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Green Synthesis Peptide-templated Gold Nanoclusters as A Novel Fluorescence Probe for Detecting Protein Kinase Activity
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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x A green method was employed for synthesizing peptidetemplated nanoclusters without strong reducing agents. Using the synthetic peptide-gold nanoclusters as fluorescence probe, a novel assay for detecting protein kinase is developed based on phosphorylation against carboxypeptidase Y digestion. Protein kinase-catalyzed phosphorylation is a process that can transfer the γ-phosphoryl from adenosine-5′-triphosphate (ATP) to a free hydroxyl group of threonine, serine or tyrosine in a peptide or protein substrate.1 It plays an especial significance to adjust cellular biological processes in many metabolic pathways. Abnormal expression of protein kinase activity often have connections with diverse diseases including cancer, HIV,2 diabetes,3 cardiac diseases4 and Alzheimer’s disease.5 Therefore, a sensitive detection method of protein kinase not only provides the information for the inchoate diagnosis of cancers, but also plays an important role in discovering cancer-related phosphorylation and screening of new drugs. Up to now, there have been reported several methods for the detection of activity of protein kinase, including colorimetric,6 radioactive,7 electrochemical,8 fluorescent9-11 and surface plasmon resonance (SPR).12 Nevertheless, most of such assays in encoded reporters need the complex labeled procedures. Hence, it is still a challenge to develop simple, sensitive, and versatile protein kinase assays. Gold nanoclusters (AuNCs), as a novel probe for fluorescence technique, have drawn great interest recently due to the outstanding properties, such as well-defined molecular structure,13 strong photoluminescence,14 good biocompatibility15 and optical chirality. Therefore, the AuNCs have been widely used in the fields of chemistry, biology, and materials. The synthesis strategies of AuNCs in water solution by some soft landings have been reported in large volumes, such as alkylthiolates16and dendrimers cavity.17 Recently, Jiang’s group developed a simple, rapid method for highly fluorescent AuNCs synthesis with different peptide templates using NaBH4 as reducing agent and 3-mercaptopropionic acid as auxiliary ligand.18 Using this AuNCs, they established the enzymeresponsive fluorescent nanocluster beacon which was highly sensitive and selective for label-free quantification of protein post-translational modifications enzymes and their inhibitors.18 However, for most of the AuNCs synthesis methods, strong reducing agents such as NaBH4 were required. Notably, the strong reducing agents would potentially be side effects in later biologic applications.19 Therefore, there is a need but a challenge
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in the development of green AuNCs synthesis method without requiring additional reducing agents. Herein, a green and simple one-step peptide biomineralization method is employed to obtain the peptide-AuNCs. In this experiment, the peptide was used as both template and reducing agent to synthetize peptide-AuNCs in this experiment. This synthetic assay is green due to the process without additional reducing agents. Furthermore, the peptide of peptide-AuNCs retains the structure and biological activity which can be phosphorylated and be cleaved by carboxypeptidase Y (CPY). Using this peptide-AuNCs as effective optical probes, we developed a fluorescence assay for monitoring kinase activity based on CPY digestion. As shown in Scheme 1, the fluorescence quenching of peptide-AuNCs occurs due to the consecutive exocleavage of peptide by CPY without peptide phosphorylation. On the contrary, when protein kinase catalyzes phosphorylation of peptide at amino residue, the phosphate group can block the digestion of CPY, and thus it prevents the fluorescence quenching of the peptide-AuNCs. Based on the fluorescence intensity change of the peptide-AuNCs, the activity of protein kinase can be facilely monitored with high sensitivity by this fluorescence method. The peptide H 2 N-CCYLRRASLG-COOH, containing two regions is originally designed. Region 1 of peptide is CCY which can reduce Au3+ into peptide-AuNCs via the phenolic group of tyrosine (Y) under alkaline conditions without additional reducing agents and capture the clusters via SH group of cysteine (C).20 Region 2 is LRRASLG, the specific peptide substrate for cAMP-dependent protein kinase (PKA), which can be phosphorylated at serine residue (S) in the presence of PKA and ATP. So, this peptide should perform the dual roles of AuNCs synthesis and protein phosphorylation. Furthermore, the peptide prevents the peptide-AuNCs from O2-mediated fluorescence
Scheme 1 Schematic representation of fluorescence assay for monitoring kinase activity based on phosphorylation against CPY digestion by using peptide-AuNCs as signal sensing probe. The inset shows the illustration of preparation peptide-AuNCs.
[journal], [year], [vol], 00–00 | 1
ChemComm Accepted Manuscript
Published on 11 May 2015. Downloaded by Fudan University on 11/05/2015 09:45:08.
Wei Song, Ru-Ping Liang, Ying Wang, Li Zhang and Jian-Ding Qiu*
ChemComm
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quenching, acting as a compacted coating layer. By comparing with the established methods,16,17 this assay protects the peptide from being destroyed by strong reducing agents and thus keeps the particular recognition function of peptide-AuNCs in following biologic applications. As a starting point of our study, the synthesized peptideAuNCs demonstrate a broad absorption band with two weak shoulders at 241 nm and 297 nm, which match the phenoxide structure of tyrosine under basic conditions (Fig. 1a).20 When excited at 340 nm, the peptide-AuNCs show a strong fluorescence emission peak at 420 nm (Fig. 1a) and keep a strong fluorescence emission in the pH range of 5.5-8.5 (Fig. S1). To optimize the condition of the synthesis process, peptide concentration was studied. The fluorescence intensity of peptideAuNCs increases with the peptide concentration and reaches a plateau at 0.5 mM (Fig. S2). Thus, the 0.5 mM peptide was chosen. A typical transmission electron microscope (TEM) image shows that the as-prepared peptide-AuNCs are well monodispersed with an average size of around 1 nm (Fig. 1b).18 Atomic force microscopy (AFM) was used to directly observe the topographic heights of peptide-AuNCs. The AFM image shows that the topographic height of peptide-AuNCs is 1.2 nm (Fig. S3). The quantum efficiency was determined to be ~14.6% using quinine sulfate as the reference (Fig. S4) and the peptide-AuNCs can keep stable for months (Fig. S5). The in-depth chemical states of the peptide-AuNCs were further characterized by X-ray photoelectron spectroscopy (XPS). The Au 4f7/2 XPS spectra for the peptide-AuNCs show two distinct components corresponding to a major species Au0 (83.7 eV) and a minor one Au+ (85.2 eV) (Fig. 1c).21 The presence of a small number of Au+ and the peak of S 2p3/2 (162.1 eV) (Fig. 1d) indicates that the strong Au-S bonds are formed.21 Hence, these results confirm the successful preparation of the peptide-AuNCs by using a single peptide sequence without any strong reducing agents. In this assay, the peptide-AuNCs is used as both the specific recognition element and response signal for determination of PKA due to the increasing fluorescence intensity of the AuNCs. The change of fluorescence intensity is because the suppression effect of phosphorylation modification on CPY digestion by the
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peptide of AuNCs reaction with PKA and ATP. The CPY is a kind of exopeptidase of hydrolyzing polypeptide chains at the Cterminal (carboxyl-terminal) end of a peptide or protein. When the peptide-AuNCs are treated with CPY, the peptide of peptideAuNCs will be literally cleaved from C-terminal into the free amino acids.6 Thus the fluorescence of peptide-AuNCs would be quenched. However, the fluorescence of peptide-AuNCs remains unchanged after phosphorylation of peptide-AuNCs by kinase, because the phosphorylated peptide-AuNCs can effectively block CPY cleavage by the bulky phosphorylated serine sites.1 Therefore, CPY is applied to recognize the kinase catalysis event due to its activity susceptible to phosphorylated modification of amino acid. A series of tests were carried out to investigate the feasibility of fluorescence mechanism of peptide-AuNCs (Fig. S6). The fluorescence intensity distinctly decreases when the peptide-AuNCs solution is incubated with CPY. No obvious fluorescence intensity recovery is observed in the presence of either single PKA or single ATP, after the peptide-AuNCs solution is treated with CPY. Only when the peptide-AuNCs solution is incubated with PKA and ATP before treated with CPY, the fluorescence intensity distinctly recovers. These results indicate that the specific and selective response to the activity of PKA via the occurrence of phosphorylation can be realized by the fluorescence signal of peptide-AuNCs system. To verify the mechanism of the suppression effect of phosphorylation modification on CPY digestion, the in-depth chemical states of the peptide-AuNCs solution with and without protein kinase in the presence of ATP and CPY were further characterized using XPS. When the peptide-AuNCs solution is treated with CPY in the absence of PKA, the XPS spectra for the peptide-AuNCs show two obvious parts corresponding to a main kind of Au+ (~91%) and a fewer one Au0 (~9%) (Fig. 2a). It demonstrates that the AuNCs losses the protection of peptide after treated with CPY in the absence of PKA, because CPY can digest the peptide of peptide-protected AuNCs into free amino acids. Then O2 is allowed to diffuse into contacting with the AuNCs directly and Au0 is reduced to Au+.18,22 Therefore, the fluorescence of the AuNCs is quenched. Amazingly, as shown in Fig. 2b, a much more percentage of Au0 (~93%) is obtained by XPS when the peptide of peptide-AuNCs is phosphorylated by PKA before treated with CPY. That is on account of the hindrance to CPY cleavage caused by the phosphate group of serine residue, which maintaining the stability of peptide-AuNCs. These results disclose that there is a close relation between the phosphorylation and the oxidation of the AuNCs, responding to the activity of PKA based on the phosphorylation-induced suppression of CPY cleavage by the fluorescence signal of peptide-AuNCs system.
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Fig. 1 (a) UV-vis absorption spectrum and fluorescence spectra of the AuNCs at different excitation wavelengths. The inset shows the photographs of an aqueous solution of peptide-AuNCs taken under visible-light irradiation (left) and UV irradiation (right) at 365 nm. (b) TEM image of the peptide-AuNCs. The inset shows the diameter distribution of the AuNCs. XPS spectra of (c) Au 4f and (d) S 2p for peptide-AuNCs.
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Fig. 2 XPS spectra of Au 4f which is AuNCs solution incubated with ATP in the absence (a) and presence (b) of PKA followed by the incubation of CPY, respectively.
This journal is © The Royal Society of Chemistry [year]
ChemComm Accepted Manuscript
DOI: 10.1039/C5CC02280K
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DOI: 10.1039/C5CC02280K
Published on 11 May 2015. Downloaded by Fudan University on 11/05/2015 09:45:08.
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This work was supported by NSFC (21265017 and 21475056) and the Program for New Century Excellent Talents in University (NCET-11-1002 and NCET-13-0848). 55
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Fig. 3 (a) The fluorescence spectra of the peptide-AuNCs solution treated with various amount of PKA, followed by the addition of 10 U mL-1 CPY. The inset shows the fluorescence color change of the peptide-AuNCs treated with various amount of PKA. (b) Plot of the relative fluorescence intensity ((I-I0)/I0) vs PKA concentration. I and I0 are the fluorescence intensity in the presence and absence of PKA, respectively.
In summary, a green synthetic procedure of the peptide-AuNCs employed peptide in the absence of strong reducing agents, which can protect the peptide from being disrupted. By using peptideAuNCs as signal sensing probe, a simple and sensitive fluorescence assay for monitoring kinase activity based on CPY digestion has been established. In comparision with the previously reported AuNCs-based protein kinase detection method,18 some improvements have been made in this assay. (1) The peptide was used as both the template and the reducing agent in synthetic procedure of peptide-AuNCs. (2) The peptideAuNCs show great stability, the fluorescence remains unchanged when the peptide of peptide-AuNCs is phosphorylated by PKA before treated with CPY. Only the fluorescence of unphosphorylated peptide-AuNCs can be quenched by CPY digestion. (3) The “turn-on” assay for detecting protein kinase is developed based on phosphorylation against CPY digestion using the peptide-AuNCs as fluorescence probe. The detection of other enzymes will be realized with more proper design of the peptide substrate sequences. What's more, this fluorescence assay can be applied to screen the inhibitor in human serum samples, which may be might be helpful for the discovery of anticancer drugs. This journal is © The Royal Society of Chemistry [year]
Department of Chemistry, Nanchang University, Nanchang 330031, China. E-mail: [email protected]. † Electronic Supplementary Information (ESI) available: Experimental details and supplementary figures.. See DOI: 10.1039/b000000x/
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1. J. Zhou, X. Xu, W. Liu, X. Liu, Z. Nie, M. Qing, L. Nie and S. Yao, Anal. Chem., 2013, 85, 5746-5754. 2. K. Shirakawa, A. Takaori-Kondo, M. Yokoyama, T. Izumi, M. Matsui, K. Io, T. Sato, H. Sato and T. Uchiyama, Nat. Struct. Mol. Biol., 2008, 15, 1184-1191. 3. H. Cho, J. Mu, J. K. Kim, J. L. Thorvaldsen, Q. Chu, E. B. Crenshaw, K. H. Kaestner, M. S. Bartolomei, G. I. Shulman and M. J. Birnbaum, Science, 2001, 292, 1728-1731. 4. Y. Wang, S. Huang, V. P. Sah, J. Ross, J. H. Brown, J. Han and K. R. Chien, J. Biol. Chem., 1998, 273, 2161-2168. 5. D. P. Hanger, H. L. Byers, S. Wray, K.-Y. Leung, M. J. Saxton, A. Seereeram, C. H. Reynolds, M. A. Ward and B. H. Anderton, J. Biol. Chem., 2007, 282, 23645-23654. 6. J. Zhou, X. Xu, X. Liu, H. Li, Z. Nie, M. Qing, Y. Huang and S. Yao, Biosens. Bioelectron., 2014, 53, 295-300. 7. C. Lehel, S. DanielIssakani, M. Brasseur and B. Strulovici, Anal. Biochem., 1997, 244, 340-346. 8. M. Wang, G.-X. Wang, F.-N. Xiao, Y. Zhao, K. Wang and X.-H. Xia, Chem. Commun., 2013, 49, 8788-8790. 9. W. Song, Y. Wang, R.-P. Liang, L. Zhang and J.-D. Qiu, Biosens. Bioelectron., 2015, 64, 234-240. 10. Y. Wang, L. Zhang, R. P. Liang, J. M. Bai and J. D. Qiu, Anal. Chem., 2013, 85, 9148-9155. 11. J. H. Kim, S. Lee, K. Kim, H. Jeon, R. W. Park, I. S. Kim, K. Choi and I. C. Kwon, Chem. Commun., 2007, 1346. 12. T. Yoshida, M. Sato, T. Ozawa and Y. Umezawa, Anal. Chem., 1999, 72, 6-11. 13. M. Zhu, C. M. Aikens, F. J. Hollander, G. C. Schatz and R. Jin, J. Am. Chem. Soc., 2008, 130, 5883-5885. 14. J. Zheng, C. Zhou, M. Yu and J. Liu, Nanoscale, 2012, 4, 4073-4083. 15. S. Y. Lin, N. T. Chen, S. P. Sun, J. C. Chang, Y. C. Wang, C. S. Yang and L. W. Lo, J. Am. Chem. Soc., 2010, 132, 8309-8315. 16. R. J. Arnold and J. P. Reilly, J. Am. Chem. Soc., 1998, 120, 15281532. 17. F. Gröhn, B. J. Bauer, Y. A. Akpalu, C. L. Jackson and E. J. Amis, Macromolecules., 2000, 33, 6042-6050. 18. Q. Wen, Y. Gu, L. J. Tang, R. Q. Yu and J. H. Jiang, Anal. Chem., 2013, 85, 11681-11685. 19. Y. Wang, Y. Cui, Y. Zhao, R. Liu, Z. Sun, W. Li and X. Gao, Chem. Commun., 2012, 48, 871-873. 20. Y. Cui, Y. Wang, R. Liu, Z. Sun, Y. Wei, Y. Zhao and X. Gao, ACS Nano, 2011, 5, 8684-8689. 21. Y. Wang, J.-T. Chen and X.-P. Yan, Anal. Chem., 2013, 85, 25292535. 22. Y. Gu, Q. Wen, Y. Kuang, L. Tang and J. Jiang, RSC Adv., 2014, 4, 13753-13756. 23. C. Liu, L. Chang, H. Wang, J. Bai, W. Ren and Z. Li, Anal. Chem., 2014, 86, 6095-6102. 24. C. Shen, X. Xia, S. Hu, M. Yang and J. Wang, Anal. Chem., 2015, 87, 693-698.
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ChemComm Accepted Manuscript
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Under the optimum conditions (Fig. S7), as demonstrated in Fig. 3a, the fluorescence intensity of AuNCs is quenched by CPY digestion in the absence of PKA. With increasing the concentration of PKA, the fluorescence intensity of AuNCs shows continuous recovering. The continuous fluorescence color change of the AuNCs can be observed under the UV lamp (inset in Fig. 3a). Fig. 3b shows the relationship between recovering in fluorescence intensity of the peptide-AuNCs and the concentration of PKA, which exhibits a linear correlation to the concentration of PKA range from 0.01 to 40 UmL-1 (Fig. 3b inset). A detection limit of 0.004 unit mL−1 (3σ) is obtained, which is lower than the results obtained by other sensors.23,24 The potential application of our method in screening protein kinase inhibitors is also confirmed in real biological systems. Ellagic acid is performed as the model inhibitor for PKA in human serum samples. As expected, the fluorescence intensity decreases gradually with increasing concentrations of ellagic acid because of the inhibition of PKA activity. The IC50 (inhibitor concentration producing 50% inhibition) of ellagic acid is determined to be 0.10 µM (Fig. S8). The result indicates that the influence of the inhibition efficiencies of inhibitor and determination of IC50 value is negligible although serum includes many varieties of proteins.
ChemComm Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: W. Song, R. Liang, Y. Wang, L. Zhang and J. Qiu, Chem. Commun., 2015, DOI: 10.1039/C5CC02280K.
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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Green Synthesis Peptide-templated Gold Nanoclusters as A Novel Fluorescence Probe for Detecting Protein Kinase Activity
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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x A green method was employed for synthesizing peptidetemplated nanoclusters without strong reducing agents. Using the synthetic peptide-gold nanoclusters as fluorescence probe, a novel assay for detecting protein kinase is developed based on phosphorylation against carboxypeptidase Y digestion. Protein kinase-catalyzed phosphorylation is a process that can transfer the γ-phosphoryl from adenosine-5′-triphosphate (ATP) to a free hydroxyl group of threonine, serine or tyrosine in a peptide or protein substrate.1 It plays an especial significance to adjust cellular biological processes in many metabolic pathways. Abnormal expression of protein kinase activity often have connections with diverse diseases including cancer, HIV,2 diabetes,3 cardiac diseases4 and Alzheimer’s disease.5 Therefore, a sensitive detection method of protein kinase not only provides the information for the inchoate diagnosis of cancers, but also plays an important role in discovering cancer-related phosphorylation and screening of new drugs. Up to now, there have been reported several methods for the detection of activity of protein kinase, including colorimetric,6 radioactive,7 electrochemical,8 fluorescent9-11 and surface plasmon resonance (SPR).12 Nevertheless, most of such assays in encoded reporters need the complex labeled procedures. Hence, it is still a challenge to develop simple, sensitive, and versatile protein kinase assays. Gold nanoclusters (AuNCs), as a novel probe for fluorescence technique, have drawn great interest recently due to the outstanding properties, such as well-defined molecular structure,13 strong photoluminescence,14 good biocompatibility15 and optical chirality. Therefore, the AuNCs have been widely used in the fields of chemistry, biology, and materials. The synthesis strategies of AuNCs in water solution by some soft landings have been reported in large volumes, such as alkylthiolates16and dendrimers cavity.17 Recently, Jiang’s group developed a simple, rapid method for highly fluorescent AuNCs synthesis with different peptide templates using NaBH4 as reducing agent and 3-mercaptopropionic acid as auxiliary ligand.18 Using this AuNCs, they established the enzymeresponsive fluorescent nanocluster beacon which was highly sensitive and selective for label-free quantification of protein post-translational modifications enzymes and their inhibitors.18 However, for most of the AuNCs synthesis methods, strong reducing agents such as NaBH4 were required. Notably, the strong reducing agents would potentially be side effects in later biologic applications.19 Therefore, there is a need but a challenge
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This journal is © The Royal Society of Chemistry [year]
in the development of green AuNCs synthesis method without requiring additional reducing agents. Herein, a green and simple one-step peptide biomineralization method is employed to obtain the peptide-AuNCs. In this experiment, the peptide was used as both template and reducing agent to synthetize peptide-AuNCs in this experiment. This synthetic assay is green due to the process without additional reducing agents. Furthermore, the peptide of peptide-AuNCs retains the structure and biological activity which can be phosphorylated and be cleaved by carboxypeptidase Y (CPY). Using this peptide-AuNCs as effective optical probes, we developed a fluorescence assay for monitoring kinase activity based on CPY digestion. As shown in Scheme 1, the fluorescence quenching of peptide-AuNCs occurs due to the consecutive exocleavage of peptide by CPY without peptide phosphorylation. On the contrary, when protein kinase catalyzes phosphorylation of peptide at amino residue, the phosphate group can block the digestion of CPY, and thus it prevents the fluorescence quenching of the peptide-AuNCs. Based on the fluorescence intensity change of the peptide-AuNCs, the activity of protein kinase can be facilely monitored with high sensitivity by this fluorescence method. The peptide H 2 N-CCYLRRASLG-COOH, containing two regions is originally designed. Region 1 of peptide is CCY which can reduce Au3+ into peptide-AuNCs via the phenolic group of tyrosine (Y) under alkaline conditions without additional reducing agents and capture the clusters via SH group of cysteine (C).20 Region 2 is LRRASLG, the specific peptide substrate for cAMP-dependent protein kinase (PKA), which can be phosphorylated at serine residue (S) in the presence of PKA and ATP. So, this peptide should perform the dual roles of AuNCs synthesis and protein phosphorylation. Furthermore, the peptide prevents the peptide-AuNCs from O2-mediated fluorescence
Scheme 1 Schematic representation of fluorescence assay for monitoring kinase activity based on phosphorylation against CPY digestion by using peptide-AuNCs as signal sensing probe. The inset shows the illustration of preparation peptide-AuNCs.
[journal], [year], [vol], 00–00 | 1
ChemComm Accepted Manuscript
Published on 11 May 2015. Downloaded by Fudan University on 11/05/2015 09:45:08.
Wei Song, Ru-Ping Liang, Ying Wang, Li Zhang and Jian-Ding Qiu*
ChemComm
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quenching, acting as a compacted coating layer. By comparing with the established methods,16,17 this assay protects the peptide from being destroyed by strong reducing agents and thus keeps the particular recognition function of peptide-AuNCs in following biologic applications. As a starting point of our study, the synthesized peptideAuNCs demonstrate a broad absorption band with two weak shoulders at 241 nm and 297 nm, which match the phenoxide structure of tyrosine under basic conditions (Fig. 1a).20 When excited at 340 nm, the peptide-AuNCs show a strong fluorescence emission peak at 420 nm (Fig. 1a) and keep a strong fluorescence emission in the pH range of 5.5-8.5 (Fig. S1). To optimize the condition of the synthesis process, peptide concentration was studied. The fluorescence intensity of peptideAuNCs increases with the peptide concentration and reaches a plateau at 0.5 mM (Fig. S2). Thus, the 0.5 mM peptide was chosen. A typical transmission electron microscope (TEM) image shows that the as-prepared peptide-AuNCs are well monodispersed with an average size of around 1 nm (Fig. 1b).18 Atomic force microscopy (AFM) was used to directly observe the topographic heights of peptide-AuNCs. The AFM image shows that the topographic height of peptide-AuNCs is 1.2 nm (Fig. S3). The quantum efficiency was determined to be ~14.6% using quinine sulfate as the reference (Fig. S4) and the peptide-AuNCs can keep stable for months (Fig. S5). The in-depth chemical states of the peptide-AuNCs were further characterized by X-ray photoelectron spectroscopy (XPS). The Au 4f7/2 XPS spectra for the peptide-AuNCs show two distinct components corresponding to a major species Au0 (83.7 eV) and a minor one Au+ (85.2 eV) (Fig. 1c).21 The presence of a small number of Au+ and the peak of S 2p3/2 (162.1 eV) (Fig. 1d) indicates that the strong Au-S bonds are formed.21 Hence, these results confirm the successful preparation of the peptide-AuNCs by using a single peptide sequence without any strong reducing agents. In this assay, the peptide-AuNCs is used as both the specific recognition element and response signal for determination of PKA due to the increasing fluorescence intensity of the AuNCs. The change of fluorescence intensity is because the suppression effect of phosphorylation modification on CPY digestion by the
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peptide of AuNCs reaction with PKA and ATP. The CPY is a kind of exopeptidase of hydrolyzing polypeptide chains at the Cterminal (carboxyl-terminal) end of a peptide or protein. When the peptide-AuNCs are treated with CPY, the peptide of peptideAuNCs will be literally cleaved from C-terminal into the free amino acids.6 Thus the fluorescence of peptide-AuNCs would be quenched. However, the fluorescence of peptide-AuNCs remains unchanged after phosphorylation of peptide-AuNCs by kinase, because the phosphorylated peptide-AuNCs can effectively block CPY cleavage by the bulky phosphorylated serine sites.1 Therefore, CPY is applied to recognize the kinase catalysis event due to its activity susceptible to phosphorylated modification of amino acid. A series of tests were carried out to investigate the feasibility of fluorescence mechanism of peptide-AuNCs (Fig. S6). The fluorescence intensity distinctly decreases when the peptide-AuNCs solution is incubated with CPY. No obvious fluorescence intensity recovery is observed in the presence of either single PKA or single ATP, after the peptide-AuNCs solution is treated with CPY. Only when the peptide-AuNCs solution is incubated with PKA and ATP before treated with CPY, the fluorescence intensity distinctly recovers. These results indicate that the specific and selective response to the activity of PKA via the occurrence of phosphorylation can be realized by the fluorescence signal of peptide-AuNCs system. To verify the mechanism of the suppression effect of phosphorylation modification on CPY digestion, the in-depth chemical states of the peptide-AuNCs solution with and without protein kinase in the presence of ATP and CPY were further characterized using XPS. When the peptide-AuNCs solution is treated with CPY in the absence of PKA, the XPS spectra for the peptide-AuNCs show two obvious parts corresponding to a main kind of Au+ (~91%) and a fewer one Au0 (~9%) (Fig. 2a). It demonstrates that the AuNCs losses the protection of peptide after treated with CPY in the absence of PKA, because CPY can digest the peptide of peptide-protected AuNCs into free amino acids. Then O2 is allowed to diffuse into contacting with the AuNCs directly and Au0 is reduced to Au+.18,22 Therefore, the fluorescence of the AuNCs is quenched. Amazingly, as shown in Fig. 2b, a much more percentage of Au0 (~93%) is obtained by XPS when the peptide of peptide-AuNCs is phosphorylated by PKA before treated with CPY. That is on account of the hindrance to CPY cleavage caused by the phosphate group of serine residue, which maintaining the stability of peptide-AuNCs. These results disclose that there is a close relation between the phosphorylation and the oxidation of the AuNCs, responding to the activity of PKA based on the phosphorylation-induced suppression of CPY cleavage by the fluorescence signal of peptide-AuNCs system.
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Fig. 1 (a) UV-vis absorption spectrum and fluorescence spectra of the AuNCs at different excitation wavelengths. The inset shows the photographs of an aqueous solution of peptide-AuNCs taken under visible-light irradiation (left) and UV irradiation (right) at 365 nm. (b) TEM image of the peptide-AuNCs. The inset shows the diameter distribution of the AuNCs. XPS spectra of (c) Au 4f and (d) S 2p for peptide-AuNCs.
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Fig. 2 XPS spectra of Au 4f which is AuNCs solution incubated with ATP in the absence (a) and presence (b) of PKA followed by the incubation of CPY, respectively.
This journal is © The Royal Society of Chemistry [year]
ChemComm Accepted Manuscript
DOI: 10.1039/C5CC02280K
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ChemComm View Article Online
DOI: 10.1039/C5CC02280K
Published on 11 May 2015. Downloaded by Fudan University on 11/05/2015 09:45:08.
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This work was supported by NSFC (21265017 and 21475056) and the Program for New Century Excellent Talents in University (NCET-11-1002 and NCET-13-0848). 55
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Fig. 3 (a) The fluorescence spectra of the peptide-AuNCs solution treated with various amount of PKA, followed by the addition of 10 U mL-1 CPY. The inset shows the fluorescence color change of the peptide-AuNCs treated with various amount of PKA. (b) Plot of the relative fluorescence intensity ((I-I0)/I0) vs PKA concentration. I and I0 are the fluorescence intensity in the presence and absence of PKA, respectively.
In summary, a green synthetic procedure of the peptide-AuNCs employed peptide in the absence of strong reducing agents, which can protect the peptide from being disrupted. By using peptideAuNCs as signal sensing probe, a simple and sensitive fluorescence assay for monitoring kinase activity based on CPY digestion has been established. In comparision with the previously reported AuNCs-based protein kinase detection method,18 some improvements have been made in this assay. (1) The peptide was used as both the template and the reducing agent in synthetic procedure of peptide-AuNCs. (2) The peptideAuNCs show great stability, the fluorescence remains unchanged when the peptide of peptide-AuNCs is phosphorylated by PKA before treated with CPY. Only the fluorescence of unphosphorylated peptide-AuNCs can be quenched by CPY digestion. (3) The “turn-on” assay for detecting protein kinase is developed based on phosphorylation against CPY digestion using the peptide-AuNCs as fluorescence probe. The detection of other enzymes will be realized with more proper design of the peptide substrate sequences. What's more, this fluorescence assay can be applied to screen the inhibitor in human serum samples, which may be might be helpful for the discovery of anticancer drugs. This journal is © The Royal Society of Chemistry [year]
Department of Chemistry, Nanchang University, Nanchang 330031, China. E-mail: [email protected]. † Electronic Supplementary Information (ESI) available: Experimental details and supplementary figures.. See DOI: 10.1039/b000000x/
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ChemComm Accepted Manuscript
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Under the optimum conditions (Fig. S7), as demonstrated in Fig. 3a, the fluorescence intensity of AuNCs is quenched by CPY digestion in the absence of PKA. With increasing the concentration of PKA, the fluorescence intensity of AuNCs shows continuous recovering. The continuous fluorescence color change of the AuNCs can be observed under the UV lamp (inset in Fig. 3a). Fig. 3b shows the relationship between recovering in fluorescence intensity of the peptide-AuNCs and the concentration of PKA, which exhibits a linear correlation to the concentration of PKA range from 0.01 to 40 UmL-1 (Fig. 3b inset). A detection limit of 0.004 unit mL−1 (3σ) is obtained, which is lower than the results obtained by other sensors.23,24 The potential application of our method in screening protein kinase inhibitors is also confirmed in real biological systems. Ellagic acid is performed as the model inhibitor for PKA in human serum samples. As expected, the fluorescence intensity decreases gradually with increasing concentrations of ellagic acid because of the inhibition of PKA activity. The IC50 (inhibitor concentration producing 50% inhibition) of ellagic acid is determined to be 0.10 µM (Fig. S8). The result indicates that the influence of the inhibition efficiencies of inhibitor and determination of IC50 value is negligible although serum includes many varieties of proteins.
