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One-step ultrasonic synthesis of graphene quantum dots with high quantum yield and its application in sensing of alkaline phosphatase
Received 00th January 2012, Accepted 00th January 2012
Yanhong Zhu, a Guangfeng Wang,a, b* Hong Jiang, a Ling Chen, a and Xiaojun
DOI: 10.1039/x0xx00000x
Zhanga,b*
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With only graphene oxide and KMnO4, the luminescent graphene quantum dots (GQDs) in high quantum yield were prepared by one-step synthesis using ultrasonication, and applied in the label-free, simple and fast fluorescence assay for alkaline phosphatase (ALP). Recently, graphene has attracted intensive interest due to its extraordinary electronic, optical, thermal and mechanical properties which benefit from its unique two-dimensional (2D) crystalline structure.1 Although it exhibits a broadband absorption ranging from 300 to 2500 nm being beneficial for ultrafast broadband photodetectors, optical luminescence is difficult to be observed in pristine graphene for the lack of a bandgap.1a, 2 One method to generate an electronic band gap in graphene is to convert graphene oxide (GO) materials into zero-dimensional graphene quantum dots (GQDs).3 GQDs have sparked significant excitement as a promising new class of fluorophores because they not only keep the excellent performance of graphene, such as high surface area, high carrier transport mobility, superior thermal/chemical stability, remarkable surface grafting property and excellent water solubility for the carboxylic acid moieties at the edge of GQDs etc.;4 but also possess high photoluminescence and slow hot-carrier relaxation, making their properties distinct from those of conventional graphene sheets due to the quantum confinement and edge effects.5 In view of the outstanding properties, GQDs have great potential applications in bio-imaging, photovoltaics, light-emitting diodes, and sensors.6 Thus, researchers have made considerable efforts to develop various chemical/physical methods for the controllable synthesis of photoluminescent GQDs. To date, a variety of methods including high-resolution electronbeam, hydrothermal, electrochemical and chemical methods have been applied to the preparation of GQDs.5, 7 However, problems such as time-consuming, low quantum yield, harsh synthetic process, and the requirement for special equipment, as well as the use of organic solvents or strong acid still existed in the reported methods.8 In 2010, Wu et al. reported a hydrothermal cleaving routes to prepare GQDs involving separate cleaving and reduction processes, which is time consuming (up to three days) and the quantum yield (QY) was as low as 8%.7d In 2011, Yang et al. prepared strongly fluorescent GQDs by one-step solvothermal method with QY high to
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11.4%. However, this approach may be limited by the use of organic solvent.9 Later, Zhang et al. proposed a water-soluble GQDs from graphite flakes using a potassium-intercalation method which not only were just in an about 9.9 wt% of the product yield but also with complex process.10 Subsequently, Peng et al. proposed oxidative methods in which carbon fibers were used to control the size of GQDs that still required a relative longer period of time (24 h).11 Very recently, Lee et al. have prepared GQDs from graphite with a shorter reaction time. Nevertheless, the use of H2SO4 and relatively poor quantum yield still limit its application.12 Intuitively it is still worth looking forward to a simple, mild and time-saving method for the synthesis of GQDs with high quantum yield. Moreover, to the best of our knowledge, most of the reported application about GQDs is based on the GQDs doped or modified nanocomposites, the application of unmodified GQDs in analytical chemistry is still in its infancy.13
Scheme 1 Schematic illustration of the synthesis procedure of GQDs (a) and ALP detection (b) based on FL quenching of GQDs through energy transfer between Cu2+ and GQDs (a).
Alkaline phosphatase (ALP), one of the most commonly used hydrolase enzyme found in various sources of mammals, has been widely used as an important biomarker for clinical diagnostics.14 The detection of ALP level is connected to several diseases, such as bone disease, ovarian and breast cancer, heart failure, liver dysfunction and leukemia.15 Due to its high sensitivity, simplicity and rapid implementation, the fluorescent method shows great advantages in the assay of ALP activity.16 Since Yan Liu and Kirk S. Schanze developed a fluorescence method for assaying ALP based on reversible binding of Cu2+ to pyrophosphate (PPi) and the fact that ALP converted PPi to phosphate (Pi), several similar fluorescence
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1.0 0.8 0.6 0.4 0.2 0.0 0
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Fig. 1 (A) HRTEM image of GQDs. Inset: Size distribution of GQDs (left) and representative images of individual GQDs (right). (B) AFM image of GQDs.
The synthesis procedure of GQDs (ESI†) is illustrated in Scheme 1(a). The GQDs were obtained from GO by ultrasonication-redox with the only employment of KMnO4 in 4h. As revealed by highresolution transmission electron microscopy (HR-TEM), the GQDs have an average lateral diameter of 3.0 nm with a narrow size distribution. The graphitic lattice of GQDs can be clearly resolved under HRTEM. The observed lattice spacing (2.10 Å) corresponds to the hexagonal lattice plane spacing of d1100 (Fig. 1A, inset). Evidently, the produced GQDs are uniform and of high crystallinity. Topographic morphology images of a typical tapping-mode, AFM image, shown in Figure 1B and Fig. S1 (ESI†), demonstrated that most of the GQDs consisted of few graphene layers with a height profile mainly within the narrow range of 0.7–3 nm, with an average
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Fig. 2 (A) XPS survey spectrum of GO and GQDs. XPS C1s analysis of (B) GO, (C) GQDs. (D) Raman spectra of GO and GQDs. (E) FTIR spectra of GQD and GO. (F) UV-vis absorption, FL excitation and emission spectra of the GQDs.
X-ray photoelectron spectroscopy (XPS) measurements were carried out to prove the composition of GQDs. As seen in Fig. 2A, the XPS showed a dominant graphitic C1s peak at 284.8 eV and O1s peak at ca. 532.2 eV for original GO and GQDs. The comparison of the high-resolution spectra of C1s (Fig. 2B, Fig. 2C) demonstrated the obvious change in carbon chemical environment from GO to GQDs. It is known that GQDs are merited with excellent stability and hydrophilicity due to the abundant hydroxyl and carboxylic groups on their surface and edges.13 This was evident by C1s XPS (Fig. 2C), which indicated GQDs were functionalized with hydroxyl, carbonyl, and carboxylic acid group. From Raman spectroscopy (Fig. 2D), we detected the D and G bands of which intensity ratio (ID/IG) was 0.92. It was found that this value was less than that of pristine GO (1:1), presumably indicating that a decrease in the fraction of aromatic sp2 domains and different degrees of GQDs oxidation increased the number of detect sites. We also sought evidence for oxygen-containing groups from IR analysis (Figure 2E). The presence of these groups made the GQDs soluble in water. Compared to the conventional graphene oxide (Figure 2E, green curve), the as-prepared GQDs have more carbonyl, carboxyl and hydroxyl groups at the edge, making the GQDs more suitable in analytical chemistry. The maximum emission intensity from GQDs achieved at 470 nm when it was excited at 380 nm (Figure 2F). A quantum yield of about 27.8% at pH 7 was calculated using Rhodamine B as the reference (Table S1, ESI), which was higher than the earlier reported literatures (the highest PL quantum yields reported for GQDs so far is 28%).19 This higher quantum yield could be attributed to the high crystallinity of GQDs and the presence of functional groups on GQD surface. It has been reported that COO-Cu2+-OH complex forms in the presence of excess Cu2+.18 Thus, we speculated that GQDs containing –COOH may have a high binding affinity to Cu2+. Inspired by the fact that PPi has a strong affinity to Cu2+,20 the chelation between Cu2+ and PPi could result in the disturbance of the interaction between Cu2+ and GQDs, leading to the fluorescence (FL) change of GQDs.
Thus we designed the detection of ALP using GQDs as fluorophore based on the coordination between Cu2+ and PPi. As shown in Scheme 1(b), in the presence of PPi, the FL intensity of GQDs was still intense because PPi could coordinate with Cu2+, and prevent
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(A)
height of approximately 0.479 nm and the resultant GQDs have narrow distribution and mostly single or double-layer GQDs. The effect of ultrasonication and its conditions on the formation of GQDs were studied. As Fig. S9 (ESI†) showed, ultrasonication played an important role in the process.
Intensity (a.u.)
methods for assaying ALP by this mechanism have been reported.17 However, although these methods based on this mechanism are straightforward and rapid, they still suffered from some limitations because most of their fluorescent probes are polyelectrolytes,17a organic dyes,17b and DNA templated metal nanoparticles17c which encountered each inevitable problems, such as laborious synthetic procedures, poor photostability, complexed labelling process, poor sensitivity or the requirement of expensive reagents. In addition, in some cases, the fluorescent assays for ALP cannot be carried out in purely aqueous solutions because of the low solubilities of fluorescent probes. Thus, the exploration of a simple, rapid, labelfree and economic method for the preparation of soluble fluorescent probes without toxicity and photo bleaching for sensitive assay of the ALP activity is necessary. It’s known that with the cavitation and vibration effects, ultrasonication can offer rapid and uniform beating for the reaction medium and thus the reaction is dramatically shortened with product yields and purities greatly improved.18 In this paper, we have successfully developed a highly efficient and “green” one-step ultrasonic strategy to synthesize GQDs with high quantum yield from GO in a short reaction time without the addition of any acid. Furthermore, a successful paradigm for exploring new and interesting application of the unmodified 0D GQDs was provided. The prepared GQDs was used non-toxic fluorescent probe in labelfree and economic sensor for rapid and sensitive detection of ALP activity in aqueous solution.
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Cu2+ from attaching on the surface of GQDs. After the addition of ALP, PPi was hydrolyzed by ALP to generate Pi, disabling the complexation between PPi and Cu2+, releasing the Cu2+. The liberated Cu2+ was in close proximity to the surface of GQDs through –COO-Cu2+-OH. Energy transfer between Cu2+ and GQDs resulted in the quenching of FL intensity of the system. The FL change of GQDs with the addition of ALP made it possible to detect ALP in the GQDs/PPi-Cu2+ system. 0.9
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Fig. 3 (A) FL quenching of GQDs containing PPi and Cu2+ in the presence of different concentrations of ALP. The concentrations of ALP were 0, 0.05, 0.1, 0.15, 0.2, 0.5, 1, 1.5, 2.5, 5, 6.5, 7.5, 8.7, 10, 20, 40, and 80 nM, respectively. (B) Plots of emission intensity change (F1/F0) vs the logarithm of the ALP concentration. The inset shows optical photos of solution of GQDs in the presence of 0.1, 0.5, 2.5, and 20 nM ALP from left to right. (C) Linear plots of emission intensity change (F1/F0) as a function of the logarithm of the ALP concentration. (D) Selectivity of the GQDs/PPi-Cu2+ system for ALP over other interfering substances.
As Cu2+ was bound to the COOH and OH of GQDs, the surface state of GQDs would change which was the base of the further ALP detection. Therefore, the interaction between Cu2+ and GQDs played an important role in the FL of GQDs. As shown in Fig. S2 (ESI†), GQDs showed strong FL intensity when they were free in an aqueous solution. However, in the presence of Cu2+, the FL of GQDs was quenched significantly. With the concentration of Cu2+ increased, the FL intensity decreased correspondingly (Fig. S3, ESI†). Then we studied the ALP-detected feasibility of the experimental principle via different measurements. The reaction between ALP and PPi was confirmed by UV-vis spectra in Figure S4A, ESI. The strong absorption peak at 274 nm was observed when ALP was mixed with PPi, due to the generation of Pi. The sole addition of ALP to the GQDs solution caused little impact on the absorbance at 350 nm (Figure S4B, ESI†). As expected, the addition of the mixture containing PPi and Cu2+ led to no obvious change of UV-vis spectrum of GQDs, due to the coordination between PPi and Cu2+ as reported before.20 In contrast, the absorbance at 350 nm slightly deceased when the ALP was added into the GQDs/PPi-Cu2+ system. Similar results were observed in FL emission spectrum (Figure S4C, ESI†) in which the FL intensity of GQDs at 470 nm quenched sharply when ALP was introduced into GQDs/PPi-Cu2+ mixture. It was most likely that ALP could hydrolyze PPi to release Cu2+, resulting in the combination of GQDs with Cu2+. Lifetime measurements were used to confirm the quenching mechanism of the system. From the results of Fig. S4D (ESI†) and the data in Table S3 (ESI†), the FL lifetime of GQDs decreased only by the addition of ALP. Therefore, it can be concluded that the ALP indeed hydrolyzed
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PPi to extricate Cu2+, and the released Cu2+ indeed combined with the surface of GQDs, resulting in a decrease in the FL lifetime of GQDs. Under the optimized conditions (Figure S6 and Fig. S7, ESI†), the sensitivity of the proposed GQDs/PPi-Cu2+ system using GQDs as fluorescent probe for ALP detection was then investigated. In the fluorescence spectra, the intensity at 470 nm decreased with the increasing concentration of ALP (Figure 3A). This was attributed to the release of Cu2+ from hydrolysis of PPi by ALP, and accompanying energy transfer from GQDs to Cu2+. The ratio of F1/F0 (where F0 and F1 is the peak intensity of GQDs before and after the introducing ALP) was employed in the quantitative analysis of ALP. A typical plot of the F1/F0 versus logarithm ALP concentration (0-80 nM) was shown in Figure 3B, and the inset image was the corresponding response to partial ALP concentrations under UV-light. Two satisfying linear relationships were obtained over the ranges of 0.05-2.5 nM and 5-10 nM with the correlation coefficients of 0.9845 and 0.9675 (Figure 3C). The results suggested that this probe could be used to detect ALP with a detection limit of 0.017 nM, as calculated by 3σ/m. To our knowledge, the linear range and the detection limit is significantly superior to or comparable with those obtained in previous reports (Table S4, ESI†). Furthermore, the quenching of FL intensity of GQDs was highly selective in the presence of ALP (78% quenching upon addition of 10 µM ALP), while other interferents (thrombin, BSA, GOx, lysozyme, ATP, avidin, and PDGF-BB) were not able to exert significant quenching effect (Figure 3D). These results indicated that our proposed method exhibited high selectivity toward ALP because of the strong affinity between PPi and ALP. The inhibitor of ALP was also investigated (Fig. S8, ESI†). The results demonstrated that this strategy can be used to study the inhibition effect of Pi on ALP activity, and it may be extended to screen other inhibitors of ALP In summary, we have developed a highly efficient and “green” one-step ultrasonic strategy to synthesize the GQDs with high quantum yield from GO without the addition of any acid in a short reaction time. The prepared GQDs were applied in the assay of the ALP activity which was cheap, simple, fast, sensitive, label-free and nontoxic. This novel approach is expected to promote the exploration of unmodified GQDs-based biosensors for target assays in biochemical and biomedical studies. Although some progress has been made, research on the application of the unmodified GQDs is still in an early stage and the full potential of this material has not yet been fathomed. Thus, we hope that this development would pave the way for extending applications of such fascinating 0D materials in various fields. This work is financially supported by the Projects (21073001, 21005001 and 21371007) from National Natural Science Foundation of China, Natural Science Foundation of Anhui (KJ2009B013Z) the project of Anhui Key Laboratory of Controllable Chemistry Reaction & Material Chemical Engineering (OFCC0905), and the Young Teacher Program of Anhui Normal University (2009xqnzc19).
Notes and references a
Anhui Key Laboratory of Chem-Biosensing, College of Chemistry and Materials Science, Center for Nanoscience and Nanotechnology, Anhui Normal University, Wuhu, 241000, P. R. China; b State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, 410082, PR China * Corresponding authors: E-mail: [email protected]; Fax: +86553-3869303; Tel: +86-553-3869303 † Electronic Supplementary Information (ESI) available: [experimental detail, EIS, contol experiments and optimization of the conditions]. See DOI: 10.1039/b000000x/
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(a) G. Eda, Y. Y. Lin, C. Mattevi, H. Yamaguchi, H. A. Chen, I. S. Chen, C. W. Chen, and M. Chhowalla, Adv. Mater. 2010, 22, 505; (b) L. F. Pan, Y. H. Li, S. Yang, P. F. Liu, M. Q. Yu, and H. G. Yang, Chem. Commun. 2014, DOI: 10.1039/c4cc05698a K. P. Loh, Q. Bao, G. Eda, and M. Chhowalla, Nat. Chem. 2010, 2, 1015. S. Neubeck, L. A. Ponomarenko, F. Freitag, A. J. M. Giesbers, U. Zeitler, S. V. Morozov, P. Blake, A. K. Geim, and K. S. Novoselov, Small 2010, 6, 1469. P. Reiss, M. Protière, and L. Li, Small 2009, 5, 154. W. Kwon, and S. W. Rhee, Chem. Commun. 2012, 48, 5256. (a) S. Zhuo, M. Shao, and S. T. Lee, ACS Nano 2012, 6, 1059−1064; (b) M. Dutta, S. Sarkar, T. Ghosh, and D. J. Basak, Phys. Chem. C. 2012, 116, 20127−20131; (c) I. Al-Ogaidi, H. Gou, Z. P. Aguilar, S. W. Guo, A. K. Melconian, A. K. A. Al-kazaz, F. Meng, and N. Q. Wu, Chem. Commun. 2014, 50, 1344. (a) L. A. Ponomarenko, F. Schedin, M. I. Katsnelson, R. Yang, E. W. Hill, K. S. Novoselov, and A. K. Geim, Science 2008, 320, 356; (b) X. Yan, X. Cui, B. Li, and L. Li, Nano Lett. 2010, 10, 1869; (c) Y. Li, Y. Hu, Y. Zhao, G. Shi, L. Deng, Y. Hou, and L. Qu, Adv. Mater. 2011, 23, 776; (d) D. G. Pan, J. C. Zhang, Z. Li, and M. H. Wu, Adv. Mater. 2010, 22, 734. Y. Dong, J. Shao, C. Chen, H. Li, R. Wang, Y. Chi, X. Lin, and G. Chen, Carbon 2012, 50, 4738–4743. S. Zhu, J. Zhang, C. Qiao, S. Tang, Y. Li, W. Yuan, B. Li, L. Tian, F. Liu, R. Hu, H. Gao, H. Wei, H. Zhang, H. Sun, and B. Yang, Chem. Commun. 2011, 47, 6858. L. Lin, and S. Zhang, Chem. Commun. 2012, 48, 10177. J. Peng, W. Gao, B. K. Gupta, Z. Liu, R. Romero-Aburto, L. Ge, L. Song, L. B. Alemany, X. Zhan, G. Gao, S. A. Vithayathil, B. A. Kaipparettu, A. A. Marti, T. Hayashi, J. J. Zhu, and P. M. Ajayan, Nano Lett. 2012, 12, 844. Y. Shin, J. Lee, J. Yang, J. Park, K. Lee, S. Kim, Y. Park, and H. Lee, Small 2014, 10, 866-870. (a) Y. Q. Dong, W. R. Tian, S. Y. Ren, R. P. Dai, Y. W. Chi, and G. N. Chen, ACS Appl. Mater. Interfaces 2014, 6, 1646−1651; (b) Y. Wang, L. Zhang, R. P. Liang, J. M. Bai, and J. D. Qiu, Anal. Chem. 2013, 85, 9148−9155; (c) S. Chen, X. Hai, X. W. Chen, and J. H. Wang, Anal. Chem. 2014, 13, 6689-6694. J. L. Millán, Purinergic Signal 2006, 2, 335−341. (a) D. Goltzman, and D. Miao, Encyclopedia Endocr. Dis. 2004, 1, 164–169; (b) R. H. Christenson, Clin. Biochem. 1997, 30, 573−593. (a) L. Jia, J. P. Xu, D. Li, S. P. Pang, Y. Fang, Z. G. Song, and J. Ji, Chem. Commun. 2010, 46, 7166. (b) Q. Chen, N. Bian, C. Cao, X. L. Qiu, A. D. Qi, and B. H. Han, Chem. Commun. 2010, 46, 4067. (c) T. I. Kim, H. Kim, Y. Choi, and Y. A. Kim, Chem. Commun. 2011, 47, 9825. (a) Y. Liu, and K. S. Schanze, Anal. Chem. 2008, 80, 8605-8612; (b) X. Su, C. Zhang, X. J. Xiao, A. Q. Xu, Z. D. Xu, and M. P. Zhao, Chem. Commun. 2013, 49, 798, (c) L. L. Zhang, J. J. Zhao, M. Duan, H. Zhang, J. H. Jiang, R. Q. Yu, Anal. Chem. 2013, 85, 3797. S. Doktycz and K. Suslick, Science 1990, 247, 1067-1069. J. Zong, S. L. Yang, A. Trinchi, S. Hardin, I. Cole, Y. H. Zhu, C. Z. Li, T. Muster, and G. Wei, Biosens. Bioelectron. 2014, 51, 330-335. L. B. Rogers, and C. A. Reynolds, J. Am. Chem. Soc. 1949, 71, 20812085.
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This article can be cited before page numbers have been issued, to do this please use: X. zhang, Y. Zhu, L. Chen, G. Wang and H. jiang, Chem. Commun., 2014, DOI: 10.1039/C4CC07449A.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
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One-step ultrasonic synthesis of graphene quantum dots with high quantum yield and its application in sensing of alkaline phosphatase
Received 00th January 2012, Accepted 00th January 2012
Yanhong Zhu, a Guangfeng Wang,a, b* Hong Jiang, a Ling Chen, a and Xiaojun
DOI: 10.1039/x0xx00000x
Zhanga,b*
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With only graphene oxide and KMnO4, the luminescent graphene quantum dots (GQDs) in high quantum yield were prepared by one-step synthesis using ultrasonication, and applied in the label-free, simple and fast fluorescence assay for alkaline phosphatase (ALP). Recently, graphene has attracted intensive interest due to its extraordinary electronic, optical, thermal and mechanical properties which benefit from its unique two-dimensional (2D) crystalline structure.1 Although it exhibits a broadband absorption ranging from 300 to 2500 nm being beneficial for ultrafast broadband photodetectors, optical luminescence is difficult to be observed in pristine graphene for the lack of a bandgap.1a, 2 One method to generate an electronic band gap in graphene is to convert graphene oxide (GO) materials into zero-dimensional graphene quantum dots (GQDs).3 GQDs have sparked significant excitement as a promising new class of fluorophores because they not only keep the excellent performance of graphene, such as high surface area, high carrier transport mobility, superior thermal/chemical stability, remarkable surface grafting property and excellent water solubility for the carboxylic acid moieties at the edge of GQDs etc.;4 but also possess high photoluminescence and slow hot-carrier relaxation, making their properties distinct from those of conventional graphene sheets due to the quantum confinement and edge effects.5 In view of the outstanding properties, GQDs have great potential applications in bio-imaging, photovoltaics, light-emitting diodes, and sensors.6 Thus, researchers have made considerable efforts to develop various chemical/physical methods for the controllable synthesis of photoluminescent GQDs. To date, a variety of methods including high-resolution electronbeam, hydrothermal, electrochemical and chemical methods have been applied to the preparation of GQDs.5, 7 However, problems such as time-consuming, low quantum yield, harsh synthetic process, and the requirement for special equipment, as well as the use of organic solvents or strong acid still existed in the reported methods.8 In 2010, Wu et al. reported a hydrothermal cleaving routes to prepare GQDs involving separate cleaving and reduction processes, which is time consuming (up to three days) and the quantum yield (QY) was as low as 8%.7d In 2011, Yang et al. prepared strongly fluorescent GQDs by one-step solvothermal method with QY high to
This journal is © The Royal Society of Chemistry 2012
11.4%. However, this approach may be limited by the use of organic solvent.9 Later, Zhang et al. proposed a water-soluble GQDs from graphite flakes using a potassium-intercalation method which not only were just in an about 9.9 wt% of the product yield but also with complex process.10 Subsequently, Peng et al. proposed oxidative methods in which carbon fibers were used to control the size of GQDs that still required a relative longer period of time (24 h).11 Very recently, Lee et al. have prepared GQDs from graphite with a shorter reaction time. Nevertheless, the use of H2SO4 and relatively poor quantum yield still limit its application.12 Intuitively it is still worth looking forward to a simple, mild and time-saving method for the synthesis of GQDs with high quantum yield. Moreover, to the best of our knowledge, most of the reported application about GQDs is based on the GQDs doped or modified nanocomposites, the application of unmodified GQDs in analytical chemistry is still in its infancy.13
Scheme 1 Schematic illustration of the synthesis procedure of GQDs (a) and ALP detection (b) based on FL quenching of GQDs through energy transfer between Cu2+ and GQDs (a).
Alkaline phosphatase (ALP), one of the most commonly used hydrolase enzyme found in various sources of mammals, has been widely used as an important biomarker for clinical diagnostics.14 The detection of ALP level is connected to several diseases, such as bone disease, ovarian and breast cancer, heart failure, liver dysfunction and leukemia.15 Due to its high sensitivity, simplicity and rapid implementation, the fluorescent method shows great advantages in the assay of ALP activity.16 Since Yan Liu and Kirk S. Schanze developed a fluorescence method for assaying ALP based on reversible binding of Cu2+ to pyrophosphate (PPi) and the fact that ALP converted PPi to phosphate (Pi), several similar fluorescence
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1.0 0.8 0.6 0.4 0.2 0.0 0
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10 15 20 Radius / nm
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Fig. 1 (A) HRTEM image of GQDs. Inset: Size distribution of GQDs (left) and representative images of individual GQDs (right). (B) AFM image of GQDs.
The synthesis procedure of GQDs (ESI†) is illustrated in Scheme 1(a). The GQDs were obtained from GO by ultrasonication-redox with the only employment of KMnO4 in 4h. As revealed by highresolution transmission electron microscopy (HR-TEM), the GQDs have an average lateral diameter of 3.0 nm with a narrow size distribution. The graphitic lattice of GQDs can be clearly resolved under HRTEM. The observed lattice spacing (2.10 Å) corresponds to the hexagonal lattice plane spacing of d1100 (Fig. 1A, inset). Evidently, the produced GQDs are uniform and of high crystallinity. Topographic morphology images of a typical tapping-mode, AFM image, shown in Figure 1B and Fig. S1 (ESI†), demonstrated that most of the GQDs consisted of few graphene layers with a height profile mainly within the narrow range of 0.7–3 nm, with an average
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Fig. 2 (A) XPS survey spectrum of GO and GQDs. XPS C1s analysis of (B) GO, (C) GQDs. (D) Raman spectra of GO and GQDs. (E) FTIR spectra of GQD and GO. (F) UV-vis absorption, FL excitation and emission spectra of the GQDs.
X-ray photoelectron spectroscopy (XPS) measurements were carried out to prove the composition of GQDs. As seen in Fig. 2A, the XPS showed a dominant graphitic C1s peak at 284.8 eV and O1s peak at ca. 532.2 eV for original GO and GQDs. The comparison of the high-resolution spectra of C1s (Fig. 2B, Fig. 2C) demonstrated the obvious change in carbon chemical environment from GO to GQDs. It is known that GQDs are merited with excellent stability and hydrophilicity due to the abundant hydroxyl and carboxylic groups on their surface and edges.13 This was evident by C1s XPS (Fig. 2C), which indicated GQDs were functionalized with hydroxyl, carbonyl, and carboxylic acid group. From Raman spectroscopy (Fig. 2D), we detected the D and G bands of which intensity ratio (ID/IG) was 0.92. It was found that this value was less than that of pristine GO (1:1), presumably indicating that a decrease in the fraction of aromatic sp2 domains and different degrees of GQDs oxidation increased the number of detect sites. We also sought evidence for oxygen-containing groups from IR analysis (Figure 2E). The presence of these groups made the GQDs soluble in water. Compared to the conventional graphene oxide (Figure 2E, green curve), the as-prepared GQDs have more carbonyl, carboxyl and hydroxyl groups at the edge, making the GQDs more suitable in analytical chemistry. The maximum emission intensity from GQDs achieved at 470 nm when it was excited at 380 nm (Figure 2F). A quantum yield of about 27.8% at pH 7 was calculated using Rhodamine B as the reference (Table S1, ESI), which was higher than the earlier reported literatures (the highest PL quantum yields reported for GQDs so far is 28%).19 This higher quantum yield could be attributed to the high crystallinity of GQDs and the presence of functional groups on GQD surface. It has been reported that COO-Cu2+-OH complex forms in the presence of excess Cu2+.18 Thus, we speculated that GQDs containing –COOH may have a high binding affinity to Cu2+. Inspired by the fact that PPi has a strong affinity to Cu2+,20 the chelation between Cu2+ and PPi could result in the disturbance of the interaction between Cu2+ and GQDs, leading to the fluorescence (FL) change of GQDs.
Thus we designed the detection of ALP using GQDs as fluorophore based on the coordination between Cu2+ and PPi. As shown in Scheme 1(b), in the presence of PPi, the FL intensity of GQDs was still intense because PPi could coordinate with Cu2+, and prevent
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height of approximately 0.479 nm and the resultant GQDs have narrow distribution and mostly single or double-layer GQDs. The effect of ultrasonication and its conditions on the formation of GQDs were studied. As Fig. S9 (ESI†) showed, ultrasonication played an important role in the process.
Intensity (a.u.)
methods for assaying ALP by this mechanism have been reported.17 However, although these methods based on this mechanism are straightforward and rapid, they still suffered from some limitations because most of their fluorescent probes are polyelectrolytes,17a organic dyes,17b and DNA templated metal nanoparticles17c which encountered each inevitable problems, such as laborious synthetic procedures, poor photostability, complexed labelling process, poor sensitivity or the requirement of expensive reagents. In addition, in some cases, the fluorescent assays for ALP cannot be carried out in purely aqueous solutions because of the low solubilities of fluorescent probes. Thus, the exploration of a simple, rapid, labelfree and economic method for the preparation of soluble fluorescent probes without toxicity and photo bleaching for sensitive assay of the ALP activity is necessary. It’s known that with the cavitation and vibration effects, ultrasonication can offer rapid and uniform beating for the reaction medium and thus the reaction is dramatically shortened with product yields and purities greatly improved.18 In this paper, we have successfully developed a highly efficient and “green” one-step ultrasonic strategy to synthesize GQDs with high quantum yield from GO in a short reaction time without the addition of any acid. Furthermore, a successful paradigm for exploring new and interesting application of the unmodified 0D GQDs was provided. The prepared GQDs was used non-toxic fluorescent probe in labelfree and economic sensor for rapid and sensitive detection of ALP activity in aqueous solution.
FL intensity (a.u.)
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Cu2+ from attaching on the surface of GQDs. After the addition of ALP, PPi was hydrolyzed by ALP to generate Pi, disabling the complexation between PPi and Cu2+, releasing the Cu2+. The liberated Cu2+ was in close proximity to the surface of GQDs through –COO-Cu2+-OH. Energy transfer between Cu2+ and GQDs resulted in the quenching of FL intensity of the system. The FL change of GQDs with the addition of ALP made it possible to detect ALP in the GQDs/PPi-Cu2+ system. 0.9
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Fig. 3 (A) FL quenching of GQDs containing PPi and Cu2+ in the presence of different concentrations of ALP. The concentrations of ALP were 0, 0.05, 0.1, 0.15, 0.2, 0.5, 1, 1.5, 2.5, 5, 6.5, 7.5, 8.7, 10, 20, 40, and 80 nM, respectively. (B) Plots of emission intensity change (F1/F0) vs the logarithm of the ALP concentration. The inset shows optical photos of solution of GQDs in the presence of 0.1, 0.5, 2.5, and 20 nM ALP from left to right. (C) Linear plots of emission intensity change (F1/F0) as a function of the logarithm of the ALP concentration. (D) Selectivity of the GQDs/PPi-Cu2+ system for ALP over other interfering substances.
As Cu2+ was bound to the COOH and OH of GQDs, the surface state of GQDs would change which was the base of the further ALP detection. Therefore, the interaction between Cu2+ and GQDs played an important role in the FL of GQDs. As shown in Fig. S2 (ESI†), GQDs showed strong FL intensity when they were free in an aqueous solution. However, in the presence of Cu2+, the FL of GQDs was quenched significantly. With the concentration of Cu2+ increased, the FL intensity decreased correspondingly (Fig. S3, ESI†). Then we studied the ALP-detected feasibility of the experimental principle via different measurements. The reaction between ALP and PPi was confirmed by UV-vis spectra in Figure S4A, ESI. The strong absorption peak at 274 nm was observed when ALP was mixed with PPi, due to the generation of Pi. The sole addition of ALP to the GQDs solution caused little impact on the absorbance at 350 nm (Figure S4B, ESI†). As expected, the addition of the mixture containing PPi and Cu2+ led to no obvious change of UV-vis spectrum of GQDs, due to the coordination between PPi and Cu2+ as reported before.20 In contrast, the absorbance at 350 nm slightly deceased when the ALP was added into the GQDs/PPi-Cu2+ system. Similar results were observed in FL emission spectrum (Figure S4C, ESI†) in which the FL intensity of GQDs at 470 nm quenched sharply when ALP was introduced into GQDs/PPi-Cu2+ mixture. It was most likely that ALP could hydrolyze PPi to release Cu2+, resulting in the combination of GQDs with Cu2+. Lifetime measurements were used to confirm the quenching mechanism of the system. From the results of Fig. S4D (ESI†) and the data in Table S3 (ESI†), the FL lifetime of GQDs decreased only by the addition of ALP. Therefore, it can be concluded that the ALP indeed hydrolyzed
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PPi to extricate Cu2+, and the released Cu2+ indeed combined with the surface of GQDs, resulting in a decrease in the FL lifetime of GQDs. Under the optimized conditions (Figure S6 and Fig. S7, ESI†), the sensitivity of the proposed GQDs/PPi-Cu2+ system using GQDs as fluorescent probe for ALP detection was then investigated. In the fluorescence spectra, the intensity at 470 nm decreased with the increasing concentration of ALP (Figure 3A). This was attributed to the release of Cu2+ from hydrolysis of PPi by ALP, and accompanying energy transfer from GQDs to Cu2+. The ratio of F1/F0 (where F0 and F1 is the peak intensity of GQDs before and after the introducing ALP) was employed in the quantitative analysis of ALP. A typical plot of the F1/F0 versus logarithm ALP concentration (0-80 nM) was shown in Figure 3B, and the inset image was the corresponding response to partial ALP concentrations under UV-light. Two satisfying linear relationships were obtained over the ranges of 0.05-2.5 nM and 5-10 nM with the correlation coefficients of 0.9845 and 0.9675 (Figure 3C). The results suggested that this probe could be used to detect ALP with a detection limit of 0.017 nM, as calculated by 3σ/m. To our knowledge, the linear range and the detection limit is significantly superior to or comparable with those obtained in previous reports (Table S4, ESI†). Furthermore, the quenching of FL intensity of GQDs was highly selective in the presence of ALP (78% quenching upon addition of 10 µM ALP), while other interferents (thrombin, BSA, GOx, lysozyme, ATP, avidin, and PDGF-BB) were not able to exert significant quenching effect (Figure 3D). These results indicated that our proposed method exhibited high selectivity toward ALP because of the strong affinity between PPi and ALP. The inhibitor of ALP was also investigated (Fig. S8, ESI†). The results demonstrated that this strategy can be used to study the inhibition effect of Pi on ALP activity, and it may be extended to screen other inhibitors of ALP In summary, we have developed a highly efficient and “green” one-step ultrasonic strategy to synthesize the GQDs with high quantum yield from GO without the addition of any acid in a short reaction time. The prepared GQDs were applied in the assay of the ALP activity which was cheap, simple, fast, sensitive, label-free and nontoxic. This novel approach is expected to promote the exploration of unmodified GQDs-based biosensors for target assays in biochemical and biomedical studies. Although some progress has been made, research on the application of the unmodified GQDs is still in an early stage and the full potential of this material has not yet been fathomed. Thus, we hope that this development would pave the way for extending applications of such fascinating 0D materials in various fields. This work is financially supported by the Projects (21073001, 21005001 and 21371007) from National Natural Science Foundation of China, Natural Science Foundation of Anhui (KJ2009B013Z) the project of Anhui Key Laboratory of Controllable Chemistry Reaction & Material Chemical Engineering (OFCC0905), and the Young Teacher Program of Anhui Normal University (2009xqnzc19).
Notes and references a
Anhui Key Laboratory of Chem-Biosensing, College of Chemistry and Materials Science, Center for Nanoscience and Nanotechnology, Anhui Normal University, Wuhu, 241000, P. R. China; b State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, 410082, PR China * Corresponding authors: E-mail: [email protected]; Fax: +86553-3869303; Tel: +86-553-3869303 † Electronic Supplementary Information (ESI) available: [experimental detail, EIS, contol experiments and optimization of the conditions]. See DOI: 10.1039/b000000x/
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