pristine graphene obtained from thermally annealing GO under the same conditions is also displayed. The spectra exhibit two remarkable peaks at around 1365 and 1596 cm−1 corresponding to the well-defined D band and G band, respectively. As is known, the G band related to the E2g vibration mode of sp2 carbon domains can be used to explain the degree of graphitization, while the D band is associated with structural defects and partially disordered structures of the sp2 domains.41 The ID/IG ratio in the Raman spectra is generally used to evaluate the disorder in the graphene materials. Although there is no significant change in the position of D and G bands, the intensity ratio of ID/IG increases from pristine graphene (0.79) to BG (0.87), clearly suggesting a broken hexagonal symmetry of the graphene, most presumably by the heterogeneous Bdopants.42 Interestingly, it is observed that both the BGQDs and B-free GQDs have an ID/IG ratio of ca. 0.7, which is much lower than that of the BG (0.87) (Figure 3b), indicating the efficient structural restoration of graphitic framework during the hydrothermal process to yield high-quality GQDs.14 In comparison with B-free GQDs, however, the BGQDs exhibit
of doped boron in the BG and BGQDs. Compared with graphene oxide (GO) involving only the core levels of C 1s and O 1s (Figure 2a), the full range XPS analysis of the resultant BGQDs sample clearly shows the presence of boron (B), carbon (C), and oxygen (O) with atomic percentages of 3.45%, 70.16%, and 26.39%, and the corresponding B 1s, C 1s, and O 1s peaks center at 190, 284, and 532 eV, respectively (Figure 2a,b), while for the BG sample, the corresponding atomic percentage of B 1s, C 1s, and O 1s is 4.25%, 80.4%, and 15.35%, respectively. The O/C atomic ratio for the BGQDs is ca. 37.6%, higher than that of the BG (ca. 19.1%), suggesting that oxidation reaction takes place during the HNO3 refluxing for the BGQDs preparation (see the Experimental Section). In addition, the B 1s peak appears at a higher binding energy (B 1s peak: 190 eV as compared to 187 eV for pure boron), which suggests that boron atoms coming from B2O3 vapor have been partly bonded to the carbon atoms in the sp2 carbon network.39,40 On the basis of the Shirley algorithm, the C 1s peak of BG can be fitted into three components (Figure 2c). The most intense peak located at 283.9 eV is assigned to sp2 hybrid carbon atoms, and small signals at higher binding energies indicate some C−O (285.5 eV) and CO (286.5 eV) species remain in the sample after thermal annealing.5 It should be noticed that the signal at 281.8 eV, corresponding to carbon atoms neighboring with boron atoms, is not observed due to the relatively low boron content.34 In comparison with BG, Figure 2d clearly reveals the wider C 1s peak of BGQDs to be associated with the more oxygen-containing groups. A highresolution spectrum of C 1s confirmed the presence of CC (284.4 eV), C−O (285.2 eV), CO (286.5 eV), and O−C O (288 eV) bonds, indicating the as-prepared BGQDs are rich in hydroxyl, carbonyl, and carboxylic acid groups on the surfaces,5,14 which is consistent with the corresponding FT-IR spectra (Figure 1d). In the high-resolution B 1s spectrum of BG (Figure 2e), the peak at 189.0 eV is attributed to the BC3 structures, while the minor peak centered at 188.0 eV may be assigned to the B4C species, which are not shown in the scheme due to its extremely low content in the sample. The most intense peak at 189.2 eV corresponds to the structure of boron atoms bonding to carbon and oxygen atoms (BC2O), suggesting a large fraction of boron exists in the form of epoxy. The signal at 191.9 eV reveals that boron atoms are surrounded by carbon and oxygen atoms (BCO2), indicating the presence of boronic acid group in this sample.34 The highresolution B 1s spectrum of BGQDs is similar to that of BG except for the increased percentage of BCO2 species (Figure 2f), which is favorable for the subsequent glucose sensing application as they act as anchoring sites for polyhydroxy4427
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Figure 4. (a) Characteristic PL response of the BGQDs with different concentrations of glucose (from 1 to 13:0, 0.1, 0.5, 1, 2, 4, 6, 8, 10, 20, 50, 100, 200 mM). The inset picture shows the PL increasing in the BGQDs solution illuminated with an UV lamp, and the homologous concentrations of glucose were 0, 2, 8, 20, and 50 mM (from left to right). (b) Plot of the PL intensity vs glucose concentration in pH 7.4 phosphate buffer. (c) PL spectra of the BGQDs in the presence of 10 mM of different saccharides. (d) PL response of the BGQDs to different saccharides (10 mM). The inset picture shows the saccharides treated BGQDs solution illuminated with an UV lamp.
a broader D band, suggesting that the intercalation of B atoms into the conjugated carbon backbone has led to somewhat disordered structures.14,41 Selective Sensing of Glucose by BGQDs. Since the asprepared BGQDs involve boronic acid structure (BCO2 species) through the B-doping, which has been proven by the XPS analysis, it is expected that the BGQDs could tightly but reversibly recognize diols, thus giving rapid and sensitive PL response to glucose. To explore this attractive possibility, we investigated the PL evolution of BGQDs in the absence and presence of different amounts of glucose. Since the pKa of BGQDs is about 3.73 (Figure S2 in the Supporting Information), BGQDs with negative charge when pH > 3.73 can reversibly react with diols of glucose, and the reaction extent could be enhanced with increasing pH in alkaline aqueous solutions (Figure S3 in the Supporting Information). When glucose is added to the BGQDs solution in pH 7.4 phosphate buffer, the PL intensity of the BGQDs linearly increases with the glucose concentration in the range of 0.1−10 mM with a detection limit of 0.03 mM (Figure 4a,b), which is comparable to or better than that of previous boronic acidbased glucose sensors.23,26,30,43−45 At a glucose concentration of 10 mM, the BGQDs become very emissive, with an intensity ∼6-fold higher than that in the absence of glucose. As expected, the sensor demonstrates a relatively wider linear range (0.05− 10 mM) and a lower detection limit (about 0.01 mM) at pH 10 phosphate buffer (Figure S4 in the Supporting Information) compared with that in pH 7.4 phosphate buffer. Considering the potential application of the proposed method in real samples (such as whole blood), we assess the sensor response in neutral pH due to its physiological relevance. In comparison with the significant changes caused by glucose, the emission spectrum of BGQDs is only slightly intensified even when a
large amount (10 mM) of fructose is added, and similar results are observed in the cases of galactose and mannose (Figure 4c,d). It should be noted that the affinity constants (Ka) for binding of the other three monosaccharides (fructose, galactose, and mannose) to the boronic acid group are all larger than that of glucose.46 This suggests that the binding affinity strength is not the decisive parameter in the selective PL recognition of BGQDs to glucose. Working Mechanism of the Unusual PL Response of BGQDs to Glucose. On the basis of the above experimental data and enlightened by previous studies of the chemical reactions of tetraphenylethene (TPE)-cored diboronic acid with glucose,45 we propose a working mechanism for the specific PL response of BGQDs to glucose as shown in Scheme 1. At a concentration of glucose higher than 0.1 mM, thanks to the accessibility of the cis-5,6-diol units of glucose by the boronate group of the negatively charged BGQDs, BGQDs− glucose aggregates may form, where two BGQDs are fastened by a glucose linker. The intramolecular rotation of the luminescence center in the aggregates involves simultaneous movements of the glucose linker and another luminescence center in the neighboring unit. The high energy barrier to such motions stiffens the aggregate structure, blocks the nonradiative relaxation channels, and populates the radiative decay, thus making the luminescence center highly emissive.45,47−50 The possibility of aggregate formation increases with increasing glucose concentration, with the PL intensity reaching its maximum at glucose of about 50 mM. Compared with glucose, fructose has no additional cis conformational diol unit to further bind with BGQDs to form aggregates though it is easier to covalently combine with BGQDs than glucose.24 This is why no obvious change in PL is observed at high concentration of fructose. Similar to the case for fructose, galactose and mannose 4428
dx.doi.org/10.1021/ac500289c | Anal. Chem. 2014, 86, 4423−4430
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Figure 5. (a) LS spectra of the BGQDs with different concentrations of glucose. The concentrations of glucose were 0, 0.1, 0.5, 1, 2, 4, 6, 8, 10, 20, 50, 100, and 200 mM (from 1 to 13). (b) Plot of LS intensity vs glucose concentration in pH 7.4 phosphate buffer. (c) TEM image of the aggregation of BGQDs with 10 mM glucose. (d) AFM image of the aggregation of BGQDs with 10 mM glucose, size: 3 × 3 μm. The inset is the height distribution of the BGQDs aggregates.
have no more cis-diol units to enhance the PL of BGQDs by cross-linking aggregation. Clearly, the existence of a pair of cisdiol units in the 1,2- and 5,6-positions in glucose is the key structural feature that confers a glucose-specific response on BGQDs. In other words, it is the aggregates of BGQDs with glucose that enhances the emission of the luminescence center. Light scattering (LS) spectra coupled TEM and AFM characterization give reliable evidence of the unusual aggregation-induced PL increasing mechanism. The LS signals of aggregated BGQDs were measured by scanning synchronously both the excitation and emission monochromators of a common spectrofluorometer. As shown in Figure 5a, the LS intensity of the solution containing BGQDs is very weak. Notably, the LS intensities at 200−500 nm for BGQDs treated with various amounts of glucose are enhanced, and the LS spectra maintain almost the same pattern as the concentration of glucose increases. It is found that the enhanced LS signals linearly increase with the increasing glucose concentration over the range of 0.1 to 10 mM with a limit of detection of around 0.06 mM (3σ) (Figure 5b). To further confirm the aggregationinduced PL increasing mechanism, we have also monitored the LS response to various amounts of glucose in pH 10 phosphate buffer. As expected, the LS intensity was enhanced with increasing glucose concentrations, demonstrating a better performance (linear range over 0.05−10 mM with a detection limit of 0.03 mM, Figure S5 in the Supporting Information) compared with that in pH 7.4 phosphate buffer, consistent with the PL results in different pH buffer solutions. TEM and AFM images gave more direct information on the aggregation of the
BGQDs. Upon interaction with 10 mM glucose, the aggregation of BGQDs was monitored, as indicated by the formation of wirelike chains (Figure 5c,d). The topographic heights of the cross-linked BGQDs are mostly between 1.0 and 1.6 nm, suggesting that most of the BGQDs aggregates are of bilayered structure. These observations support our suggestion that the PL increase should be ascribed to the formation of BGQDs aggregates as a result of the intense and exclusive combination of boronate group on the BGQDs surfaces with glucose, offering strong support to the proposed working mechanism shown in Scheme 1.
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CONCLUSION In summary, we report on a novel hydrothermal approach for the cutting of BG into BGQDs; the boronic acid groups on the BGQDs surfaces could provide important application in glucose selective sensing. It works on the basis of a conceptually new mechanism: the PL of BGQDs is enhanced by the restriction of the intramolecular rotations activated by the peculiar BGQDs−glucose interactions. In contrast to the conventional “aggregation-induced quenching” mode, which is not preferred in practice because there are a variety of ligands or solvents that may interfere with quenching, leading to “false positive”, the reaction of the two cis-diol units in glucose with the two boronic acid groups on the BGQDs surfaces creates structurally rigid BGQDs−glucose aggregates, resulting in a great boost in the PL intensity. The present sensing process excludes any saccharide with only one cis-diol unit, as manifested by the high specificity of BGQDs for glucose over 4429
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its close isomeric cousins fructose, galactose, and mannose. It is reasonable to believe that the doping of boron can introduce the GQDs to a new kind of surface state, which offers great scientific insights to the PL enhancement mechanism after treatment with glucose.
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ASSOCIATED CONTENT
S Supporting Information *
The stability of BGQDs (Figure S1); the isoelectric point of BGQDs (Figure S2); the effects of pH values on the PL response to the glucose (Figure S3); the PL as well as the LS response of the BGQDs to different concentrations of glucose in pH 10 phosphate buffer (Figures S4 and S5). This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Author Contributions §
L.Z. and Z.-Y.Z. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (21163014, 21105044, and 21265017) and the Program for New Century Excellent Talents in University (NCET-11-1002, NCET-13-0848).
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