Silver Nanoclusters-Based Fluorescence Assay of Protein Kinase

Chemistry and Chemical Engineering, Central South University, Changsha 410083, China. Anal. Chem. , 2015, 87 (1), pp 693–698. DOI: 10.1021/ac503...
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Silver Nanoclusters-Based Fluorescence Assay of Protein Kinase Activity and Inhibition Congcong Shen, Xiaodong Xia, Shengqiang Hu, Minghui Yang,* and Jianxiu Wang* College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China S Supporting Information *

ABSTRACT: A simple and sensitive fluorescence method for monitoring the activity and inhibition of protein kinase (PKA) has been developed using polycytosine oligonucleotide (dC12)templated silver nanoclusters (Ag NCs). Adenosine-5′triphosphate (ATP) was found to enhance the fluorescence of Ag NCs, while the hydrolysis of ATP to adenosine diphosphate (ADP) by PKA decreased the fluorescence of Ag NCs. Compared to the existing methods for kinase activity assay, the developed method does not involve phosphorylation of the substrate peptides, which significantly simplifies the detection procedures. The method exhibits high sensitivity, good selectivity, and wide linear range toward PKA detection. The inhibition effect of kinase inhibitor H-89 on the activity of PKA was also studied. The sensing protocol was also applied to the assay of drug-stimulated activation of PKA in HeLa cell lysates.

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Metal nanoclusters (MNCs) are a type of new luminescent probes that are composed of a few to roughly a hundred metal atoms.16,17 The MNCs exhibit strong photoluminescence, large Stokes shift, good photostability, and biocompatibility.18 Silver nanoclusters (Ag NCs) are one of the most widely used MNCs due to their facile synthesis and tunable emission properties.19 These NCs possess wide applications in biosensing and bioimaging.20,21 In this study, a simple Ag NCs-based fluorescence method for monitoring the PKA activity and inhibition has been developed. The fluorescence of polycytosine oligonucleotide (dC12)-templated Ag NCs was found to be significantly increased in the presence of ATP. However, the hydrolysis of ATP by PKA to ADP leads to the decrease of the fluorescence of Ag NCs. Upon incorporation of the PKA inhibitor H-89, the fluorescence of Ag NCs was restored. The feasibility of the method for the detection of PKA in cell lysates has been demonstrated. The sensing protocol thus holds great promise for monitoring of PKA activity and screening of kinase-related drugs.

rotein kinase (PKA) is capable of transferring phosphate groups from nucleoside triphosphates (usually adenosine5′-triphosphate, ATP) to specific amino acids, thus catalyzing the phosphorylation of proteins.1,2 Phosphorylation usually leads to the functional change of the target proteins, which plays a critical regulatory role in many fundamental biological processes, such as cellular signal communications, metabolic pathways, and neural activities.3,4 Aberrations in the expression of PKA can cause abnormal protein phosphorylation processes, and even diseases, such as diabetes, Alzheimer’s disease, and cancers.5,6 As a result, the development of sensitive and simple methods for monitoring PKA activity and inhibition is of great importance for clinical diagnosis, biomedical research, and kinase-related drug discovery.7,8 Typically, the assay of PKA activity utilized the substrate peptides that were specific to the target kinase.9,10 Upon phosphorylation, the interaction of the phosphopeptides with various signal probes was monitored.11,12 Various methods, such as electrochemistry, fluorescence, quartz crystal microbalance, and surface plasmon resonance have been proposed for assay of PKA activity.9,12−15 Among those, the fluorescencebased method is attractive due to its high sensitivity, simplicity, and high-throughput capability. In such assay, the reaction of the phosphopeptides with fluorescence probes leads to the change of the fluorescence properties. For example, the conjugates of peptide−graphene quantum dots (GQDs) were used as a signal probe for photoluminescence assay of PKA activity.14 The phosphorylated peptide−GQD conjugates were aggregated in the presence of Zr4+, thus quenching the fluorescence of GQDs. However, these methods are based on the phosphorylation of the substrate peptides, which are expensive, and require rather complex procedures. Therefore, simple, cost-effective, and label-free assays of PKA activity are much preferred. © XXXX American Chemical Society

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EXPERIMENTAL SECTION Materials and Apparatus. DNA with a sequence of 5′CCCCCCCCCCCC-3′ (dC12) was synthesized and purified by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). AgNO3 (99.99%) and NaBH4 (98%) were purchased from Sigma-Aldrich. Adenosine-5′-triphosphate (ATP) was obtained from Generay Biotech Co., Ltd. (Shanghai, China). Adenosine5′-diphosphate disodium salt (ADPNa2), forskolin, 3-isobutyl1-methylxantine (IBMX), and H-89 were acquired from Received: September 17, 2014 Accepted: December 9, 2014

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RESULTS AND DISCUSSION Using Ag NCs as a fluorescence probe, a simple method for assay of PKA activity that avoids the traditional peptide phosphorylation process has been developed. The schematic representation of the assay procedures is shown in Scheme 1.

Beyotime Institute of Biotechnology (Shanghai, China). Cyclic adenosine 3′,5′-monophosphate (cAMP)-dependent protein kinase (PKA) was obtained from New England Biolabs Inc. (Beverly, MA). A concentration of 10 mM phosphate-buffered saline (PBS, pH = 7.4) was used throughout the experiments. Other reagents were of analytical grade and used without further purification. All stock solutions were prepared with double-distilled water filtered by Milli-Q (Millipore, Billerica, MA). The fluorescence spectra were recorded on a fluorescence spectrophotometer (Hitachi, F-4600) using a 350 μL quartz cell. The UV−vis spectra were obtained using a UV−vis spectrophotometer (Shimadzu, UV-2450). Mass spectra were determined by LC−MS (Agilent 1100 liquid chromatograph, Thermo Finnigan mass spectrometer). Circular dichroism (CD) spectra were collected on a Jasco-815 spectropolarimeter (Jasco, Japan). Electron microscopic characterization was carried out on a Titan G2 60-300 transmission electron microscope (TEM, FEI, USA). X-ray photoelectron spectroscopy (XPS) spectra were recorded on an ESCALAB 250Xi spectrometer (Thermo Fisher, U.S.A.). Synthesis of Water-Soluble Ag NCs. DNA-stabilized Ag NCs were synthesized according to the previous report.22 Typically, 18 μL of 1 mM AgNO3 was mixed with 30 μL of 100 μM DNA (molar ratio of Ag+/DNA = 6:1) and stirred on a circular oscillator for 30 min. Next, 18 μL of freshly prepared 1 mM NaBH4 (molar ratio of Ag+/NaBH4 = 1:1) was quickly added into the mixture to reduce Ag+. The final solution was incubated at 4 °C in the dark for 3 h before use. Measurement of PKA Activity and Inhibition. To study ATP-enhanced fluorescence of Ag NCs, different concentrations of ATP were added into 50 μL of Ag NCs solution. After reaction for 10 min at room temperature, the fluorescence of the respective solutions was recorded. For measurement of PKA activity, different concentrations of PKA were mixed with 80 μM ATP and incubated at 37 °C for 1 h. The resulting solution was then added into 50 μL of Ag NCs and the fluorescence was recorded. For assay of PKA inhibition, 0.1 U/μL of PKA was mixed with 80 μM ATP and the kinase inhibitor H-89 with various concentrations for 1 h. The inhibition of PKA activity was then measured. Kinase Activity Assay in HeLa Cell Lysates. HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (1:1) supplemented with 10% fetal bovine serum. The cells were incubated under a humidified atmosphere containing 5% CO2 at 37 °C in a cell culture box for 3−4 days. The culture medium was then replaced by 4 mL of serum-free medium before stimulation. The 10 μL PBS solutions with various concentrations of forskolin and IBMX were added into the medium to activate the intracellular PKA. For comparison, 10 μL of PBS was added into the medium as a control. After stimulation for 30 min, the cultured cells were centrifuged to remove the nutrient solution and then broken with an ultrasonic liquid processor for 2 min. The cell lysates were centrifuged again and ready for use. To detect PKA activity in HeLa cell lysates, 80 μM ATP was added into the cell lysates and incubated at 37 °C for 60 min. The control experiments were conducted using unstimulated cell lysates or stimulated cell lysates in the presence of 10 μM kinase inhibitor H-89. Upon completion of the above procedures, 50 μL of the respective solutions was mixed with 50 μL Ag NCs for fluorescence detection.

Scheme 1. Schematic Representation of the Ag NCs-Based Fluorescence Assay of PKA Activity

The dC12-templated Ag NCs display fluorescence emission at 635 nm when excited at 565 nm (Figure 1A).23 However, the addition of ATP into the Ag NCs solution greatly enhanced the fluorescence of Ag NCs. It can be seen that the fluorescence intensity increased with the increase of ATP concentration, while the emission wavelength remained unchanged. The fluorescence enhancement reached a constant value at about 10 min, and a linear relationship between the fluorescence intensity and ATP concentrations ranging from 0.1 to 80 μM was obtained (inset of Figure 1A). At 80 μM, the fluorescence intensity of Ag NCs increased about 2.5 times. Further increase of the ATP concentration to 100 μM results in a slight decrease of the fluorescence intensity, which might be ascribed to the aggregation of Ag NCs induced by higher concentrations of ATP. Control experiment was also performed with the addition of 80 μM ADP (the hydrolysis product of ATP), and little fluorescence enhancement was attained (Figure 1B). A slight red-shift of about 5 nm was obtained with the addition of ADP, while no red-shift was observed in the case of ATP. Since ATP and ADP bear different negative charges, the red-shift might be ascribed to the change of the microenvironment of Ag NCs. The fluorescence enhancement with the addition of 80 μM ATP was about 15 times higher than that in the case of 80 μM ADP. The effect of other compounds, such as adenosine monophosphate (AMP), guanosine triphosphate (GTP), adenosine, cytidine triphosphate (CTP), and thymidine triphosphate (TTP) on the fluorescence of Ag NCs was also tested. The results indicated that 80 μM AMP and CTP caused a little decrease of the fluorescence of Ag NCs, while other compounds have no obvious influence on the fluorescence of Ag NCs (Supporting Information, Figure S1). In addition, about the salt effect on ATP-induced fluorescence enhancement, we found that the addition of 0.9% NaCl also resulted in a decrease of the fluorescence of Ag NCs (Supporting Information, Figure S2) To study the mechanism of fluorescence enhancement of Ag NCs with the addition of ATP, the Ag NCs in the absence and presence of ATP were characterized by different techniques. The Ag NCs exhibit two absorbance peaks at 442 and 550 nm (curve a, Figure 2), being in accordance with the electronic transitions for small silver clusters.23 The addition of 80 μM B

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Figure 1. (A) Fluorescence spectra of Ag NCs in the presence of different concentrations of ATP, from a to g: 0, 10, 20, 30, 40, 60, 80 μM. The inset shows the dependence of the fluorescence change on the concentration of ATP. The fluorescence change was denoted as the difference of the fluorescence intensity in the presence and absence of different concentrations of ATP. (B) Fluorescence spectra of Ag NCs in the absence (a) and presence (b) of 80 μM ADP.

of DNA−Ag NCs (Supporting Information, Figure S3).24 In the XPS spectra, the peak for Ag 3d5/2 shifted from 368.3 to 368.1 eV after the addition of ATP into Ag NCs (Supporting Information, Figure S4). On the other hand, the surface chemistry of Ag NCs was also studied by XPS.25 After the addition of ATP, the binding energy of Ag NCs at around 1137 eV was changed into two components (Supporting Information, Figure S4). One component may be originated from the inner Ag atoms and the other from the surface Ag atoms, indicating the capping of Ag NCs with ATP.26 In addition, the fluorescence lifetime of the Ag NCs is around 0.1 ns, which does not change after the addition of ATP.21 From the above characterization, we can conclude that the enhancement of fluorescence of Ag NCs was mainly due to the decreased size of Ag NCs after the addition of ATP. Similarly, the enhancement in the fluorescence of Ag NCs in the presence of guanine-rich DNA sequences and thiolated molecules has also been reported.27−29 After demonstrating the capability of ATP to enhance the fluorescence of Ag NCs, we used this method for PKA assay. Since PKA can hydrolyze ATP into ADP, the fluorescence decrease was then expected via incorporation of PKA. The hydrolysis of ATP by PKA was first characterized by LC−MS. For the mixture of PKA and ATP, the peak at m/z 426 from ADP was observed, which was confirmed by the sample containing only ADP (Supporting Information, Figure S5). These data prove the formation of ADP via hydrolysis of ATP by PKA. For fluorescence assay of PKA using Ag NCs, several control experiments were conducted. For example, the addition of PKA only into the Ag NCs did not result in fluorescence change (Supporting Information, Figure S6). No fluorescence change was also observed with the addition of denatured PKA (denatured at 50 °C) into the Ag NCs in the presence of ATP (Supporting Information, Figure S7). However, the incorporation of PKA into the mixture of Ag NCs and ATP results in a decrease in the fluorescence intensity (Supporting Information, Figure S7). The above results indicate that the hydrolysis of ATP could cause the fluorescence change of Ag NCs, thus providing the possibility for assay of PKA. The effect of ATP concentration on PKA assay was examined, and a concentration of 80 μM was selected for

Figure 2. UV−vis spectra of Ag NCs in the absence (a) and presence (b) of 80 μM ATP.

ATP resulted in an increase of the adsorption intensity (curve b, Figure 2). In addition, a slight red-shift of the 550 nm peak was observed. The TEM image of the Ag NCs indicated that the size of Ag NCs was not uniform with the presence of relatively large nanoparticles of size around 4−5 nm (Figure 3A). However, after the addition of ATP, it can be seen that the Ag NCs became more uniform and the size was decreased to around 2 nm (Figure 3B). The circular dichroism (CD) spectra show that the addition of ATP resulted in no structural changes

Figure 3. TEM images of Ag NCs in the absence (A) and presence (B) of 80 μM ATP. C

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prevented by select PKA inhibitors. H-89, a potential and cellpermeable PKA inhibitor, was used as a model system.31,32 A solution of 0.1 U/μL PKA was initially treated with various concentrations of H-89 for 1 h, and then the activity of the treated PKA was tested. As displayed in Figure 6A, with the increase of the concentrations of H-89, the fluorescence intensity of Ag NCs increased, which revealed the inhibition of PKA activity. When the concentration of H-89 reached 30 μM, the activity of PKA was totally inhibited and the fluorescence intensity of Ag NCs was close to that without PKA. A sigmoidal profile was attained when plotting the fluorescence intensity versus H-89 concentrations (Figure 6B). These results clearly indicate that the developed method is capable of screening kinase inhibitors in a simple and sensitive manner. Due to the important role of PKA in cell signaling and signal transduction, the activity of PKA in cells is highly regulated. Therefore, the detection of PKA activity in cell lysates is much important for monitoring the regulation of kinases in cell systems. It has been reported that the combination of forskolin (an activator of adenylyl cyclase) and IBMX (a phosphodiesterase inhibitor) can efficiently increase the intracellular levels of cAMP, leading to the activation of cAMP-dependent PKA.33 We then investigated whether the Ag NCs-based assay could be applied to the detection of PKA activity in cell lysates. HeLa cells were selected and treated with various concentrations of forskolin and IBMX for activation of PKA (the concentrations of forskolin and IBMX are shown in the inset of Figure 7A). The PKA activity in cell lysates was then evaluated by the proposed assay (Figure 7A). For cell lysates without drug stimulation (sample no. 0), the fluorescence of the mixture of Ag NCs and ATP remains almost unchanged, indicating that the method is largely free from the matrix effect of the cell lysates. However, the fluorescence intensity decreased with the addition of drug-stimulated HeLa cell lysates into the Ag NCs solution, and the fluorescence change increased with the increasing concentrations of forskolin/IBMX. The relationship between the fluorescence change and the drug concentrations (Figure 7B) indicates that the kinase activity in cell lysates initially displayed a quick increase with the increase of the drug concentrations, and then the increase was slowed down. With the addition of 10 μM H-89 into the stimulated cell lysates, the fluorescence of the Ag NCs was almost restored to that in the case of cell lysates without drug stimulation, again proving the inhibition effect of H-89 on the activity of PKA (Figure 7A). Thus, the activation of PKA in HeLa cells by drugs is easily determined by examining the fluorescence change of Ag NCs, providing a viable alternative for in vitro detection of cellular kinase activity.

higher sensitivity (Supporting Information, Figure S8). Under the optimized experimental conditions, the fluorescence intensity of Ag NCs decreased with the increase of PKA concentrations (Figure 4) and a linear relationship between the

Figure 4. (A) Fluorescence spectra of the mixture of Ag NCs and 80 μM ATP in the presence of different concentrations of PKA, from a to i: 0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2 U/μL. The inset is the calibration curve of the assay of PKA. The fluorescence change was denoted as the difference of the fluorescence intensity in the absence and presence of different concentrations of PKA.

fluorescence change and the logarithm of PKA concentrations ranging from 0.001 to 2 U/μL was attained (inset of Figure 4). The detection limit was estimated to be 0.5 mU/μL. Such a detection level is comparable with those of PKA assay based on rolling circle amplification and quantum dots amplification (0.5 and 0.47 mU/μL, respectively).30,31 Via the “mix and detect” format, the whole assay can be finished in approximately 1 h. The feasibility of the method for PKA assay was further demonstrated by examining the reproducibility and selectivity of the assay. The relative standard deviations were determined to be 1.2% and 2.1% for 0.1 and 1 U/μL PKA (n = 5), respectively, thus proving the reliability of the method. To evaluate the specificity of the assay, different proteins or enzymes, such as bovine serum albumin (BSA), glucose oxidase (GOx), and alcohol dehydrogenase (ADH), were tested (Figure 5). In comparison with that in the case of PKA, only slight fluorescence change was attained for BSA, GOx, and ADH. These results suggest that the proposed method possesses high selectivity to PKA assay. The proposed method can be applied to the screening of PKA inhibitors because the hydrolysis of ATP by PKA is

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CONCLUSIONS A simple, sensitive, and selective Ag NCs-based fluorescence method for assay of protein kinase activity and inhibition has been proposed. The detection was based on the ATP-induced fluorescence enhancement of Ag NCs and then the hydrolysis of ATP by PKA which lowered the fluorescence intensity. A select PKA inhibitor, H-89, was used as a model system for assay of PKA inhibition. The developed method was applied to the investigation of drug-induced PKA activation in HeLa cells, providing a promising means for screening of kinase-related drugs. The assay adopts a “mix and detect” format, which is simple and cost-effective. The proposed method avoids the

Figure 5. Sensitivity of the method for assay of 0.1 U/μL of PKA, GOx, ADH, and 2% BSA. D

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Figure 6. (A) Fluorescence spectra of Ag NCs in the presence of 0.1 U/μL PKA, 80 μM ATP, and H-89 with various concentrations. (B) Dose− response curve of inhibition of PKA activity by H-89.

Figure 7. (A) Fluorescence spectra recorded with the addition of the cell lysates stimulated by different concentrations of drugs (forskolin/IBMX) into the mixture of Ag NCs and 80 μM ATP. (B) The relationship between the fluorescence change and the concentrations of the added forskolin. (3) Zhou, X.; Xing, D.; Zhu, D.; Jia, L. Anal. Chem. 2008, 81, 255− 261. (4) Xu, X.; Zhou, J.; Liu, X.; Nie, Z.; Qing, M.; Guo, M.; Yao, S. Anal. Chem. 2012, 84, 4746−4753. (5) Shults, M. D.; Imperiali, B. J. Am. Chem. Soc. 2003, 125, 14248− 14249. (6) Wang, C.-L.; Wei, L.-Y.; Yuan, C.-J.; Hwang, K. C. Anal. Chem. 2012, 84, 971−977. (7) Patterson, H.; Nibbs, R.; McInnes, I.; Siebert, S. Clin. Exp. Immunol. 2014, 176, 1−10. (8) Proctor, A.; Herrera-Loeza, S. G.; Wang, Q.; Lawrence, D. S.; Yeh, J. J.; Allbritton, N. L. Anal. Chem. 2014, 86, 4573−4580. (9) Wang, Z. H.; Sun, N.; He, Y.; Liu, Y.; Li, J. H. Anal. Chem. 2014, 86, 6153−6159. (10) Zhou, J.; Xu, X.; Liu, W.; Liu, X.; Nie, Z.; Qing, M.; Nie, L.; Yao, S. Anal. Chem. 2013, 85, 5746−5754. (11) Li, T.; Liu, X.; Liu, D.; Wang, Z. Anal. Chem. 2013, 85, 7033− 7037. (12) Kerman, K.; Song, H.; Duncan, J. S.; Litchfield, D. W.; Kraatz, H.-B. Anal. Chem. 2008, 80, 9395−9401. (13) Xu, S.; Liu, Y.; Wang, T.; Li, J. Anal. Chem. 2010, 82, 9566− 9572. (14) Wang, Y.; Zhang, L.; Liang, R.-P.; Bai, J.-M.; Qiu, J.-D. Anal. Chem. 2013, 85, 9148−9155. (15) Yoshida, T.; Sato, M.; Ozawa, T.; Umezawa, Y. Anal. Chem. 2000, 72, 6−11. (16) Shang, L.; Dong, S. J.; Nienhaus, G. U. Nano Today 2011, 6, 401−418. (17) Xu, H. X.; Suslick, K. S. Adv. Mater. 2010, 22, 1078−1082. (18) Lu, Y. Z.; Chen, W. Chem. Soc. Rev. 2012, 41, 3594−3623. (19) Diez, I.; Ras, R. H. A. Nanoscale 2011, 3, 1963−1970.

traditional peptide phosphorylation protocols, serving as a good alternative for protein kinase activity assay.

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ASSOCIATED CONTENT

S Supporting Information *

Additional figures showing characterization of the fluorescence enhancement of Ag NCs by ATP, the hydrolysis of ATP by PKA, the detection of PKA activity, and the effect of ATP concentration on the sensitivity of PKA detection. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Phone: +86 731 88836356. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are thankful for the support of this work by the National Key Basic Research Program of China (2014CB744502) and the National Natural Science Foundation of China (nos. 21105128, 21375150, and 21175156).

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