A Label-Free Homogeneous Electrochemical Sensing Platform for

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A Label-Free Homogeneous Electrochemical Sensing Platform for Protein Kinase Assay Based on Carboxypeptidase Y–Assisted Peptide Cleavage and Vertically Ordered Mesoporous Silica Films Jinquan Liu, Hong Cheng, Dinggeng He, Xiaoxiao He, Kemin Wang, Qiaoqiao Liu, Shuaiqi Zhao, and Xudong Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01739 • Publication Date (Web): 08 Aug 2017 Downloaded from http://pubs.acs.org on August 9, 2017

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Analytical Chemistry

A Label-Free Homogeneous Electrochemical Sensing Platform for Protein Kinase Assay Based on Carboxypeptidase Y–Assisted Peptide Cleavage and Vertically Ordered Mesoporous Silica Films Jinquan Liu, Hong Cheng, Dinggeng He, Xiaoxiao He,* Kemin Wang,* Qiaoqiao Liu, Shuaiqi Zhao, Xudong Yang State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, College of Chemistry and Chemical Engineering, Hunan University, Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, Changsha 410082, China. * E-mail: [email protected]. Phone: +86-731-8882-1566 * E-mail: [email protected]. Phone: +86-731-8882-3073 ABSTRACT: Herein, a simple, robust, and label-free homogeneous electrochemical sensing platform was constructed for the detection of protein kinase activity and inhibition by integration of carboxypeptidase Y (CPY)-assisted peptide cleavage reaction and vertically ordered mesoporous silica films (MSFs). In this sensing platform, the substrate peptide composed of kinase specific recognized sequence and multiple positively charged arginine (R) residues was ingeniously designed. In the presence of protein kinase, the substrate peptide was phosphorylated and then immediately resisted CPY cleavage. The phosphorylated peptide could be effectively adsorbed on the negatively charged surface of MSFs modified indium-tin oxide (ITO) electrode (MSFs/ITO) by noncovalent electrostatic attraction. The adsorbed peptide was subsequently used as a hamper to prevent the diffusion of electroactive probe (FcMeOH) to the electrode surface through the vertically aligned nanopores, resulting in a detectable reduced of electrochemical signal. As demonstrated for the feasibility and universality of the sensing platform, both of protein kinase A (PKA) and casein kinase II (CK2) were selected as the models, and the detection limits were determined to be 0.083 UmL-1 and 0.095 UmL-1, respectively. This sensing platform had the merits of simplicity, easy manipulation and improved phosphorylation and cleavage efficiency, which benefited from homogeneous solution reactions without sophisticated modification or immobilization procedures. In addition, given the key role of inhibition and protein kinase activity detection in cell lysates, this proposed sensing platform showed great potential in kinase-related bioanalysis and clinical biomedicine.

Phosphorylation of protein, catalyzed by protein kinase with the addition of phosphate group from adenosine-5triphosphate (ATP), is an important biological process that is involved in signal transduction, gene expression, metabolism, and cell proliferation.1, 2 It has been demonstrated that nearly 30% of human proteins are regulated by phosphorylation.3 Aberrant protein kinase could change the normal protein phosphorylation networks, which is profoundly related with the pathogenesis of numerous diseases including cancer,4 Alzheimer’s disease5 and diabetes.6 In this case, an accurate detection of protein kinase activity and their inhibitor is important for the research of protein kinase-related clinical diagnosis and drug discovery, as well as for further understanding the fundamental biological metabolism processes. Nowadays, there has been a growing interest in developing amounts of techniques for the detection of protein kinases activity, including 32P radioactive,7 surface-enhanced Raman spectroscopy (SERS),8 mass spectroscopy (MS),9 quartz crystal microbalance (QCM),10 colorimetric,11, 12 fluorescence, 13-16 photoelectrochemical17 and electrochemical sensing18-20 and so on. Among all the existing techniques, great efforts

have been focused on electrochemical sensing because of the advantages of rapid response, ease of miniaturization, and good sensitivity and selectivity. To date, the strategies for developing of electrochemical sensing for protein kinase activity analysis were generally employed by ATP labeled with ferrocene or thiol which modified on the substrate peptide for the period of phosphorylation processes.21, 22 These strategies could provide effective methods for protein kinase detection. However, the labeling of ATP tended to significantly increase the experimental complexity and cost. To avoid the defect of the additional modification of ATP, the electro-catalyzed tyrosine oxidation was developed. 23, 24 Unfortunately, the employment of this strategy exhibited highly specific and could only detect the activity of tyrosine kinase, which restricted its extensive application. Apart from these electrochemical strategies, some metallic ions such as Ti4+, Zr4+ and Phos-tag (i.e., Zn2+ or Mn2+) were also employed on account of their fantastic feature of the specific binding with phosphate groups.25-27 In this strategy, the metallic ions could specifically mediated reaction between the phosphorylated peptide and the signal transition probe. The signal transition probe consisted of some phosphorylated

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binding receptor then could be self-assembled onto the surface of the electrode. It was likely that this strategy could offer a new means for highly sensitive and universal detection of protein kinases without ATP labeling. Essentially, all of the aforementioned electrochemical strategies were performed in heterogeneous systems. The phosphorylation was obtained on the surface of the electrode, suffering from the immobilization of substrate peptide on the electrode surface and relatively low phosphorylation efficiency resulted from local steric hindrance. Hence, it was highly desirable to develop more efficient and convenient immobilization-free electrochemical strategy for the detection of protein kinases activity. Homogeneous electrochemical strategy, with this in mind, was effective options that it can eliminate the tedious probe immobilization processes and avoid the steric hindrance occurred on the interface between the solution and electrode surface.28 Very recently, homogeneous electrochemical strategy has been developed for various targets detection such as enzyme,29-32 small molecules,33 ions,34 nucleic acid35 and so on. For example, Xuan et al. constructed a homogeneous electrochemical strategy for the detection of target DNA.35 Our group developed a novel homogeneous electrochemical sensing for the firstly detection of casein kinase II (CK2) activity and inhibition by taking advantage of the specific binding of the phosphate groups with TiO2/MWCNT nanocomposites.31 Indeed, these homogeneous electrochemical strategies have exhibited effective detection for targets. However, the developed homogeneous electrochemical strategies were mainly realized by the labeling of substrates. In this context, the development of label-free homogeneous electrochemical strategy was highly desirable for the applications in target assay, especially for protein kinase. Mesoporous silica films (MSFs), as recently fascinating porous materials, have attracted extensive attention owing to their well-ordered and vertically aligned nanopores on solid substrates.36-39 Particularly, MSFs possess unrivalled candidates for label-free electrochemical and electrochemiluminescence sensing, because they could effectively enable fast mass transfer for signal-generating molecular into or out of the underlying electrode surface and dramatically enhance the signal-to noise ratio.40-42 Lately, Fernández et al. reported a novel electrochemical sensing to detect transglutaminase (TGase) activity based on TGasecontrolled diffusion of Fe(CN)63-/4- through aminofunctionalized MSFs.43 Yan et al. have built a cost-effective and simple electrochemical sensing for the direct detection of redox-active organic analytes in complex media without sample pre-treatment by vertically ordered MSFs and surfactant micelles (OSM@SM).44 Meanwhile, our group constructed a simple electrochemiluminescence aptasensor platform for label-free detection of enzyme, small molecules, and ion by integration of vertically ordered MSFs and aptamer-gated systems.45 These MSFs-assisted label-free electrochemical and electrochemiluminescence sensing methods have realized the detection of various targets with rapidity and good efficiency. To date, however, the use of MSFs for homogeneous electrochemical sensing construction has not been reported yet, as well as the detection for protein kinases activity. Thus, it is highly encouraged to adopt MSFs based homogeneous electrochemical sensing platform to

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further improve the efficiency and universality of protein kinase activity and inhibition assay. Herein, an effective homogeneous electrochemical sensing platform for the simple, label-free and robust detection of protein kinase activity and inhibits was developed by coupling with vertically ordered MSFs and carboxypeptidase Y (CPY)assisted selective peptide cleavage. In this sensing platform, the MSFs could be adopted as nanochannels to allow the electroactive probe (FcMeOH) transport into the underlying electrode surface. The CPY was a kind of proteinase with hydrolytic activity towards peptide bonds at the carboxylterminal (C-terminal) end of substrate peptide.14,18 The presence of protein kinase could lead to the phosphorylation of the substrate peptide. Then, the phosphorylated peptide can effectively resist CPY cleavage and be adsorbed on the surface of MSFs modified indium-tin oxide (ITO) electrode (MSFs/ITO) to prevent the diffusion of FcMeOH. Thus, an ideal decrease of electrochemical signals was obtained. Notably, the proposed sensing platform involved homogeneous protein kinase-catalyzed phosphorylation and CPY-catalyzed cleavage reactions where the enzyme reacted with a dissolved substrate peptide in aqueous media. As such, the substrate peptide was undemanding, easy synthesis and did not need any sophistical labeling or chemical modification. This design then offered a general homogeneous electrochemical sensing platform to realize detection of different protein kinase activity by virtue of altering the specific positively charged substrate peptide sequences. Here, the PKA and CK2 were respectively used as model protein kinase for verifying the adaptability and versatility of the homogeneous electrochemical platform. Moreover, the successful assays of inhibitions and protein kinase activity in cell lysates have further indicated that this developed homogeneous electrochemical sensing platform exhibited great potential in kinase-related clinical diagnosis and biochemical fundamental.

EXPERIMENTAL SECTION Chemicals and Materials. PKA, CK2 and T4 polynucleotide kinase (T4 PNK) were purchased from New England Biolabs (Beverly, MA, USA). The PKA specific substrate peptide (SPepPKA, NH2-RRRRRRRRRRLRRASLG-COOH) and CK2 specific substrate peptide (S-PepCK2, NH2RRRRRRRRRRRADDSDDDDD-COOH) were synthesized by Chinapeptides Co. Ltd. (Shanghai, China). ATP, CPY and human serum albumin (HSA) were obtained from Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). Ferrocene methanol (FcMeOH), adenosine 3’, 5’-cyclic monophosphate sodium salt monohydrate (cAMP) and 4,5,6,7Tetrabromobenzotriazole (TBB) were purchased from SigmaAldrich (St. Louis, MO, USA). Ellagic acid was purchased from TCI Development Co., Ltd. (Shanghai, China). Ncetyltrimethylammonium bromide (CTAB) and tetraethylorthosilicate (TEOS, 98%) were obtained from Alfa Aesar (Ward Hill, MA, USA). Forskolin and 3-isobutyl-1methylxantine (IBMX) were purchased from J&K Chemical Co. Ltd (Beijing, China). The other chemicals were commercially obtained from Reagent & Glass Apparatus Corporation of Changsha, which were used without further purification or treatment.

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Preparation of MSFs Modified Electrode. The MSFs/ITO was prepared by growing vertically aligned MSFs on the conducting ITO glass (1×1.5 cm, surface resistivity ≤ 10 Ω) according to the previous reported Stöber-solution growth approach.46 Firstly, the ITO glass was respectively sonicated in acetone, ethanol, and water for 15 min. The cleaned ITO glass was then immersed into precursor mixture containing 35 mL of water, 15 mL of ethanol, 0.08 g of CTAB, 5 µL of ammonia aqueous solution (25 wt. %) and 40 µL of TEOS. For growing of MSFs, the ITO glass should immerse in the precursor solution under quiescent condition at 60 °C. After 48 h, the surfactant-templated MSFs modified ITO electrode was obtained. It was further cleaned with ethanol and water and dried overnight at 60 °C. Finally, the electrode with surfactanttemplated MSFs on it was dunked into 0.1 M HCl of ethanol solution under stirring for 15 min so that the CTAB surfactants could be removed, and then the MSFs/ITO was obtained. Protein Kinase-Catalytic Phosphorylation in Solution. The phosphorylation reaction for PKA was performed in 50 µL of reaction buffer (50 mM Tris-HCl, 10 mM MgCl2, pH 7.4) containing 1 mM ATP, 10 mM cAMP, 20 µM S-PepPKA and a varying amount of PKA at 30 °C for 60 min. For CK2, 50 µL of reaction buffer (20 mM Tris-HCl, 50 mM KCl, 10 mM MgCl2, pH 7.5) containing 1 mM ATP, 20 µM S-PepCK2 and different activity of CK2 were incubated at 25 °C for 60 min. After phosphorylation, the reaction solutions were hydrolyzed by 5 UmL-1 CPY at 25 °C for 30 min, and then dropped onto the MSFs/ITO for 2 h in atmosphere ambient. Finally, the electrode was washed thoroughly with buffer and measured by electrochemical measurement. For inhibitor detection, different concentrations of ellagic acid or TBB were introduced into PKA or CK2 reaction buffer. The following procedures were employed in the same procedures as those for detection of protein kinase activity. Cell Culture and Lysate Preparation. In order to test the capability of the developed sensing in cell lysates, HeLa (human cervical cancer cell) and MCF-7 (human breast cancer cell) were incubated under a humidified atmosphere with 5% CO2 at 37 °C in DMEM medium supplemented with 10% fetal bovine serum. Meanwhile, after 4 h incubation in serum-free medium replacing the culture medium, the MCF-7 cells were treated with some forskolin and IBMX or ellagic acid to stimulate intracellular PKA activity. Subsequently, the cultured cells (~1.5×106 cells) were washed and centrifuged three times with phosphate buffer (10 mM, pH 7.4). After removing the supernatant, 400 µL lysis buffers were then treated with the resulted precipitated cells for 30 min at 4 °C and the cells lysates were centrifuged at 12000 rpm at 4 °C for 20 min. The final obtained supernatant was stored at -20 °C for use. Electrochemical Detection. The electrochemical detection was achieved on CHI660A electrochemical workstation (Shanghai Chenhua Instrument Co. Ltd., China) with a conventional three-electrode system consisting of ITO working electrode, a saturated calomel reference electrode (SCE), and a platinum counter electrode. Differential pulse voltammetry (DPV) was carried out in 10 mM Tris-HCl (pH 7.4), 0.1 M KCl containing 0.5 mM FcMeOH over the potential range from 0 to 0.6 V.

Scheme 1. Schematic diagram for the label-free homogeneous electrochemical sensing platform for protein kinase assay based on CPY-assisted peptide cleavage and vertically ordered MSFs. Apparatus and Characterization. Transmission electron microscopy (TEM) image was employed on Tecnai G2 20 STwin (FEI, Czech Republic) operated at 200 kV. The TEM samples were obtained by mechanically scraping small pieces of the MSFs from the ITO surface. The cross-sectional scanning electron microscope (SEM) image was taken on JSM-6700F (JEOL, Japan). X-ray diffraction (XRD) pattern was obtained on Scintag XDS-2000 powder diffractometer (Cupertino, Canada).

RESULTS AND DISCUSSION The Principle of Protein Kinase Activity Detection. As shown in Scheme 1, the label-free homogeneous electrochemical sensing platform for the detection of protein kinase activity was just developed based on CPY-assisted selective peptide cleavage and the vertically ordered MSFs grown in suit of the ITO surface. In this strategy, an artificially synthesized substrate peptide was designed. This substrate peptide consisted of a protein kinase specific recognized sequence and an arginine (R) residues-rich sequence. The R residues-rich sequence could ensure the peptide net charge being positive both before and after phosphorylation. In the absence of protein kinase, the substrate peptide would be literally cleaved from C-terminal by CPY into free amino acids which would be unable to adsorb on the surface of MSFs. Thus, a large amount of free FcMeOH in solution exhibited great diffusivity toward the MSFs/ITO, resulting in a large electrochemical signal. However, when substrate peptide was phosphorylated by protein kinase in homogeneous enzyme reaction mixture, it would remarkably resist CPY cleavage. Then the un-cleaved phosphorylated peptide would tightly adsorb on the surface of MSFs/ITO through the non-covalent electrostatic attraction between multiple R residues and negatively charged MSFs/ITO surface. As a result, the diffusion current of FcMeOH was greatly reduced. The reduced electrochemical signal was related to the activity of the protein kinase. Therefore, taking advantage of CPYassisted selective peptide cleavage and the vertically ordered MSFs, protein kinase specific to the substrate peptide could be detected. In this work, PKA was used as the model to demonstrate the feasibility and applicability of the

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Figure 1. (A) TEM and (B) cross-sectional SEM image of the MSFs grown on ITO electrode by the Stöber-solution growth approach.

homogeneous electrochemical sensing platform. Furthermore, the universality of the sensing platform was also verified by using CK2 as the example. Characterization of MSFs Grown on ITO Electrode. To characterize the morphology and structure of MSFs grown onthe ITO electrode, the TEM, SEM and XRD were performed, respectively. As displayed in Figure 1A, the TEM images of MSFs moved from ITO electrode showed highly ordered of pores over the entire film and the size of pores were about 2~3 nm which were in common with previous reports.47 In addition, according to cross-sectional SEM image investigation (Figure 1B), it was clearly observed that the MSFs exhibited a uniform film thickness and the thickness was roughly estimated at 90 nm. Meanwhile, from XRD result confirmed in Figure S1, no out-of-plane XRD reflection were observed, verifying the vertically distribution of pores on the MSFs.46 Afterwards, DPV was employed to determine the efficiency of signal molecular transport across the vertically aligned MSFs by using FcMeOH as electroactive probe. As shown in Figure S2, a high electrochemical signal was observed at the bare ITO electrode (curve a). While surfactanttemplated MSFs was grown on the surface of ITO electrode, an obviously decreased electrochemical signal was achieved (curve b), which indicated that the high coverage of the electrode surface with surfactant-templated MSFs. What’ more, the response shifted towards anodic potentials was also obtained corresponding to the FcMeOH which likely accumulated in the surfactant phase35. After surfactant removal, an increased signal was observed due to the mesoporous of MSFs was opened (curve c), demonstrated that MSFs can effectively allow the FcMeOH transport into the underlying ITO electrode. In a word, these results clearly demonstrated that the vertically aligned MSFs grown on the surface of the ITO electrode have been successfully prepared. Feasibility Investigation of the Sensing. In the proposed sensing, the DPV was chosen to verify the feasibility of the homogeneous electrochemical sensing and PKA was used to be the first type of model target. As illustrated in Figure 2, the MSFs/ITO exhibits a high electrochemical signal (curve a). Upon the addition of S-PepPKA, the electrochemical signal was significantly decreased (curve b). The results showed that the S-PepPKA could be tightly adsorbed on the surface of MSFs/ITO and prevent the diffusion of FcMeOH into the electrode. When S-PepPKA was treated with both PKA and CPY, the electrochemical signal was also decreased obviously (curve c), indicating that the phosphorylated peptide could be resistant to digestion by CPY and adsorbed on the surface of

Figure 2. DPV response of MSFs/ITO (a) incubated with SPepPKA (b) and kinase-catalyzed phosphorylation solution in the presence (c) and absence (d) of PKA after treating with CPY for 30 min in 10 mM Tris-HCl (pH 7.4) containing 0.1 M KCl and 0.5 mM FcMeOH. The MSFs/ITO incubated without S-PepPKA but with PKA and CPY (e). The ITO electrode incubated with unphosphorylated (f) and phosphorylated (g) S-PepPKA after treating with CPY. The DPV parameters used were as followed: increment potential 0.004 V, amplitude 0.05 V, pulse width: 0.05 s, quiet time 2 s and pulse period 0.2 s.

MSFs/ITO. It caused the closing of the nanopores and then decreased the diffusion of FcMeOH. In contrast, the treatment of S-PepPKA with only CPY resulted in a little decrease in electrochemical signal (curve d), which attributed to the cleavage of the substrate peptide by CPY. Further control experiment was carried out by treating the MSFs/ITO with PKA and CPY but without S-PepPKA. In comparison, a slight reduced of electrochemical intensity was observed (curve e). It was demonstrated that both of PKA and CPY themselves had little distractions for the diffusion of FcMeOH. In addition, we also ran a control experiment just by using bare ITO electrode instead of MSFs/ITO. In this situation, a high electrochemical signal was still observed, in which S-PepPKA was incubated with CPY but without PKA (curve f). On the contrary, when the peptide was phosphorylated by PKA, a reduced in electrochemical signal could be also detected (curve g). Although the result indicated that PKA could also be detected without the help of MSFs, the electrochemical sensing of MSFs/ITO had better signal-noise ratios (up to 1.9-fold increase, the inset in Figure 2). All of the results clearly

Figure 3. (A) DPV signals corresponding to the assay of PKA activity with various activity (from 0, 0.1, 1, 5, 10, 50, 100, 200 UmL-1, respectively). (B) Relative current decrement ratio (I0-I)/I0 as a function of PKA activity. Inset shows the linear relationship between current decrement ratio and the logarithm of PKA activity in the range of 0.1-50 UmL-1.

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Figure 5. (A) DPV signals corresponding to the assay of CK2 activity with various activity (from 0, 0.1, 0.5 1, 5, 10, 50, 100, 200 UmL-1, respectively). (B) Relative current decrement ratio (I0-I)/I0 as a function of CK2 activity. Inset shows the linear relationship between current decrement ratio and the logarithm of CK2 activity in the range of 0.1-50 UmL-1. Figure 4. Inhibition of the PKA activity by ellagic acid. The concentrations of ellagic acid were 0, 1, 2, 4, 6, 10 and 15 µM, respectively.

verified that the proposed sensing could effectively and sensitively profile the determination of PKA activity. Detection of Activity and Inhibition of PKA. Under the optimized conditions (20 µM of substrate peptide and 5 UmL-1 CPY, Figure S3), the relationship between the electrochemical signal and PKA activity was illustrated in Figure 3A. It can be seen that the electrochemical signal gradually decreased accordingly with the activity of PKA ranged from 0 to 200 UmL-1. Figure 3B showed the relationship between current decrement ratio and the activity of PKA. Here, the current decrement ratio was defined as (I0 - I)/I0, where I0 and I were electrochemical signal obtained in the absence and presence of target. It can be seen that current decrement ratio increased linearly with the logarithm of PKA activity. The regression equation can be represented as (I0 - I)/I0= 0.22 + 0.14 lg(C / UmL-1) with the correlation coefficient of R = 0.965. The linear range for PKA activity was obtained from 0.1 to 50 UmL-1. The detection limit was 0.083 UmL-1 (S/N = 3), which was better than some of previous reported method (Table S1). In addition, the assay time of the proposed sensing was about 4 h, which was also shorter than some of reported method for PKA activity detection (Table S1). These results showed that our homogeneous electrochemical sensing exhibited a good sensitivity for PKA. To confirm whether the proposed sensing could be applicable to quantitatively detection the inhibition of PKA activity, ellagic acid was selected owning to its cell-permeable antioxidant and anticarcinogenic characteristics.47 The inhibition experimental was performed by co-incubating 50 UmL-1 PKA with a varying amount of ellagic acid. As shown in Figure 4, as the ellagic acid concentration was increased, there was a decrease in current decrement ratio and the IC50 value (the half maximal inhibitory concentration) was estimated to be 1.65 µM which was comparable to that reported in the literature.25 The result verified that the developed sensing could be applied in the field of screening the kinase inhibitor. Detection of Activity and Inhibition of CK2. To confirm a generality of the homogeneous electrochemical sensing, we chose CK2 as another model target by changing the S-PepPKA as S-PepCK2 with the same principle. The feasibility of CK2 activity detection was illustrated in Figure S4. As expected,

the electrochemical signal obviously decreased upon phosphorylation by CK2. However, a high electrochemical signal was observed when the phosphorylation was not happened. The DPV response to different activity of CK2 was displayed in Figure 5A. The decreased DPV peak currents were observed with the increasing activity of CK2 in the range from 0 to 200 UmL-1. As depicted in Figure 5B, the current decrement ratio emerged a clear increased with improving CK2 activity from 0.1 to 200 U/mL. The increase of current decrement ratio was proportional to the logarithm value of CK2 activity in the range of 0.1 to 50 UmL-1. The linear regression equation was (I0 - I)/I0 = 0.21 + 0.17 lg(C / UmL-1) (correlation coefficient was 0.985). Meanwhile, the detection limit and assay time were 0.095 U mL-1 and 4 h, respectively. As shown in Table S2, the presented sensing have an ideal detection limit and assay time for the detection of CK2 activity by compared with that of previous reported literatures. Subsequently, the inhibition activity of TBB for CK2 was also investigated. As presented in Figure S5A, the current decrement ratio decreased rapidly with increasing concentration of TBB. The IC50 value of TBB was determined to be 6 µM. This fact was further implied that the developed electrochemical sensing has an excellent ability sensing in screening the activity of the kinase inhibitor. Selectivity and Reproducibility of the Sensing. In order to further evaluate the selectivity of the developed sensing, we firstly selected PKA as the model target and several control proteins including CK2, T4 PNK and HSA as the possible interferents. For investigating it, the S-PepPKA was incubated with the PKA reaction buffer containing 50 U/mL PKA or 50 U/mL other control proteins. As shown in Figure S6, the current decrement ratio of the sensing fabricated with CK2, T4 PNK and HSA were much lower than that of PKA, indicating that CK2, T4 PNK and HSA showed no effect on PKA detection. In addition, the current decrement ratio of the sensing by mixing 50 U/mL PKA and S-PepCK2 was also observed, and it was lower than that of PKA mixed with SPepPKA. All of the results could clearly verify that the proposed sensing possessed high detection selectivity. Meantime, the reproducibility of the homogeneous electrochemical sensing was firstly examined by detecting 10 UmL-1 of PKA and the relative standard deviation (RSD) was 11.8% obtained for five measurements. In addition, the reproducibility of MSFs/ITO and peptide adsorption on the surface of MSFs/ITO were also investigated and the RSD for

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developed homogeneous electrochemical sensing was feasible to analyze intracellular protein kinase activities in vitro.

CONCLUSION

Figure 6. Relative current decrement ratio of the homogeneous electrochemical sensing in response to different PKA activation levels in cell lysates induced by activators. The inset shows the concentration of the activators for cell lysate samples from 1 to 3.

five determinations were 6.7% and 8.5%, respectively. These results suggested an acceptable reproducibility of the proposed sensing. Application in Complex Biological Samples. Firstly, in order to demonstrate the possible existence of protein adsorption in complex biological samples, we performed the experiment by incubating with HeLa cell lysates on the surface of MSFs/ITO. As can be seen in Figure S7, there was a high electrochemical signal detected on the MSFs/ITO (curve a). In comparison, the electrochemical signal of the MSFs/ITO incubated with HeLa cell lysates has a weak decreased (curve b). These phenomena revealed that there was a negligible protein adsorption in complex biological samples. Subsequently, to evaluate the feasibility of the developed homogeneous electrochemical sensing in complex biological samples, the activity detection of PKA and CK2 in HeLa cell lysates was investigated, respectively. In this experimental, the protein kinase in HeLa cell lysates were performed by adding a certain activity of protein kinase in ten times diluted HeLa cell lysates and then tested using the developed homogeneous electrochemical sensing. As shown in Table S3, the mean recovery of each samples were between 88% and 115%, suggesting the good accuracy and potential application of protein kinase activity assay in complex samples. Furthermore, our proposed homogeneous electrochemical sensing was also exploited to investigate the endogenous protein kinase activity in cell lysates. Several reports have proved that the PKA activity in human cells could be efficiently up-regulated through extracellular stimulation by forskolin and IBMX.14 Hence, the MCF-7 cell was chosen and treated with some forskolin combined with IBMX or ellagic acid for activation or inhibition of the intracellular PKA. As illustrated in Figure 6, it was observed that the control MCF-7 cell lysates without stimulations exhibited a low current decrement ratio, indicating that the PKA activity in MCF-7 cell lysates was likely to be less. Upon the adding of forskolin and IBMX, an obvious increase in current decrement ratio was found, which attributed to the activation of PKA activity in cell lysate. In contrast, a lower current decrement ratio could be obtained with the treatment of ellagic acid corresponding to the inhibition of PKA activity. These results indicated that the

In conclusion, this work presented a novel, simple, and label-free homogeneous electrochemical sensing platform for convenient quantitative assay of protein kinase activity by employing CPY-assisted selective peptide cleavage reaction and vertically ordered MSFs. Compared to the conventional heterogeneous electrochemical sensing, our proposed sensing involved homogeneous protein kinase-catalyzed phosphorylation in the solution with simple procedures. As such, the developed sensing could display high efficiency of phosphorylation and avoid sophisticated substrate peptide immobilization processes. Importantly, the substrate peptide in this sensing possessed the advantages of easy synthesis and did not need any sophistical labeling or chemical modification. It was indicated that this sensing can easily expand to monitor various protein kinases just by simply altering specific positively charged substrate peptide sequences. The universality of this homogeneous electrochemical sensing has been verified by successful monitor of PKA and CK2. The results illustrated that the proposed sensing showed high sensitivity for PKA and CK2, and the limit of detection were 0.083 UmL-1 and 0.095 UmL-1, respectively. Moreover, given the important roles of protein kinase activity detection in cell lysates and inhibitor screening, the MSFs based homogeneous electrochemical sensing platform could be applied in kinaserelated bioanalysis and clinical biomedicine.

ASSOCIATED CONTENT Supporting Information Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. XRD analysis result of MSFs, DPV analysis result of MSFs, optimization of experimental conditions, feasibility investigation for CK2, inhibition of the activity of CK2, the selectivity test, DPV analysis result of MSFs/ITO incubated with HeLa cell lysates, comparison of different sensing for PKA activity detection, comparison of different sensing for CK2 activity detection, and protein kinases activity assay in 10 times diluted HeLa cell lysates. (PDF)

AUTHOR INFORMATION Corresponding Author * To whom correspondence should be addressed. Tel: 86-73188821566; Fax: 86-731-88821566; E-mail: [email protected]. [email protected].

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

Acknowledgment This work was supported in part by the National Natural Science Foundation of China (21675046, 21190044, and 21221003) and Graduate Student Research Innovation Project of Hunan Province (CX2015B078).

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REFERENCES (1) López-Otín, C.; Hunter, T. Nat. Rev. Cancer 2010, 10, 278-292. (2) Cohen, P. Nat. Rev. Drug Discov. 2002, 1, 309-315. (3) Manning, G.; Whyte1, D. B.; Martinez, R.; Hunter, T.; Sudarsanam, S. Science 2002, 298, 1912–1934. (4) Griner, E. M.; Kazanietz, M. G. Nat. Rev. Cancer 2007, 7, 281294. (5) Cai, Z. Y,; Yan, L. J.; Li, K. Neuromol. Med. 2012, 14, 1–14. (6) Amrutkar, M.; Cansby, E.; Chursa, U.; Chanclón, B.; Ståhlman, M.; Fridén, V.; Wickman, A.; Smith, U.; Bäckhed, F.; Borén, J.; Howell, B. W.; Mahlapuu, M. Diabetes 2015, 64, 2791-2804. (7) Park, S. H.; Ko, K. C.; Gwon, H. J. J. Labelled Comod. Rad. 2007, 50, 724-728. (8) Siddhanta, S.; Karthigeyan, D.; Kundu, P. P.; Kundu, T. K.; Narayana, C. RSC Adv. 2013, 3, 4221-4230. (9) Cho, Y. L.; Kim, Y. P.; Son, J. G.; Son, M.; Lee, T. G. Anal. Chem. 2017, 89, 799-806. (10) Xu, X. H.; Zhou, J.; Liu, X.; Nie, Z.; Qing, M.; Guo, M. L.; Yao, S. Z. Anal. Chem. 2012, 84, 4746-4753. (11) Lowe, S. B.; Dick, J. A. G.; Cohen, B. E.; Stevens, M. M. ACS Nano 2012, 6, 851-857. (12) Zhou, J.; Xu, X. H.; Liu, X.; Li, H.; Nie, Z.; Qing, M.; Huang, Y.; Yao, S. Z. Biosens. Bioelectron. 2014, 53, 295-300. (13) Wu, S.; Kong, X. J.; Cen, Y.; Yu, R. Q.; Chu, X. Chem. Commun. 2016, 52, 776-779. (14) Yin, C.; Wang, M.; Lei, C. Y.; Wang, Z.; Li, P.; Li, Y.; Li, W.; Huang, Y.; Nie, Z.; Yao, S. Z. Anal. Chem. 2015, 87, 6311-6318. (15) Zhang, L.; Song, W.; Liang, R. P.; Qiu, J. D. Anal. Chem. 2016, 88, 11460-11467. (16) Zhou, J.; Xu, X. H.; Liu, W.; Liu, X.; Nie, Z.; Qing, M.; Nie, L. H.; Yao, S. Z. Anal. Chem. 2013, 85, 5746-5754. (17) Li, X.; Zhu, L. S.; Zhou, Y. L.; Yin, H. S.; Ai, S. Y. Anal. Chem. 2017, 89, 2369-2376. . (18) Yin, H. S.; Wang, X. X.; Guo, Y. L.; Zhou Y. L; Ai, S. Y. Biosens. Bioelectron. 2015, 66, 77-83. (19) Shen, C. C.; Li, X. Z.; Rasooly, A.; Guo, L. Y.; Zhang, K.; Yang, M. H. Biosens. Bioelectron. 2016, 85, 220-225. (20) Sun, K.; Chang, Y.; Zhou, B.; Wang, X; Liu, L. Int. J. Nanomed. 2017, 12, 1905-1915. (21) Martic, S.; Labib, M.; Freeman, D.; Kraatz, H. B. Chem. Eur. J. 2011, 17, 6744-6752. (22) Liu J. Q.; He, X. X.; Wang, K. M.; Wang, Y. H.; Yan, G. P.; Mao, Y. F. Talanta 2014, 129, 328-335. (23) Wang, C. L.; Wei, L. Y.; Yuan, C. J.; Hwang, K. C. Anal. Chem. 2012, 84, 971-977. (24) Yang, Y.; Guo, L. H.; Qu, N.; Wei, M. Y.; Zhao, L .X.; Wan, B. Biosens. Bioelectron. 2011, 28, 284-290. (25) Wang, Z.H.; Sun, N.; He, Y.; Liu, Y.; Li, J. H. Anal. Chem. 2014, 86, 6153-6159. (26) Miao, P.; Ning, L. M.; Li, X. X.; Li, P. F.; Li, G. X. Bioconjugate Chem. 2012, 23, 141-145. (27) Yin, H. S.; Sun, B.; Dong, L. F.; Li, B. C.; Zhou, Y. L.; Ai, S. Y. Biosens. Bioelectron. 2015, 64, 462-468. (28) Zhang, F. T.; Cai, L. Y.; Zhou , Y. L.; Zhang, X. X.; TraceTrend. Anal. Chem. 2016, 85, 17-32. (29) Wei, X. F.; Ma, X. M.; Sun, J. J.; Lin, Z. Y.; Guo, L. H.; Qiu, B.; Chen, G. N. Anal. Chem. 2014, 86, 3563-3567. (30) Liu, X. J.; Li, W.; Hou, T.; Dong, S. S.; Yu, G. H.; Li, F. Anal. Chem. 2015, 87, 4030-4036. (31) He, X. X.; Chen, Z. F.; Wang, Y. H.; Wang, K. M.; Su, J.; Yan; G. P. Biosens. Bioelectron. 2012, 35, 134-139. (32) Shin, I.; Chand, R.; Lee, S. W.; Rhee, H. W.; Kim, Y. S.; Hong, J. I. Anal. Chem. 2014, 86, 10992-10995. (33) Liu, S. F.; Wang, Y.; Zhang, C. X.; Lin, Y.; Li, F. Chem. Commun. 2013, 49, 2335-2337. (34) Xuan, F.; Luo, X. T.; Hsing, I. M. Anal. Chem. 2013, 85, 45864593.

(35) Xuan, F.; Luo, X. T.; Hsing, I. M. Anal. Chem. 2012, 84, 52165220. (36) Yan, F.; Lin, X. Y.; Su, B. Analyst 2016, 141, 3482-3495. (37) Walcarius, A.; Sibottier, E.; Etienne, M.; Ghanbaja, J. Nat. Mater. 2007, 6, 602-608. (38) Urbanova, V. Walcarius, A. Z. Anorg. Allg. Chem. 2014, 640, 537-546. (39) Duc, Y. L.; Gilles, A.; Mihai, S.; Rouessac, V.; Tingry, S.; Barboiu, M. Chem. Commun., 2013, 49, 3667-3669. (40) Saadaoui, M.; Fernández, I.; Sánchez, A.; Díez, P.; Campuzano, S.; Raouafi, N.; Villalonga, R. Electrochem. Commun. 2015, 58, 5761. (41) Wu, M. S.; Sun, X. T.; Zhu, M. J.; Chen, H. Y.; Xu, J. J. Chem. Commun. 2015, 51, 14072-14075. (42) Mathieu, E.; Aurélie, G.; Emilie, S.; Alain, W. J. Nanosci. Nanotechnol. 2009, 9, 2398-2406. (43) Fernández, I.; Sánchez, A.; Díez, P.; Martínez-Ruiz, P.; Pierro, P. D.; Porta, R.; Villalonga, R.; Pingarrón, J. M. Chem. Commun. 2014, 50, 13356-13358. (44) Yan, F. Zheng, W. J.; Yao, L. N.; Su, B. Chem. Commun. 2015, 51, 17736-17739. (45) Liu, J.Q.; He, D. G.; Liu, Q. Q.; He, X. X.; Wang, K. M.; Yang, X.; Shangguang, J. F.; Tang, J. L.; Mao, Y. F. Anal. Chem. 2016, 88, 11707-11713. (46) Teng, Z. G.; Zheng, G. F.; Dou, Y. Q.; Li, W.; Mou, C. Y.; Zhang, X. H.; Asiri, A. M.; Zhao, D. Y. Angew. Chem. 2012, 124, 2215 -2219. (47) Cozza, G.; Bonvini, P.; Zorzi, E.; Poletto, G. J. Med. Chem. 2006, 49, 2363-2366.

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