Graphene Oxide–Peptide Nanocomplex as a Versatile Fluorescence

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Graphene Oxide−Peptide Nanocomplex as a Versatile Fluorescence Probe of Protein Kinase Activity Based on Phosphorylation Protection against Carboxypeptidase Digestion Jiang Zhou,†,§ Xiahong Xu,†,‡,§ Wei Liu,† Xin Liu,† Zhou Nie,*,† Meng Qing,† Lihua Nie,† and Shouzhuo Yao† †

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, China ‡ College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China S Supporting Information *

ABSTRACT: The research on complicated kinomics and kinase-target drug discovery requires the development of simple, cost-effective, and multiplex kinase assays. Herein, we propose a novel and versatile biosensing platform for the detection of protein kinase activity based on graphene oxide (GO)−peptide nanocomplex and phosphorylation-induced suppression of carboxypeptidase Y (CPY) cleavage. Kinasecatalyzed phosphorylation protects the fluorophore-labeled peptide probe against CPY digestion and induces the formation of a GO/peptide nanocomplex resulting in fluorescence quenching, while the nonphosphopeptide is degraded by CPY to release free fluorophore as well as restore fluorescence. This GO-based nanosensor has been successfully applied to sensitively detect two model kinases, casein kinase (CKII) and cAMP−dependent protein kinase (PKA) with low detection limits of 0.0833 mU/μL and 0.134 mU/μL, respectively. The feasibility of this GO-based sensor was further demonstrated by the assessment of kinase inhibition by staurosporine and H-89, in vitro kinase assay in cell lysates, and simultaneous detection of CKII and PKA activity. Moreover, the GO-based fluorescence anisotropy (FA) kinase assay has been also developed using GO as a FA signal amplifier. The proposed sensor is homogeneous, facile, universal, label-free, and applicable for multiplexed kinase assay, presenting a promising method for kinase-related biochemical fundamental research and inhibitor screening. nonredundant phosphorylation sites identified in ca. ∼18 000 proteins (www.phosphosite.org). However, to date, most of these phosphorylation sites are still functionally uncharacterized, and their relationships with the specifically responsible kinases remain unidentified. The consensus peptide sequence flanking the phosphorylatable residues of the substrate is crucial for the selectivity and specificity of the kinase recognition, which is important for the identification of the linking of phosphorylation sites and corresponding kinases. Based on the profile of substrate recognition sequence, Ser/Thr kinases can be classified into three main categories: basophilic kinases, proline-directed kinases, and acidophilic/phosphate directed kinases.9 Due to the big challenge in phosphoproteomics and kinomics to explore the complex network between vast substrate proteins and their cognate kinases, versatile methods

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ntracellular phosphorylation by protein kinase is the major signal transduction mechanism, which plays a pivotal role in various vital biological processes including cell cycle progression, cell differentiation, metabolism, gene expression, and apoptosis.1−3 It has been demonstrated that aberrant protein kinase activity or specificity is closely relevant to severe illnesses such as cancer, diabetes, Alzheimer’s disease, restenosis, immune deficiencies, endocrinological disorders, or cardiovascular diseases.4−8 For this reason, protein kinase regulation constitutes a crucial area in therapeutic development, and protein kinases have become one of the major therapeutic targets over the past 10 years. Indeed, it is estimated that over 25% of drug development efforts target the development of protein kinase inhibitors. Protein kinase catalyzes the transfer of the γ-phosphoryl from adenosine-5′-triphosphate (ATP) to a free hydroxyl group of serine, threonine, or tyrosine in a peptide or protein substrate. The protein kinase superfamily constitutes about 2% of the human genome and includes at least 500 kinds of kinases. These kinases are responsible for more than 170 000 © XXXX American Chemical Society

Received: February 1, 2013 Accepted: May 23, 2013

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phosphorylated peptide fragment binding on GO causing the quenching of fluorescence. Due to its phosphorylation-specific inhibition and residue-nonspecific cleavage, this CPY-dependent phosphorylation recognition presents a versatile strategy compatible with various kinases with different substrate sequence selectivity, which has been demonstrated by using casein kinase (CKII) and cAMP-dependent protein kinase (PKA) as typical models of acidophilic/phosphate directed kinase and basophilic kinase, respectively, in this study. Furthermore, the feasibility of this GO-based sensing platform has also been demonstrated in fluorescence anisotropy (FA) and multiplexed kinase assays. Because of its potency, simplicity, and versatility, this GO−peptide kinase nanosensor not only shows great potential in multiplexed kinase assay and inhibitor screening but also extends the GO-based biosensing applications into a new field.

to probe kinase activity compatible with various kinases and substrates are increasing in demand. Traditional methods for assessing kinase acitivities use [γ-32P]ATP where transfer of a radioactive γ-phosphoryl moiety from ATP to the substrate is quantified by scintillation counting.10,11 Radiometric methods are general and versatile but hampered by its complicated multisteps procedure and unhealthy radioactive waste. Current methods commonly rely on the use of specialized biological reagents such as phosphopeptide-recognized antibodies or a protein binding domain (e.g., SH2 and 14−3−3 domains).12−17 These methods are highly specific so that their suitable targets are restricted by the availability of the recognition protein. Other problems include the high cost and inconsistent batch-to-batch quality of these proteins. Recently, the self-reported fluorescent peptide probes by rational design have been reported as a promising technique to assay kinase activity.18−21 However, these methods require sophisticated synthesis and case-to-case screening from synthesized peptide candidates for specific kinases, making them less accessible for general laboratories. Hence, it is still a challenge to develop facile, cost-effective, and versatile protein kinase assays. Graphene oxide (GO), a single-atomic-layered two-dimensional carbon nanomaterial, is a promising material for use in biosensors due to its superb electrical, mechanical, and optical properties.22−25 Because of the efficient quenching effect of GO on the photoluminance of the fluorophore and the controllable adsorption property of biomacromolecules on the GO surface, GO has attracted increasing attention in fluorescence-based biosensing. Recently, increasing efforts have focused on the DNA-related biosensors to detect nucleic acids, proteins, ions, and small molecules by DNA recognition and GO−DNA interaction.26−30 By comparison, the application of GO− peptide nanocomposites in a fluorescence biosensor is much less explored, and only a few reports have appeared to date. Qu’s group first reported the ultrasensitive detection of cyclin A2 using GO and an FITC-labeled peptide probe.31 Ye et al. have comprehensively investigated the interactions between various amino acids and GO and developed a novel GO-based biosensing platform using peptides as probes to detect protease activity.32 Furthermore, some novel fluorescence protease assays have been successively developed for detecting the activity of matrix metalloproteinase, thrombin, and apoptosisrelated caspase relying on the nanocomposites of GO and peptides labeled by FITC or quantum dots as well as the GO− peptide covalent nanoconjugates.33−35 Notably, the available analytes of these methods are restricted in the proteases and peptide-binding proteins. However, to the best of our knowledge, so far no GO-based fluorescent detection platform has been employed for detecting activities of post-translational modification enzymes, such as protein kinases. Herein, we propose the proof-of-principle of a novel and versatile fluorescent peptide/GO platform for sensing the activity and inhibition of protein kinases. The measurement of protein kinase activity is based on the suppression effect of phosphorylation modification on carboxypeptidase Y (CPY) digestion. The CPY is a protease capable of hydrolyzing peptide bonds at the carboxyl-terminal (C-terminal) end of a protein or peptide.36 Without peptide phosphorylation, sequential exocleavage of fluorophore-labeled peptide by CPY causes the release of fluorophores and unaffected fluorescence by GO treatment. Oppositely, phosphorylation at the amino residue can block the digestion of carboxypepetidase37 and results in

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EXPERIMENTAL SECTION Materials and Reagents. Graphite oxide dispersion (0.5 mg/mL) was purchased from XF Nano (Nanjing, China). Peptides, FITC-RRRADDSDDDDD, FITCRRRADDpSDDDDD, FITC-LRRASLG, and rhodamine B (RB)-LRRASLG were purchased from GL Biochem (Shanghai, China). Casein kinase (CKII) and cAMP-dependent protein kinase (PKA, catalytic subunit) were obtained from New England Biolabs (Beverly, MA, USA). Carboxypeptidase Y, staurosporine, forskolin, and 3-isobutyl-1-methylxantine (IBMX) were purchased from Sigma-Aldrich (St. Louis, MO, USA). ATP was purchased from Generay Biotech (Shanghai, China). H-89 was obtained from EMD Biosciences (Calbiochem-Novabiochem. La Jolla, CA, USA). The improved Bradford protein assay dye reagent kit was obtained from Sangon (Shanghai, China). Other reagents including bovine serum albumin (BSA), Tris, glycerol, DTT, EDTA, and Triton X-100 were purchased from Bio Basic (Ontario, Canada). Human breast cancer cells (MCF-7) were obtained from the Cell Bank of Xiangya Central Experiment Laboratory of Central South University (Changsha, China). All solutions were prepared using ultrapure water (18.3 MΩ·cm) from the Millipore Milli-Q system. Fluorescence measurements were performed on a Synergy Mx multimode microplate reader (BioTek). All samples were illuminated at an excitation wavelength of 480 nm, and the fluorescence emission was scanned from 500 to 700 nm at 25 °C. Except for the specific cases mentioned in the text, the fluorescence intensity of all the spectra was measured as the emission intensity at the maximum emission peak. The fluorescence measurements were performed three times for each sample (n = 3). Fluorescence anisotropy measurements were carried out on a QuantaMasterTM 4 fluorescence spectrometer (PTI). Cell-breaking was conducted by a JY92IIN ultrasonic cell disruption system (Scientz, Ningbo, China). An atomic force microscope (AFM, PICO-5, Agilent) was used to characterize the morphology of graphene oxide combined with peptide. Circular dichroism (CD) spectral measurements were performed using a JASCO J-815 spectropolarimeter (Tokyo, Japan) and a 1 mm path length rectangular quartz cuvette. The spectra were obtained at room temperature over the wavelength range of 190−300 nm in 0.1 nm intervals. Fluorescence Quenching of FITC Molecules and FITCpeptide by Graphene Oxide. Six microliters of GO (45 μg/ mL) was added into 60 μL of FITC-peptide (10 μM in 20 mM Tris-HCl, 10 mM MgCl2, 50 mM KCl, pH 7.5) in one well of a B

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Scheme 1. Schematic Illustration of the Protein Kinase Activity Assay Based on GO/Peptide Nanocomplex and Suppression of Phosphorylation to Carboxypeptidase Y Cleavage

The detailed experimental descriptions about the detection of protein kinase inhibition and data analysis of fluorescence anisotropy are given in the Supporting Information. MCF-7 Cell Culture and Lysate Preparation. MCF-7 breast cancer cells (1 × 106 cells) were supplemented with 10% fetal bovine serum, MEM nonessential amino acid solution (0.1 mM), 1% insulin-transferrin-selenium-A supplement, penicillin (100 U/mL), streptomycin (100 mg/mL), and amphotericin B (0.25 mg/mL). The cells were incubated under a humidified atmosphere containing 5% CO2 at 37 °C. The culture medium was replaced with a serum-free medium 4 h before stimulation. Solutions of Fsk and IBMX with defined concentrations in DMSO were added to the medium to activate intracellular PKA. DMSO (equal volume) was added to the medium for unstimulated samples. Thirty minutes after stimulation, the cultured cells were removed by scraping and lysed in Dulbecco’s phosphate-buffered saline (D-PBS) by sonication (200 W) for 2 s × 60 times at an interval of 3 s for each time. The sample was centrifuged for 60 min at 22 000 rpm and 4 °C, and the resulting supernatants were transferred to freezing tubes and stored at −20 °C for experiments. The total protein concentration of cell lysate was assessed by using the improved Bradford protein assay dye reagent kit with BSA as the standard. Finally, an aliquot of the cell extract was mixed with Bradford reagent and detected as described above. Its total protein concentration was then calculated by reference to the calibration curve. In subsequent experiments for the kinase assay in cell lysate, the total protein concentration of cell lysate was diluted to 8 μg/mL.

96-well microplate and then mixed for 1 min with the multimode microplate reader. The fluoresence intensity was scanned from 500 to 700 nm at an excitation wavelength of 480 nm at 25 °C. To discuss the effect of the concentration of GO, the FITC-peptide (10 μM) and FITC (10 μM), respectively, were mixed with various concentrations of GO: 0, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 μg/mL. Hydrolyzation of Peptide by CPY. One hundred microliters of reaction solution containing CPY (3.29 U/mL) and peptide (10 μM) was incubated for 30 min at 25 °C. Then 60 μL of reacted solution was moved out and mixed with 6 μL of GO (45 μg/mL) for 1 min, and the fluorescent emission spectrum was scanned with the multimode microplate reader. A range of concentrations (0−13.16 U/mL) of CPY was employed to optimize the quantity of CPY. Detection of Protein Kinase Activity and Inhibition. The reaction solution for protein kinase CKII contained 10 μM FITC-peptide (FITC-RRRADDSDDDDD), 0.1 mM ATP, 1 U/μL CKII, 20 mM Tris-HCl, 10 mM MgCl2, and 50 mM KCl (pH 7.5), and the reaction solution for PKA contained 10 μM FITC-peptide (FITC-LRRASLG), 0.1 mM ATP, 1 U/μL PKA, 50 mM Tris-HCl, and 10 mM MgCl2 (pH 7.5). Both the reaction solutions (100 μL) were incubated for 1 h at 30 °C for phosphorylation and then hydrolyzed by 3.29 U/mL CPY for 30 min at 25 °C, respectively. Then 60 μL of hydrolyzed solution was moved out and mixed with 6 μL of GO (45 μg/ mL) for 1 min. The resulting mixture was measured by the fluorescence detection using a multimode microplate reader or by fluorescence anisotropy detection using a fluorescence spectrometer. C

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concentration. This result indicates the strong fluorescent quenching of FITC by GO due to the peptide-caused proximity. Because the consequence of CPY cleavage in our assay is peptide-bound and free FITC for phospho and nonphospho peptides, respectively, 45 μg/mL GO was chosen for the following kinase assay because it yields the maximal discrepancy in fluorescence between peptide-bound and free FITC. Suppression of Carboxypeptidase Y Digestion by Peptide Phosphorylation. It has been reported that phosphorylation of peptides greatly suppresses their proteolytic degradation by endoproteases and exopeptidases, probably due to steric hindrance of the bulky phosphate group to substrateenzyme interaction.37−39 Thus, protein kinase-catalyzed phosphorylation of the peptide can be monitored by the dramatically increased resistance of phorphorylated peptide to protease cleavage. Considering the significant difference in adsorption ability on GO between peptide-bound FITC and free FITC, we chose CPY, a presentative exopeptidase, rather than endoprotease in this assay because it is capable of thorough and nonspecific degradation of the whole peptide to release FITC. To demonstrate that CPY can discriminate the phosphopeptides from nonphospho counterparts, we used GO to investigate the digesting effect of CPY on the S-pep and Ppep. As shown in Figure 1, after the S-pep was treated by 3.29

RESULTS AND DISCUSSION The Detection Mechanism of GO−Peptide Nanobiosensor for Kinase Activity Assay. GO has been regarded as a suitable matrix to load peptides through the stacking interactions of its largely hydrophobic basal plane with aromatic and hydrophobic peptide residues and the electrostatic interactions of its ionizable edges with charged peptidic residues. Scheme 1 shows the mechanism of the digestive suppression-based peptide-GO biosensor for kinase activity assay proposed in this work. Casein kinase (CKII) is an important protein kinase that was found to phosphorylate more than 160 different proteins. As a typical acidophilic/phosphate directed kinase, CKII prefers carboxylic acid residues in its consensus sequence. Carboxypeptidase Y was used to discriminate between the phosphorylated and unphosphorylated substrate peptides. Without kinase treatment, CPY sequentially cleaves the FITC-labeled substrate peptide (Spep) of CKII, FITC-RRRADDSDDDDD, from the carboxyl end and finally releases the FITC bound at the amino end, inducing negligible fluorescence quenching by GO due to free FITC without peptide conjugation. The phosphorylated peptide (P-pep, FITC-RRRADDpSDDDDD) catalyzed by CKII in the presence of ATP, however, is resistant to digestion by the protease CPY and would readily bind to GO, resulting in the immediate fluorescence quenching of P-pep. Thus, the fluorescence intensity measured in the assay is inversely correlated with kinase CKII activity. Moreover, to prove the versatility of this assay, the feasibility of this method for PKA, a typical basophilic kinase which preferentially phosphorylates substrates with basic residues, was also tested by using PKAspecific substrate FITC-LRRASLG. Characterization of the Fluorescent Properties of Peptide Binding on GO. Since the fluorescence quenching by peptide binding on GO is the precondition of this assay, at first we characterized and optimized the adsorption effect of the substrate peptide (S-pep) on GO. AFM was adopted to visually examine the binding of peptide and GO. Figure S1A shows highly dispersed pieces of GO with a lateral width of 200−700 nm and a thickness of 1 ± 0.2 nm, which matches well with the reported apparent thickness of GO.27 The peptide/GO composite (Figure S1B) exhibits dispersed piece-like structures with an increased thickness of 2 ± 0.5 nm, indicating that the adsorbed peptide layer is formed on GO as expected. The adsorption of S-pep on GO was further assessed by a CD experiment. The GO/S-pep complex was prepared by centrifugation to dispose unbound S-pep. Both the S-pep and GO/S-pep complex exhibit similar CD spectra with a conspicuous negative peak at 197 nm and a positive broad peak around 216 nm (Figure S2), which is the signature of a random coil/unfolded peptide structure. This suggests that the S-pep was adsorbed on GO without a remarkable conformation change. It has been reported that the fluorescence emission of dye-labeled peptide is quenched due to the energy-transfer to the GO when peptides are adsorbed on the GO surface.32 The effect of GO concentration on the fluorescence of FITC-labeled peptide (FITC-RRRADDSDDDDD, the S-pep of the CKII) has been investigated. As shown in Figure S3, when the concentration of GO increased, the fluorescent intensity of peptide-conjugated FITC at 520 nm dramatically decreased even to 15% of its initial intensity at a concentration of GO of 50 μg/mL; on the contrary, the free FITC molecules retained 90% of the initial fluorescent emission at the same

Figure 1. Fluorescence emission spectra of (1) 10 μM FITC-labeled substrate peptide (S-pep) and (2) 10 μM FITC-labeled phosphorylated peptide (P-pep) mixed with 45 μg/mL of the GO complex after incubation with 3.29 U/mL CPY for 30 min, (3) S-pep, or (4) P-pep mixed with the GO complex without digestion by CPY.

U/mL CPY for 30 min and then mixed with GO, a minor change of fluorescence emission took place compared to its initial emission, indicating that effective digestion of the peptide by CPY yielded free FITC insusceptible to GO quenching. However, the P-pep treated by CPY and GO largely decreased the fluorescence emission due to the fluorescence quenching of FITC by GO assisted by the binding of peptide, thus demonstrating the effective resistance of peptide phosphorylation to the CPY digestion. The concentration of CPY has been optimized for full digestion of 10 μM S-pep. As shown in Figure S4, the fluorescence intensity of obtained peptide increased along with the increase of the CPY concentration until it reached 3.29 U/ mL, followed by a minor decrease at higher CPY concentrations. Thus, we chose 3.29 U/mL of CPY for the following digestive experiments. D

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Detection of the Activity of CKII Kinase by Carboxypeptidase Y Digestion. On the basis of the phosphorylation-mediated resistance to proteolytic degradation and GO−peptide fluorescent sensing platform, we expected to develop a novel and potent analysis protocol for detecting protein kinase activity. FITC-labeled S-pep was phosphorylated by its specific protein kinase CKII and then sequentially treated by CPY and GO. The resulting fluorescence emission spectra are shown in Figure 2. After phosphorylation by CKII, the

Figure 2. Fluorescence detection of CKII kinase activity by the GO/ peptide system. (1) 10 μM S-pep was phosphorylated by 1 U/μL CKII in the presence of 0.1 mM ATP, (2) 1 without ATP, (3) 1 with the inhibitor staurosporine, 5 μM, (4) 10 μM S-pep incubated with control protein kinase PKA (1 U/μL) and ATP, (5) 10 μM control peptide (kemptide) incubated with 1 U/μL CKII and ATP. Figure 3. (A) Fluorescence emission spectra of GO/peptide system in response to different concentrations of CKII. (B) Fluorescence response of the GO/peptide system as a function of the concentration of CKII (from 0 to 833.33 mU/μL).

fluorescence intensity of the FITC-labeled peptide was decreased by binding with GO which caused a dramatic quenching through efficient energy-transfer from FITC to GO (spectrum 1). By contrast, high intensity remained when phosphorylation was carried out by CKII without the coreactant ATP (spectrum 2) or with 5 μM staurosporine, a broad-spectrum inhibitor of protein kinase (spectrum 3). In the presence of control protein kinase PKA, no obvious fluorescence quenching was observed (spectrum 4). A similar response was obtained using FITC-labeled kemptide, the specific substrate of PKA, incubated with CKII (spectrum 5). These results indicated that the fluorescence signal of this peptide/GO system can specifically and selectively respond to the occurrence of phosphorylation, demonstrating the feasibility of this assay. The detection of CKII activity was quantitatively investigated by incubation of the S-pep with different amounts of CKII (0− 833.33 mU/μL) in the TBS buffer (20 mM Tris-HCl, 10 mM MgCl2, 50 mM KCl, pH 7.5) at 30 °C for 1 h. After being digested by CPY and bound to GO, the relative fluorescence signals were recorded by microplate reader. The fluorescence intensity decreased along with the increase of CKII concentration (Figure 3A), since the higher the phosphorylation degree, the less the peptide digestion, the more the GObound phosphorylated peptide, and the less the fluorescence intensity. The logarithmic plot of the fluorescence intensity versus the CKII concentration indicated an EC50 value (enzyme concentration at which 50% substrate is converted) of 8.80 mU/μL with a minimum detectable concentration of 0.0833 mU/μL (Figure 3B). This clearly implied that the proposed protocol is competent for the CKII activity analysis.

Fluorescence Anisotropy Detection of Kinase Activity Using CPY-assisted GO−peptide Biosensor. The fluorescence anisotropy (FA) measurement provides the rotation information of a fluorophore in its microenvironment, which is closely related to the size, shape, and interaction process of the fluorophore.40,41 The remarkable difference of binding ability on GO of free FITC and peptide-conjugated FITC could be sensitively reflected by FA because of the significant molecular weight increase after attachment on the GO surface. Hence, FA has been implemented to further investigate the phosphorylation-mediated peptide−GO interaction and corresponding kinase activity. As shown in Figure 4A, the original FA value of FITC−peptide is 0.0514, and it decreases to 0.0313 after CPY cleavage, implying the liberation of free FITC. However, the addition of GO causes a significant FA value increase of FITC− peptide to 0.3314, indicating that the rotational diffusion of labeled FITC is enormously hampered by the attachment of peptide on GO. After the GO/peptide sensor underwent the CKII phosphorylation and sequential CPY treatment, the FA value remained high, though a bit smaller than that of the GO/ peptide nanocomposite, probably due to a relatively weak interaction resulting from the degradation of the upstream part of the phosphorylation site, and a 6.48-fold increase of the FA value was observed compared with free FITC. This indicates that a large dynamic range can be obtained by FA measurement using this GO/peptide sensor. Further quantitative measurements were conducted using different concentrations of CKII, E

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the other hand, we also estimated the inhibiting capability of H89. As shown in Figure S5B, we found a similar increase of fluorescence intensity in the concentration range of H-89 from 0 to 200 μM and obtained the IC50 of 1.18 μM, which is comparable to that reported.43 Hence, the proposed protocol is feasible in the analysis and screening of the small molecule inhibitors. Application of GO−Peptide Sensor in Detection of PKA Activity. To demonstrate the generality of our assay, we applied the peptide/GO system to detect protein kinase PKA, a presentative basophilic Ser/Thr kinase. Because PKA plays a key role in abnormal proliferation of cancer cells, it is hyperactivated in many cancer cells or tissues (such as melanoma, lung, or breast cancer). Therefore, monitoring of PKA activity is considered to be useful for cancer diagnosis.44−47 A PKA-specific substrate peptide (LRRASLG, kemptide) labeled by FITC has been exploited to realize a sensitive PKA analysis. As shown in Figure S6, the fluorescence intensity of the peptide/GO system obviously decreased as expected upon phosphorylation by PKA (spectrum 1), but a high fluorescence signal remained in the absence of ATP (spectrum 2) or PKA (spectrum 4) and the presence of inhibitor staurosporine (spectrum 3). Correspondingly, the FA value of CPY-digested peptide in the presence of GO is very low, while it increases 9.42-fold upon PKA phosphorylation (Figure S7). All of these validated that PKA-catalyzed phosphorylation could effectively hinder the digestion of CPY and lead to the formation of a peptide/GO nanocomposite. Quantitative analysis of PKA activity was conducted using a variety of PKA samples with different concentrations (Figure 5A). The fluorescence at 520 nm dynamically increased with the increasing PKA concentration in the range from 0 to 1000 mU/μL. The EC50 value was determined to be 14.52 mU/μL, and the detection limit of PKA was 0.134 mU/μL estimated from 3 (Sb/m), where Sb is the standard deviation of the fluorescence signal in the absence of PKA (n = 10) and m is the slope of the analytical curve in the linear range. Moreover, the fluorescence anisotropy (FA) measurement was also applied to detect PKA activity because of its significant dynamic range. Compared with fluorescence detection, the FA detection by this GO−peptide sensor is more sensitive and can detect the activity of a very low amount of PKA with an EC50 of 39.54 mU/μL and a detection limit of 0.0783 mU/μL (Figure 5C), which is much lower than that of many previously reported kinase assays.48−50 In addition, the inhibition of PKA by the broad-spectrum inhibitors staurosporine and H-89 was also evaluated by this method. As shown in Figure S8, IC50 values of 34.03 nM and 43.58 nM were obtained for staurosporine and H-89, respectively, which well agree with the previously reported ones.51,52 The lower IC50 value of H-89 for PKA compared with that for CKII implied that H-89 is much more potent for PKA than CKII, which is in accordance with previously reported results.43 Therefore, the application of a peptide/GO system in the detection of PKA activity has been successfully achieved, indicating that the phosphorylation suppression of CPY digestion incorporated with GO−peptide interaction is a versatile mechanism for kinase activity sensing. Detection of Protein Kinase Activity in Cancer Cell Lysate. Protein kinases are important regulators in various cell signal pathways, and the switch of their activity modulates a series of fundamental cell events, such as transcription, apoptosis, and differentiation. The analysis method of protein kinase activity in a cytosolic environment is essential to

Figure 4. (A) Fluorescence anisotropy values of (1) S-pep and (2) Spep mixed with GO, (3) S-pep treated by 3.29 U/mL CPY for 30 min and then mixed with GO, and (4) S-pep phosphorylated by 1 U/μL CKII and then treated by CPY and GO. (B) The FA value change of the GO/peptide system as a function of the CKII concentration (from 0 to 833.33 mU/μL).

and the results are plotted in Figure 4B. The FA value of the sensor increases with the CKII concentration from 0 to 833.33 mU/μL, and the minimum detectable concentration of CKII was 0.0833 mU/μL with an EC50 of 7.84 mU/μL. Therefore, this GO-based biosensor is potent for the fluorescence anisotropy assay of kinase activity. Screening of Inhibitors Based on Resisted Carboxypeptidase Y Digestion by Phosphorylation. The screening and analysis of small molecule inhibitors is of high importance in the development and therapeutic application of kinase-targeted anticancer medicine. We examined whether the proposed assay is applicable for detecting small molecule inhibitors using staurosporine and H-89, two general inhibitors for the protein kinase family, as examples. Briefly, a series of concentrations of inhibitors was added into the CKII reaction solution before the phosphorylation. After the CPY digestion and binding of GO, the fluorescence signals were collected, as shown in Figure S5A. We found that the fluorescence obviously increased along with the increase of the staurosporine concentrations (0−20 μM) and reached the maximum at 5 μM. The IC50 of staurosporine (the concentration at the 50% inhibiting) was 94 nM, estimated from the plot of fluorescence intensity versus the logarithmic concentration of staurosporine, which is in agreement with the previously reported value.42 On F

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thus activate PKA. Hence, we activated varying degrees of PKA activities through adding different concentrations of forskolin and IBMX to stimulate MCF-7 cell and then lysated the activated cells and obtained the supernates for detections. Afterward, FITC-kemptide was sequentially exposed to the lysates with different activating levels for phosphorylation, digested by CPY, and bound to GO. The fluorescence signals were recorded before and after the binding to GO, respectively. Accordingly, the activity of PKA was estimated by the fluorescent quenching calculated by (F0 − F)/F0, where F0 and F were the fluorescence intensity before and after the binding to GO, respectively. As shown in Figure 6, we found

Figure 6. Fluorescence quenching signal of the GO/peptide system in response to different PKA activation levels in cell lysates induced by different concentrations of activators forskolin/IBMX. The inset shows the concentration of the activators for cell lysate samples from 1 to 6.

the fluorescent quenching gradually increased along with the increase of the concentration of activators until sample 5 with CFsk = 10 μM and CIBMX = 20 μM. The fluorescence response induced by activated cell lysate could be completely inhibited by inhibitor H-89 (Figure S9), demonstrating that this response is directly derived from kinase activity. Therefore, the activation of kinase activity in cell lysate can be efficiently detected by our GO−peptide biosensor, indicating that the selectivity and specificity of this sensor is competent in a complex cytoplasmic environment. Simultaneous Detection of CKII and PKA Activity by the GO-Based Kinase Assay. It has been demonstrated that GO possesses a high quenching efficiency to various fluorescence labels; thus the proposed method is expected to be a versatile platform for simultaneous detection of multiple kinases. To validate this idea, we used this GO-based method to simultaneously probe CKII and PKA activity using two cognate substrate peptide probes labeled by FITC and RB, respectively. Fluorescence spectra of the sensors exposed to the mixture of two peptides before and after kinase-catalyzed phosphorylation were separately obtained from the FITC detection window (λex = 480 nm, emission spectra from 500 to 630 nm) and RB detection window (λex = 550 nm, emission spectra from 570 to 650 nm), respectively. As shown in Figure 7, the typical signaloff response of the FITC-labeled probe to CKII activity in the wavelength region from 500 to 630 nm was not interfered with by the PKA activity and its RB-tagged probe (A) and vice versa (B). Moreover, the FA measurement using the developed sensor was also feasible for two kinase assays via respective

Figure 5. (A) Fluorescence emission spectra of the GO/peptide system in response to different concentrations of PKA. (B) Fluorescence response of the GO/peptide system as a function of concentration of PKA (from 0 to 1000 mU/μL). (C) The FA value change of the GO/peptide system as a function of concentration of PKA (from 0 to 1000 mU/μL).

understanding the function of kinases in cell signal transduction. Here, the proposed method has been implemented to investigate the kinase activity in MCF-7 breast cancer cell lysate. Several reports have proved the PKA activity can be efficiently up-regulated by the external activating reagents of forskolin and IBMX.53,54 The reason is that the forskolin as an adenosine acid cyclization enzyme activator and IBMX as a phosphodiesterase inhibitor can largely increase the intracellular level of cyclic adenosine monophosphate (cAMP) and G

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for phosphorylation recognition is much more cost-effective, stable, and versatile than phosphorylation-specific antibodies or protein domains, and the materials used in this method are easily accessible by general laboratories without sophistical modification and synthesis. Moreover, this sensor broadened the territory of the biosensing applications of graphene oxide into the protein post-translational modification research. Because the proposed approach is applicable to multiwell assay, multiplex kinase assay, and cell lysate detection, our GO−peptide kinase activity biosensor shows great potential in high-throughput screening for kinase-targeted drug discovery and kinase analysis in cell signal pathways.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information including some experimental detail and extensive figures as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86-731-88821626. Fax: +86-731-88821848. E-mail: [email protected]. Author Contributions §

These two authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Basic Research Program of China (973 Program, Nos. 2009CB421601 and 2011CB911002), the National Natural Science Foundation of China (Nos. 21222507 and 21175036 to Z. N., 21075031 to S.Z.Y., and 21205106 to X.H.X), the Foundation for Innovative Research Groups of NSFC (Grant 21221003), the Program for New Century Excellent Talents in University (NCET-10-0366), and the Ph.D. Programs Foundation of the Ministry of Education of China (No. 20120161110025).

Figure 7. Simultaneous detection of CKII and PKA activity by the GO/peptide system using FITC-labeled CKII S-pep and RB-labeled PKA S-pep. Fluorescence spectra for the mixture of GO and two kinds of S-pep without (a) or with (b) treatments by two cognate kinases. (A) FITC detection window (excited at 480 nm) and (B) the rhodamine B detection window (excited at 550 nm). The inset shows corresponding fluorescence anisotropy responses.

measurement of FA values of the FITC emission at 520 nm (excitation at 480 nm, inset of A) and RB emission at 580 nm (excitation at 550 nm, inset of B). Therefore, these results implied that the developed approach provides a good prospect for multiplex in vitro kinase assays.

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CONCLUSIONS In summary, a sensitive and versatile fluorescent peptide/GO system for probing the activity and inhibition of protein kinases has been developed. The sensing process of this kinase activity sensor composed by a novel kinase recognition strategy relied on phosphorylation protection against carboxypeptidase Y digestion and a GO−peptide interaction-based signal transduction. The versatility of this system has been proved by successful detection of two presentative kinases, CKII and PKA, with different consensus substrate sequences, which is attributed to the nonspecific cleavage of CPY and efficient adsorption capacity of GO to the peptide. Besides the fluorescence detection, this method also presents a potent technique for fluorescence anisotropy (FA) kinase assay due to its large dynamic range and ultrahigh sensitivity derived from significant enhancement of molecular weight by GO binding. Compared with the existing kinase activity assays, using CPY H

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