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Phosphorylation-regulated crosslinking of gold nanoparticles: a new strategy for colorimetric detection of protein kinase activity Sujuan Sun,a Haixia Shen,b Chenghui Liu*a and Zhengping Lia Accurate and rapid detection of protein kinase activities is of great significance because protein kinases play important regulatory roles in many vital biological processes. Herein, we wish to report a facile colorimetric protein kinase assay based on the phosphorylation-tuned crosslinking of gold nanoparticles (GNPs) by using protein kinase A (PKA) as a proof-of-concept target. In this new strategy, a biotinylated peptide (biotin-LRRASLG) is used as the PKA-specific substrate. When mixed with streptavidin-functionalized GNPs (STV-GNPs), the positively charged biotin-peptide will combine with different GNPs both through the specific STV–biotin binding and through electrostatic interactions, which will lead to the crosslinking and coagulation of GNPs. In contrast, under the catalysis of PKA, the biotin-peptide will be
Received 13th May 2015, Accepted 23rd June 2015
phosphorylated at the serine residue and its net charge will be obviously altered, which may significantly weaken the electrostatic interaction between the phosphopeptide and GNPs and thus effectively prevent
DOI: 10.1039/c5an00963d
the STV-GNPs from crosslinking and settlement. Therefore, by viewing the color changes of the GNPs,
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the PKA activity can be easily detected by the naked eye.
Introduction It is well-recognized that post-translation modifications (PTM) may extensively extend the diversity of the protein functionality.1,2 Protein phosphorylation catalyzed by protein kinases (PKs) is one of the most important PTM mechanisms in the process of cellular signal transduction. As such, PKs are known to play important regulatory roles in diverse biological processes such as signal transduction, immune regulation, cell apoptosis, proliferation and differentiation and some important metabolic pathways in the human body.3–6 It has been revealed that the abnormal expression level of PKs and the unusual phosphorylation state of proteins are closely related to the development of many diseases. So PKs have been considered as an important family of molecular targets for the discovery of new targeted drugs. Therefore, the development of simple and sensitive PK assays capable of monitoring the PK activity and identifying potential inhibitors is highly desired not only for the fundamental biological research of signal transduction, but also for the clinical diagnosis and drug discovery.
a Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710119, Shaanxi Province, P. R. China. E-mail: [email protected] b Basic Experimental Teaching Centre, Shaanxi Normal University, Xi’an 710062, Shaanxi Province, P. R. China
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Up to now, a variety of PK assays have been established by using various kinds of analytical techniques such as electrochemistry,7–9 fluorescence,10–12 flow cytometry,13 radioactive methods,14,15 quartz-microbalance,16 mass spectrometry,17,18 magnetic resonance imaging19,20 and electrochemiluminescence.21 Although these methods have made great advances in PK analysis, most of them generally require labor-intensive operation or expensive/sophisticated instruments that are only available in specialized laboratories. To overcome these shortcomings, gold nanoparticle (GNP)-based colorimetric sensing strategies, which enable the facile detection of various biomolecules by the naked eye without the requirement of sophisticated instruments,22,23 have attracted much attention for visual detection of protein kinase activities.24–28 The Brust group and Stevens group have separately developed colorimetric kinase assays based on the kinase-induced crosslinking of GNPs.25,26 For example, Brust and coworkers have prepared two kinds of functionalized GNPs, one is modified with kinase-specific substrate peptides and the other is capped with avidin.25 In their design, biotin-labeled ATP is used during the kinase reaction, so that the phosphorylated GNPs with the biotin label will further bind with the avidin-functionalized GNPs to form a crosslinking network of GNPs accompanied by obvious color changes. Similarly, Stevens and coworkers also established an attractive strategy for the detection of tyrosine protein kinase activity based on the phosphorylation-induced crosslinking of two kinds of GNPs functionalized with
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kinase-specific peptides and antiphosphotyrosine antibodies,26 respectively. These crosslinking strategies by using two types of functionalized GNPs are robust and highly specific for PK analysis, but they also suffer from the complicated functionalization of GNPs since the two kinds of GNPs should be modified respectively with the substrate peptide and avidin (or antibodies). Although the protocols for capping GNPs with avidin or antibodies have been well-established, the properties of substrate peptide motifs specific to different PKs may vary significantly, some of which themselves may cause the aggregation of GNPs irrespective of the kinase-catalyzed phosphorylation and thus may interfere with the PK analysis. Recently, the Katayama group has proposed a GNP-based noncrosslinking strategy for evaluating PK activities,27,28 where the non-crosslinking aggregation of unmodified GNPs can be tuned by the phosphorylation-induced net charge change of the substrate peptide. However, it should be noted that the aggregation of unmodified GNPs is also highly susceptible to the amino acid sequences of the substrate peptide as well as the ionic strength of the reaction media during the PK-catalyzed phosphorylation reaction, which may significantly interfere with the detection of PK activity and thus greatly limit the wide application of the non-crosslinking strategy. Hence, it is still highly desirable to further improve the GNP-based colorimetric kinase assays with more simple procedures and high specificity towards the PK-induced peptide phosphorylation against the undesired salt-induced interference. Herein, we wish to report a new GNP-based crosslinking strategy for convenient and sensitive detection of PK activity and inhibition by taking protein kinase A (PKA) as a proof-ofconcept target. Compared with the GNP crosslinking-based protein kinase assays by using two kinds of functionalized GNPs, only one type of streptavidin-functionalized GNP (STV-GNPs) is needed in this work, which can be facilely prepared with mature and simple procedures. On the other hand, by using the STV-GNPs as the sensing elements, STV can effectively stabilize the GNPs and protect GNPs from undesired salt-induced aggregation, making the STV-GNPs specifically responsive to the PKA-catalyzed phosphorylation reaction. In this design, a biotinylated and positively charged peptide (biotin-LRRASLG) is used as the PKA-specific substrate, which can lead to the crosslinking and coagulation of STV-GNPs through both STV-biotin binding and electrostatic interaction. In contrast, under the catalysis of PKA, the biotin-peptide will be phosphorylated and its net charge will be obviously altered, which may prevent the STV-GNPs from crosslinking and settlement. Therefore, by viewing the color changes of the GNPs, the PKA activity can be easily detected by the naked eye.
Experimental section Materials and reagents cAMP-dependent protein kinase A (PKA, catalytic subunit) was purchased from New England Biolabs. Streptavidin (STV) was obtained from Promega Corporation. H-89 was purchased
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from EMD Bioscience and ATP was from Sangon Biotech (Shanghai, China). Biotin-LRRASLG and non-biotinylated LRRASLG specific for PKA were custom synthesized by GL Biochem (Shanghai, China). HAuCl4·4H2O was supplied by Sinopharm (Shanghai, China). All of the other reagents used in this work were of analytical grade and used as received without further purification. Preparation of GNPs and STV-GNPs GNPs with an average diameter of ∼13 nm were prepared by citrate reduction according to the literature methods.29,30 Generally, 100 mL water which contained 4.2 mL of 1% HAuCl4 was added into a three-necked flask, and the solution was heated to reflux under stirring. After refluxing for 15 min, 10 mL of trisodium citrate (38.8 mM) was rapidly added into the boiling HAuCl4 solution and then the mixture was kept boiling for another 30 min. Finally, the solution was removed from heat and cooled to room temperature under stirring. The sizes of the as-prepared GNPs are about 13 nm which exhibit a characteristic absorption peak at ∼520 nm. For the preparation of STV-GNP conjugates, the pH of the as-synthesized GNPs was firstly adjusted to ∼9.0 by using 0.1 M Na2CO3. Then STV (final concentration of 40 μg mL−1) was added into 5 mL of GNP solution and this mixture was incubated at room temperature for 1 h. Afterwards, the GNPs were centrifuged and washed three times at 4 °C to remove the unbound STV. Finally, the as-prepared STV-GNP bioconjugates were dispersed in 5 mL of sterilized water and stored at 4 °C for subsequent use. Standard assay procedures for the detection of PKA activity Typically, in a total 100 μL of PKA reaction buffer (50 mM TrisHCl, 10 mM MgCl2, pH 7.5), 1.5 μM of biotin-peptide was mixed with 12 μM of ATP and series dilutions of PKA. The mixture was incubated at 37 °C for 60 min under shaking. Subsequently, an equal volume (100 μL) of STV-GNPs was mixed with the PKA reaction system and incubated at room temperature for 2 h. Finally, the color changes of the supernatant of all samples were analyzed by visual inspection and UV-vis spectroscopy.
Results and discussion Design principle of the proposed colorimetric kinase assay Fig. 1 illustrates the work principle of the proposed colorimetric protein kinase assay based on the peptide phosphorylation-regulated crosslinking of GNPs. Protein kinase A (PKA), a protein kinase critical to memory formation, is selected as a proof-of-concept target. Correspondingly, a biotinylated kemptide (biotin-LRRASLG), which is positively charged (+2 net charge) in the Tris-HCl reaction buffer, is utilized as the PKAspecific substrate. The citrate-capped 13 nm GNPs are functionalized with a layer of STV, the dosage of which is selected to be capable of efficiently stabilizing GNPs against undesired salt-induced precipitation but won’t completely block the
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Fig. 1
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Schematic illustration of the new GNP-based colorimetric PKA assay.
negatively charged citrate-capped surface of GNPs. On this occasion, the biotin group of the substrate peptide will combine with one GNP through the STV-biotin interaction while the positively charged peptide may also bind with another GNP via an electrostatic force, which may result in the formation of cross-linked network aggregates of GNPs accompanied by distinct color changes. In contrast, in the presence of PKA, the biotin-LRRASLG will be phosphorylated through the PKA-catalyzed transfer of γ-phosphoryl of ATP to the hydroxyl group of serine. Consequently, the negatively charged phosphate group of the biotin-phosphopeptide will no longer combine with the GNPs through electrostatic force and therefore efficiently prevent the GNPs from crosslinking and aggregation, and so the red color of GNPs will be maintained. In this way, visual detection of the PKA activity can be realized by recording the color changes of GNPs. Optimization of the STV dosage functionalized on GNPs surface Fig. 2a shows a typical TEM image of the GNPs used in this work, from which one can see that the as-prepared GNPs display spherical shapes with an average diameter of ∼13 nm. Furthermore, the zeta potential of the GNPs is tested to be −55.7 mV, indicating that the GNPs are negatively charged due to the citrate layer on their surface. According to the design principle of the proposed kinase assay illustrated in Fig. 1, the functionalization of GNPs with STV is essential for the crosslinking of GNPs, which can also well stabilize the GNPs since GNPs are susceptible to salt-induced precipitation. On the other hand, if excessive STV is coated on the surface of GNPs, the negative charges of citrate on the surface of GNPs will be efficiently blocked. One can also see from Fig. 1 that the negative charges of citrate-capped GNPs are also critical for the proposed PKA assay. Since the amount of STV modified on the GNPs may greatly influence the performance of the proposed PKA assay, we firstly optimized the STV dosage for GNP functionalization by a flocculation test.31
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Fig. 2 (a) Representative TEM image of the GNPs; (b–c) optimization of the STV dosage for GNP functionalization. (b) Photographs of GNPs mixed with different dosages of STV before (top panel) and after the addition of 10% NaCl (bottom panel). The amount of STV (μg mL−1, from left to right): 0, 2, 4, 8, 15, 20, 40, 80, 100, 150; (c) the plot between the corresponding absorbance of these samples at 520 nm and the concentrations of STV.
As we know, both the colloidal GNPs and STV-GNPs have a characteristic absorbance peak centered at 520 nm. However, when a certain amount of NaCl is introduced, the unmodified GNPs will be aggregated immediately accompanied by the redshift of the absorbance peak and a sharp decrease of absorbance at 520 nm. In contrast, the functionalization of GNPs with STV can protect the GNPs from salt-induced aggregation. The more STV coated on the surface of GNPs, the more monodisperse and red-color GNPs maintained with stable and characteristic absorbance at 520 nm against the high salt media. Based on these phenomena, series dilutions of STV were incubated with 1.0 mL of GNPs for 1 h, and then 30 μL of 10% NaCl was added to each sample. After the incubation at room temperature for 5 min, the coagulation of GNPs reached equilibrium and then the samples were detected with a UV–vis
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spectrophotometer at 520 nm. It can be seen from Fig. 2b that at low STV concentrations, the colors of GNPs will rapidly turn blue or purple upon the addition of NaCl solution. In contrast, high concentrations of STV can effectively stabilize the GNPs and maintain their intrinsic red color. Correspondingly, one can see from Fig. 2c that the absorbance of GNPs at 520 nm increases gradually with the increase of STV concentration from 0 to 40 μg mL−1, indicating that the STV is not enough and thus GNPs cannot be fully protected from the NaClinduced coagulation. However, when the amount of STV is higher than 40 μg mL−1, the GNPs exhibit stable deep red color upon the addition of NaCl, and the absorbance of GNPs at 520 nm will remain almost stable, indicating that STV is enough to fully stabilize GNPs against the salt-induced color changes. In this study, the ideal dosage of STV coated on the GNPs should be capable of efficiently stabilizing the GNPs against undesired salt-induced precipitation but won’t be excessive to completely block the negatively charged citratecapped surface of GNPs. So 40 µg mL−1 STV is selected as the optimal dosage for coating GNPs. Verification of the feasibility and sensing mechanism of the proposed PKA assay Under the optimized STV dosage, we then examined the feasibility of the proposed GNP-based colorimetric strategy for PKA analysis. As show in Fig. 3a, when the STV-GNPs are mixed with the blank sample without PKA, the color of GNPs immediately turns purple due to the biotin-peptide-directed aggregation of GNPs. After standing for 2 h, almost all of the STV-GNPs precipitate to the bottom of the tube and the supernatant become colorless and transparent. In contrast, in the presence of PKA, a portion of the biotin-peptide will be phosphorylated which will prevent the GNPs from aggregation and settlement. One can see from Fig. 3a that after incubating the GNPs with the PKA reaction system for 2 h, the red-colored GNPs remaining in the supernatant increase gradually with the increase of PKA concentration, making the red color of the supernatant more and more deep that can be clearly identified by the naked eye. However, when the biotin-peptide is treated with PKA but without adding ATP, the phosphorylation of peptide will not occur since ATP is the source of the phosphate group. After incubation with such reaction samples for 2 h, as shown in Fig. 3b, all of the GNPs will settle to the bottom of the tubes irrespective of the PKA concentration, leaving the supernatant colorless. These results clearly prove that the inhibition of GNP precipitation and the maintenance of red color of GNPs are solely originated from PKA-catalyzed phosphorylation of biotin-peptides but not from PKA molecules themselves. Furthermore, in order to fully elucidate the sensing mechanism of this GNP-based kinase assay, several control experiments are also conducted. As displayed in Fig. 3c, if STV-GNPs are further blocked with 0.5% BSA (other conditions are the same as those for Fig. 3a), the biotin-peptide-induced precipitation of the GNPs are significantly reduced because the negative charges on the GNP surface may be efficiently enclosed by
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Fig. 3 Verification of the feasibility and sensing mechanism of the proposed PKA assay. Reaction components: (a) STV-GNPs incubated with PKA reaction samples (the PKA activities are 0, 0.005 and 0.05 U μL−1 from left to right in all of the images); (b) STV-GNPs incubated with PKA reaction samples without adding ATP; (c) STV-GNPs blocked by BSA and then incubated with PKA reaction samples; (d) STV-GNPs mixed with PKA reaction samples by using a non-biotinylated peptide; (e) BSA-GNPs incubated with PKA reaction samples. The top panel of each image displays the photographs of the GNPs immediately mixed with the kinase reaction system, and the bottom panels are the corresponding mixtures after standing for 2 h. Other experimental conditions are the same as the standard assay procedures described in the Experimental section.
excess BSA. As a result, the PKA-induced color changes will not be clearly identified by the naked eye. Similarly, the results shown in Fig. 3d and e manifest that if a non-biotinylated peptide is used for the kinase reaction, or STV-GNPs are substituted by BSA-capped GNPs, neither crosslinking-induced precipitation nor PKA dosage-responsive color changes of GNPs can be observed. All of these results shown in Fig. 3 clearly demonstrate that the crosslinking and coagulation of GNPs are undoubtedly driven by both the biotin-STV interaction and electrostatic interaction between the biotin-peptide and STV-GNPs, and the PKA-catalyzed phosphorylation of biotinpeptide can efficiently prevent the GNPs from settlement and thus maintain the red color of the monodisperse GNPs, which enables the visual detection of PKA activity. Analytical performance of the colorimetric assay for detection of PKA activity The analytical performance of the GNP-based colorimetric method for the detection of PKA activity is investigated by
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both visual detection and UV-vis spectra. Fig. 4a shows the photographs of STV-GNPs after mixing with a series of kinase reactions treated with different concentrations of PKA. As can be seen that immediately after the mixing, the color of the mixtures exhibits a gradual purple-to-red change with the increase of PKA activity. Fascinatingly, after standing for 2 h, the crosslinked GNPs will be completely precipitated to the bottom of the tube and the red color of monodisperse GNPs remaining in the supernatant will remain stable for at least several hours. More importantly, the color depth of the red GNPs remaining in the supernatant is proportionally correlated with the PKA activity. It can be seen from the bottom panel of Fig. 4a that the red colors of GNPs will become deeper gradually with increased PKA activity, and the color depth responsive to PKA activities from 0.0005 U μL−1 to 0.05 U μL−1 can be discriminated distinctly by the naked eye. Fig. 4b shows the corresponding absorption spectra of the red GNPs supernatant, where the absorbance at 520 nm increases gradually as the PKA activity increases from 0.0005 U μL−1 to 0.05 U μL−1, and then the absorbance will remain stable when the PKA activity is further elevated. Moreover, as displayed in Fig. 4c, there is a good linear relationship between the absorbance at 520 nm (Abs520) and the PKA activity in the range of 0.0005 to 0.02 U μL−1. The correlation equation is Abs520 = 0.134 + 54.7 × Cpka (U μL−1) with a regression coefficient (R) of 0.9972. From the results shown in Fig. 4, it can be seen that as low as 0.0005 U μL−1 PKA can be clearly discriminated from the blank control either by visual detection or through absorption spectra. As we know, the detection limits of PKA by using several recently reported electrochemical or fluorometric
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kinase assays typically fall in the range of 0.0001–0.0005 U μL−1.10,32–34 So, it can be found that neither expensive instruments nor sophisticated procedures are required in our GNPbased colorimetric assay, but its sensitivity for detection of PKA activity is comparable with those of most existing PKA protocols. Furthermore, we have also examined the applicability of the GNP-based colorimetric strategy for PKA analysis in complex cell lysates. In this study, MCF-7 cells were cultured with/or without forskolin/IBMX stimulation, and the PKA activities in these cell lysates were evaluated respectively by the proposed method. The stimulation of MCF-7 cells by forskolin/IBMX can effectively activate the cellular cAMP-dependent PKA.35,36 The results shown in Fig. 5 indicate that the cell lysate of MCF-7 cells without forskolin/IBMX stimulation produces only a weak response, indicating a relatively low level of PKA activity. However, the cell lysate stimulated with 10 μM of forskolin and 20 μM of IBMX results in the obvious red color of GNPs with strong absorbance at 520 nm. In addition, if the forskolin/ IBMX-treated cell lysate is pre-incubated with H-89 (a potent PKA inhibitor) during the phosphorylation reaction, no red color can be observed, indicating that the cell lysate-induced color and absorbance responses are indeed resulted from the drug-activated PKA but not from other factors. These results suggest that the GNP-based colorimetric strategy is applicable for in vitro detection of protein kinase activities in complex biological samples. Detection of the inhibition of PKA activity To investigate whether the GNP-based colorimetric assay can be further employed to assess the inhibition of PKs which is
Fig. 4 Analytical performance of the proposed colorimetric assay for the detection of PKA activity. (a) Photographs of the GNPs after mixing with a series of kinase reactions treated with different concentrations of PKA. Top panel, immediately after mixing; Bottom panel, 2 h later; (b) corresponding absorption spectra of the samples in the bottom panel of image (a); (c) the relationship between the absorbance at 520 nm and the PKA activity. The error bars represent the standard deviation of three replicates for each data point. Other conditions are the same as those described in the Experimental section.
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Fig. 5 Absorption spectra of the proposed colorimetric assay system for the detection of PKA activities in different types of MCF-7 cell lysates. (1), blank control without any cell lysate; (2), unstimulated cell lysate; (3), 10 μM forskolin/20 μM IBMX-stimulated cell lysate; (4), 10 μM forskolin/20 μM IBMX-stimulated cell lysate pre-mixed with 5 μM H-89. (inset) corresponding photographs. The total protein concentration for each type of MCF-7 cell lysate is fixed at 2 μg mL−1 and other conditions are all performed according to the standard assay procedures stated in the Experimental section.
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Fig. 6 The results of PKA inhibition study. (a) Photographs of the colorimetric assay system in the presence of different concentrations of H-89 (0–10 µM) by fixing PKA activity at 0.02 U µL−1; (b) The corresponding absorption spectra of the samples in the bottom panel of a; (c) the relationship between the absorbance at 520 nm and H-89 concentrations on the logarithm scale. The error bars represent the standard deviation of three replicates for each data point.
essential for the screening of potential small-molecule inhibitor drugs, a proof-of-concept inhibition study for PKA is performed in this study by using H-89, a well-known small molecule capable of inhibiting PKA activity, as a model inhibitor. The experiments are conducted following essentially the same procedures as the PKA assay, except for the pre-incubating of a fixed PKA concentration (0.02 U µL−1) with series dilutions of H-89 (0–10 µM). Fig. 6 exhibits the inhibitory results of H-89. It can be obviously observed that the deep red color and the absorbance of GNPs decrease gradually with increasing concentrations of H-89 because of the inhibition of PKA activity and thus the low levels of peptide phosphorylation, which may accelerate the crosslinking and coagulation of GNPs. The relationship between the absorbance at 520 nm and H-89 concentrations on the logarithm scale is plotted in Fig. 6c, from which the IC50 value is determined to be 102 nM, which agrees well with the literature values.10,37 Therefore, the GNP-based colorimetric assay is suitable for the inhibition study of PKs, and may also find potential applications in screening effective PK inhibitors as potential targeted drugs.
Conclusions In summary, we have developed a new colorimetric strategy for the detection of protein kinase activity based on the peptide phosphorylation-regulated crosslinking and aggregation of GNPs. This new kinase assay shows several distinct advantages. Firstly, the GNP-based colorimetric assay is extremely simple because neither expensive instruments nor sophisticated procedures are required. Only by simply incubating the protein kinase reaction sample with the STV-GNPs for 2 h can the color changes of GNPs be identified by the naked eye. Sec-
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ondly, for previously reported GNP crosslinking-based colorimetric kinase assays, two types of GNPs functionalized respectively with the substrate peptide and avidin (or antibodies) are required. The enzyme reaction on the surface of GNPs may be quite different from those performed in homogeneous physiological media. In this study, the GNPs only need to be functionalized with STV through a rather simple procedure and the kinase reaction is conducted under the optimal physiological conditions in homogeneous solution, which is more suitable for the study of enzyme–substrate specific interaction. Thirdly, as we know, the charge of a peptide can be exactly tuned by adding (+)- or (−)-charged amino acid residues to a kinase-specific core peptide motif. Therefore, by rationally tuning the net charges of other peptide substrates to be similar to that of the kemptide used in this work, the proposed strategy may be easily extended to the detection of different PKs. Moreover, despite its simple design, the sensitivity of the proposed assay is satisfactory and it has been successfully applied to the inhibition study and kinase detection in complex biosamples, suggesting that the GNP-based colorimetric assay may find potential applications for PK analysis in signal transduction pathways and kinaserelated drug discovery.
Acknowledgements This work was supported by the National Natural Science Foundation of China (21335005), the Fundamental Research Funds for the Central Universities (GK201402051 and GK201501003), the Natural Science Foundation of Shaanxi Province (2014JQ2058) and the Program for Innovative Research Team in Shaanxi Province (No. 2014KCT-28).
Notes and references 1 B. A. Gibson and W. Lee Kraus, Nat. Rev. Mol. Cell Biol., 2012, 13, 411–424. 2 C. Janke and J. C. Bulinski, Nat. Rev. Mol. Cell Biol., 2011, 12, 773–786. 3 G. Manning, D. B. Whyte, R. Martinez, T. Hunter and S. Sudarsanam, Science, 2002, 298, 1912–1934. 4 P. A. Futreal, A. Kasprzyk, E. Birney, J. C. Mullikin, R. Wooster and M. R. Stratton, Nature, 2001, 409, 850–852. 5 D. E. Kalume, H. Molina and A. Pandey, Curr. Opin. Chem. Biol., 2003, 7, 64–69. 6 C. Bertolotto, P. Abbe, T. J. Hemesath, K. Bille, D. E. Fisher, J. P. Ortonne and R. Ballotti, J. Cell Biol., 1998, 142, 827– 835. 7 K. Kerman, M. Chikae, S. Yamamura and E. Tamiya, Anal. Chim. Acta, 2007, 588, 26–33. 8 C. Wang, L. Wei, C. Yuan and K. C. Hwang, Anal. Chem., 2012, 84, 971–977. 9 K. Kerman and H. B. Kraatz, Biosens. Bioelectron., 2009, 24, 1484–1489.
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Published on 24 June 2015. Downloaded by University of Connecticut on 07/07/2015 15:31:54.
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10 J. Bai, C. Liu, T. Yang, F. Wang and Z. Li, Chem. Commun., 2013, 49, 3887–3889. 11 J. Zhou, X. Xu, W. Liu, X. Liu, Z. Nie, M. Qing, L. Nie and S. Yao, Anal. Chem., 2013, 85, 5746–5754. 12 L. Wang, Y. Yang and C. Zhang, Anal. Chem., 2015, 87, 4696–4703. 13 W. Ren, C. Liu, S. Lian and Z. Li, Anal. Chem., 2013, 85, 10956–10961. 14 B. E. Turk, J. E. Hutti and L. C. Cantley, Nat. Protoc., 2006, 1, 375–379. 15 B. T. Houseman, J. H. Huh, S. J. Kron and M. Mrksich, Nat. Biotechnol., 2002, 20, 270–274. 16 X. Xu, J. Zhou, X. Liu, Z. Nie, M. Qing, M. Guo and S. Yao, Anal. Chem., 2012, 84, 4746–4753. 17 Y. P. Kim, E. Oh, Y. H. Oh, D. W. Moon, T. G. Lee and H. S. Kim, Angew. Chem., Int. Ed., 2007, 46, 6816– 6819. 18 Y. Yu, R. Anjum, K. Kubota, J. Rush, J. Villen and S. P. Gygi, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 11606–11611. 19 G. V. Maltzahn, D. H. Min, Y. Zhang, J. H. Park, T. J. Harris, M. Sailor and S. N. Bhatia, Adv. Mater., 2007, 19, 3579– 3583. 20 M. G. Shapiro, J. O. Szablowski, R. Langer and A. Jasanoff, J. Am. Chem. Soc., 2009, 131, 2484–2486. 21 Z. Zhao, X. Zhou and D. Xing, Biosens. Bioelectron., 2012, 31, 299–304. 22 Z. Fu, X. Zhou and D. Xing, Sens. Actuators, B, 2013, 182, 633–641. 23 Z. Fu, X. Zhou and D. Xing, Methods, 2013, 64, 260– 266.
This journal is © The Royal Society of Chemistry 2015
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24 Z. Wang, J. Lee, A. R. Cossins and M. Brust, Anal. Chem., 2005, 77, 5770–5774. 25 Z. Wang, R. Lévy, D. G. Fernig and M. Brust, J. Am. Chem. Soc., 2006, 128, 2214–2215. 26 S. Gupta, H. Andresen, J. E. Ghadiali and M. M. Stevens, Small, 2010, 6, 1509–1513. 27 J. Oishi, Y. Asami, T. Mori, J. H. Kang, M. Tanabe, T. Niidome and Y. Katayama, ChemBioChem, 2007, 8, 875– 879. 28 J. Oishi, Y. Asami, T. Mori, J. H. Kang, T. Niidome and Y. Katayama, Biomacromolecules, 2008, 9, 2301–2308. 29 D. Liu, W. Chen, K. Sun, K. Deng, W. Zhang, Z. Wang and X. Jiang, Angew. Chem., Int. Ed., 2011, 50, 4103–4107. 30 R. Jin, G. Wu, Z. Li, C. A. Mirkin and G. C. Schatz, J. Am. Chem. Soc., 2003, 125, 1643–1654. 31 Z. Li, Y. Wang, C. Liu and Y. Li, Anal. Chim. Acta, 2005, 551, 85–91. 32 J. Bai, Y. Zhao, Z. Wang, C. Liu, Y. Wang and Z. Li, Anal. Chem., 2013, 85, 4813–4821. 33 P. Miao, L. Ning, X. Li, P. Li and G. Li, Bioconjugate Chem., 2012, 23, 141–145. 34 X. Xu, Z. Nie, J. Chen, Y. Fu, W. Li, Q. Shen and S. Yao, Chem. Commun., 2009, 6946–6948. 35 C. Liu, L. Chang, H. Wang, J. Bai, W. Ren and Z. Li, Anal. Chem., 2014, 86, 6095–6102. 36 W. Yang, X. Lu, Y. Wang, S. Sun, C. Liu and Z. Li, Sens. Actuators, B, 2015, 210, 508–512. 37 S. Shiosaki, T. Nobori, T. Mori, R. Toita, Y. Nakamura, C. W. Kim, T. Yamamoto, T. Niidome and Y. Katayama, Chem. Commun., 2013, 49, 5592–5594.
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Phosphorylation-regulated crosslinking of gold nanoparticles: a new strategy for colorimetric detection of protein kinase activity Sujuan Sun,a Haixia Shen,b Chenghui Liu*a and Zhengping Lia Accurate and rapid detection of protein kinase activities is of great significance because protein kinases play important regulatory roles in many vital biological processes. Herein, we wish to report a facile colorimetric protein kinase assay based on the phosphorylation-tuned crosslinking of gold nanoparticles (GNPs) by using protein kinase A (PKA) as a proof-of-concept target. In this new strategy, a biotinylated peptide (biotin-LRRASLG) is used as the PKA-specific substrate. When mixed with streptavidin-functionalized GNPs (STV-GNPs), the positively charged biotin-peptide will combine with different GNPs both through the specific STV–biotin binding and through electrostatic interactions, which will lead to the crosslinking and coagulation of GNPs. In contrast, under the catalysis of PKA, the biotin-peptide will be
Received 13th May 2015, Accepted 23rd June 2015
phosphorylated at the serine residue and its net charge will be obviously altered, which may significantly weaken the electrostatic interaction between the phosphopeptide and GNPs and thus effectively prevent
DOI: 10.1039/c5an00963d
the STV-GNPs from crosslinking and settlement. Therefore, by viewing the color changes of the GNPs,
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the PKA activity can be easily detected by the naked eye.
Introduction It is well-recognized that post-translation modifications (PTM) may extensively extend the diversity of the protein functionality.1,2 Protein phosphorylation catalyzed by protein kinases (PKs) is one of the most important PTM mechanisms in the process of cellular signal transduction. As such, PKs are known to play important regulatory roles in diverse biological processes such as signal transduction, immune regulation, cell apoptosis, proliferation and differentiation and some important metabolic pathways in the human body.3–6 It has been revealed that the abnormal expression level of PKs and the unusual phosphorylation state of proteins are closely related to the development of many diseases. So PKs have been considered as an important family of molecular targets for the discovery of new targeted drugs. Therefore, the development of simple and sensitive PK assays capable of monitoring the PK activity and identifying potential inhibitors is highly desired not only for the fundamental biological research of signal transduction, but also for the clinical diagnosis and drug discovery.
a Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710119, Shaanxi Province, P. R. China. E-mail: [email protected] b Basic Experimental Teaching Centre, Shaanxi Normal University, Xi’an 710062, Shaanxi Province, P. R. China
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Up to now, a variety of PK assays have been established by using various kinds of analytical techniques such as electrochemistry,7–9 fluorescence,10–12 flow cytometry,13 radioactive methods,14,15 quartz-microbalance,16 mass spectrometry,17,18 magnetic resonance imaging19,20 and electrochemiluminescence.21 Although these methods have made great advances in PK analysis, most of them generally require labor-intensive operation or expensive/sophisticated instruments that are only available in specialized laboratories. To overcome these shortcomings, gold nanoparticle (GNP)-based colorimetric sensing strategies, which enable the facile detection of various biomolecules by the naked eye without the requirement of sophisticated instruments,22,23 have attracted much attention for visual detection of protein kinase activities.24–28 The Brust group and Stevens group have separately developed colorimetric kinase assays based on the kinase-induced crosslinking of GNPs.25,26 For example, Brust and coworkers have prepared two kinds of functionalized GNPs, one is modified with kinase-specific substrate peptides and the other is capped with avidin.25 In their design, biotin-labeled ATP is used during the kinase reaction, so that the phosphorylated GNPs with the biotin label will further bind with the avidin-functionalized GNPs to form a crosslinking network of GNPs accompanied by obvious color changes. Similarly, Stevens and coworkers also established an attractive strategy for the detection of tyrosine protein kinase activity based on the phosphorylation-induced crosslinking of two kinds of GNPs functionalized with
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kinase-specific peptides and antiphosphotyrosine antibodies,26 respectively. These crosslinking strategies by using two types of functionalized GNPs are robust and highly specific for PK analysis, but they also suffer from the complicated functionalization of GNPs since the two kinds of GNPs should be modified respectively with the substrate peptide and avidin (or antibodies). Although the protocols for capping GNPs with avidin or antibodies have been well-established, the properties of substrate peptide motifs specific to different PKs may vary significantly, some of which themselves may cause the aggregation of GNPs irrespective of the kinase-catalyzed phosphorylation and thus may interfere with the PK analysis. Recently, the Katayama group has proposed a GNP-based noncrosslinking strategy for evaluating PK activities,27,28 where the non-crosslinking aggregation of unmodified GNPs can be tuned by the phosphorylation-induced net charge change of the substrate peptide. However, it should be noted that the aggregation of unmodified GNPs is also highly susceptible to the amino acid sequences of the substrate peptide as well as the ionic strength of the reaction media during the PK-catalyzed phosphorylation reaction, which may significantly interfere with the detection of PK activity and thus greatly limit the wide application of the non-crosslinking strategy. Hence, it is still highly desirable to further improve the GNP-based colorimetric kinase assays with more simple procedures and high specificity towards the PK-induced peptide phosphorylation against the undesired salt-induced interference. Herein, we wish to report a new GNP-based crosslinking strategy for convenient and sensitive detection of PK activity and inhibition by taking protein kinase A (PKA) as a proof-ofconcept target. Compared with the GNP crosslinking-based protein kinase assays by using two kinds of functionalized GNPs, only one type of streptavidin-functionalized GNP (STV-GNPs) is needed in this work, which can be facilely prepared with mature and simple procedures. On the other hand, by using the STV-GNPs as the sensing elements, STV can effectively stabilize the GNPs and protect GNPs from undesired salt-induced aggregation, making the STV-GNPs specifically responsive to the PKA-catalyzed phosphorylation reaction. In this design, a biotinylated and positively charged peptide (biotin-LRRASLG) is used as the PKA-specific substrate, which can lead to the crosslinking and coagulation of STV-GNPs through both STV-biotin binding and electrostatic interaction. In contrast, under the catalysis of PKA, the biotin-peptide will be phosphorylated and its net charge will be obviously altered, which may prevent the STV-GNPs from crosslinking and settlement. Therefore, by viewing the color changes of the GNPs, the PKA activity can be easily detected by the naked eye.
Experimental section Materials and reagents cAMP-dependent protein kinase A (PKA, catalytic subunit) was purchased from New England Biolabs. Streptavidin (STV) was obtained from Promega Corporation. H-89 was purchased
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from EMD Bioscience and ATP was from Sangon Biotech (Shanghai, China). Biotin-LRRASLG and non-biotinylated LRRASLG specific for PKA were custom synthesized by GL Biochem (Shanghai, China). HAuCl4·4H2O was supplied by Sinopharm (Shanghai, China). All of the other reagents used in this work were of analytical grade and used as received without further purification. Preparation of GNPs and STV-GNPs GNPs with an average diameter of ∼13 nm were prepared by citrate reduction according to the literature methods.29,30 Generally, 100 mL water which contained 4.2 mL of 1% HAuCl4 was added into a three-necked flask, and the solution was heated to reflux under stirring. After refluxing for 15 min, 10 mL of trisodium citrate (38.8 mM) was rapidly added into the boiling HAuCl4 solution and then the mixture was kept boiling for another 30 min. Finally, the solution was removed from heat and cooled to room temperature under stirring. The sizes of the as-prepared GNPs are about 13 nm which exhibit a characteristic absorption peak at ∼520 nm. For the preparation of STV-GNP conjugates, the pH of the as-synthesized GNPs was firstly adjusted to ∼9.0 by using 0.1 M Na2CO3. Then STV (final concentration of 40 μg mL−1) was added into 5 mL of GNP solution and this mixture was incubated at room temperature for 1 h. Afterwards, the GNPs were centrifuged and washed three times at 4 °C to remove the unbound STV. Finally, the as-prepared STV-GNP bioconjugates were dispersed in 5 mL of sterilized water and stored at 4 °C for subsequent use. Standard assay procedures for the detection of PKA activity Typically, in a total 100 μL of PKA reaction buffer (50 mM TrisHCl, 10 mM MgCl2, pH 7.5), 1.5 μM of biotin-peptide was mixed with 12 μM of ATP and series dilutions of PKA. The mixture was incubated at 37 °C for 60 min under shaking. Subsequently, an equal volume (100 μL) of STV-GNPs was mixed with the PKA reaction system and incubated at room temperature for 2 h. Finally, the color changes of the supernatant of all samples were analyzed by visual inspection and UV-vis spectroscopy.
Results and discussion Design principle of the proposed colorimetric kinase assay Fig. 1 illustrates the work principle of the proposed colorimetric protein kinase assay based on the peptide phosphorylation-regulated crosslinking of GNPs. Protein kinase A (PKA), a protein kinase critical to memory formation, is selected as a proof-of-concept target. Correspondingly, a biotinylated kemptide (biotin-LRRASLG), which is positively charged (+2 net charge) in the Tris-HCl reaction buffer, is utilized as the PKAspecific substrate. The citrate-capped 13 nm GNPs are functionalized with a layer of STV, the dosage of which is selected to be capable of efficiently stabilizing GNPs against undesired salt-induced precipitation but won’t completely block the
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Fig. 1
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Schematic illustration of the new GNP-based colorimetric PKA assay.
negatively charged citrate-capped surface of GNPs. On this occasion, the biotin group of the substrate peptide will combine with one GNP through the STV-biotin interaction while the positively charged peptide may also bind with another GNP via an electrostatic force, which may result in the formation of cross-linked network aggregates of GNPs accompanied by distinct color changes. In contrast, in the presence of PKA, the biotin-LRRASLG will be phosphorylated through the PKA-catalyzed transfer of γ-phosphoryl of ATP to the hydroxyl group of serine. Consequently, the negatively charged phosphate group of the biotin-phosphopeptide will no longer combine with the GNPs through electrostatic force and therefore efficiently prevent the GNPs from crosslinking and aggregation, and so the red color of GNPs will be maintained. In this way, visual detection of the PKA activity can be realized by recording the color changes of GNPs. Optimization of the STV dosage functionalized on GNPs surface Fig. 2a shows a typical TEM image of the GNPs used in this work, from which one can see that the as-prepared GNPs display spherical shapes with an average diameter of ∼13 nm. Furthermore, the zeta potential of the GNPs is tested to be −55.7 mV, indicating that the GNPs are negatively charged due to the citrate layer on their surface. According to the design principle of the proposed kinase assay illustrated in Fig. 1, the functionalization of GNPs with STV is essential for the crosslinking of GNPs, which can also well stabilize the GNPs since GNPs are susceptible to salt-induced precipitation. On the other hand, if excessive STV is coated on the surface of GNPs, the negative charges of citrate on the surface of GNPs will be efficiently blocked. One can also see from Fig. 1 that the negative charges of citrate-capped GNPs are also critical for the proposed PKA assay. Since the amount of STV modified on the GNPs may greatly influence the performance of the proposed PKA assay, we firstly optimized the STV dosage for GNP functionalization by a flocculation test.31
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Fig. 2 (a) Representative TEM image of the GNPs; (b–c) optimization of the STV dosage for GNP functionalization. (b) Photographs of GNPs mixed with different dosages of STV before (top panel) and after the addition of 10% NaCl (bottom panel). The amount of STV (μg mL−1, from left to right): 0, 2, 4, 8, 15, 20, 40, 80, 100, 150; (c) the plot between the corresponding absorbance of these samples at 520 nm and the concentrations of STV.
As we know, both the colloidal GNPs and STV-GNPs have a characteristic absorbance peak centered at 520 nm. However, when a certain amount of NaCl is introduced, the unmodified GNPs will be aggregated immediately accompanied by the redshift of the absorbance peak and a sharp decrease of absorbance at 520 nm. In contrast, the functionalization of GNPs with STV can protect the GNPs from salt-induced aggregation. The more STV coated on the surface of GNPs, the more monodisperse and red-color GNPs maintained with stable and characteristic absorbance at 520 nm against the high salt media. Based on these phenomena, series dilutions of STV were incubated with 1.0 mL of GNPs for 1 h, and then 30 μL of 10% NaCl was added to each sample. After the incubation at room temperature for 5 min, the coagulation of GNPs reached equilibrium and then the samples were detected with a UV–vis
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spectrophotometer at 520 nm. It can be seen from Fig. 2b that at low STV concentrations, the colors of GNPs will rapidly turn blue or purple upon the addition of NaCl solution. In contrast, high concentrations of STV can effectively stabilize the GNPs and maintain their intrinsic red color. Correspondingly, one can see from Fig. 2c that the absorbance of GNPs at 520 nm increases gradually with the increase of STV concentration from 0 to 40 μg mL−1, indicating that the STV is not enough and thus GNPs cannot be fully protected from the NaClinduced coagulation. However, when the amount of STV is higher than 40 μg mL−1, the GNPs exhibit stable deep red color upon the addition of NaCl, and the absorbance of GNPs at 520 nm will remain almost stable, indicating that STV is enough to fully stabilize GNPs against the salt-induced color changes. In this study, the ideal dosage of STV coated on the GNPs should be capable of efficiently stabilizing the GNPs against undesired salt-induced precipitation but won’t be excessive to completely block the negatively charged citratecapped surface of GNPs. So 40 µg mL−1 STV is selected as the optimal dosage for coating GNPs. Verification of the feasibility and sensing mechanism of the proposed PKA assay Under the optimized STV dosage, we then examined the feasibility of the proposed GNP-based colorimetric strategy for PKA analysis. As show in Fig. 3a, when the STV-GNPs are mixed with the blank sample without PKA, the color of GNPs immediately turns purple due to the biotin-peptide-directed aggregation of GNPs. After standing for 2 h, almost all of the STV-GNPs precipitate to the bottom of the tube and the supernatant become colorless and transparent. In contrast, in the presence of PKA, a portion of the biotin-peptide will be phosphorylated which will prevent the GNPs from aggregation and settlement. One can see from Fig. 3a that after incubating the GNPs with the PKA reaction system for 2 h, the red-colored GNPs remaining in the supernatant increase gradually with the increase of PKA concentration, making the red color of the supernatant more and more deep that can be clearly identified by the naked eye. However, when the biotin-peptide is treated with PKA but without adding ATP, the phosphorylation of peptide will not occur since ATP is the source of the phosphate group. After incubation with such reaction samples for 2 h, as shown in Fig. 3b, all of the GNPs will settle to the bottom of the tubes irrespective of the PKA concentration, leaving the supernatant colorless. These results clearly prove that the inhibition of GNP precipitation and the maintenance of red color of GNPs are solely originated from PKA-catalyzed phosphorylation of biotin-peptides but not from PKA molecules themselves. Furthermore, in order to fully elucidate the sensing mechanism of this GNP-based kinase assay, several control experiments are also conducted. As displayed in Fig. 3c, if STV-GNPs are further blocked with 0.5% BSA (other conditions are the same as those for Fig. 3a), the biotin-peptide-induced precipitation of the GNPs are significantly reduced because the negative charges on the GNP surface may be efficiently enclosed by
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Fig. 3 Verification of the feasibility and sensing mechanism of the proposed PKA assay. Reaction components: (a) STV-GNPs incubated with PKA reaction samples (the PKA activities are 0, 0.005 and 0.05 U μL−1 from left to right in all of the images); (b) STV-GNPs incubated with PKA reaction samples without adding ATP; (c) STV-GNPs blocked by BSA and then incubated with PKA reaction samples; (d) STV-GNPs mixed with PKA reaction samples by using a non-biotinylated peptide; (e) BSA-GNPs incubated with PKA reaction samples. The top panel of each image displays the photographs of the GNPs immediately mixed with the kinase reaction system, and the bottom panels are the corresponding mixtures after standing for 2 h. Other experimental conditions are the same as the standard assay procedures described in the Experimental section.
excess BSA. As a result, the PKA-induced color changes will not be clearly identified by the naked eye. Similarly, the results shown in Fig. 3d and e manifest that if a non-biotinylated peptide is used for the kinase reaction, or STV-GNPs are substituted by BSA-capped GNPs, neither crosslinking-induced precipitation nor PKA dosage-responsive color changes of GNPs can be observed. All of these results shown in Fig. 3 clearly demonstrate that the crosslinking and coagulation of GNPs are undoubtedly driven by both the biotin-STV interaction and electrostatic interaction between the biotin-peptide and STV-GNPs, and the PKA-catalyzed phosphorylation of biotinpeptide can efficiently prevent the GNPs from settlement and thus maintain the red color of the monodisperse GNPs, which enables the visual detection of PKA activity. Analytical performance of the colorimetric assay for detection of PKA activity The analytical performance of the GNP-based colorimetric method for the detection of PKA activity is investigated by
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both visual detection and UV-vis spectra. Fig. 4a shows the photographs of STV-GNPs after mixing with a series of kinase reactions treated with different concentrations of PKA. As can be seen that immediately after the mixing, the color of the mixtures exhibits a gradual purple-to-red change with the increase of PKA activity. Fascinatingly, after standing for 2 h, the crosslinked GNPs will be completely precipitated to the bottom of the tube and the red color of monodisperse GNPs remaining in the supernatant will remain stable for at least several hours. More importantly, the color depth of the red GNPs remaining in the supernatant is proportionally correlated with the PKA activity. It can be seen from the bottom panel of Fig. 4a that the red colors of GNPs will become deeper gradually with increased PKA activity, and the color depth responsive to PKA activities from 0.0005 U μL−1 to 0.05 U μL−1 can be discriminated distinctly by the naked eye. Fig. 4b shows the corresponding absorption spectra of the red GNPs supernatant, where the absorbance at 520 nm increases gradually as the PKA activity increases from 0.0005 U μL−1 to 0.05 U μL−1, and then the absorbance will remain stable when the PKA activity is further elevated. Moreover, as displayed in Fig. 4c, there is a good linear relationship between the absorbance at 520 nm (Abs520) and the PKA activity in the range of 0.0005 to 0.02 U μL−1. The correlation equation is Abs520 = 0.134 + 54.7 × Cpka (U μL−1) with a regression coefficient (R) of 0.9972. From the results shown in Fig. 4, it can be seen that as low as 0.0005 U μL−1 PKA can be clearly discriminated from the blank control either by visual detection or through absorption spectra. As we know, the detection limits of PKA by using several recently reported electrochemical or fluorometric
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kinase assays typically fall in the range of 0.0001–0.0005 U μL−1.10,32–34 So, it can be found that neither expensive instruments nor sophisticated procedures are required in our GNPbased colorimetric assay, but its sensitivity for detection of PKA activity is comparable with those of most existing PKA protocols. Furthermore, we have also examined the applicability of the GNP-based colorimetric strategy for PKA analysis in complex cell lysates. In this study, MCF-7 cells were cultured with/or without forskolin/IBMX stimulation, and the PKA activities in these cell lysates were evaluated respectively by the proposed method. The stimulation of MCF-7 cells by forskolin/IBMX can effectively activate the cellular cAMP-dependent PKA.35,36 The results shown in Fig. 5 indicate that the cell lysate of MCF-7 cells without forskolin/IBMX stimulation produces only a weak response, indicating a relatively low level of PKA activity. However, the cell lysate stimulated with 10 μM of forskolin and 20 μM of IBMX results in the obvious red color of GNPs with strong absorbance at 520 nm. In addition, if the forskolin/ IBMX-treated cell lysate is pre-incubated with H-89 (a potent PKA inhibitor) during the phosphorylation reaction, no red color can be observed, indicating that the cell lysate-induced color and absorbance responses are indeed resulted from the drug-activated PKA but not from other factors. These results suggest that the GNP-based colorimetric strategy is applicable for in vitro detection of protein kinase activities in complex biological samples. Detection of the inhibition of PKA activity To investigate whether the GNP-based colorimetric assay can be further employed to assess the inhibition of PKs which is
Fig. 4 Analytical performance of the proposed colorimetric assay for the detection of PKA activity. (a) Photographs of the GNPs after mixing with a series of kinase reactions treated with different concentrations of PKA. Top panel, immediately after mixing; Bottom panel, 2 h later; (b) corresponding absorption spectra of the samples in the bottom panel of image (a); (c) the relationship between the absorbance at 520 nm and the PKA activity. The error bars represent the standard deviation of three replicates for each data point. Other conditions are the same as those described in the Experimental section.
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Fig. 5 Absorption spectra of the proposed colorimetric assay system for the detection of PKA activities in different types of MCF-7 cell lysates. (1), blank control without any cell lysate; (2), unstimulated cell lysate; (3), 10 μM forskolin/20 μM IBMX-stimulated cell lysate; (4), 10 μM forskolin/20 μM IBMX-stimulated cell lysate pre-mixed with 5 μM H-89. (inset) corresponding photographs. The total protein concentration for each type of MCF-7 cell lysate is fixed at 2 μg mL−1 and other conditions are all performed according to the standard assay procedures stated in the Experimental section.
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Fig. 6 The results of PKA inhibition study. (a) Photographs of the colorimetric assay system in the presence of different concentrations of H-89 (0–10 µM) by fixing PKA activity at 0.02 U µL−1; (b) The corresponding absorption spectra of the samples in the bottom panel of a; (c) the relationship between the absorbance at 520 nm and H-89 concentrations on the logarithm scale. The error bars represent the standard deviation of three replicates for each data point.
essential for the screening of potential small-molecule inhibitor drugs, a proof-of-concept inhibition study for PKA is performed in this study by using H-89, a well-known small molecule capable of inhibiting PKA activity, as a model inhibitor. The experiments are conducted following essentially the same procedures as the PKA assay, except for the pre-incubating of a fixed PKA concentration (0.02 U µL−1) with series dilutions of H-89 (0–10 µM). Fig. 6 exhibits the inhibitory results of H-89. It can be obviously observed that the deep red color and the absorbance of GNPs decrease gradually with increasing concentrations of H-89 because of the inhibition of PKA activity and thus the low levels of peptide phosphorylation, which may accelerate the crosslinking and coagulation of GNPs. The relationship between the absorbance at 520 nm and H-89 concentrations on the logarithm scale is plotted in Fig. 6c, from which the IC50 value is determined to be 102 nM, which agrees well with the literature values.10,37 Therefore, the GNP-based colorimetric assay is suitable for the inhibition study of PKs, and may also find potential applications in screening effective PK inhibitors as potential targeted drugs.
Conclusions In summary, we have developed a new colorimetric strategy for the detection of protein kinase activity based on the peptide phosphorylation-regulated crosslinking and aggregation of GNPs. This new kinase assay shows several distinct advantages. Firstly, the GNP-based colorimetric assay is extremely simple because neither expensive instruments nor sophisticated procedures are required. Only by simply incubating the protein kinase reaction sample with the STV-GNPs for 2 h can the color changes of GNPs be identified by the naked eye. Sec-
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ondly, for previously reported GNP crosslinking-based colorimetric kinase assays, two types of GNPs functionalized respectively with the substrate peptide and avidin (or antibodies) are required. The enzyme reaction on the surface of GNPs may be quite different from those performed in homogeneous physiological media. In this study, the GNPs only need to be functionalized with STV through a rather simple procedure and the kinase reaction is conducted under the optimal physiological conditions in homogeneous solution, which is more suitable for the study of enzyme–substrate specific interaction. Thirdly, as we know, the charge of a peptide can be exactly tuned by adding (+)- or (−)-charged amino acid residues to a kinase-specific core peptide motif. Therefore, by rationally tuning the net charges of other peptide substrates to be similar to that of the kemptide used in this work, the proposed strategy may be easily extended to the detection of different PKs. Moreover, despite its simple design, the sensitivity of the proposed assay is satisfactory and it has been successfully applied to the inhibition study and kinase detection in complex biosamples, suggesting that the GNP-based colorimetric assay may find potential applications for PK analysis in signal transduction pathways and kinaserelated drug discovery.
Acknowledgements This work was supported by the National Natural Science Foundation of China (21335005), the Fundamental Research Funds for the Central Universities (GK201402051 and GK201501003), the Natural Science Foundation of Shaanxi Province (2014JQ2058) and the Program for Innovative Research Team in Shaanxi Province (No. 2014KCT-28).
Notes and references 1 B. A. Gibson and W. Lee Kraus, Nat. Rev. Mol. Cell Biol., 2012, 13, 411–424. 2 C. Janke and J. C. Bulinski, Nat. Rev. Mol. Cell Biol., 2011, 12, 773–786. 3 G. Manning, D. B. Whyte, R. Martinez, T. Hunter and S. Sudarsanam, Science, 2002, 298, 1912–1934. 4 P. A. Futreal, A. Kasprzyk, E. Birney, J. C. Mullikin, R. Wooster and M. R. Stratton, Nature, 2001, 409, 850–852. 5 D. E. Kalume, H. Molina and A. Pandey, Curr. Opin. Chem. Biol., 2003, 7, 64–69. 6 C. Bertolotto, P. Abbe, T. J. Hemesath, K. Bille, D. E. Fisher, J. P. Ortonne and R. Ballotti, J. Cell Biol., 1998, 142, 827– 835. 7 K. Kerman, M. Chikae, S. Yamamura and E. Tamiya, Anal. Chim. Acta, 2007, 588, 26–33. 8 C. Wang, L. Wei, C. Yuan and K. C. Hwang, Anal. Chem., 2012, 84, 971–977. 9 K. Kerman and H. B. Kraatz, Biosens. Bioelectron., 2009, 24, 1484–1489.
This journal is © The Royal Society of Chemistry 2015
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Published on 24 June 2015. Downloaded by University of Connecticut on 07/07/2015 15:31:54.
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10 J. Bai, C. Liu, T. Yang, F. Wang and Z. Li, Chem. Commun., 2013, 49, 3887–3889. 11 J. Zhou, X. Xu, W. Liu, X. Liu, Z. Nie, M. Qing, L. Nie and S. Yao, Anal. Chem., 2013, 85, 5746–5754. 12 L. Wang, Y. Yang and C. Zhang, Anal. Chem., 2015, 87, 4696–4703. 13 W. Ren, C. Liu, S. Lian and Z. Li, Anal. Chem., 2013, 85, 10956–10961. 14 B. E. Turk, J. E. Hutti and L. C. Cantley, Nat. Protoc., 2006, 1, 375–379. 15 B. T. Houseman, J. H. Huh, S. J. Kron and M. Mrksich, Nat. Biotechnol., 2002, 20, 270–274. 16 X. Xu, J. Zhou, X. Liu, Z. Nie, M. Qing, M. Guo and S. Yao, Anal. Chem., 2012, 84, 4746–4753. 17 Y. P. Kim, E. Oh, Y. H. Oh, D. W. Moon, T. G. Lee and H. S. Kim, Angew. Chem., Int. Ed., 2007, 46, 6816– 6819. 18 Y. Yu, R. Anjum, K. Kubota, J. Rush, J. Villen and S. P. Gygi, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 11606–11611. 19 G. V. Maltzahn, D. H. Min, Y. Zhang, J. H. Park, T. J. Harris, M. Sailor and S. N. Bhatia, Adv. Mater., 2007, 19, 3579– 3583. 20 M. G. Shapiro, J. O. Szablowski, R. Langer and A. Jasanoff, J. Am. Chem. Soc., 2009, 131, 2484–2486. 21 Z. Zhao, X. Zhou and D. Xing, Biosens. Bioelectron., 2012, 31, 299–304. 22 Z. Fu, X. Zhou and D. Xing, Sens. Actuators, B, 2013, 182, 633–641. 23 Z. Fu, X. Zhou and D. Xing, Methods, 2013, 64, 260– 266.
This journal is © The Royal Society of Chemistry 2015
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24 Z. Wang, J. Lee, A. R. Cossins and M. Brust, Anal. Chem., 2005, 77, 5770–5774. 25 Z. Wang, R. Lévy, D. G. Fernig and M. Brust, J. Am. Chem. Soc., 2006, 128, 2214–2215. 26 S. Gupta, H. Andresen, J. E. Ghadiali and M. M. Stevens, Small, 2010, 6, 1509–1513. 27 J. Oishi, Y. Asami, T. Mori, J. H. Kang, M. Tanabe, T. Niidome and Y. Katayama, ChemBioChem, 2007, 8, 875– 879. 28 J. Oishi, Y. Asami, T. Mori, J. H. Kang, T. Niidome and Y. Katayama, Biomacromolecules, 2008, 9, 2301–2308. 29 D. Liu, W. Chen, K. Sun, K. Deng, W. Zhang, Z. Wang and X. Jiang, Angew. Chem., Int. Ed., 2011, 50, 4103–4107. 30 R. Jin, G. Wu, Z. Li, C. A. Mirkin and G. C. Schatz, J. Am. Chem. Soc., 2003, 125, 1643–1654. 31 Z. Li, Y. Wang, C. Liu and Y. Li, Anal. Chim. Acta, 2005, 551, 85–91. 32 J. Bai, Y. Zhao, Z. Wang, C. Liu, Y. Wang and Z. Li, Anal. Chem., 2013, 85, 4813–4821. 33 P. Miao, L. Ning, X. Li, P. Li and G. Li, Bioconjugate Chem., 2012, 23, 141–145. 34 X. Xu, Z. Nie, J. Chen, Y. Fu, W. Li, Q. Shen and S. Yao, Chem. Commun., 2009, 6946–6948. 35 C. Liu, L. Chang, H. Wang, J. Bai, W. Ren and Z. Li, Anal. Chem., 2014, 86, 6095–6102. 36 W. Yang, X. Lu, Y. Wang, S. Sun, C. Liu and Z. Li, Sens. Actuators, B, 2015, 210, 508–512. 37 S. Shiosaki, T. Nobori, T. Mori, R. Toita, Y. Nakamura, C. W. Kim, T. Yamamoto, T. Niidome and Y. Katayama, Chem. Commun., 2013, 49, 5592–5594.
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