Biosensors and Bioelectronics 63 (2015) 26–32
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A sensitive electrochemical biosensor for detection of protein kinase A activity and inhibitors based on Phos-tag and enzymatic signal amplification Huanshun Yin, Mo Wang, Bingchen Li, Zhiqing Yang, Yunlei Zhou n, Shiyun Ai n College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 20 April 2014 Received in revised form 26 June 2014 Accepted 8 July 2014 Available online 11 July 2014
A simple, highly sensitive and selective electrochemical assay is developed for the detection of protein kinase A (PKA) activity based on the specific recognition utility of Phos-tag for kinase-induced phosphopeptides and enzymatic signal amplification. When the substrate peptide was phosphorylated by PKA reaction, they could specifically bind with Phos-tag-biotin in the presence of Zn2 þ through the formation of a specific noncovalent complex with the phosphomonoester dianion in phosphorylated peptides. Through the further specific interaction between biotin and avidin, avidin functionalized horseradish peroxidase (HRP) can be captured on the electrode surface. Under the catalytic effect of HRP, a sensitive electrochemical signal for benzoquinone was obtained, which was related to PKA activity. Under the optimal experiment conditions, the proposed electrochemical method presented dynamic range from 0.5 to 25 unit/mL with low detection limit of 0.15 unit/mL. This new detection strategy was also successfully applied to analyze the inhibition effect of inhibitors (ellagic acid and H-89) on PKA activity and monitored the PKA activity in cell lysates. Therefore, this Phos-tag-based electrochemical assay offers an alternative platform for PKA activity assay and inhibitor screening, and thus it might be a valuable tool for development of targeted therapy and clinical diagnosis. & 2014 Elsevier B.V. All rights reserved.
Keywords: Protein kinase A Phos-tag-biotin Enzymatic signal amplification Elelctrochemical biosensor Inhibitors
1. Introduction Protein kinases can catalyze the phosphorylation of serine, threonine and tyrosine residues in peptides and proteins by transferring phosphate groups from adenosine-triphosphate (ATP) to amino acids, which play critical roles in intracellular signal transduction and the regulation of cellular functions, such as metabolism, gene expression, and apoptosis, cell cycle progression and differentiation (Cohen, 2002; Kalume et al., 2003; Manning et al., 2002). More importantly, aberrant expression of protein kinases has been involved in several diseases including cancer (Griner and Kazanietz, 2007), HIV (Critchfield et al., 1997), and Alzheimer's disease (Flajolet et al., 2007). The quantitative analysis of kinases and their potential inhibitors is not only necessary for basic biology to clarify molecular mechanisms of signal transduction but also valuable for protein kinase-targeted drug discovery and therapy. Therefore, protein kinases have been regarded as the major targets for drug discovery (Zhang et al., 2009) and as biomarkers for cancer diagnosis (Kang et al., 2009). Based on it, there is an increase in demand to develop simple and n
Corresponding authors. Tel.: þ 86 538 8249248; fax: þ86 538 8242251. E-mail addresses: [email protected] (Y. Zhou), [email protected] (S. Ai).
http://dx.doi.org/10.1016/j.bios.2014.07.016 0956-5663/& 2014 Elsevier B.V. All rights reserved.
rapid detection methods for protein kinase activity, especially in cells or cellular lysates. The generally used techniques for the measurement of protein kinase activities mostly relied on radioisotopic methods which used radioactive ATP for monitoring phosphorylation reactions (Hayslett et al., 1995; Lehel et al., 1997). Although these methods could sensitively detect protein kinase activity, they depended on specific instruments and suffered from a high risk of radioactive contamination. To eliminate the problems that are associated with radioactive methods, many other methods have been developed, such as resonance light scattering (Li et al., 2013; Wang et al., 2005), colorimetric assay (Wang et al., 2006), fluorescence method (Bai et al., 2013; Xu et al., 2011; Zhou et al., 2013), photoluminescent (Wang et al., 2013), electrochemiluminescence (Chen et al., 2013; S. Xu et al., 2012; Zhao et al., 2012), quantitative mass spectrometry (Ji et al., 2012), Raman spectroscopic assay (Li et al., 2009), surface plasmon resonance imaging technique (Inamori et al., 2005). However, these methods have the disadvantages of expensive and complicated instruments, sophisticated operating procedures, time-consuming, tedious sample treatment. Therefore, the development of sensitive, reliable, and in-expensive method for protein kinase assays and inhibitor screening still remains a great challenge.
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Electrochemical methods have been demonstrated to be simpler and more cost-effective than previous methods for determining the activity of protein kinases (Chiku et al., 2010; Freeman et al., 2007; Ji et al., 2009; Kerman and Kraatz, 2009; Li et al., 2014; Song et al., 2008; Wang et al., 2012, 2011, 2010; Xu et al., 2009). One of the key factors for electrochemical assay of protein kinase activity based on the catalytic phosphorylation of substrate peptide is the effective recognition of phosphate group. Recently, a kind of commercially specific phosphate-binding reagent, named as Phos-tag, has attracted more attentions on its application in protein phosphorylation detection and protein kinase activity assay (Kinoshita et al., 2009, 2013, 2012). In the presence of Zn2 þ or Mn2 þ , the Phos-tag can form a specific noncovalent complex with the phosphomonoester dianion in phosphorylated peptides or proteins containing phospho-Ser, phospho-Thr, phospho-Tyr, and phospho-His residues at neutral pH (Kinoshita and Kinoshita-Kikuta, 2011). Though Phos-tag shows great potential in protein kinase activity assay, the electrochemical methods based on phos-tag has yet been reported until now. Herein, we propose the proof-of-principle of a novel and versatile electrochemical sensing platform for assaying the activity and inhibition of protein kinases A (PKA), which is based on its catalytic effect on substrate peptide and the phosphate group recognition ability of Phos-tag. The detection mechanism was shown in Scheme 1. With the peptide phosphorylation catalyzed by PKA, the biotin functionalized Phos-tag (Phos-tag-biotin) could be captured on the electrode surface through the specific recognition effect of Phos-tag towards phosphate group. Then, avidin functionalized horseradish peroxidase (avidin-HRP) could be further modified on the electrode surface through the immunoreaction between avidin and biotin, and the HRP molecule could catalyze H2O2 to chemically oxidize hydroquinone, which would result in a strong electrochemical reduction current of the oxidative product of benzoquinone. Because this response is related to the phosphorylation level cayalyzed by PKA, this electrochemical signal was used to detect the PKA activity. It can also provide a simple, sensitive, selective, and universal platform for other kinases activity assay. Moreover, the developed method is also successfully applied to detect PKA activity in cell lysates.
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2. Experimental 2.1. Reagents The substrate peptide (kemptide, H-CGGALRRASLG-NH2), the control peptide (H-CGGALRRAALG-NH2), avidin-HRP, adenosine 5′-triphosphate (ATP) disodium salt hydrate and the modified Bradford protein assay kit were purchased from Sangon Biotech (Shanghai) Co., Ltd. Mercaptopropionic acid (MPA) was purchased from Fluka. cAMP-dependent protein kinase A (PKA) catalytic subunit was purchased from New England Biolabs Ltd. (Beverly, MA). Biotinylated Phos-tag™ was obtained from Wako Pure Chemical Industries, Ltd. (Japan). Tris(2-carboxyethyl) phosphine hydrochloride (TCEP), tris(hydroxymethyl)aminomethane (Tris), EDTA, ellagic acid and chloroauric acid (HAuCl4) were purchased from Aladdin (shanghai, China). H-89 was obtained from EMD Millipore Corporation (Billerica, MA, USA). The buffer solutions employed in this study are as follows. PKA storage buffer, 20 mM Tris–HCl, 50 mM NaCl, 1 mM EDTA, 2.0 mM DTT and 10% glycerol, pH 7.5. PKA reaction buffer, 50 mM Tris–HCl, 70 μM ATP, 10 mM MgCl2, pH 7.5. Phos-tag-biotin reaction buffer, 10 mM Tris–HCl (pH 7.0) containing 0.1 M NaCl, 0.1% Tween-20, 0.4 mM Zn(NO3)2. avidin-HRP reaction buffer, 10 mM PBS (pH 7.5). Washing buffer, 10 mM Tris–HCl (pH 7.5) containing 0.05% tween20. Cell lysis buffer, 10 mM PBS (pH 7.4) containing 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2, 2.0 mM DTT, 1 mM sodium orthovanadate, 80 mM b-glycerophosphate, 3.0 μg/mL pepstatin A, 5.0 μg/ mL aprotinin, and 1.0 mM phenylmethylsulfonyl fluoride (PMSF). All reagents were analytically pure grade. All of the solution and redistilled deionized water used were autoclaved. 2.2. Preparation of AuNPs/GCE Glassy carbon electrode (GCE) was polished with 0.3 and 0.05 μm alumina slurry, followed by successive sonication in double distilled deionized water, ethanol and double distilled deionized water for 5 min. The cleaned GCE was immersed into 3.0 mM HAuCl4 solution containing 0.1 M KNO3, then the gold nanoparticles (AuNPs) were electrochemically deposited on GCE surface over 200 s at 0.2 V. Thus, the modified electrode was obtained as AuNPs/GCE.
Scheme 1. The schematic representation of the Phos-tag-based electrochemical biosensor for PKA activity assay.
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2.3. Peptide immobilization and phosphorylation
2.5. Inhibition
The AuNPs/GCE was incubated with 10 μL of substrate peptide (0.4 μM, dissolved in 10 mM Tris–HCl containing 10 mM TCEP, 10 mM MgCl2 and 50 mM KCl, pH 7.0) for 12 h in a humidified chamber with 99% humidity at ambient temperature. Then, the peptide modified electrode (noted as Peptide/AuNPs/GCE) was rinsed with washing buffer for three times to remove the unimmobilized peptides. The Peptide/AuNPs/GCE was then immersed into 10 μL of MPA solution (33 μM) for 1 h to occupy the spare space on the electrode surface and induce peptide chain to orientate orderly, avoiding their inclining onto the electrode surface (Pavlov et al., 2004). Finally, the electrode was immersed into the washing buffer for 1 h, so that tween-20 in the buffer blocked the adsorption of free PKA enzyme and replaced nonspecific proteins by occupying the spare sites of the electrode (Steinitz, 2000; Wang et al., 2010). PKA-catalyzed phosphorylation was performed according to the manufacturer's protocol. Briefly, 10 μL of PKA reaction solution containing different concentrations of PKA was dropped on the electrode surface and incubated for 50 min at 37 °C in a humidified chamber. After that, the electrode was rinsed thoroughly with the washing buffer. The obtained electrode was noted as P-Peptide/ AuNPs/GCE.
For investigating the inhibition effect of ellagic acid and H-89 on PKA activity, different concentrations of inhibitors were introduced into the PKA reaction solution and the Peptide/AuNPs/GCE was incubated with this solution for 50 min at 37 °C. Then the electrode was rinsed thoroughly using the washing buffer. The following operation was same as that in Section 2.4. 2.6. Electrochemical detection All the electrochemical measurements were carried out with a CHI660C electrochemical workstation (Austin, USA). A conventional three-electrode electrochemical cell with a Pt-wire auxiliary electrode, a saturated calomel electrode as reference electrode, and a modified GCE as working electrode was used for all the electrochemical measurements. Differential pulse voltammetry (DPV) was performed in 10 mM PBS (0.1 M, pH 7.4) containing 0.4 mM hydroquinone and 0.4 mM H2O2.The parameters of DPV are as follows: initiative potential, 0.5 V, final potential, 0.2 V, step potential, 0.004 V, amplitude, 0.05 V, pulse width, 0.05 s, pulse period, 0.2 s, quiet time, 2 s. Electrochemical impedance measurements were carried out in 0.1 M KCl solution containing 5.0 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1, molar ratio). The electrochemical impedance spectra (EIS) were recorded in the frequency range of 105–10 1 Hz.
2.4. Phos-tag-biotin recognition and avidin-HRP immobilization
2.7. Cell culture and lysate preparation
For recognizing and capturing the phosphorylation site, 10 μL of Phos-tag-biotin reaction buffer containing 20 μM Phos-tag-biotin was casted on the phosphorylated peptide modified electrode surface and incubated for 30 min in a humidified chamber. Then, the electrode was rinsed with washing buffer for three times (the obtained electrode was noted as Phos-tag/P-Peptide/AuNPs/GCE). Afterwards, the electrode was further incubated with 10 μL of 10 mM PBS (pH 7.4) containing 0.01 mg/mL avidin-HRP for 1 h in a humidified chamber. Finally, the electrode (named as HRP/Phostag/P-Peptide/AuNPs/GCE) was rinsed with washing buffer for three times and stored in refrigerator at 4 °C in 10 mM Tris–HCl (pH 7.0) before use.
Cells were cultured in DMEM (Dulbecco's modified Eagle's medium) (Gibco-BRL, USA) supplemented with 10% (v/v) foetal bovine serum (Invitrogen, New Zealand) in a CO2 (5%) incubator at 37 °C. The cultured cells were washed three times with normal saline. Then the cells were lysed according to previous reports with some modifications (Zhou et al., 2013). In brief, the cells (105 cells) in 0.3 mL lysis buffer were sonicated for 2 s 60 times at 4 °C with an interval of 3 s for each time. The homogenates were centrifuged at 13 000 rpm for 30 min at 4 °C using an Eppendorf centrifuge (Eppendorf, Germany). Then, the obtained supernatants were transferred to a freezing centrifuge tube (Axygen, USA) and stored at 20 °C in a refrigerator. The total protein concentration in cell lysate was evaluated by modified Bradford protein assay kit
Fig. 1. (A) Nyquist plots of different electrodes in 5 mM Fe(CN)6 3 − /4 − containing 0.1 M KCl. (a) GCE, (b) AuNPs/GCE, (c) Peptide/AuNPs/GCE, (d) P-Peptide/AuNPs/GCE, (e) Phos-tag/P-Peptide/AuNPs/GCE, (f) HRP/Phos-tag/P-Peptide/AuNPs/GCE. (B) Differential pulse voltammograms of different electrodes in 0.1 M PBS (pH 7.4) containing 0.4 mM H2O2 and 0.4 mM hydroquinone. (a) Phos-tag/P-Peptide/AuNPs/GCE, (b) HRP/Phos-tag/P-Peptide/AuNPs/GCE, (c) Peptide/AuNPs/GCE incubated with Phos-tag-biotin and avidin-HRP successively, (d) P-Peptide/AuNPs/GCE incubated with avidin-HRP. PKA concentration, 25 unit/mL.
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according to the manufacturer's recommendation. All cell lysates were diluted to 10 μg/mL total protein concentration for kinase activity assay.
3. Results and discussion 3.1. Biosensor characterization The preparation process of the biosensor was monitored by EIS. As shown in Fig. 1A, the interface electron transfer resistance (Ret) decreased significantly and only a straight line was observed in the whole frequency region after AuNPs were electrodeposited on GCE surface (curve b), indicating the good conductivity of AuNPs. However, in this work, the role of AuNPs was not only located at the increase of conductivity, but also employed as an immobilization medium for peptide. The Ret increased when the substrate peptides were assembled on the electrode surface through the formation of Au–S bond (curve c), where the S was supplied by the cysteine residue at the C-end of substrate peptide. This increase could be attributed to the immobilized peptides, which blocked the diffusion of the redox probe of Fe(CN)6 3 − /4 − and increased the interface electron transfer resistance. Afterwards, the Ret value further increased after the phosphorylation reaction catalyzed by PKA (curve d) and the capture of biotinylated Phos-tag through the specific interaction between Phos-tag and phosphate group (curve e). Subsequently, the immobilization of avidin-HRP caused a further increase on interface electron transfer resistance (curve f). It could be explained as the fact that the large size of avidin-HRP hindered the diffusion of redox probe towards electrode surface and increased the electron transfer resistance. Through the variation of interface electron resistance, it can be confirmed that the biosensor was successfully fabricated. 3.2. Detection feasibility In order to prove the principle of the prepared biosensor on PKA activity assay, the electrochemical behavior of the biosensor was recorded in 10 mM PBS (0.1 M, pH 7.4) containing 0.4 mM hydroquinone and 0.4 mM H2O2 after different treatment processes. As seen in Fig. 1B, only a weak reduction peak for benzoquinone was obtained using Peptide/AuNPs/GCE (curve a) as working electrode. This weak reduction peak could be explained as the low concentration of benzoquinone, indicating a weak oxidation ability and slow oxidation rate of H2O2 towards
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hydroquinone in the absence of HRP. After the Peptide/AuNPs/ GCE was incubated with PKA, Phos-tag-biotin and avidin-HRP successively (HRP/Phos-tag/P-Peptide/AuNPs/GCE was fabricated), the electrochemical reduction peak of benzoquinone enhanced significantly (curve b). In the presence of PKA, the serine residue in substrate peptide was phosphorylated, and the phosphate group was specifically recognized by Phos-tag-biotin. Then, through the specific interaction between biotin and avidin, avidin-HRP was further assembled on the electrode surface. Under the catalytic effect of HRP on the chemical oxidation of hydroquinone by H2O2, more benzoquinone was produced, which resulted in an improved electrochemical response. For further confirming that the increased current was depended on the phosphorylation reaction, Peptide/AuNPs/GCE was incubated with Phos-tag-biotin and avidin-HRP, and then the DPV response was recorded. As shown in curve c, the reduction peak current of benzoquinone was much lower than that obtained at HRP/Phos-tag/P-Peptide/AuNPs/GCE. Because Peptide/AuNPs/GCE was not incubated with PKA, the substrate peptide was not phosphorylated. In the absence of phosphate group, Phos-tag-biotin could not be captured on the electrode surface through the specific recognition ability towards phosphate group, which led to the failing immobilization of HRP. Moreover, the reduction current of curve c was closed to that obtained at Peptide/AuNPs/GCE, which further demonstrated the unsuccessful immobilization of Phos-tag-biotin and avidin-HRP due to the lack of phosphorylation process. Therefore, one can conclude that the increase of the reduction peak current in curve b is triggered by the phosphorylation reaction. This detection strategy can be used to detect PKA activity based on the current change. In order to testify the specific recognition effect of Phostag towards phosphate group, another control experiment was performed. After Peptide/AuNPs/GCE was phosphorylated by PKA, the electrode was directly incubated with HRP. The DPV result was shown as curve d. It was clear that the reduction peak current was more weaker than that of curve b and closed to curve a, indicating a low concentration of benzoquinone, which could be explained as the failed immobilization of HRP due to the absence of Phos-tagbiotin. Thus, Phos-tag-biotin, as specific recognition reagent for phosphate group, played very vital role in PKA activity assay because it was acted as a ‘bridge’ to link phosphate group and HRP. Based on it, the detection strategy based on enzymatic signal amplification can be successful achieved.
Fig. 2. (A) The differential pulse voltammograms of the biosensors incubated with differential concentrations of PKA. a–h: 0, 0.5, 1, 2, 5, 10, 15, 20, 25 unit/mL. (B) The dependence of the electrochemical reduction peak current on the logarithm value of PKA concentration.
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3.3. PKA activity assay 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. Under the optimum experimental conditions (see Supporting information), the performance of the PKA activity assay was carried out by exposing the substrate peptide on the biosensor to different concentrations of PKA under the same experimental conditions. With boosting the PKA concentration from 0.5 to Table 1 Comparison of the proposed method with previous reports on PKA activity detection. Method
Linear range (unit/mL)
LOD (unit/mL)
Refs.
Electrochemistry Electrochemistry Electrochemistry Electrochemistry QCM Fluorescence Fluorescence Fluorescence Fluorescence ECL ECL ECL Electrochemistry
– – – 5–500 0.64–22.33
10 0.15 0.2 0.5 0.061 0.47 0.1 0.134 0.5 0.07 0.005 0.005 0.15
Kerman et al. (2007) Xu et al. (2009) Ji et al. (2009) Miao et al. (2012) X. Xu et al. (2012) Xu et al. (2011) Bai et al. (2013) Zhou et al. (2013) Tan et al. (2013) Chen et al. (2010) Zhao et al. (2012) Chen et al. (2013) This work
0.5–500 – – 0.07–32 0.01–50 0.01–1 0.5–25
25 unit/mL, the electrochemical reduction signal of benzoquinone increased gradually (Fig. 2A). As shown in Fig. 2B, the electrochemical reduction peak current was proportional to the logarithm value of PKA concentration. The linear regression equation could be expressed as Ipc ¼ 0.32 log cþ 3.51 (R ¼0.9959) and the detection limit was estimated to be 0.15 unit/mL (S/N ¼3). Compared with other electrochemical methods, quartz crystal microbalance (QCM) and fluorescent methods as listed in Table 1, our results present rational linear range and competitive detection limit. However, the detection sensitivity of our work is lower than that at electrochemiluminescence (ECL). But our work has its own advantages, such as simple operation, low cost, cheap and portable instrument. And this method should be an alternative for protein kinase activity assay. For evaluating the selectivity of the fabricated biosensor, substrate peptide (S-peptide) and control peptide (C-peptide) were immobilized on AuNPs/GCE, respectively. Then, the electrodes were incubated with PKA (25 unit/mL), followed by Phos-tagbiotin and avidin-HRP as described in Sections 2.3 and 2.4. Finally, the DPV was carried out as described in Section 2.6. For substrate peptide, the electrochemical reduction peak current was 10.92 μA, which was greatly higher than that for the control peptide (1.35 μA). In addition, the reduction peak current for the control peptide was closed to that obtained at substrate peptide modified AuNPs/GCE (Peptide/AuNPs/GCE), which further proved that the phosphorylation reaction did not happen due to the absence of phosphorylation site for PKA in the control peptide. These results demonstrated that the developed approach has good detection selectivity for PKA. The reproducibility of the electrochemical biosensor was also assessed by measuring 10 unit/mL PKA with
Fig. 3. Differential pulse voltammograms of the fabricated biosensor after inhibition by ellagic acid (A) and H-89 (C). (B) and (D) were the inhibition effect of ellagic acid (B) and H-89 (D) on PKA activity.
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five biosensors. The relative standard deviation value was found to be 8.35%, suggesting acceptable reproducibility. 3.4. Inhibition assay Since abnormal expression of PKA has been implicated in a number of diseases including cancers, the identification of PKA inhibitors is valuable for protein kinase-targeted drug discovery and relative diseases therapy. Thus, the PKA activities were evaluated with different concentrations of potential PKA inhibitors, and the half-maximum inhibition value of IC50 was also calculated. Here, H-89 and ellagic acid were used as the model inhibitors, which were potent and cell-permeable inhibitor of PKA. As shown in Fig. 3A, the electrochemical reduction signal of benzoquinone decreased with increasing the concentration of H-89, which revealed the inhibition of PKA activity and low level of substrate peptide phosphorylation. The relationship between inhibition ratio and H-89 concentration was presented in Fig. 3B. The IC50 value (the half maximum inhibitory concentration) was calculated to be 28 nM, which was well consistent with some of previous reports (Bai et al., 2013; Xu et al., 2009). Fig. 3C illustrates the effect of ellagic acid on the PKA activity. The electrochemical reduction signal decreased with increasing the concentration of ellagic acid from 1.0 to 30 μM, which reflected the inhibitory activity of ellagic acid towards PKA. Through the plot in Fig. 3D on the relationship between inhibitory ratio and ellagic concentration, the IC50 value of ellagic acid was calculated to be 2.77 μM,
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which was in agreement with previous reports (Chen et al., 2013; Wang et al., 2010; Xu et al., 2010). These results clearly proved that the developed bioassay is of great potential in screening PKA inhibitors. The fabricated biosensor could be also applied in the discovery of new pharmaceuticals for therapy of some diseases, which are caused by the abnormal expression of protein kinase. 3.5. Detection of PKA activity in cancer cell lysate Because protein kinases play crucial roles in intracellular signaling pathways, a practical method for assaying kinase activity in complex biological samples can contribute significantly to understand the function of kinases in cell signal transduction. Moreover, in order to further explore the applicability of the developed biosensor, PKA activity in different cell lysates were measured. In this case, the substrate peptide was reacted with two kinds of cell lysates including normal hepatic cell (L-02 cell) lysates and hepatoma carcinoma cell (HepG2 cell) lysates, respectively. Then, the substrate peptide was further incubated with Phos-tag-biotin and avidin-HRP successively as described in Section 2.4. Finally, the DPV was performed. As can be seen from Fig. 4A, the electrochemical response of the substrate peptide, incubated with HepG2 cell lysates, was obviously higher than that for L-02 cell lysates, indicating that the PKA activity inHepG2 cell was higher than that in L02 cell. High PKA activity can lead to more phosphorylation events, which might be one of the reasons for cancer. For further evaluating the applicability of the developed
Fig. 4. (A) Differential pulse voltammograms of the biosensor incubated with L02 (b) and HepG2 (a) cell lysates. (B) The differential pulse voltammograms of the biosensor incubated with L02 cell lysate (a), a þ0.5 μM ellagic acid (b), and aþ 0.5 μM H-89 (c). (C) The differential pulse voltammograms of the biosensor incubated with HepG2 cell lysate (a), aþ 0.5 μM ellagic acid (b), and a þ0.5 μM H-89 (c). (D) Histogram for the electrochemical response of the biosensor incubated with different cell lysates and different inhibitors.
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approach for PKA-target drug screening, the substrate peptide on the biosensor was simultaneously incubated with cell lysate and PKA inhibitor (0.5 μM ellagic acid and 0.5 μM H-89, respectively). The corresponding differential pulse voltammograms are illustrated in Fig. 4B and C. As illustrated in Fig. 4D, the electrochemical reduction signal decreased when different PKA inhibitors were introduced into L02 cell lysate and the substrate peptide was incubated with it. In addition, we also found that the current decrease level of the biosensor incubated with the mixed incubation solution of L02 cell lysate and H-89 was higher than that at L02 cell lysate and ellagic acid, which indicated the stronger inhibition ability of H-89 than ellagic acid. Similar results were also obtained when the substrate peptide was incubated with HepG2 cell lysate and different PKA inhibitors. Based on the above research, one can conclude that the developed approach is work well in complex biological system, and it is feasible to analyze intracellular cell kinase activities in vitro and screen kinase-target drugs.
4. Conclusion In summary, a simple and sensitive electrochemical method was developed for protein kinase activity and inhibition assay. Based on the specific recognition ability of Phos-tag for kinaseinduced phosphopeptides and HRP-catalyzed signal amplification, the detection sensitivity of this work was higher than most of other electrochemical methods. This proposed approach was also successfully applied to analyze PKA activity in cell lysates and evaluate the inhibition effect of PKA inhibitors, revealing significant potential for drug discovery, kinase-related diseases diagnose and therapy, kinase function analysis in signal-transduction pathways. However, compared with ECL technique, the detection limit obtained in this work was still needed to be improved. Thus, in the following work, we will develop new and effective signal amplification strategy combining with electrochemical or photoelectrochemical detection technique.
Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 21105056 and 21375079), China Postdoctoral Science Foundation (2014M550369) and the Project of Development of Science and Technology of Shandong Province, China (No. 2013GZX20109).
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.07.016.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
A sensitive electrochemical biosensor for detection of protein kinase A activity and inhibitors based on Phos-tag and enzymatic signal amplification Huanshun Yin, Mo Wang, Bingchen Li, Zhiqing Yang, Yunlei Zhou n, Shiyun Ai n College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 20 April 2014 Received in revised form 26 June 2014 Accepted 8 July 2014 Available online 11 July 2014
A simple, highly sensitive and selective electrochemical assay is developed for the detection of protein kinase A (PKA) activity based on the specific recognition utility of Phos-tag for kinase-induced phosphopeptides and enzymatic signal amplification. When the substrate peptide was phosphorylated by PKA reaction, they could specifically bind with Phos-tag-biotin in the presence of Zn2 þ through the formation of a specific noncovalent complex with the phosphomonoester dianion in phosphorylated peptides. Through the further specific interaction between biotin and avidin, avidin functionalized horseradish peroxidase (HRP) can be captured on the electrode surface. Under the catalytic effect of HRP, a sensitive electrochemical signal for benzoquinone was obtained, which was related to PKA activity. Under the optimal experiment conditions, the proposed electrochemical method presented dynamic range from 0.5 to 25 unit/mL with low detection limit of 0.15 unit/mL. This new detection strategy was also successfully applied to analyze the inhibition effect of inhibitors (ellagic acid and H-89) on PKA activity and monitored the PKA activity in cell lysates. Therefore, this Phos-tag-based electrochemical assay offers an alternative platform for PKA activity assay and inhibitor screening, and thus it might be a valuable tool for development of targeted therapy and clinical diagnosis. & 2014 Elsevier B.V. All rights reserved.
Keywords: Protein kinase A Phos-tag-biotin Enzymatic signal amplification Elelctrochemical biosensor Inhibitors
1. Introduction Protein kinases can catalyze the phosphorylation of serine, threonine and tyrosine residues in peptides and proteins by transferring phosphate groups from adenosine-triphosphate (ATP) to amino acids, which play critical roles in intracellular signal transduction and the regulation of cellular functions, such as metabolism, gene expression, and apoptosis, cell cycle progression and differentiation (Cohen, 2002; Kalume et al., 2003; Manning et al., 2002). More importantly, aberrant expression of protein kinases has been involved in several diseases including cancer (Griner and Kazanietz, 2007), HIV (Critchfield et al., 1997), and Alzheimer's disease (Flajolet et al., 2007). The quantitative analysis of kinases and their potential inhibitors is not only necessary for basic biology to clarify molecular mechanisms of signal transduction but also valuable for protein kinase-targeted drug discovery and therapy. Therefore, protein kinases have been regarded as the major targets for drug discovery (Zhang et al., 2009) and as biomarkers for cancer diagnosis (Kang et al., 2009). Based on it, there is an increase in demand to develop simple and n
Corresponding authors. Tel.: þ 86 538 8249248; fax: þ86 538 8242251. E-mail addresses: [email protected] (Y. Zhou), [email protected] (S. Ai).
http://dx.doi.org/10.1016/j.bios.2014.07.016 0956-5663/& 2014 Elsevier B.V. All rights reserved.
rapid detection methods for protein kinase activity, especially in cells or cellular lysates. The generally used techniques for the measurement of protein kinase activities mostly relied on radioisotopic methods which used radioactive ATP for monitoring phosphorylation reactions (Hayslett et al., 1995; Lehel et al., 1997). Although these methods could sensitively detect protein kinase activity, they depended on specific instruments and suffered from a high risk of radioactive contamination. To eliminate the problems that are associated with radioactive methods, many other methods have been developed, such as resonance light scattering (Li et al., 2013; Wang et al., 2005), colorimetric assay (Wang et al., 2006), fluorescence method (Bai et al., 2013; Xu et al., 2011; Zhou et al., 2013), photoluminescent (Wang et al., 2013), electrochemiluminescence (Chen et al., 2013; S. Xu et al., 2012; Zhao et al., 2012), quantitative mass spectrometry (Ji et al., 2012), Raman spectroscopic assay (Li et al., 2009), surface plasmon resonance imaging technique (Inamori et al., 2005). However, these methods have the disadvantages of expensive and complicated instruments, sophisticated operating procedures, time-consuming, tedious sample treatment. Therefore, the development of sensitive, reliable, and in-expensive method for protein kinase assays and inhibitor screening still remains a great challenge.
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Electrochemical methods have been demonstrated to be simpler and more cost-effective than previous methods for determining the activity of protein kinases (Chiku et al., 2010; Freeman et al., 2007; Ji et al., 2009; Kerman and Kraatz, 2009; Li et al., 2014; Song et al., 2008; Wang et al., 2012, 2011, 2010; Xu et al., 2009). One of the key factors for electrochemical assay of protein kinase activity based on the catalytic phosphorylation of substrate peptide is the effective recognition of phosphate group. Recently, a kind of commercially specific phosphate-binding reagent, named as Phos-tag, has attracted more attentions on its application in protein phosphorylation detection and protein kinase activity assay (Kinoshita et al., 2009, 2013, 2012). In the presence of Zn2 þ or Mn2 þ , the Phos-tag can form a specific noncovalent complex with the phosphomonoester dianion in phosphorylated peptides or proteins containing phospho-Ser, phospho-Thr, phospho-Tyr, and phospho-His residues at neutral pH (Kinoshita and Kinoshita-Kikuta, 2011). Though Phos-tag shows great potential in protein kinase activity assay, the electrochemical methods based on phos-tag has yet been reported until now. Herein, we propose the proof-of-principle of a novel and versatile electrochemical sensing platform for assaying the activity and inhibition of protein kinases A (PKA), which is based on its catalytic effect on substrate peptide and the phosphate group recognition ability of Phos-tag. The detection mechanism was shown in Scheme 1. With the peptide phosphorylation catalyzed by PKA, the biotin functionalized Phos-tag (Phos-tag-biotin) could be captured on the electrode surface through the specific recognition effect of Phos-tag towards phosphate group. Then, avidin functionalized horseradish peroxidase (avidin-HRP) could be further modified on the electrode surface through the immunoreaction between avidin and biotin, and the HRP molecule could catalyze H2O2 to chemically oxidize hydroquinone, which would result in a strong electrochemical reduction current of the oxidative product of benzoquinone. Because this response is related to the phosphorylation level cayalyzed by PKA, this electrochemical signal was used to detect the PKA activity. It can also provide a simple, sensitive, selective, and universal platform for other kinases activity assay. Moreover, the developed method is also successfully applied to detect PKA activity in cell lysates.
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2. Experimental 2.1. Reagents The substrate peptide (kemptide, H-CGGALRRASLG-NH2), the control peptide (H-CGGALRRAALG-NH2), avidin-HRP, adenosine 5′-triphosphate (ATP) disodium salt hydrate and the modified Bradford protein assay kit were purchased from Sangon Biotech (Shanghai) Co., Ltd. Mercaptopropionic acid (MPA) was purchased from Fluka. cAMP-dependent protein kinase A (PKA) catalytic subunit was purchased from New England Biolabs Ltd. (Beverly, MA). Biotinylated Phos-tag™ was obtained from Wako Pure Chemical Industries, Ltd. (Japan). Tris(2-carboxyethyl) phosphine hydrochloride (TCEP), tris(hydroxymethyl)aminomethane (Tris), EDTA, ellagic acid and chloroauric acid (HAuCl4) were purchased from Aladdin (shanghai, China). H-89 was obtained from EMD Millipore Corporation (Billerica, MA, USA). The buffer solutions employed in this study are as follows. PKA storage buffer, 20 mM Tris–HCl, 50 mM NaCl, 1 mM EDTA, 2.0 mM DTT and 10% glycerol, pH 7.5. PKA reaction buffer, 50 mM Tris–HCl, 70 μM ATP, 10 mM MgCl2, pH 7.5. Phos-tag-biotin reaction buffer, 10 mM Tris–HCl (pH 7.0) containing 0.1 M NaCl, 0.1% Tween-20, 0.4 mM Zn(NO3)2. avidin-HRP reaction buffer, 10 mM PBS (pH 7.5). Washing buffer, 10 mM Tris–HCl (pH 7.5) containing 0.05% tween20. Cell lysis buffer, 10 mM PBS (pH 7.4) containing 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2, 2.0 mM DTT, 1 mM sodium orthovanadate, 80 mM b-glycerophosphate, 3.0 μg/mL pepstatin A, 5.0 μg/ mL aprotinin, and 1.0 mM phenylmethylsulfonyl fluoride (PMSF). All reagents were analytically pure grade. All of the solution and redistilled deionized water used were autoclaved. 2.2. Preparation of AuNPs/GCE Glassy carbon electrode (GCE) was polished with 0.3 and 0.05 μm alumina slurry, followed by successive sonication in double distilled deionized water, ethanol and double distilled deionized water for 5 min. The cleaned GCE was immersed into 3.0 mM HAuCl4 solution containing 0.1 M KNO3, then the gold nanoparticles (AuNPs) were electrochemically deposited on GCE surface over 200 s at 0.2 V. Thus, the modified electrode was obtained as AuNPs/GCE.
Scheme 1. The schematic representation of the Phos-tag-based electrochemical biosensor for PKA activity assay.
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2.3. Peptide immobilization and phosphorylation
2.5. Inhibition
The AuNPs/GCE was incubated with 10 μL of substrate peptide (0.4 μM, dissolved in 10 mM Tris–HCl containing 10 mM TCEP, 10 mM MgCl2 and 50 mM KCl, pH 7.0) for 12 h in a humidified chamber with 99% humidity at ambient temperature. Then, the peptide modified electrode (noted as Peptide/AuNPs/GCE) was rinsed with washing buffer for three times to remove the unimmobilized peptides. The Peptide/AuNPs/GCE was then immersed into 10 μL of MPA solution (33 μM) for 1 h to occupy the spare space on the electrode surface and induce peptide chain to orientate orderly, avoiding their inclining onto the electrode surface (Pavlov et al., 2004). Finally, the electrode was immersed into the washing buffer for 1 h, so that tween-20 in the buffer blocked the adsorption of free PKA enzyme and replaced nonspecific proteins by occupying the spare sites of the electrode (Steinitz, 2000; Wang et al., 2010). PKA-catalyzed phosphorylation was performed according to the manufacturer's protocol. Briefly, 10 μL of PKA reaction solution containing different concentrations of PKA was dropped on the electrode surface and incubated for 50 min at 37 °C in a humidified chamber. After that, the electrode was rinsed thoroughly with the washing buffer. The obtained electrode was noted as P-Peptide/ AuNPs/GCE.
For investigating the inhibition effect of ellagic acid and H-89 on PKA activity, different concentrations of inhibitors were introduced into the PKA reaction solution and the Peptide/AuNPs/GCE was incubated with this solution for 50 min at 37 °C. Then the electrode was rinsed thoroughly using the washing buffer. The following operation was same as that in Section 2.4. 2.6. Electrochemical detection All the electrochemical measurements were carried out with a CHI660C electrochemical workstation (Austin, USA). A conventional three-electrode electrochemical cell with a Pt-wire auxiliary electrode, a saturated calomel electrode as reference electrode, and a modified GCE as working electrode was used for all the electrochemical measurements. Differential pulse voltammetry (DPV) was performed in 10 mM PBS (0.1 M, pH 7.4) containing 0.4 mM hydroquinone and 0.4 mM H2O2.The parameters of DPV are as follows: initiative potential, 0.5 V, final potential, 0.2 V, step potential, 0.004 V, amplitude, 0.05 V, pulse width, 0.05 s, pulse period, 0.2 s, quiet time, 2 s. Electrochemical impedance measurements were carried out in 0.1 M KCl solution containing 5.0 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1, molar ratio). The electrochemical impedance spectra (EIS) were recorded in the frequency range of 105–10 1 Hz.
2.4. Phos-tag-biotin recognition and avidin-HRP immobilization
2.7. Cell culture and lysate preparation
For recognizing and capturing the phosphorylation site, 10 μL of Phos-tag-biotin reaction buffer containing 20 μM Phos-tag-biotin was casted on the phosphorylated peptide modified electrode surface and incubated for 30 min in a humidified chamber. Then, the electrode was rinsed with washing buffer for three times (the obtained electrode was noted as Phos-tag/P-Peptide/AuNPs/GCE). Afterwards, the electrode was further incubated with 10 μL of 10 mM PBS (pH 7.4) containing 0.01 mg/mL avidin-HRP for 1 h in a humidified chamber. Finally, the electrode (named as HRP/Phostag/P-Peptide/AuNPs/GCE) was rinsed with washing buffer for three times and stored in refrigerator at 4 °C in 10 mM Tris–HCl (pH 7.0) before use.
Cells were cultured in DMEM (Dulbecco's modified Eagle's medium) (Gibco-BRL, USA) supplemented with 10% (v/v) foetal bovine serum (Invitrogen, New Zealand) in a CO2 (5%) incubator at 37 °C. The cultured cells were washed three times with normal saline. Then the cells were lysed according to previous reports with some modifications (Zhou et al., 2013). In brief, the cells (105 cells) in 0.3 mL lysis buffer were sonicated for 2 s 60 times at 4 °C with an interval of 3 s for each time. The homogenates were centrifuged at 13 000 rpm for 30 min at 4 °C using an Eppendorf centrifuge (Eppendorf, Germany). Then, the obtained supernatants were transferred to a freezing centrifuge tube (Axygen, USA) and stored at 20 °C in a refrigerator. The total protein concentration in cell lysate was evaluated by modified Bradford protein assay kit
Fig. 1. (A) Nyquist plots of different electrodes in 5 mM Fe(CN)6 3 − /4 − containing 0.1 M KCl. (a) GCE, (b) AuNPs/GCE, (c) Peptide/AuNPs/GCE, (d) P-Peptide/AuNPs/GCE, (e) Phos-tag/P-Peptide/AuNPs/GCE, (f) HRP/Phos-tag/P-Peptide/AuNPs/GCE. (B) Differential pulse voltammograms of different electrodes in 0.1 M PBS (pH 7.4) containing 0.4 mM H2O2 and 0.4 mM hydroquinone. (a) Phos-tag/P-Peptide/AuNPs/GCE, (b) HRP/Phos-tag/P-Peptide/AuNPs/GCE, (c) Peptide/AuNPs/GCE incubated with Phos-tag-biotin and avidin-HRP successively, (d) P-Peptide/AuNPs/GCE incubated with avidin-HRP. PKA concentration, 25 unit/mL.
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according to the manufacturer's recommendation. All cell lysates were diluted to 10 μg/mL total protein concentration for kinase activity assay.
3. Results and discussion 3.1. Biosensor characterization The preparation process of the biosensor was monitored by EIS. As shown in Fig. 1A, the interface electron transfer resistance (Ret) decreased significantly and only a straight line was observed in the whole frequency region after AuNPs were electrodeposited on GCE surface (curve b), indicating the good conductivity of AuNPs. However, in this work, the role of AuNPs was not only located at the increase of conductivity, but also employed as an immobilization medium for peptide. The Ret increased when the substrate peptides were assembled on the electrode surface through the formation of Au–S bond (curve c), where the S was supplied by the cysteine residue at the C-end of substrate peptide. This increase could be attributed to the immobilized peptides, which blocked the diffusion of the redox probe of Fe(CN)6 3 − /4 − and increased the interface electron transfer resistance. Afterwards, the Ret value further increased after the phosphorylation reaction catalyzed by PKA (curve d) and the capture of biotinylated Phos-tag through the specific interaction between Phos-tag and phosphate group (curve e). Subsequently, the immobilization of avidin-HRP caused a further increase on interface electron transfer resistance (curve f). It could be explained as the fact that the large size of avidin-HRP hindered the diffusion of redox probe towards electrode surface and increased the electron transfer resistance. Through the variation of interface electron resistance, it can be confirmed that the biosensor was successfully fabricated. 3.2. Detection feasibility In order to prove the principle of the prepared biosensor on PKA activity assay, the electrochemical behavior of the biosensor was recorded in 10 mM PBS (0.1 M, pH 7.4) containing 0.4 mM hydroquinone and 0.4 mM H2O2 after different treatment processes. As seen in Fig. 1B, only a weak reduction peak for benzoquinone was obtained using Peptide/AuNPs/GCE (curve a) as working electrode. This weak reduction peak could be explained as the low concentration of benzoquinone, indicating a weak oxidation ability and slow oxidation rate of H2O2 towards
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hydroquinone in the absence of HRP. After the Peptide/AuNPs/ GCE was incubated with PKA, Phos-tag-biotin and avidin-HRP successively (HRP/Phos-tag/P-Peptide/AuNPs/GCE was fabricated), the electrochemical reduction peak of benzoquinone enhanced significantly (curve b). In the presence of PKA, the serine residue in substrate peptide was phosphorylated, and the phosphate group was specifically recognized by Phos-tag-biotin. Then, through the specific interaction between biotin and avidin, avidin-HRP was further assembled on the electrode surface. Under the catalytic effect of HRP on the chemical oxidation of hydroquinone by H2O2, more benzoquinone was produced, which resulted in an improved electrochemical response. For further confirming that the increased current was depended on the phosphorylation reaction, Peptide/AuNPs/GCE was incubated with Phos-tag-biotin and avidin-HRP, and then the DPV response was recorded. As shown in curve c, the reduction peak current of benzoquinone was much lower than that obtained at HRP/Phos-tag/P-Peptide/AuNPs/GCE. Because Peptide/AuNPs/GCE was not incubated with PKA, the substrate peptide was not phosphorylated. In the absence of phosphate group, Phos-tag-biotin could not be captured on the electrode surface through the specific recognition ability towards phosphate group, which led to the failing immobilization of HRP. Moreover, the reduction current of curve c was closed to that obtained at Peptide/AuNPs/GCE, which further demonstrated the unsuccessful immobilization of Phos-tag-biotin and avidin-HRP due to the lack of phosphorylation process. Therefore, one can conclude that the increase of the reduction peak current in curve b is triggered by the phosphorylation reaction. This detection strategy can be used to detect PKA activity based on the current change. In order to testify the specific recognition effect of Phostag towards phosphate group, another control experiment was performed. After Peptide/AuNPs/GCE was phosphorylated by PKA, the electrode was directly incubated with HRP. The DPV result was shown as curve d. It was clear that the reduction peak current was more weaker than that of curve b and closed to curve a, indicating a low concentration of benzoquinone, which could be explained as the failed immobilization of HRP due to the absence of Phos-tagbiotin. Thus, Phos-tag-biotin, as specific recognition reagent for phosphate group, played very vital role in PKA activity assay because it was acted as a ‘bridge’ to link phosphate group and HRP. Based on it, the detection strategy based on enzymatic signal amplification can be successful achieved.
Fig. 2. (A) The differential pulse voltammograms of the biosensors incubated with differential concentrations of PKA. a–h: 0, 0.5, 1, 2, 5, 10, 15, 20, 25 unit/mL. (B) The dependence of the electrochemical reduction peak current on the logarithm value of PKA concentration.
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3.3. PKA activity assay 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. Under the optimum experimental conditions (see Supporting information), the performance of the PKA activity assay was carried out by exposing the substrate peptide on the biosensor to different concentrations of PKA under the same experimental conditions. With boosting the PKA concentration from 0.5 to Table 1 Comparison of the proposed method with previous reports on PKA activity detection. Method
Linear range (unit/mL)
LOD (unit/mL)
Refs.
Electrochemistry Electrochemistry Electrochemistry Electrochemistry QCM Fluorescence Fluorescence Fluorescence Fluorescence ECL ECL ECL Electrochemistry
– – – 5–500 0.64–22.33
10 0.15 0.2 0.5 0.061 0.47 0.1 0.134 0.5 0.07 0.005 0.005 0.15
Kerman et al. (2007) Xu et al. (2009) Ji et al. (2009) Miao et al. (2012) X. Xu et al. (2012) Xu et al. (2011) Bai et al. (2013) Zhou et al. (2013) Tan et al. (2013) Chen et al. (2010) Zhao et al. (2012) Chen et al. (2013) This work
0.5–500 – – 0.07–32 0.01–50 0.01–1 0.5–25
25 unit/mL, the electrochemical reduction signal of benzoquinone increased gradually (Fig. 2A). As shown in Fig. 2B, the electrochemical reduction peak current was proportional to the logarithm value of PKA concentration. The linear regression equation could be expressed as Ipc ¼ 0.32 log cþ 3.51 (R ¼0.9959) and the detection limit was estimated to be 0.15 unit/mL (S/N ¼3). Compared with other electrochemical methods, quartz crystal microbalance (QCM) and fluorescent methods as listed in Table 1, our results present rational linear range and competitive detection limit. However, the detection sensitivity of our work is lower than that at electrochemiluminescence (ECL). But our work has its own advantages, such as simple operation, low cost, cheap and portable instrument. And this method should be an alternative for protein kinase activity assay. For evaluating the selectivity of the fabricated biosensor, substrate peptide (S-peptide) and control peptide (C-peptide) were immobilized on AuNPs/GCE, respectively. Then, the electrodes were incubated with PKA (25 unit/mL), followed by Phos-tagbiotin and avidin-HRP as described in Sections 2.3 and 2.4. Finally, the DPV was carried out as described in Section 2.6. For substrate peptide, the electrochemical reduction peak current was 10.92 μA, which was greatly higher than that for the control peptide (1.35 μA). In addition, the reduction peak current for the control peptide was closed to that obtained at substrate peptide modified AuNPs/GCE (Peptide/AuNPs/GCE), which further proved that the phosphorylation reaction did not happen due to the absence of phosphorylation site for PKA in the control peptide. These results demonstrated that the developed approach has good detection selectivity for PKA. The reproducibility of the electrochemical biosensor was also assessed by measuring 10 unit/mL PKA with
Fig. 3. Differential pulse voltammograms of the fabricated biosensor after inhibition by ellagic acid (A) and H-89 (C). (B) and (D) were the inhibition effect of ellagic acid (B) and H-89 (D) on PKA activity.
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five biosensors. The relative standard deviation value was found to be 8.35%, suggesting acceptable reproducibility. 3.4. Inhibition assay Since abnormal expression of PKA has been implicated in a number of diseases including cancers, the identification of PKA inhibitors is valuable for protein kinase-targeted drug discovery and relative diseases therapy. Thus, the PKA activities were evaluated with different concentrations of potential PKA inhibitors, and the half-maximum inhibition value of IC50 was also calculated. Here, H-89 and ellagic acid were used as the model inhibitors, which were potent and cell-permeable inhibitor of PKA. As shown in Fig. 3A, the electrochemical reduction signal of benzoquinone decreased with increasing the concentration of H-89, which revealed the inhibition of PKA activity and low level of substrate peptide phosphorylation. The relationship between inhibition ratio and H-89 concentration was presented in Fig. 3B. The IC50 value (the half maximum inhibitory concentration) was calculated to be 28 nM, which was well consistent with some of previous reports (Bai et al., 2013; Xu et al., 2009). Fig. 3C illustrates the effect of ellagic acid on the PKA activity. The electrochemical reduction signal decreased with increasing the concentration of ellagic acid from 1.0 to 30 μM, which reflected the inhibitory activity of ellagic acid towards PKA. Through the plot in Fig. 3D on the relationship between inhibitory ratio and ellagic concentration, the IC50 value of ellagic acid was calculated to be 2.77 μM,
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which was in agreement with previous reports (Chen et al., 2013; Wang et al., 2010; Xu et al., 2010). These results clearly proved that the developed bioassay is of great potential in screening PKA inhibitors. The fabricated biosensor could be also applied in the discovery of new pharmaceuticals for therapy of some diseases, which are caused by the abnormal expression of protein kinase. 3.5. Detection of PKA activity in cancer cell lysate Because protein kinases play crucial roles in intracellular signaling pathways, a practical method for assaying kinase activity in complex biological samples can contribute significantly to understand the function of kinases in cell signal transduction. Moreover, in order to further explore the applicability of the developed biosensor, PKA activity in different cell lysates were measured. In this case, the substrate peptide was reacted with two kinds of cell lysates including normal hepatic cell (L-02 cell) lysates and hepatoma carcinoma cell (HepG2 cell) lysates, respectively. Then, the substrate peptide was further incubated with Phos-tag-biotin and avidin-HRP successively as described in Section 2.4. Finally, the DPV was performed. As can be seen from Fig. 4A, the electrochemical response of the substrate peptide, incubated with HepG2 cell lysates, was obviously higher than that for L-02 cell lysates, indicating that the PKA activity inHepG2 cell was higher than that in L02 cell. High PKA activity can lead to more phosphorylation events, which might be one of the reasons for cancer. For further evaluating the applicability of the developed
Fig. 4. (A) Differential pulse voltammograms of the biosensor incubated with L02 (b) and HepG2 (a) cell lysates. (B) The differential pulse voltammograms of the biosensor incubated with L02 cell lysate (a), a þ0.5 μM ellagic acid (b), and aþ 0.5 μM H-89 (c). (C) The differential pulse voltammograms of the biosensor incubated with HepG2 cell lysate (a), aþ 0.5 μM ellagic acid (b), and a þ0.5 μM H-89 (c). (D) Histogram for the electrochemical response of the biosensor incubated with different cell lysates and different inhibitors.
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approach for PKA-target drug screening, the substrate peptide on the biosensor was simultaneously incubated with cell lysate and PKA inhibitor (0.5 μM ellagic acid and 0.5 μM H-89, respectively). The corresponding differential pulse voltammograms are illustrated in Fig. 4B and C. As illustrated in Fig. 4D, the electrochemical reduction signal decreased when different PKA inhibitors were introduced into L02 cell lysate and the substrate peptide was incubated with it. In addition, we also found that the current decrease level of the biosensor incubated with the mixed incubation solution of L02 cell lysate and H-89 was higher than that at L02 cell lysate and ellagic acid, which indicated the stronger inhibition ability of H-89 than ellagic acid. Similar results were also obtained when the substrate peptide was incubated with HepG2 cell lysate and different PKA inhibitors. Based on the above research, one can conclude that the developed approach is work well in complex biological system, and it is feasible to analyze intracellular cell kinase activities in vitro and screen kinase-target drugs.
4. Conclusion In summary, a simple and sensitive electrochemical method was developed for protein kinase activity and inhibition assay. Based on the specific recognition ability of Phos-tag for kinaseinduced phosphopeptides and HRP-catalyzed signal amplification, the detection sensitivity of this work was higher than most of other electrochemical methods. This proposed approach was also successfully applied to analyze PKA activity in cell lysates and evaluate the inhibition effect of PKA inhibitors, revealing significant potential for drug discovery, kinase-related diseases diagnose and therapy, kinase function analysis in signal-transduction pathways. However, compared with ECL technique, the detection limit obtained in this work was still needed to be improved. Thus, in the following work, we will develop new and effective signal amplification strategy combining with electrochemical or photoelectrochemical detection technique.
Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 21105056 and 21375079), China Postdoctoral Science Foundation (2014M550369) and the Project of Development of Science and Technology of Shandong Province, China (No. 2013GZX20109).
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.07.016.
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