Electrogenerated Chemiluminescence Bioassay of Two Protein

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Electrogenerated Chemiluminescence Bioassay of Two Protein Kinases Incorporating Peptide Phosphorylation and Versatile Probe Xia Liu, Manman Dong, Honglan Qi, Qiang Gao, and Chengxiao Zhang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 12 Aug 2016 Downloaded from http://pubs.acs.org on August 12, 2016

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Analytical Chemistry

Electrogenerated Chemiluminescence Bioassay of Two Protein Kinases Incorporating Peptide Phosphorylation and Versatile Probe Xia Liu, Manman Dong, Honglan Qi*, Qiang Gao, Chengxiao Zhang Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an, 710062, P.R. China

Corresponding author. Tel: +86-29-81530726; Fax: +86-29-81530727. E-mail address: [email protected] (H. L. Qi).

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Abstract A sensitive electrogenerated chemiluminescence (ECL) bioassay was developed for the detection of two protein kinases incorporating the peptide phosphorylation and a versatile ECL probe. Cyclic adenosine monophosphate-dependent protein kinase (PKA) and casein kinase II (CK2) were used as proof-of-concept targets whilst a PKA-specific

peptide

(CLRRASLG)

and

a

CK2-specific

peptide

(CRRRADDSDDDDD) were used as the recognition substrates. Taking advantage of the ability of protein A binding with the Fc region of a variety of antibodies with high affinity, a ruthenium derivative-labeled protein A was utilized as a versatile ECL probe for bioassay of multiple protein kinases. A specific peptide substrate toward target protein kinase was firstly self-assembled on the surface of gold electrode and then serine in the specific peptide on the electrode was phosphorylated by target protein kinase in the presence of adenosine-5’-triphosphate. After recognition of the phosphorylated peptide by monoclonal anti-phosphoserine antibody, the versatile ECL probe was specifically bound to the anti-phosphoserine antibody on the electrode surface. The ECL bioassay was developed successfully in the individual detection of PKA and CK2 with detection limit of 0.005 U/mL and 0.004 U/mL, respectively. In addition, the ECL bioassay was applied to quantitative analysis of the kinase inhibitors and monitoring drug-triggered kinase activation in cell lysates. Moreover, an ECL imaging bioassay using electron-multiplying charged coupled device as detector on the gold electrode array was developed for the simultaneous detection of PKA and CK2 activity from 0.01 U/mL to 0.4 U/mL, respectively, at one time. This work demonstrates that the ingenious design and use of a versatile ECL probe are promising to simultaneous detection of multiple protein kinases and screening of kinase inhibitor.

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Introduction Protein kinase is a kinase enzyme that modifies target proteins by transferring phosphate groups from nucleoside-5’ triphosphate (ATP) to serine, tyrosine, or threonine residues within their proteins (phosphorylation).1 Protein phosphorylation catalyzed by protein kinases plays vital regulatory roles in many vital biological processes.2,3 The over-expression of protein kinases and the following abnormal protein phosphorylation are related with diseases, such as aggressive tumor, diabetes, and Alzheimer’s disease, etc.4,5 Therefore, the development of sensitive and selective method for monitoring target kinase activities is necessary to understand the relationship between kinase activities and intracellular signaling pathways.

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Radioactive assay is a conventional method for the detection of protein kinase activities using γ-32P-ATP during the phosphorylation reaction. 7 The limit of radioactive assay is obvious with unhealthy radioactive waste and complicated procedure. Immunoassays coupled with colorimetric,

8 , 9

fluorescence,

10

and

electrochemical technique 11 are alternative approaches to radioactive assay by employing the labeled phospho-specific antibodies as molecule recognition elements to specifically recognize phosphorylated products produced by kinase reaction. However, most of the labeled methods are limited in multiplexed bioassay since they require the phospho-specific antibodies which are labeled with different signal reagents for multiple protein kinases. It is reported that at least 500 kinds of protein kinases have been found and are related with more than 170, 000 non-redundant phosphorylation sites in ca. 18 000 proteins. 12 Many fundamental biochemical processes in intracellular signaling pathways are associated with multiple protein kinases. 13 ,14 Simultaneous bioassay for multiple protein kinases is increasing in demand due to its advantages, such as short analytical time, small sampling volume,

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low cost, and high efficiency compared with one target assay. Immunoassays coupled with mass spectroscopy 15,16 fluorescence2 and electrochemical techniques 17 have been developed for measuring multiple kinase activity. Unfortunately, some of them were limited in linear range, sensitivity, and sometimes, the complicated synthesis of probes was needed. Therefore, it is a challenge to explore novel strategy for further improvement of simplicity, sensitivity and accuracy of the bioassay of multiple kinases. Electrogenerated chemiluminescence (ECL) is one kind of chemiluminescence phenomenon generated by electrochemical reaction. ECL methods have many advantages, such as high sensitivity, low background, simplified optical setup and good temporal and spatial control.18,19,20,21,22 ECL methods are recently receiving increasing attention in the assay of protein kinase based on the detection of ECL signal generated by phospho-specific antibodies labeled with ECL emitter,23,24 Ti4+ and Zr4+ coordinated with ECL emitter,25,26,27,28 or gold nanoparticles attached with ECL emitter 29 with good performance. However, these methods are limited to bioassay of multiple protein kinases because of one ECL probe for one target, which makes the procedure both complicated and expensive. We recently developed an ECL biosensor for the detection of protein kinase using Ru(bpy)32+ functionalized gold nanoparticles,30 in which one ECL probe was used to detect two targets. However, adenosine-5’-(γ-thio)-triphosphate needs to be used as a co-substrate and the synthesis of functionalized gold nanoparticles should be carefully controlled. The coupling of biotin-avidin interaction e.g. binding of Ru(bpy)32+ complex modified streptavidin as one ECL signal probe with biotinylated secondary antibodies is an alternative promising approach in the detection of multiple targets. 31 However, biotinylated secondary antibodies were needed to be synthesized. Therefore, the development of

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bioassay with simplicity and easy-manipulation is still needed for the detection of multiple protein kinases. Here, an ECL bioassay was proposed for the detection of two protein kinases incorporating a peptide phosphorylation and a versatile ECL probe. Protein A, a cell wall component of Staphylococcus aureus, can bind with the fragment crystallizable (Fc) region of immunoglobulin G with a high affinity.32,33 It has been used as an affinity protein in piezoelectric crystal biosensor for the determination of immunoglobulins34 and competitive phase-separation immunoassay of paclitaxel.35 Taking advantage of the ability of protein A binding with a variety of antibodies with high affinity, here, protein A was chosen as a carrier for ECL emitter and an affinity protein

for

the

antibody.

The

[bis(2,2’-bipyridine)-4,4’-dicarboxybipyridine-ruthenium bis(hexafluorophosphate),

ruthenium -N-succinimidyl

complex ester-

the cation was abbreviated as Ru1] used as ECL emitter

was labeled onto protein A to form Ru1-labeled protein A (abbreviated as Ru1-protein A). Ru1-protein A is utilized as a versatile ECL probe which can specifically bind with the anti-phosphoserine antibodies in this work for the bioassay of multiple protein kinases. Cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and casein kinase II (CK2) are chosen as proof-of-concept targets since these protein kinases are two classical protein kinases implicated in diseases.36,37 Specific peptide substrates CLRRASLG for PKA 38 and CRRRADDSDDDDD for CK2 39 were designed as molecule recognition substrate, respectively. The specific peptide substrates used in this work containe a cysteine residue to facilitate self-assembling the peptide on the surface of gold electrode, and a serine residue to be phosphorylated by the corresponding protein kinase (PKA/CK2) in the presence of ATP. On the basis of the facts that Ru1-protein A serves as both an ECL emitter and efficient recognizing

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element for anti-phosphoserine antibody, an ECL bioassay of multiple protein kinases is facilely developed, as shown in Figure 1. The specific peptide was self-assembled on the gold electrode surface through thiol-gold bond. In the presence of target protein kinase (PKA/CK2) and ATP, the phosphate group of ATP was selectively transferred to hydroxyl group of the serine residue of desired specific peptide on the electrode. After monoclonal anti-phosphoserine antibody was selectively bound to the phosphorylated peptide, the Ru1-protein A was finally bound to the bounded anti-phosphoserine antibody, resulting in ECL emission in the presence of tripropylamine (TPA). In the proposed strategy, one selective reaction was involved in the phosphorylation reaction, and foremost, two versatile binding reactions including an anti-phosphoserine antibody binding with the phosphorylated peptide and a Ru1-protein A binding with the antibody were cleverly used. Therefore, our ECL bioassay provides a versatile approach in the detection processes for the detection of multiple protein kinases using only one ECL probe. The characteristics of the ECL bioassay and the analytical performance of individual and simultaneous determination of PKA and CK2 are investigated. In addition, the quantitative analysis of kinase inhibition and monitoring drug-triggered kinase activation in cell lysates are carried out. Experimental section Reagents and apparatus PKA and CK2 were purchased from New England Biolabs (USA). Specific peptide substrates CLRRASLG (Figure S-1) for PKA, CRRRADDSDDDDD (Figure S-2) for CK2 and non-specific peptide CGPLGVRGK were obtained from Shanghai Apeptide

Co.,

ltd

(China).

ATP,

4,4’,5,5’,6,6’-hexahydroxydiphenic

acid

2,6,2’,6’-dilactone (C14H6O8, ellagic acid), protein A, alkaline phosphatase (ALP),

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monoclonal

anti-phosphoserine

antibody

produced

in

mouse

(Ab),

4,5,6,7-tetrabromobenzotriazole (TBB), 6-mercapto-1-hexanol (MCH), forskolin, 3-isobutyl-1-methylxantine (IBMX), and Ru1 were obtained from Sigma-Aldrich (USA). Bovine serum albumin was purchased from Xi'an Wolsen Bio-technology Co., Ltd (China). Other reagents with analytical grade were obtained from Sinopharm Chemical Reagent Co., Ltd (China) and Millipore Milli-Q water was used in this work. ECL measurements were done on MPI-A ECL detector (Xi’an Remax Analysis Instruments Co.Ltd, China), in which a photomultiplier tube (PMT) is biased on 900V; or home-made ECL imaging detector. Two types of working electrode, including a gold electrode (2.0 mm diameter), or four gold electrode array (1.0 mm diameter) were used. A platinum wire was used as counter electrode and an Ag/AgCl (saturated KCl) was used as reference electrode. Synthesis of Ru1-protein A The synthesis of Ru1-labeled protein A (Ru1-protein A) as ECL probe was done according to a procedure in ref.40 In brief, 500 µL of 1×10-2 M Ru1 prepared with DMSO was added into 2 mL of 2.4×10-5 mol/L protein A in 20 mM Tris-HCl (pH 7.4) under stirring for 24 h at 4 0C. The Ru1-protein A was purified by dialysis using MD36-1Da molecular (2000) weight cutoff membrane. The dialyzed solution was stored at 4 0C as ECL probe. The concentration of Ru1-protein A was calculated to be 2.0×10-5 M according to the absorption of Ru1 at 457 nm. Fabrication of ECL biosensor array An ECL biosensor was fabricated by dipping a gold electrode in 20 µL of 1.14×10-5 M PKA specific peptide solution or 1.39×10-5 M CK2 specific peptide solution for 1 h, and then blocked with 1 mM MCH for 30 min. The

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peptide-assembled electrode was then thoroughly rinsed with washing buffer (10 mM Tris–HCl containing 0.05% tween-20, pH 7.4). For the fabrication of ECL peptide-based biosensor array, a four gold electrode array was used as working electrode array, in which four gold working electrodes were named as W1, W2, W3 and W4, respectively (Figure S-3). W1 and W3 were modified with PKA specific peptide while W2 and W4 were modified with CK2 specific peptide. 20 µL of 1.14×10-5 M PKA specific peptide solution and 1.39×10-5 M CK2 specific peptide solution was attentively drop-coated onto the surface of desired gold electrode for 1 h at room temperature, respectively. After that, 100 µL of 1 mM MCH solution was dropped onto all resulting electrode for 30 min. At each step, the electrode was washed with washing buffer. ECL Measurements Each fabricated peptide-based biosensor was treated with following processes by the mixture of ATP and protein kinase (PKA or CK2 alone, or the mixture PKA and CK2), anti-phosphoserine antibody, Ru1-protein A, step by step (Scheme 1). The peptide-assembled electrode was firstly incubated with different concentration of target PKA, or CK2 or the mixture of PKA and CK2 in the presence of 75 µM ATP at 30 °C for 1 h, and then the resulted electrode was incubated with 100 µL of 1:100 diluted monoclonal anti-phosphoserine antibody (20 mM Tris-HCl containing 0.8% NaCl, pH 7.5) for 30 min. Finally, the resulted electrode was incubated with 100 µL of 1.0×10-6 M Ru1-protein A for 1 h. After every step, the electrodes were washed with washing buffer. Two ECL detection models, including a PMT detector and electron-multiplying charged coupled device (EMCCD) detector, were used to separately and simultaneously quantify of the activity of two protein kinases. Separated ECL

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measurement was done at +1.2 V in 50 mM Tris–HCl-50 mM TPA (pH 7.4) using one ECL peptide-based biosensor as working electrode and PMT detector. The increased ECL intensity (∆I=Is –I0) is used as analytical signal for the quantification of PKA or CK2, where I0 is the ECL intensity in the absence of target kinase and Is is the ECL intensity in the presence of target kinase. For evaluation of kinase inhibitor, different concentrations of inhibitors with 0.4 U/mL PKA or 0.3 U/mL CK2 was assayed using the above separated ECL procedures. The cell lysates were prepared by a literature reported method41,42 and the assay of protein kinase in the cell lysates was assayed according to the above separated ECL procedures. Simultaneous ECL measurement of PKA and CK2 was done in 50 mM Tris–HCl -50 mM TPA (pH 7.4) at +1.2 V using four electrode array as working electrode and EMCCD detector with an exposure time of 11 s. The gray value analysis of EMCCD images was done using an ImageJ software. The increased ECL count (∆A=As –A0) is used to assay the activity of PKA and CK2, where A0 and AS are the ECL count in the absence and presence of protein kinase. Results and Discussion Characterization of the ECL probe and the ECL biosensor In this work, protein A, as a carrier for the ECL emitter and an affinity protein for the anti-phosphoserine antibody, was covalently coupled with Ru1 via amide reaction to synthesis an ECL probe (Ru1-protein A). The characteristic UV-Vis absorption peaks of Ru1-protein A appeared at 245 nm, 286 nm and 457 nm, respectively (Figure S-4). The label ratio of Ru1 to protein A in Ru1-protein A conjugate was estimated to be 10:1 by the absorption value for Ru1 at 457 nm, and protein A at 245 nm, indicating that the amplification function of protein A as a carrier is obvious. A slight red shift in fluorescent emission spectra was observed by a

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comparison of Ru1-protein A (λem=642 nm) with Ru1 (λem= 627 nm) (see Fig S-5 in supporting information). A comparison of the ECL response (Figure S-6, A) and the corresponding cyclic voltammogram (Figure S-6, B) of Ru1-protein A with that of Ru1 indicated that the ECL probe and Ru1 exhibited similar responses.18-22 The successful labeling of Ru1 on protein A, therefore is evident. The fabrication processes of an ECL biosensor and the phosphorylation by PKA in the presence of ATP using PKA as model target were characterized using electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy (XPS) respectively.

Electrochemical

impedance

spectroscopy

in

the

presence

of

ferri/ferrocyanide showed that the charge-transfer resistance (Rct) was 180 Ω and 6500 Ω at bare and peptide-assembled gold electrode, respectively. The Rct decreased to 3200 Ω after the peptide-assembled electrode was blocked with MCH. The increase and the decrease in Rct are ascribed to the successful self-assembly of the specific peptide onto gold electrode and removing nonspecifically adsorbed peptide by MCH, respectively (Figure S-7). In X-ray photoelectron spectra of the phosphorylated peptide modified gold film (Figure S-8), the presence of S 2p peak at 162.7 eV, N 1s peak at 400.0 eV, P 2p peaks at 131.9 eV and 133.5 eV provide unambiguous evidence for that the peptide has successfully immobilized onto gold electrode by thiol-gold bond and the phosphorylation by PKA in the presence of ATP is efficient.43,44 In order to explore whether Ru1-protein A could be used as a versatile ECL probe to detect two protein kinases, the ECL responses of the constructed biosensors were examined for the individual detection of PKA and CK2, respectively. Figure 2A shows the ECL intensity vs potential profiles of the PKA specific peptide-based biosensor to blank, 0.03 U/mL PKA and 0.2 U/mL PKA, respectively. The ECL

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biosensor shows a low ECL signal in the absence of PKA (a, 2169), attributed to nonspecifically adsorbed Ru1-protein A. As the activity of PKA is increased from 0.03 U/mL to 0.2 U/mL, the ECL intensity greatly increases from 4548 (d) to 8269 (e). This result is attributed to the fact that there exist more phosphorylated peptide and then more Ru1-protein A onto the surface of electrode. Additionally, low ECL signals for 0.2 U/mL PKA were observed in the case of the PKA specific peptide biosensor in the absence of anti-phosphoserine antibody (2079, b) and PKA specific peptide was replaced by a non-specific peptide CGPLGVRGK as substrate for fabrication of the biosensor (1903, c), respectively. Similar experimental phenomena were also observed for the CK2 specific peptide biosensor to blank, 0.03 U/mL CK2 and 0.2 U/mL CK2 (Figure 2B). The obtained data demonstrate that Ru1-protein A can be used as a versatile ECL probe to detect two protein kinases. Individual detection of two protein kinases The ECL method was firstly employed to detect separately two protein kinases using the versatile ECL probe. Firstly, the experimental conditions, including buffer solution, applied potential, phosphorylation time and temperature were optimized using PKA as target kinase. 50 mM tris-HCl (pH 7.4) buffer is used instead of classical phosphate buffered saline (PBS) since phosphate in PBS has a potential interference on the recognition reactions. 1.2 V as applied potential, 60 min as phosphorylation time and 30 °C as phosphorylation temperature were employed as the optimal conditions (Figure S-9). Under the optimal conditions, analytical performance for the individual detection of two protein kinases was examined using a PMT detector. Figure 3A shows the ECL intensity vs time profiles of PKA specific peptide-based biosensor for the detection of PKA. From Figure 3A, we can see that the ECL intensity increases with the increase of activity of PKA from 0.01 U/mL to

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0.5 U/mL. The linear regression equation was ΔI = 20924C + 2021 (unit of C is U/mL, r=0.9977). The detection limit was calculated to be 0.005 U/mL for PKA (S/N= 3). The relative standard deviation (RSD, n=5) was 4.4% for 0.3 U/mL PKA. Figure 3B shows the ECL intensity vs time profiles of CK2 specific peptide-based biosensor for the detection of CK2. The ECL intensity linearly increases with the increase of the activity of CK2 from 0.01 U/mL to 0.4 U/mL. The linear regression equation was ∆I = 22627C+2097 (unit of C is U/mL, r=0.9941). The detection limit was 0.004 U/mL for CK2. RSD (n=6) was 3.5% for 0.1 U/mL CK2. The wider linear ranges and the low detection limits of the developed ECL bioassay for two protein kinase, therefore, are successfully achieved. The selectivity of the ECL method was assessed using PKA specific peptide-based biosensor and CK2 specific peptide-based biosensor for PKA, CK2, and the interference proteins, such as BSA and ALP. For PKA specific peptide-based biosensor, a significant increase of the ECL intensity for PKA (∆I=10614) was observed, while the increase of the ECL intensity from interference proteins was much lower for CK2 (∆I=404), BSA (∆I=277), and ALP (∆I=185). For CK2 specific peptide-based biosensor, CK2 induced an increase of ECL intensity (∆I=11147), compared with PKA (∆I=300), BSA (∆I=115), and ALP (∆I=265) (Figure S-10). We can conclude that the sufficient selectivity in the developed strategy is feasible to detection of PKA or CK2. Evaluation of kinase inhibitors and detection of the protein kinases in cell lysates Evaluation of small-molecule inhibitors of the protein kinase is of great importance in drug discovery. In order to examine the potential applications in the screening of kinase inhibitor, the developed ECL method was used to evaluate kinase inhibitors by detecting PKA activity in the presence of ellagic acid (a cell-permeable

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PKA inhibitor45) and CK2 activity in the presence of TBB (a cell-permeable CK2 inhibitor46), respectively. The ECL intensity vs time profiles for ellagic acid in the presence of 0.4 U/mL PKA showed a decrease in the ECL intensity with increasing the concentrations of ellagic acid (Figure S-11A). The half-maximal inhibition concentration (IC50) of ellagic acid was 3.78 µM, similar with that using electrochemical method (IC50 =2.77 µM).45 For TBB in the presence of 0.4 U/mL CK2, a decrease in the ECL intensity occurred with increasing TBB concentration (Figure S-11 B). The IC50 of TBB is estimated to be 0.70 µM for CK2, which is similar with that using ECL method (IC50 =0.58 µM).30 The obtained data clearly indicate the feasibility of the ECL bioassay in screening inhibitors for PKA and CK2. To evaluate the potential applicability of our ECL strategy in the detection of protein kinases activity in biological systems, we detected the PKA activity and CK2 activity in cell lysates using two constructed biosensors, respectively. It was reported that the intracellular levels of cAMP-dependent PKA increased in cell lysates after the cells was stimulated by the combination of forskolin and IBMX.47 In this work, HeLa cells were chosen as model cells and were stimulated with forskolin and IBMX. Figure 4 show the ECL intensity vs time profiles obtained from four cases at PKA specific peptide-based biosensor and CK2 specific peptide-based biosensor, respectively. The observed ECL intensities (curve b) in HeLa cell lysates were obviously higher than (curve a) that in the blank medium, attributed to the fact that both PKA activity and CK2 activity in the HeLa cell lysates were higher than that in the blank medium.48 After HeLa cells were stimulated with forskolin and IBMX, the activity of PKA increased with concentration of forskolin and IBMX while there was no obvious change on the activity of CK2 (curve c, d). This is attributed to the fact that a stimulation of the cells with forskolin and IBMX can increase the PKA activity

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and cannot increase the CK2 activity.49,50 It can be concluded that the developed ECL approach can be used to assay PKA and CK2 in biological samples, and the selective analysis of the PKA activities of cells in vitro is feasible. Simultaneous detection of two protein kinases using ECL imaging The ECL imaging technique can provide not only intensity-based quantitative information at a single electrode, but also the qualitative information including emission intensity (gray level) and shape at multiple electrodes. The ECL imaging technique has been used in the characterization of electrode surface,51,52 investigation of DNA damage,53 and multi-analyte immunoassays,54 and cell assay.55,56,57 Here, a four gold working electrode array (Fig. 5 A) was used to fabricate ECL peptide-based biosensor array for simultaneous detection of two protein kinases on one shot using EMCCD detector. At first, the ECL images at four working gold electrode array were checked in 1 µM Ru(bpy)32+-50 mM TPA (Figure S-12). The obtained images indicate that spatially resolved individual ECL spot images of each electrode can be obtained using EMCCD detector, which is consistent with the pattern of four gold electrode array. A non-uniform ECL emission is attributed to the spatial heterogeneity of electron transfer at the surface of the gold electrode 58 and non-uniform current distribution at gold electrode. 59 The ECL count at each electrode in 1 µM Ru(bpy)32+-50 mM TPA was calculated to be 3.75×105, 3.76×105, 4.10×105 and 4.00×105, respectively. The obtained RSD of 3.8% demonstrates that the fabricated gold electrode array has a good ECL reproducibility. In order to examine the feasibility of the simultaneous detection of PKA and CK2 on one shot, a peptide-based biosensor array was fabricated, in which W1 and W3 were modified with PKA specific peptide and W2 and W4 were modified with CK2 specific peptide, respectively, as showed in Figure 5A. Figure 5 (B to D) shows the

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ECL images of the biosensor array for the detection of PKA alone, CK2 alone, and the mixture of PKA and CK2, respectively. In the detection of PKA and CK2 alone, ECL emissions are only observed at W1 and W3 in Figure 5B and at W2 and W4 in Figure 5C. In the simultaneous detection of PKA and CK2, ECL emissions can be clearly observed at four electrode surfaces in Figure 5D. In each case, a perfectly localized and well-defined ECL imaging signal was observed, indicating where ECL active surfaces were resulting from ECL probe. The results indicate that there is no obvious cross-talk between the examined biosensors and the phosphate recognition for different targets is only from immobilized different specific peptide on each sensing platform. Therefore, simultaneous detection of two protein kinsaes at one times by collecting multiple ECL images on the ECL peptide-based biosensor array is feasible. The quantitative imaging bioassay of PKA and CK2 were carried out with various activities of two protein kinases with the ECL biosensor array. Figure 6 shows the ECL imagings and calibration curves for PKA and CK2. Two distinguishable and separately signals were observed in presence of two analytes and the ECL counts were directly related to the activity of two protein kinases (Figure 6A). The ECL counts increased with the increase of the activity of PKA and CK2, respectively. The ECL counts is linear with the activity of PKA from 0.01 U/mL to 0.4 U/mL (Figure 6B). The ECL counts is related with the activity of CK2 from 0.01 U/mL to 0.4 U/mL (Figure 6C). The linear regression equations are A=3.4×105lgCPKA+7.8×105(R=0.9984) and A=6.2×105lgCCK2+1.5×106 (R=0.9974) for PKA and CK2 (unit of C is U/mL), respectively. The detection limits are 0.008 U/mL for PKA and CK2. Thus, the ECL imaging bioassay can be used to simultaneous detection of two protein kinases in a single run at the designed

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biosensor array and using the developed versatile ECL probe. Conclusion An ECL bioassay method for the detection of two protein kinases was developed incorporating the phosphorylation of peptide and ruthenium complex functionalized protein A as a versatile ECL probe. The ECL method has been developed successfully for the evaluation of kinase inhibitor and the detection of PKA and CK2 activity in living cell lysates. An ECL imaging bioassay using an ECL peptide-based biosensor array and EMCCD detector was developed for the simultaneous detection of PKA and CK2 from 0.01 U/mL to 0.4 U/mL at one time using the versatile ECL probe. The concept may also be expanded to detection of protein phosphorylation and other protein kinases based on the peptide containing threonine or tyrosine which is taken as phosphorylated site. This work demonstrates that the ingenious design and use of a versatile ECL probe have great potential in multiplexed bioassay of multiple protein kinases and profiling of kinase inhibitor, which can open a new door toward the development of detection method for other biomarkers on biochips.

Additional information The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. Experimental methods, chemical structure of the peptides, scheme and photograph of ECL cell, UV-Vis spectrum, fluorescence spectrum, Nyquist plots of impedance spectra, XPS, optimization of condition, cyclic voltammograms, ECL profiles, and ECL imaging. Corresponding Author [email protected]

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Figures and Captions

Figure 1 The schematic diagram of ECL bioassay of protein kinases

Figure 2 ECL intensity vs potential profiles of the ECL biosensors in 50 mM Tris-HCl buffer containing 50 mM TPA (pH 7.4) with a scan rate of 50 mV/s. (A) PKA specific peptide-based biosensor before (a) and after reaction with 0.03 U/mL PKA (d) and 0.2 U/mL PKA (e), respectively. (B) CK2 specific peptide-based biosensor before (a) and after reaction with 0.03 U/mL CK2 (d) and 0.2 U/mL CK2 (e), respectively. (b) the specific peptide-based biosensor for the detection of 0.2 U/mL PKA (A) or 0.2 U/mL CK2 (B) in the absence of anti-phosphoserine antibody. (c) the non-specific peptide-based biosensor for the detection of 0.2 U/mL PKA (A) or 0.2 U/mL CK2 (B). 20


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Figure 3 ECL intensity vs time profiles for the detection of different activities of PKA (A) and CK2 (B). (A) (a) 0 U/mL; (b) 0.01 U/mL; (c) 0.03 U/mL; (d) 0.05 U/mL; (e) 0.1 U/mL; (f) 0.2 U/mL; (g) 0.3 U/mL; (h) 0.4 U/mL; (i) 0.5 U/mL; (B) (a) 0 U/mL; (b) 0.01 U/mL; (c) 0.03 U/mL; (d) 0.05 U/mL; (e) 0.1 U/mL; (f) 0.2 U/mL; (g) 0.3 U/mL; (h) 0.4 U/mL. Insert, calibration curve of PKA and CK2. The measurement conditions: 50 mM Tris-HCl containing 50 mM TPA (pH 7.4), applied potential, 1.2V.

Figure 4 ECL intensity vs time profiles obtained from the PKA specific peptide-based biosensor for the detection of PKA (A) and from the CK2 specific peptide-based biosensor for the detection of CK2 (B) in the absence of the cell lysate (a), in presence of the cell lysate without stimulation (b), and the cell lysate with stimulation by 10 µM forskolin and 20 µM IBMX(c), and the cell lysate with stimulation by 25 µM forskolin and 50 µM IBMX (d) The measurement conditions are same as Figure 3.

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Figure 5 (A) Scheme of four gold electrode array. (B-D) ECL imagings of the ECL peptide-based biosensor array for the detection of 0.2 U/mL PKA (B), 0.2 U/mL CK2(C), 0.2 U/mL PKA + 0.2 U/mL CK2 (D) obtained with EMCCD detector (iXon+ DU-897 Andor EMCCD, Andor Technology Ltd., Northern Ireland). The measurement conditions are 50 mM Tris-HCl containing 50 mM TPA (pH 7.4), applied potential, 1.2 V, exposure time 11 s.

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Figure 6 (A) ECL imagings of the ECL peptide-based biosensor array for the detection of different activities of PKA and CK2 obtained with EMCCD detector. Calibration curves of PKA (B) and CK2 (C). The measurement conditions are same as Figure 5.

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for TOC only

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Electrogenerated Chemiluminescence Bioassay of Two Protein

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