Aptameric Peptide for One-Step Detection of Protein Kinase

Apr 26, 2012 - Herein, we present a novel one-step strategy to detect protein kinase by using a kinase-specific aptameric peptide-functionalized quart...
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Aptameric Peptide for One-Step Detection of Protein Kinase Xiahong Xu, Jiang Zhou, Xin Liu, Zhou Nie,* Meng Qing, Manli Guo, and Shouzhuo Yao State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, People's Republic of China S Supporting Information *

ABSTRACT: Protein kinases are significant regulators in the cell signal pathway, and it is difficult to achieve quick kinase detection because traditional kinase assays normally rely on a time-consuming kinase phosphorylation process. Herein, we present a novel one-step strategy to detect protein kinase by using a kinase-specific aptameric peptide-functionalized quartz crystal microbalance (QCM) electrode, in which the detection can be finished in less than 10 min. A peptide kinase inhibitor (IP20) was used as the aptameric peptide because of its selective and strong interaction with the target protein kinase (cyclic adenosine monophosphate-dependent protein kinase A, PKA), high stability, and ease of inexpensive synthesis, presenting a new direct recognition element for kinase. The aptameric peptide was immobilized on the Au-coated quartz electrode through dual-thiol anchoring and the binding of His-tagged peptide with a nitrilotriacetic acid/Ni(II) complex, fabricating a highly specific and stable detection platform. The interaction of aptameric peptide with kinase was monitored with the QCM in real time, and the concentration of protein kinase was sensitively measured by the frequency response of the QCM with the low detection limit for PKA at 0.061 mU μL−1 and a linear range from 0.64 to 22.33 mU μL−1. This method is rapid and reagentless and does not require a phosphorylation process. The versatility of our aptameric peptide-based strategy has also been demonstrated by the application in kinase assay using electrochemical impedance spectroscopy. Moreover, this method was successfully applied to detect the forskolin/3-isobutyl-1-methylxanthine-stimulated activation of PKA in cell lysate.

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the phosphorylation-induced aggregation of metal nanoparticles8,10,22 or quantum dots,23 a DNA-based electrochemical method,24 and a phosphorylation-responsive field-effect transistor.25 Actually, all of the above-mentioned methods for detecting protein kinase involve a kinase-catalyzed phosphorylation process of the peptide substrate, which requires a relatively long reaction time (generally about 1 h) to accumulate phosphorylated product for effective detection. Except a few real-time monitoring methods, most of these methods use end point assays where the detection is conducted after the termination of the phosphorylation reaction. Some methods involve a multistep detection process including incubation, washing, and signal generation and amplification, which make the whole assay laborious and time-consuming. Therefore, developing new methods to rapidly detect kinase, especially one-step detection, is significant for the fast diagnosis, but it is still a big challenge. DNA/RNA aptamers have been recently used for protein detection and exhibit some merits including high specificity and affinity for the target, ease of synthesis and modification, and good chemical/thermal stability.26,27 Unfortunately, only a limited number of protein-recognizing DNA/RNA aptamers

rotein phosphorylation by kinase is one of the most important post-translational modifications and plays an essential role in many cellular processes, such as cell cycle, growth, apoptosis, and differentiation.1−3 The protein kinase superfamily accounts for nearly 2% of the human genome coding for more than 500 kinases. Aberrant phosphorylation and abnormal expression of kinase have been implicated in the pathogenesis of many diseases including cancer, diabetes, and Alzheimer’s disease.4 A growing interest in developing methods for detecting protein kinases has recently culminated for biochemical investigation and clinical use. Protein kinase has now become the second most important group of drug targets, after G-protein-coupled receptors. The traditional method for assessing protein kinase activities relies on a radioactive adenosine triphosphte (ATP) analogue and the autoradiography technique.5,6 Owing to its hazardous effect, the radiometric method has largely been replaced by a number of colorimetric,7−11 fluorescent,12,13 electrogenerated chemiluminescent,14 and electrochemical approaches.15−17 Most of these methods depend on the immunoassays using probe-labeled antibodies specific to the phosphorylated product,18 the transfer of the labeling γ-phosphoryl moiety from ATP to the peptide,7,14,15,19,20 a chemically modified substrate peptide with an optical or electrochemical tag,13,21 or monitoring the depletion of ATP as a result of phosphorylation.10 Recently, some label-free biosensors for protein kinase have been developed, such as optical methods involving © 2012 American Chemical Society

Received: January 18, 2012 Accepted: April 26, 2012 Published: April 26, 2012 4746

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activity assay kit. Especially, the feasibility of this novel strategy was also proven by monitoring the stimulator-triggered PKA activation in cell lysates, indicating the high selectivity of the aptameric peptide to differentiate the activated PKA from the inactive form of PKA. Unlike previously reported kinase assays, the aptameric peptide−kinase recognition does not involve a phosphorylation process; thus, it is rapid and reagentless to obtain the detection results in one step (less than 10 min).

are currently available, and few of them target protein kinase. Peptide aptamers are artificial proteins in which a specific short peptide domain with affinity for a target protein is displayed from a scaffold protein.28,29 Peptide aptamers have been applied in the detection of cyclin-dependent kinase 2 (CDK2).30 However, the affinity of peptide aptamers depends on the conformation-constrained effect of the scaffold protein, which would restrict their application due to the chemical/thermal susceptibility of the protein and sophisticated production and modification by protein engineering. Hence, a kinaserecognizing element possessing the advantages of both DNA/ RNA aptamers and peptide aptamers would be an ideal candidate for rapid kinase detection. The substrate specificity of protein kinases is attributed to the recognition motif, which comprises particular amino acids flanking the phosphorylation site. Peptides analogous to this recognition motif have the potential to be substrate competitive inhibitors. The first example of these inhibitors is the natural protein kinase A (PKA) inhibitor protein, PKI, which contains an RRNAI motif resembling the RRXS recognition sequence of PKA, except that the phospho-acceptor serine is replaced by alanine (underlined).31−33 A synthetic 20-residue peptide, IP20, derived from the corresponding “substrate-like” sequence of PKI can specifically recognize the free catalytic subunit of PKA, the activated form of PKA in the cell, and shows a much higher affinity compared to the substrate peptide, according to the fact that IP20 is a highly potent inhibitor of PKA with Ki = 2.3 nM and the Km of the substrate kemptide is 5 μM.34 Due to their high affinity and specificity, these protein kinase peptide inhibitors have the potential to be aptamer-mimicking recognition elements for active protein kinases. Similar to the DNA/RNA aptamer, these short peptide inhibitors are chemically/thermally stable and can be artificially synthesized and modified. Moreover, peptide inhibitors targeting protein kinases present a ubiquitous mechanism for regulating the activity of various protein kinases; thus, the scope of application of these peptide inhibitor-based aptamers can be comparable to that of the peptide aptamer. Hence, the kinase peptide inhibitor, termed the aptameric peptide here, would be an attractive alternative biorecognition element against protein kinase in a biosensor. Nevertheless, to the best of our knowledge, the protein kinase sensor based on these aptameric peptides is relatively unexplored. Herein, we describe a novel one-step strategy for rapid detection of protein kinase based on the quartz crystal microbalance (QCM) to monitor the interaction of aptameric peptide and kinase. The QCM is a reliable and effective method to detect biomolecular interaction with high sensitivity and without using labeling.35−38 Cyclic adenosine 3′,5′-monophosphate (cAMP)-dependent protein kinase (PKA) was used as a model kinase, and IP20 was exploited as the aptameric peptide against PKA. The immobilization strategy of IP20 on a Au-coated quartz crystal relies on the specific interaction of the polyhistidine tag with the metal ion, producing a highly oriented peptide assembly and avoiding nonspecific protein adsorption. The frequency of the QCM crystal sensitively responds to the amount of kinase bound to the surface, allowing us to monitor the binding of PKA in real time and quantitatively determine the concentration of PKA. Because the target recognized by the aptameric peptide is the activated form of PKA, the determined concentration of active PKA corresponds to its kinase activity, which was demonstrated by the comparison with the results of the commercial kinase

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EXPERIMENTAL SECTION Chemicals. Dihydrolipoic acid (DHLA), 1-mercaptohexane (MCH), forskolin (Fsk), 3-isobutyl-1-methylxanthine (IBMX), and nitrilotriacetic acid (NTA) were purchased from SigmaAldrich (St. Louis, MO). Cyclic adenosine 3′,5′-monophosphate-dependent PKA (catalytic subunit, MW = 40 000, mass concentration 2.08 mg mL−1, activity concentration:127.6 U μL−1) was purchased from Promega (Madison, WI). To facilitate the comparison with the commercial kinase activity assay, the activity concentration was used in all kinase experiments. Casein kinase II (CKII) was obtained from New England Biolabs (Beverly, MA). Hexahistidine-tagged aptameric peptide for PKA (His6-IP20) (HHHHHHTTYADFIASGRTGRRNAIHD), control peptide (His 6 -GA 20 ) (HHHHHHGGGGRTGRRNAIHDILVSSA), cysteine-tagged aptameric peptide (Cys-IP20) (CYADFIASGRTGRRNAIHD), 1-ethyl-3-(3′-(dimethylamino)propyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were purchased from GL Biochem Ltd. (Shanghai, China). ATP was purchased from Generay (Shanghai, China). Protease inhibitor and an improved Bradford protein assay dye reagent kit were obtained from Sangon (Shanghai, China). NiCl2 was purchased from Sinopharm (Shanghai, China). Other reagents including bovine serum albumin (BSA), thrombin, lysozyme, Tris, glycerol, dithiothreitol (DTT), and ethylenediaminetetraacetic acid (EDTA) were purchased from Bio Basic (Ontario, Canada). A commercial kinase activity assay kit (ProFluor PKA assay) was purchased from Promega. All solutions were prepared using ultrapure water (18.3 MΩ·cm) from a Millipore Milli-Q system. Immobilization of Aptameric Peptide (IP20) on the QCM Au Electrode. A computer-interfaced Maxtek RQCM quartz crystal microbalance research system equipped with an AT-cut 9 MHz piezoelectric quartz crystal (PQC) (12.5 mm in diameter) was used in the QCM experiment. Before the fabrication of the peptide-based QCM sensor, the QCM Au electrode was first cleaned with piranha, a 3:1 mixture of concentrated H2SO4 and 30% H2O2, rinsed with ethanol, and then rinsed thoroughly with water. The assembly procedures of the aptameric peptide-immobilized QCM were as follows. The QCM Au electrode was immersed in 1 mM DHLA alcoholic solution for self-assembly to obtain the DHLA/QCM Au electrode. NHS (5 mM) and EDC (2.5 mM) in 0.1 M 2(N-morpholino)ethanesulfonic acid (MES) buffer (pH 6.0) were dropped onto the electrode for activation of DHLA carboxylic acids. Further QCM/DHLA/NTA modification was carried out by adding NTA/Ni2+ solution to the Au electrode of the QCM through amidation for 1 h. The NTA/Ni2+ complex was preprepared by reaction of NTA with an excess of NiCl2 in 20 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) in aqueous solution. The nickel excess was precipitated by adjusting the pH to 10, and the hydroxide was removed by filtration through a 0.2 μm membrane (Millipore). The amidation was terminated by adding 50 mM 4747

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minutes after stimulation, the cultured cells were removed by scraping and lysed in Dulbecco’s phosphate-buffered saline (DPBS) including protease inhibitor by sonication (200 W) for 2 s 60 times at an interval of 3 s 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 the 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. In subsequent experiments for kinase assay in cell lysate, the total protein concentration of cell lysate was diluted to 12.5 μg mL−1. Kinase Activity Measurement with the Fluorescent PKA Activity Assay Kit. The ProFluor PKA assay system was used as a reference method to assay PKA kinase activity. The assay was conducted following the protocol in the manufacturer’s technical manual. A 25 μL volume of kinase solution containing different concentrations of PKA (0−1276.0 mU μL−1) and 10 μM PKA R110 substrate in 1× reaction buffer was added to a black 96-well microplate. Then 25 μL of 100 μM ATP solution in 1× reaction buffer was added. The plate was shaken for 15 s and incubated for 20 min at room temperature. After incubation, 25 μL of protease solution was added to all wells. The resulting solution was mixed and incubated for 30 min at room temperature. Finally, 25 μL of stabilizer solution was added to all wells. The fluorescence signal was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. For kinase activity assay with cell lysates, the experiments were conducted under the above-mentioned conditions, except for the involvement of different cell lysate samples in the kinase solutions.

Tris−HCl buffer onto the QCM electrode for 30 min, and the amino group of excess Tris reacted with all NHS esters remaining on the surface of the QCM. The resulting NTA/ DHLA/QCM electrode was treated with a hexahistidine-tagged peptide (His6-IP20) solution (0.1 mM) in Tris-buffered saline (TBS; 20 mM Tris−HCl, pH 7.5) and left for 6 h at 4 °C to obtain the IP20/NTA/DHLA/QCM Au electrode. The QCM sensor functionalized by the cysteine-IP20 (CysIP20) peptide was prepared as follows: The cleaned QCM Au electrode was treated with 0.1 mM cysteine-IP20 peptide solution and left for 6 h at 4 °C, rinsed with TBS buffer several times, and then immersed into a 0.1 mM ethanolic solution of MCH for 10 min to obtain the Cys-IP20/QCM Au electrode. Electrochemical Measurement. All electrochemical experiments were carried out on a CHI660A electrochemical workstation (CH Instrument Co., China). A homemade threeelectrode electrolytic cell was used in the experiments. A gold electrode (2 nm in diameter), a saturated calomel electrode (SCE), and a platinum gauze electrode served as the working electrode, the reference electrode, and the counter electrode, respectively. All the potential values in this paper refer to the SCE. Prior to the electrochemical measurement, the gold electrode was subjected to potential cycling between 0 and 1.5 V in a 0.50 M H2SO4 aqueous solution until the cyclic voltammogram of a clean polycrystalline gold electrode was reproducibly obtained. For each step of immobilization, electrochemical impedance spectroscopy (EIS) measurements were performed in the presence of equimolar 5 mM [Fe(CN)6]3−/4− in TBS. Electrochemical detection was performed with EIS with the following parameters: scan rate 0.1 V s−1, initial potential 0.2 V, frequency 100 kHz to 0.05 Hz, amplitude 0.005 V. All the impedance spectra were plotted in the form of Nyquist plots. Specific Recognition and Detection of PKA by the QCM. The IP20-immobilized QCM resonator was soaked beforehand in kinase binding buffer (50 mM Tris−HCl, pH 7.5, 10 mM MgCl2). Then the resonant frequency of the QCM was defined as the zero position after equilibrium (ca. 10−20 min). The frequency decrease of the QCM resonator, in response to the addition of PKA solutions of varying concentrations, was then recorded over time. Measurements were performed at 25 °C. For comparison, the resonant frequencies of the other control QCM electrodes were determined following the same steps. BSA (5 μM), lysozyme (0.25 U μL−1), thrombin (0.5 μM), and CKII (0.25 U μL−1) were used as the reference proteins to characterize the nonspecific adsorption of the IP20-immobilized surface. The real time detection process was carried out with the QCM. MCF-7 Cell Culture and Lysate Preparation. MCF-7 breast cancer cells (1 × 106 cells) were supplemented with 10% fetal bovine serum, Mimimum Eagle's essential medium (MEM) nonessential amino acid solution (0.1 mM), 1% insulin−transferrin−selenium A supplement, penicillin (100 U mL−1), streptomycin (100 mg mL−1), and amphotericin B (0.25 mg mL−1). The cells were incubated under a humidified atmosphere containing 5% CO2 at 37 °C. The culture medium was replaced by serum-free medium 4 h before stimulation. Solutions of Fsk and IBMX with defined concentration in dimethyl sulfoxide (DMSO) were added to the medium to activate intracellular PKA (final concentrations of forskolin and IBMX are given in Figure 4 B, inset). DMSO (equal volume) was added to the medium for unstimulated samples. Thirty

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RESULTS AND DISCUSSION Construction and Characterization of the Aptameric Peptide-Immobilized Surface. The binding affinity, orientation, and surface coverage of immobilized peptide recognition elements are crucial to the sensitivity and specificity of the peptide-functionalized QCM sensor. In this work, the aptameric peptide IP20, specific for protein kinase A, was tagged with six histidines (His-tag) at the N-terminus for its immobilization on a gold QCM surface. As shown in Scheme 1A, the immobilization strategy of His-tagged IP20 was elaborately designed on the basis of two features: (1) strong and specific binding of the His-tag with nickel(II)/nitrilotriacetic acid (Ni2+/NTA) and (2) dithiolated NTA ligand derived from DHLA possessing enhanced gold surface-binding stability through dithiol−gold bonds. The self-assembled monolayer (SAM) of dithiol-functionalized NTA on the QCM gold surface allowed His-tagged IP20 specific tethering in an oriented layer, which is favorable for selective binding of PKA and avoiding nonspecific protein adsorption. The IP20 aptameric peptide can selectively bind the catalytic unit of PKA, due to its “substrate-like” RRNAI site, which shows strong cohesion with PKA through occupation of the substrate binding site.32 The peptide immobilization and the binding of PKA can be sensitively detected by the QCM with a PQC as its central sensing element. The QCM can detect an electrode mass change down to the nanogram level, and the mass effect can be depicted by the Sauerbrey equation:38 Δf0 = −2f0g 2 4748

Δm Δm = ( −2.264 × 10−6)f0g 2 A ρμ A

(1)

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In the Nyquist plots of impedance spectra, an increase of the diameter of the semicircle reflects an increase of the interfacial electron-transfer resistance (Ret).41,42 Figure S2 (Supporting Information) shows the electrochemical EIS results of the bare Au (1), Au/DHLA (2), Au/DHLA/NTA (3), and Au/DHLA/ NTA/IP20 (4). It is noted that the bare electrode showed a very small semicircle domain (Ret = ∼0.2 kΩ, curve 1), indicating a very fast electron-transfer process of [Fe(CN)6]3−/4−. The selfassembly of negatively charged DHLA on the electrode surface effectively repelled the [Fe(CN)6]3−/4− anions and thus led to an enhanced electron-transfer resistance (a significant increase in Ret to ∼120 kΩ, curve 2). Then the NTA/Ni(II) complex caused a decrease of Ret to ∼12 kΩ (curve 3). It is also found that the binding of His6-IP20 by NTA/Ni(II) to the electrode surface led to a significant decrease in Ret (∼7.5 kΩ, curve 4) because of the positively charged arginine residues of the peptide sequence drawing [Fe(CN)6]3−/4− anions to the electrode surface. All these experimental results demonstrate that the sensing interface has been fabricated successfully according to Scheme 1. Rapid Detection of PKA by the IP20-Modified QCM. Conventional methods for detecting protein kinase are based on measuring kinase-catalyzed phosphorylation. Most of them involve two steps, including a phosphorylation step and a signal report step, making the whole assay inevitably time-consuming. Moreover, ATP and Mg2+, essential for phosphorylation, and the reagents for signal report are normally required in these methods, which increase the expense of the assays. Taking the aptameric peptide as the protein kinase probe, we represented a one-step strategy for rapid detection of protein kinase, which can be expected to be time-saving and reagentless. Furthermore, due to the real-time feature of QCM analysis, the merit of this QCM-based kinase assay is not only to quantitatively detect the amount of kinase via the recognition of the aptameric peptide, but also to monitor the binding process of the aptameric peptide with kinase in real time. The preparatory IP20/NTA/DHLA-functionalized QCM was used for monitoring the recognition of the aptameric peptide IP20 toward PKA through the “RRNAI” motif as the PKAbinding site (as step 3 shown in Scheme 1B). Figure 1 shows the typical frequency change for the IP20-immobilized QCM resonator in response to addition of PKA (curve 1). A continuous decrease of frequency was observed due to binding

Scheme 1. Schematic Illustration of the Aptameric Peptide (IP20)−PKA Conjugate as a Sensing Platform To Monitor Kinase: (A) Construction of the Dithiolated NTA/Ni2+ Interface for Specific Immobilization of Histidine-Tagged IP20 and Recognition of PKA by Aptameric Peptide,a (B) Immobilization Steps for the Aptameric Peptide to the QCM Au Electrode and the Recognition of PKA by the IP20Immobilized QCM Resonator

a

The RRNAI motif is the PKA-binding site of IP20.

where f 0g (Hz) is the QCM resonant frequency in the fundamental mode in air, Δm (g) is the electrode mass, A (cm2) is the piezoelectrically active area, ρ (=2.648 g cm−3) is the density of the crystal, and μ (=2.947 × 1011 g cm−1 s−2) is the shear modulus of quartz. The detailed immobilization process is outlined in Scheme 1B. The fabrication of the DHLA−NTA/Ni2+ interface is shown as step 1. At first, the QCM Au electrode was coated by DHLA to form a stable SAM, which was monitored with the QCM (as shown in Figure S1A in the Supporting Information). The DHLA assembly caused a frequency change of ∼−90 Hz, and the final frequency decrease (Δf 0) of ∼29.8 Hz in air indicated that about 163 ± 42 ng cm−2 of DHLA was immobilized on the Au surface according to the Sauerbrey equation. Then the terminal carboxylate group of surfaceconfined DHLA was covalently conjugated with the free amino group of the NTA/Ni(II) complex, causing a frequency change of ∼−184 Hz (Figure S1B). The NTA/Ni(II) complex termination contains two free coordination sites occupied by water molecules that can be replaced by histidine residues,39,40 so aptameric peptide IP20 bearing a six-histidine tag could be immobilized specifically to obtain an IP20-functionalized QCM (shown as step 2). The immobilization process of IP20 was monitored with the QCM in real time (Figure S1C). Peptide aptamer His6-IP20 anchoring to the surface of the NTA/ DHLA/Au QCM resulted in a gradual decrease of the frequency response, demonstrating that the binding of the hexahistidine-tagged peptide with the NTA surface was effective. We also employed electrochemical EIS to investigate the characteristics of the aptameric peptide-modified gold surface.

Figure 1. Real-time frequency response of the QCM surface with IP20 (1) or GA20 (2) or without IP20 modification (3) to the recognition of PKA (22.33 mU μL−1). 4749

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of PKA to IP20. After addition of 22.33 mU μL−1, the frequency change reached a platform with Δf = ∼−340 Hz at 4 min, which indicated that the binding of PKA finished rapidly in a short time. In accordance with our expectation, this one-step strategy takes only 4 min to obtain the signal readout of aptameric peptide−kinase interaction as well as to achieve protein kinase detection without addition of any other reagents. The duration of this one-step kinase assay is remarkably shorter than that of the previously reported methods relying on a phosphorylation process. Another peptide (His6-GA20) with the same electrostatic charge and similar amino acid sequence but much weaker affinity for PKA compared to IP20 (Ki of 75 μM for GA20 vs Ki of 2.3 nM for IP20) was used as a control peptide. Only a slight response (Δf ≈ 25 Hz) was induced (curve 2), indicating that the high affinity of IP20 for PKA is the prerequisite for the selective recognition and sensitive detection of kinase by the aptameric peptide. No obvious frequency change was observed on the QCM surface without peptide (curve 3). These results clearly indicate the specific peptide− protein interaction between the aptameric peptide (IP20) and protein kinase PKA. Detection of the protein kinase was further studied with different concentrations of PKA (0−1276 mU μL−1). As shown in Figure 2A, the detection of PKA by the aptameric peptidemodified QCM was monitored by the frequency change in real time, and all signal readouts for varying concentrations of PKA were achieved in several minutes. This QCM sensor is sensitive

and has a relatively obvious frequency response (Δf = ∼−80 Hz) even at a subnanomolar level of PKA (0.64 mU μL−1). Increasing the concentration of PKA from 0 to 89.32 mU μL−1 resulted in a significant increase of the frequency change from 0 to ∼−592 Hz. Further increasing the concentration of PKA (from 89.32 to 1276 mU μL−1) induced a moderate increase of the frequency change (from ∼−592 Hz to ∼−700 Hz), indicating that the IP20-functionalized sensor surface became completely bound by, and saturated with, PKA. Figure 2B shows the frequency response of the IP20-immobilized QCM as a function of the concentration of PKA (ranging from 0 to 1276 mU μL−1). A linear relationship between the frequency response and the concentration of PKA was obtained in the range from 0.64 to 22.33 mU μL−1. The linear relationship can be represented as −Δf = 10.18c + 90.49 with a correlation coefficient R2 = 0.992, where Δf is the frequency response (Hz) and c is the kinase concentration (mU μL−1). The sensitivity of the QCM sensor was 10.18 Hz/(mU μL−1), and the detection limit of PKA was 0.061 mU μL−1 (signal-to-noise ratio of 3). This demonstrates that the proposed aptameric peptidemodified QCM sensor can be employed for highly sensitive protein kinase detection over a wide concentration range and in a short time. To compare the aptameric peptide-based method with the traditional kinase activity assay, a commercial kinase activity assay kit was employed as a reference method. As shown in Figure S3 (Supporting Information), the frequency response curve as a function of the logarithmic PKA concentration obtained with the IP20-modified QCM electrode can perfectly match the fluorescence signal curve of the PKA assay kit. The EC50 (the amount of kinase needed to achieve 50% of the maximum signal) of the IP20-modified QCM electrode is 33.64 mU μL−1, which is very close to that of the commercial kit (31.36 mU μL−1). Additionally, the frequency response of the IP20-modified QCM electrode to the thermal inactivated PKA was comparable to that of the enzyme storage buffer, which corresponds to no fluorescence signal of the PKA activity assay in response to the thermal inactivated PKA (as shown in Figure S4, Supporting Information). These results effectively demonstrate that, although without a phosphorylation process, the aptameric peptide-based method also can reflect the kinase activity level through specific and quantitative measurement of the active form of PKA. Moreover, compared to about 1 h of detection time and the multistep process required in the commercial PKA activity assay, the aptameric peptide-based QCM method exhibited great advantages in its quick detection (less than 10 min), reagentlessness, and add-and-read feature. The feasibility of the aptameric peptide as a kinase recognition element was also checked by measuring the EIS response of the IP20-immobilized Au electrode with respect to the kinase concentration. Figure S5A (Supporting Information) shows the Nyquist plots for the faradic impedance of the IP20immobilized Au electrode against different concentrations of PKA. With increasing concentration of PKA, the electrontransfer resistance (ΔRet) of the electrode significantly increased (from 7.5 to 22 kΩ) because the introduction of large protein molecules via aptameric peptide−kinase interaction inhibited the electron transfer of the redox probe on the surface. The concentration of PKA can be quantified by the ΔRet change of the electrode, and the related ΔRet changes as a function of PKA concentration are plotted in Figure S5B. The resistance change increases with increasing kinase concentration ranging from 0 to 85 mU μL−1 and reaches a plateau

Figure 2. (A) Real-time frequency response of the IP20-immobilized QCM resonator to different concentrations of PKA (from 0 to 89.32 mU μL−1). (B) Change in the frequency response as a function of the PKA kinase concentration. The inset is a linear plot of the dependence of the change in frequency responses on the concentration of PKA. 4750

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above 85 mU μL−1. The linear relationship is ΔRet = 145.1c + 75.52 (from 10.6 to 35.4 mU μL−1, R2 = 0.998), and the detection limit for PKA is 0.44 mU μL−1. Hence, besides the QCM, the aptameric peptide-based EIS measurement is also a potent method for label-free detection of protein kinase. Specificity, Stability, and Regeneration of the IP20Immobilized QCM. To investigate the specificity of the proposed method, the responses of the aptameric peptideimmobilized QCM electrode to other proteins, such as BSA, lysozyme, thrombin, and CKII, were compared with that to PKA. As shown in Figure 3, PKA provoked an evident decrease

instability of the Cys-IP20 QCM in the presence of thiol, possibly hampering its application in PKA sensing. Oppositely, our ditholated ligand-based immobilization strategy showed effective resistance to desorption and substitution by DTT because of the multiple bonds of each ligand with the gold surface, which is reliable and suitable for kinase detection in biological media. The His-tag−Ni/NTA system is practically attractive because this interaction can be easily renewed by treatment with imidazole, EDTA, or low pH. This feature allows us to fabricate the reusable sensor interface easily. Treatment of the kinasebound peptide layer with 0.5 M imidazole led to complete dissociation of the kinase and aptameric peptide from the surface. Addition of Ni2+ and His-tagged IP20 resulted in regeneration of the sensor interface. The regenerated IP20modified QCM exhibited a frequency shift similar to that of the original QCM sensor in response to 89.32 mU μL−1 PKA, and after four cycles of regeneration, the frequency shift only decreased 7.8% in comparison with that of the original sensor. Protein Kinase Detection in Cell Lysate. Protein kinases have crucial and multifaceted effects on signal transduction and cell signaling; therefore, the generation and amount of their active form are highly regulated in a cell. The activation or inactivation of kinases caused by extracellular stimulation can trigger a series of important cellular processes, including transcription, apoptosis, and differentiation.3 Thus, kinase assays available for detection in cell lysate are significant for the study of kinase regulation in the cell system. We examined whether our method can be applied to detect kinase in cell lysate. PKA in human cells could be activated by stimulation with Fsk and IBMX. Fsk is a compound capable of stimulating adenylate cyclase and elevating cyclic AMP levels in the cell, and IBMX can inhibit cAMP phosphodiesterase, which eliminates cyclic AMP. As shown in Scheme 2, in the absence

Figure 3. Real-time frequency response of the IP20-immobilized QCM resonator to PKA (51.04 mU μL−1), BSA (5 μM), lysozyme (0.25 U μL−1), thrombin (0.5 μM), and CKII (0.25 U μL−1).

in the frequency response after addition, owing to the rapid binding with the peptide aptamer IP20, but BSA, thrombin, and lysozyme provoked only an inconspicuous frequency change, while another kinase, CKII, whose representative peptide substrate sequence is RRRADDSDDDDD, gave a negligible frequency decrease, indicating that the aptameric peptide is a highly selective recognizer of its target kinase. To ensure the enhanced stability of the dithiolate ligand SAM, we investigated the performance of the IP20-immobilized QCM in the presence of thiolated species. Thiolated species are ubiquitous in the cytoplasm and the storage buffer of commercially available kinases. It has been reported that the SAM of monothiolated ligands on a gold surface are prone to partial desorption and ligand exchange in biological media.43 A His-tagged IP20−NTA/Ni−DHLA-immobilized QCM was compared with a Cys-IP20-immobilized QCM in the presence of DTT, a model thiol. As shown in Figure S6 (Supporting Information), the His6-IP20 SAM immobilized on the QCM by DHLA and His-tag−Ni/NTA exhibited a frequency response of −367 Hz to 51.04 mU μL−1 PKA, and no obvious frequency shift was observed when DTT was added. The Cys-IP20 SAMcoated QCM showed a significantly smaller frequency response to the same concentration of PKA (Δf = ∼−173 Hz) compared to the His-tagged IP20−NTA/Ni−DHLA QCM. Moreover, the addition of DTT induced gradual desorption of the Cys-IP20 SAM with an increase of frequency, resulting from the replacement of cysteine by DTT and subsequent dissociation of the peptide or peptide−PKA complex.44 After the gradual frequency increase for 30 min, a frequency decay was followed, probably due to the immobilization of the excess DTT onto the QCM gold surface. This frequency fluctuation indicates the

Scheme 2. Schematic Representation of the Stimulation of PKA in MCF-7 Cancer Cell Lysate by Cyclic AMP Which Was Aroused through Activator Fsk and IBMXa

a

The active catalytic subunits of PKA were then recognized by the aptameric peptide (IP20)-immobilized QCM resonator for real-time detection.

of cAMP, PKA exists predominantly as an inactive tetrameric holoenzyme composed of two catalytic (C) and two regulatory (R) subunits. In the presence of cAMP, each R subunit binds two molecules of cAMP at separate allosteric binding sites and the holoenzyme dissociates into an R subunit dimer and two monomeric C subunits. The free C subunit is an active form of PKA which is able to phosphorylate specific serine and threonine residues in related protein substrates. Fsk and 4751

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that of IP20 (Ki of 2.3 nM). The nonpeptide controls (DHLAmodified QCM surface) showed nearly no response to stimulated cell lysate (curve d) or unstimulated cell lysate (curve e). Meanwhile, we investigated the dose effect of stimulators (Fsk and IBMX). MCF-7 breast carcinoma cells were stimulated with Fsk and IBMX of gradually increasing concentrations (the concentrations of Fsk and IBMX are shown in the inset table in Figure 4B), and the amount of activated PKA in stimulated MCF-7 cell lysate was monitored. As depicted in Figure 4B, the frequency change of the IP20immobilized QCM increased with increasing concentration of stimulators, and the frequency change nearly reached a plateau when the concentrations of Fsk and IBMX were higher than 25 and 50 μM, respectively. This stimulator-dose-dependent curve of the frequency signal was very similar to that of the fluorescence signal obtained with the PKA activity assay kit for the same cell lysate samples (as shown in Figure S7, Supporting Information), indicating that the aptameric peptide-based method can not only selectively differentiate the activated PKA, the free catalytic subunit of PKA, from inactive PKA holoenzyme in the complex cytoplasmic environment, but also reliably reflect the intracellular PKA activity level via direct detection of active PKA. Therefore, the stimulator-triggered activation of PKA in cells was successfully detected via the aptameric peptide-modified QCM, demonstrating this sensing platform is feasible for in vitro cell kinase assay.

IBMX work together to induce a remarkable increase of cAMP, leading to subsequent activation of PKA. The high affinity of the aptameric peptide IP20 for the C subunit of PKA allows the IP20-functionalized QCM surface to potently capture the free C subunit of PKA, and the corresponding frequency response can be recorded for measurement of active PKA in cell lysate. As shown in Figure 4A, the activation of PKA in MCF-7 cells by

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CONCLUSIONS This study has demonstrated for the first time the use of aptameric peptide−protein recognition in QCM technology for detecting protein kinase A. Because of its high affinity for the catalytic subunit of PKA, the protein kinase inhibitor peptide IP20 as an aptameric peptide presented a novel and potent recognition element to detect active PKA. This aptameric peptide-based method is rapid (less than 10 min), facile, and reagentless due to its one-step detection without a phosphorylation process, exhibiting unique advantages over conventional kinase assays which are time-consuming (normally above 1 h), need multiple steps, and require addition of several reagents in a phosphorylation and detection process. Because of its direct detection of active PKA, this method showed results comparable to those of a commercial kinase activity assay, demonstrating it is capable of reflecting in part the kinase activity level. The piezoelectric kinase sensor is capable of sensitively detecting protein kinase A with a detection limit of 0.061 mU μL−1 and monitoring the binding of PKA and the aptameric peptide in real time. To the best of our knowledge, this is the first kinase assay relying on the QCM technique. The rational strategy for immobilization of the aptameric peptide composed of the selective binding of a His-tag with Ni2+/NTA and the DHLA-derived dithiolate ligand possessing strong interaction with the gold surface endowed this peptidefunctionalized QCM with high stability and specificity, even in the complex cell lysate. The rapid detection of active PKA in stimulated cell lysate revealed the feasibility of our method in real cell samples and the potentiality for biochemical investigation of signal transduction. Since the aptameric peptide with a His-tag is potent for PKA recognition and easy to immobilize, its application in another label-free technique, electrochemical impedance spectroscopy, has also been achieved, and it may also be exploited in (surface plasmon resonance) kinase sensing. Furthermore, an increasing number of aptameric peptides targeting various protein kinases have

Figure 4. (A) Real-time frequency response of the IP20-immobilized (a) or GA20-immobilized (b) QCM to stimulated MCF-7 cancer cell lysate, the IP20-immobilized QCM to unstimulated cell lysate (c), and the DHLA-modified QCM to stimulated cell lysate (d) or unstimulated cell lysate (e) (Fsk, 25 μM; IBMX, 50 μM). (B) Bar chart of the frequency response of the IP20-immobilized QCM to different concentrations of activator Fsk/IBMX mixtures. The inset shows the concentration of the cell lysate samples from 1 to 6; the blank was treated with unstimulated cell lysate.

these stimulators (25 μM Fsk and 50 μM IBMX) was clearly detected by the IP20-functionlized QCM surface, and a large frequency shift (Δf 0 = ∼−1039 Hz) was observed (curve a), but the QCM surface modified by the control peptide exhibited a negligible frequency shift response to stimulated cell lysate (curve b), indicating that (i) the remarkable QCM signal is due to a selective and stable combination of stimulator-activated PKA and its specific aptameric peptide IP20 and (ii) the nonspecific binding effect of the cell lysate sample on the peptide-modified QCM is insignificant. There was a slight frequency shift (Δf 0 = ∼−119 Hz) of the IP20-immobilized QCM in response to the cell lysate without treatment of stimulators (curve c). We prefer to attribute this slight QCM response to the minute amount of free C subunit of PKA in a nonstimulated cell rather than the replacement of the R subunit of PKA by IP20 to bind with the C subunit of PKA because the R subunit affinity for the C subunit (Kd of 0.39 nM) exceeds 4752

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been discovered and identified in biochemical studies;32 therefore, this aptameric peptide-based method shows promise as a versatile way to assay kinases with therapeutic significance. This proof-of-principle work will potentially aid the future creation of highly multiplexed aptameric peptide microarrays using a broad range of label-free optical or electronic assays for simultaneous determination of multikinases in cells.

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ASSOCIATED CONTENT

S Supporting Information *

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

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-731-88821626. Fax: +86-731-88821848. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Grants 21175036, 21075031, 20905024, and 20975032), the National Basic Research Program of China (973 Program, Grants 2011CB911002 and 2009CB421601), and the Program for New Century Excellent Talents in University (Grant NCET-10-0366).

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