Biosensors and Bioelectronics 70 (2015) 54–60
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A highly sensitive electrochemiluminescence assay for protein kinase based on double-quenching of graphene quantum dots by G-quadruplex–hemin and gold nanoparticles Jinquan Liu, Xiaoxiao He n, Kemin Wang n, Dinggeng He, Yonghong Wang, Yinfei Mao, Hui Shi, Li Wen State Key Laboratory of Chemo/Biosensing and Chemometrics, Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, College of Biology, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China
art ic l e i nf o
a b s t r a c t
Article history: Received 31 December 2014 Received in revised form 9 March 2015 Accepted 10 March 2015 Available online 11 March 2015
A highly sensitive electrochemiluminescence (ECL) strategy was developed for the protein kinase A (PKA) activity and inhibition assay based on double-quenching of graphene quantum dots (GQDs) ECL by G-quadruplex–hemin DNAzyme and gold nanoparticles (AuNPs). In this strategy, the GQDs were modified onto the indium-tin oxide (ITO) electrode and further assembled with substrate peptide of target protein kinase through covalent coupling, which can exhibit high and stable ECL signal. The AuNPs, functionalized with the phosphorylated DNA and G-quadruplex–hemin DNAzyme via Au–S chemistry, were selected as quenching probes. In the presence of PKA, the peptide on the electrode was phosphorylated and the AuNPs functionalized with the phosphorylated DNA and G-quadruplex–hemin DNAzyme were subsequently integrated onto the phosphorylated peptide by Zr4 þ . Owing to the reduction of coreactant H2O2 resulting from G-quadruplex–hemin DNAzyme catalytic reaction and the ECL energy transfer (ECL-RET) between AuNPs and GQDs, the ECL intensity of GQDs was significantly decreased. By taking advantage of the double-quenching effect, this assay can detect PKA with a linear range of 0.05 to 5 U mL 1 and a detection limit of 0.04 U mL 1. In addition, the PKA inhibition assay and interferences experiments of CK2 and T4 PNK have been studied respectively. This assay was also successfully applied to PKA assay in serum samples and cell lysates, indicating that the developed method have the potential applications in protein kinase-related biochemical fundamental research and clinical diagnosis. & 2015 Elsevier B.V. All rights reserved.
Keywords: Electrochemiluminescence Graphene quantum dots Protein kinase A Gold nanoparticles G-quadruplex–hemin DNAzyme
1. Introduction Phosphorylation of proteins, chemically adding phosphate groups to the proteins by kinase enzymes, will result in a functional change of the target protein (substrate) by changing enzyme activity, cellular location, or association with other proteins (Nestler and Greengard, 1983), which plays important roles in numerous biological systems, including metabolism regulation, signal transduction, genes expression, cell proliferation, differentiation and apoptosis (López-Otín and Hunter, 2010; Cohen, 2002). The human genome contains about 500 protein kinase genes and they constitute about 2% of all human genes. According to its action on specific amino acids, the protein kinases can be divided n
Corresponding authors. Fax: þ 86 731 88821566. E-mail addresses: [email protected] (X. He), [email protected] (K. Wang). http://dx.doi.org/10.1016/j.bios.2015.03.026 0956-5663/& 2015 Elsevier B.V. All rights reserved.
into the following kinds: serine/threonine-specific protein kinases, tyrosine-specific protein kinases, histidine-specific protein kinases, and so on (Adams, 2001). Protein kinases A (PKA), is a kind of serine/threonine-specific protein kinases that modulate the phosphorylation of proteins by transferring the ATP terminal phosphates to the serine, or threonine residues. The activation of the PKA could mediate diverse cellular responses to external signals such as proliferation, ion transport, regulation of metabolism and gene transcription (Sims et al., 2013; Dupré et al., 2014). It has been reported that the abnormal expression and changing of PKA might have implications for many human diseases such as the carney complex, dilated cardiomyopathy and Huntington's disease (Moujalled et al., 2010; Lin et al., 2013). In this case, the assay of the PKA activity and potential inhibitors screening are of immediate significance for biochemical fundamental research and clinical diagnostics. Up to now, various methods have been designed for assaying
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activity of protein kinases including 32P radioactive (Park et al., 2007), surface-enhanced Raman spectroscopy (SERS) (Siddhanta et al., 2013), resonance-light scattering (RLS) (Li et al., 2010), mass spectroscopy (MS) (Shigaki et al., 2006), colorimetric sensing (Kitazaki et al., 2012; Zhou et al., 2014), fluorescence (Bai et al., 2013; Freeman et al., 2010; Wang et al., 2014), quartz crystal microbalance (QCM) (Xu et al., 2012), electrochemical (Ji et al., 2009; Martić et al., 2012; Wang et al., 2014, 2010; Yin et al., 2015) and electrochemiluminescence (ECL) technique (Chen et al., 2013). In particular, the ECL technique, a light emission process in a redox reaction of electrogenerated reactants, has recently attracted increasing concern for protein kinase analysis owing to its intrinsic advantages of low cost, wide range of analytes, rapid response, high sensitivity, good stability, and low background (Richter, 2004). For example, Xu et al. (2010) developed a novel ECL biosensor for protein kinase activity and inhibition assay using gold (nanoparticles) AuNPs as signal transduction probes. The AuNPs were assembled onto the electrode based on the introduction of thiol group during the kinase catalyzed-phosphorylation process. Due to its good conductivity, large surface area, and excellent electroactivity to luminol oxidization, low detection limit of 0.07 U mL 1 PKA had been obtained. Zhao et al. (2012) reported an ECL biosensor for protein kinase activities and inhibition monitoring based on the magnetic beads (MB) technology and signal enhancement of AuNPs. In this work, the coupling of AuNPs carrying lots of ECL compounds resultstris-(2, 2′-bipyridyl) ruthenium (TBR) and the MB surfaces used for separation and reaction with unmodified ECL electrode detection greatly improved the quality of kinase activity assay. Indeed, these reported ECL methods provided simple and highly sensitive strategies for PKA kinase activity assay and inhibition monitoring. However, the luminescence reagents involved in the ECL based protein kinase biosensors are confined to molecular reagents, which unfortunately suffer from expensive and/or poisonous reagents or complex modification (Tang et al., 2014). In recent years, semiconductor quantum dots (QDs), as new arising ECL indicators, have received great attention (Gill et al., 2008; Zhang et al., 2012). The QDs could generate cathodic emission in the presence of different coreactants such as oxygen, hydrogen peroxide or peroxydisulfate. By taking advantages the
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peroxidase-like property of G-quadruplex–hemin DNAzyme to catalyze the reduction of H2O2 or dissolved oxygen, QDs-based ECL biosensors have been developed and used for the ECL detection of DNA, ATP, and α-fetoprotein (Zhou et al., 2013; Liu et al., 2014a; Lin et al., 2011). Although these QDs indicators involved ECL biosensors displayed sensitive and selective detection and have a promising application for ultrasensitive analysis in biomedicine, the serious health and environmental concerns raised from cadmium-based QDs is still a challenge. Graphene quantum dots (GQDs), as recently emerging carbon-based materials, are grapheme sheets smaller than 100 nm (Dong et al., 2012). Due to quantum confinement and edge effects, GQDs are attracting extensive attention in the fields of photovoltaic devices, bioimaging, biosensing and drug delivery as they are superior to traditional semiconductor QDs in terms of chemical inertness, low toxicity, good biocompatibility, excellent solubility and stable photoluminescence (Li et al., 2013). Apart from their photoluminescence properties, it's very exciting to see that GQDs also show some brilliant ECL properties with H2O2 as coreactant (Lu et al., 2013). In addition, it has been recently reported the ECL signal GQDs could be quenched by AuNPs due to the occurrence of an electrochemiluminescence resonance energy transfer (ECL-RET) between the GQDs and the AuNPs (Lu et al., 2014). Thus, it is very encouraged to develop GQDs indicator based ECL strategy for PKA activity and inhibition assay. Herein, for the first time, an effective ECL assay for high sensitive detection of PKA activity and inhibition was constructed based on double-quenching of GQDs ECL by G-quadruplex–hemin DNAzyme and AuNPs. As shown in Scheme 1, the assay platform was just developed by assembling the substrate peptide to the electrode which was functionalized with GQDs through covalent coupling. In this strategy, the peptide/GQDs modified electrode showed a strong and stable ECL emission in the presence of coreactant H2O2 at the moment. After the substrate peptide was phosphorylated by PKA using ATP as the co-substrate, AuNPs functionalized with the phosphorylated linker DNA and G-quadruplex–hemin DNAzyme were attached on the electrode surface by the Zr4 þ -mediated reaction between the phosphorylated peptide and the phosphorylated linker DNA. The G-quadruplex–hemin DNAzyme on the surface of AuNPs could catalyze the reduction of
Scheme 1. Schematic illustration of ECL assay for PKA based on double-quenching of GQDs by pDNA@DNAzyme-AuNPs.
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coreactant H2O2 and then reduced the ECL intensity of GQDs. Meanwhile, the ECL intensity was also quenched due to the presence of ECL-RET between the proximal AuNPs and GQDs at a relative short distance. As a joint result of the DNAzyme catalytic reaction and ECL-RET analytical technique, the present ECL assay opened up a new way and showed an excellent analytical performance for the high sensitive detection of PKA activity and inhibition. Moreover, the PKA assay in serum samples and cell lysates was also indicated that the proposed method have the promise for potential applications in protein kinase-related biochemical fundamental research and clinical diagnosis.
2. Experimental 2.1. Chemicals and materials Protein kinase A (PKA) was obtained from Ray Biotech, Inc. (Specific activity is 415,000,000 U/mg). Casein kinase II (CK2) and T4 polynucleotide kinase (T4 PNK) were obtained from New England Biolabs, Inc. Adenosin triphosphate (ATP), bovine serum albumin (BSA) and fetal bovine serum (FBS) were obtained from Dingguo Biological Products Company (China). Adenosine 3′, 5′cyclic monophosphate sodium salt monohydrate (cAMP), ZrOCl2, 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich. Hydrogen tetrachloroaurate trihydrate (HAuCl4 3H2O) was purchased from Beijing Chemicals (Beijing, China). Ellagic acid and tris (2-carboxyethyl) phosphine hydrochloride (TCEP) were purchased from TCI Development Co. Ltd. (Shanghai, China). Citric acid (CA) was obtained from Shanghai Reagent (Shanghai, China). Sodium dodecylsulfate (SDS) and hydrogen peroxide (H2O2) were obtained from Sinopharm Chemical Reagent Co. Ltd. All other chemicals were obtained from Reagent & Glass Apparatus Corporation of Changsha and were of analytical grade used without further purification. The washing buffer was 10 mM Tris–HCl containing 0.05% tween-20, pH 7.4. Ultrapure water (Milli-Q 18.2 MΩ, Millipore System Inc.) was used for all the experiment. The peptide and oligonucleotides were purchased and purified by Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. (China). The sequences of peptide and oligonucleotides used in this experimental were given below Substrate peptide: CLRRASIG. Phosphorylated linker DNA: 5′-SH-C6-AAAAAAGTGT GTGTGTGTGTGTGTG TG-P-3′. G-quadruplex: 5′-SH-C6-AAAAAAGGGTTGGGCGGGATGGGT-3′. Phosphorylated G-quadruplex: 5′AAAAAAGGGTTGGGCGGGATGGGT-3′. Indium tin oxide (ITO)-coated (thickness, 100 nm; resistance, 15 Ω/square) aluminosilicate glass slides were purchased from Zhuhai Kaivo Optolelectronic Technology Co. Ltd. (Zhuhai, China).
2.3. Preparation of DNA functionalized AuNPs The phosphorylated linker DNA and G-quadruplex modified AuNPs were prepared as literatures with little modification (Alhasan et al., 2012). Firstly, 50 μL 5′-terminal disulfide groups of the DNA strands were deprotected by 10 mM TCEP for 30 min, and then added to 450 μL citrate-capped 13 nm AuNPs solutions (the final phosphorylated linker DNA concentration was 2 mM and G-quadruplex concentration was 4 mM) for 2 h at room temperature. Next, the mixed solution was salted to 0.1 M NaCl in PBS (10 mM, 0.01% SDS, pH 7.4,) and incubated for an additional 2 h at room temperature. Two aliquots of NaCl were added with an incubation time of 1 h between each addition to achieve a final concentration of 0.3 M. After incubation overnight at room temperature with gentle shaking, the AuNPs solution was centrifuged (15,000 rpm, 30 min at 4 °C) in order to remove excessive DNA. The precipitate was washed with 0.1 M NaCl, 10 mM phosphate buffer (pH 7.4) solution, recentrifuged, and finally dispersed in 10 mM phosphate buffer (pH 7.4) containing 0.1 M KCl. The phosphorylated linker DNA and G-quadruplex modified AuNPs were characterized by UV–vis spectra (Fig. S1). Finally, a 50 μL portion of 5 μM hemin was added to the resulting DNA modified AuNPs solutions to form the phosphorylated linker DNA and G-quadruplex–hemin DNAzyme modified AuNPs (pDNA@DNAzyme-AuNPs) and then stored at 4 °C for further use. 2.4. Preparation of the ECL electrode and phosphorylation of substrate peptide The ITO electrode (1 cm 1.5 cm each) was ultrasonically cleaned with acetone, ethanol, and water subsequently and dried at room temperature. Then 20 μL GQDs solution was coated on the ITO electrode and dried in shade at room temperature. To further conjugate the substrate peptide onto the ECL electrode, the GQDs modified ITO electrode was dropped on 20 mL EDC/NHS mixture (200 mM EDC and 50 mM NHS) for 1 h at 4 °C to activate the terminal carboxylic acid groups of GQDs. The activated GQDs modified electrode was then soaked in a PBS (50 mM, pH 7.4) solution containing 0.3 μM amino terminated substrate peptide overnight at 4 °C. Finally, the electrode was dipped in 20 μL of 1% BSA solution at 4 °C for 1 h to block possible remaining active sites against nonspecific adsorption. The electrode surface was rinsed with blank buffer after each step to remove nonspecifically adsorbed species. A PKA-catalyzed phosphorylation reaction was then performed by adapting the manufacturer's protocol. Briefly, 20 μL reaction mixtures (40 mM Tris–HCl and 20 mM MgCl2, pH 7.4) containing the final assay concentration of 200 μM cAMP, 80 μM ATP and a desired amount of PKA were added onto the substrate peptide modified ITO electrode surface at 30 °C for 1 h. Then the electrode was washed with blank buffer to remove the excess reagents. For the PKA inhibitor assay, the same experiments were performed, except for different concentrations of ellagic acid were added in the PKA reaction mixture.
2.2. Preparation of GQDs The GQDs were prepared by directly pyrolyzing CA according to previous report (Dong et al., 2012). Briefly, 2 g CA was heated to liquated at 200 °C. After further heated, the color of the liquid was changed from colorless to pale yellow, and then orange, it indicated that the GQDs were formed. The resulted orange liquid was then added drop by drop into 100 mL of 10 mg/mL NaOH solution under vigorous stirring. After the pH value was adjusted to 7.0, the solution changed to greenish yellow. The final obtained GQDs solution was stored in the refrigerator (4 °C) and ready for use.
2.5. Linkage of phosphorylated peptide with pDNA@DNAzymeAuNPs by Zr4 þ The above mentioned phosphorylated peptide modified electrode was then treated with 20 μL 1 mM Zr4 þ solution for 1 h at room temperature. After incubation, the electrode was rinsed thoroughly with blank buffer solution, followed by dipping twice in water. After being dried with nitrogen, the electrode was coated with 20 μL pDNA@DNAzyme-AuNPs at room temperature for 1 h. The resulted electrode was washed with washing buffer and dried by nitrogen and then used for the ECL measurement.
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2.6. Preparation of cell lysates To demonstrate the application of this ECL kinase assay in cell lysates, normal cells HEK293, lung cells A549 and breast cells MCF7 were incubated in DMEM medium supplemented with 10% FBS under a humidified atmosphere containing 5% CO2 at 37 °C, respectively. Subsequently, the cells solution ( 1.5 106 cells) was centrifuged and washed three times with PBS buffer (10 mM, pH 7.4). After the supernatant was removed, the precipitated cells were treated with 200 μL lysis buffer at 4 °C for 30 min. Finally, the cells lysate were centrifuged (12,000 rpm, 20 min at 4 °C) and then the supernatant were stored at 20 °C for further use. For detection of the kinase activity in complex biological samples, the cell lysates rather than PKA were added into desired volumes phosphorylation reaction solutions. 2.7. ECL and electrochemical measurements ECL signals were measured with MPI-E ECL analyzer (Remax Electronic Instrument Limited Co., Xi’ an, China) with the voltage of the photomultiplier tube (PMT) set at 800 V. The ECL assay was carried out in 4 mL solution (0.1 M PBS, 0.1 M KCl, pH 8.0) with 1 mM H2O2 as coreactant at a potential from 0 V to 2.0 V and a scan rate of 0.1 V/s. The electrochemical impedance spectroscopy (EIS) was obtained on a CHI 660 A electrochemical workstation (Shanghai Chenhua Instrument Corporation, China) in PBS buffer (10 mM PBS, containing 5 mM [Fe(CN)6]3 /4 and 0.1 M KCl, pH ¼7.4) in the frequency range from 0.01 Hz to 100 kHz. Both ECL and EIS measurements were investigated on a three-electrode system with ITO electrode as the working electrode and a platinum wire and an Ag/AgCl (saturated KCl solution) as the counter and reference electrode, respectively. 2.8. Apparatus and characterization Transmission electron microscopy (TEM) images were performed on JEM-3010 (JEOL, Japan). Atomic force microscopy (AFM) images were obtained in ScanAsyst mode with MultiMode-8 (Bruker, Germany). Fluorescence measurement was performed using an F-7000 spectrofluorometer (Hitachi, Japan). The UV–vis absorption spectrum was collected on UV-2600 spectrophotometer (Shimadzu, Japan). The Fourier transform infrared (FTIR) spectrum was recorded with TENSOR27 FT-IR spectrometer
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(Bruker, Germany).
3. Results and discussion 3.1. Characterization of GQDs To observe the morphology and microstructure of the GQDs, the TEM and AFM images were acquired. TEM images showed that the diameters and uniformity of GQDs were not evenly distributed but mainly dispersed in the range of 6–12 nm with average diameter of 10 nm (Fig. 1A), which was similar to previous reports (Dong et al., 2012). The AFM results indicated that the topographic height of the GQDs were mostly between 1.2 and 3.6 nm (Fig. 1B), suggesting that most of GQDs were single layered or bilayered. The FTIR spectrum (Fig. S2A) exhibited characteristic absorption bands of hydroxyl functional group at 3423 cm 1, COO stretching vibrations at 1608 cm 1 and 1400 cm 1, indicating that the GQDs possess excellent solubility in water. The UV–vis and PL spectra were also used to characterize the formation of GQDs (Fig. S2B). The synthesized GQDs showed a broad UV–vis absorption band at 362 nm (curve a), and the PL spectrum of the GQDs solution showed a strong emission peak at 470 nm under the excitation at 320 nm (curve b) and the maximum emission wavelength was not changed with different batches of samples. 3.2. Characterization of the ITO Electrodes modification EIS, as an effective method for probing the interfacial properties of modified electrode, was employed to confirm the fabrication process of the ITO electrode modification using [Fe (CN) 6] 3 /4 as redox probe. Fig. 2A shows the nyquist plots of EIS observed upon the stepwise fabrication processes. The bare ITO electrode given a very small semicircular domain (curve a) and the resistances (Ret) value of bare ITO electrode was about 34.15 Ω. Because of the GQDs can promote electron-transfer processes, upon the GQDs assembled on electrode surface, the Ret value decrease to 26.42 Ω (curve b). After the substrate peptide and BSA were self-assembled onto the electrode, the Ret increased to 55.89 Ω (curve c). This is ascribed to the poor conductivity of substrate peptide and BSA. Due to the electrostatic repulsion between the negative charged phosphorylation sites and redox probe, the Ret was increased to 58.89 Ω after the phosphorylation by PKA (curve d). Subsequently,
Fig. 1. (A) The TEM image of GQDs. (B) AFM image and the corresponding height profiles of GQDs.
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Fig. 2. (A) The EIS of bare ITO electrode (a) GQDs modified electrode, (b) peptide and BSA modified electrode, (c) phosphorylated peptide modified electrode before, (d) and after, (e) the assembly of pDNA@DNAzyme-AuNPs in 0.01 M PBS solution containing 5 mM [Fe(CN)6]3 /4 electroactive probes within the range from 0.1 Hz to 100 KHz, the amplitude of the applied sine wave potential was 5 mV and quiet time was 2 s. (B) ECL-potential curves of peptide/GQDs modified ITO (a) peptide phosphorylated, (b) or unphosphorylated, (c) by PKA after treatment with pDNA@DNAzyme-AuNPs mediated by Zr4 þ and the Zr4 þ mediated phosphorylated DNAzyme, (d) or phosphorylated linker DNA modified AuNPs and (e) assembled phosphorylated peptide modified electrode. ECL conditions: the voltage of PMT was 800 V and amplifier gain was set at 4. Scan rate: 100 mV/s.
when the pDNA@DNAzyme-AuNPs was assembled onto the electrode by the linkage of Zr4 þ , the Ret emerged a further increase corresponding to the large amount of negatively charged DNA linked on the AuNPs (curve e), and the Ret value was 84.88 Ω. The EIS results proved that the modification process of the electrode was achieved successfully. The AFM image of the resulting pDNA@DNAzyme-AuNPs bound to the phosphorylated peptide modified electrode was shown in Fig. S3. It is noted that a number of pDNA@DNAzymeAuNPs with height of 15 nm were uniformly distributed on the surface of the electrode, and no aggregation was found (Fig. S3A). However, only little pDNA@DNAzyme-AuNPs were observed on the surface of the control electrode which was without PKA treatment (Fig. S3B). This is probably due to nonspecific absorption of pDNA@DNAzyme-AuNPs. As the result of AFM, the pDNA@DNAzyme-AuNPs could be successfully attached to the phosphorylated peptide modified electrode. The ECL behaviors of the GQDs and mechanism of the proposed ECL assay was shown in supporting information (Fig. S4). 3.3. Feasibility investigation of the ECL assay After confirmation the modification of the electrode and the conjugation of pDNA@DNAzyme-AuNPs onto the phosphorylated peptide, the feasibility of this ECL assay was then investigated by the ECL emission. As it is shown in Fig. 2B, the peptide/GQDs modified electrode exhibited a strong ECL signal with 1 mM H2O2 as coreactant at the applied potential range of 0 to 2.0 V (curve a). In the presence of PKA, the ECL intensity significantly decreased upon phosphorylation by PKA with the linkage of pDNA@DNAzyme-AuNPs by Zr4 þ (curve b). In comparison, a weak decrease in ECL intensity can be observed in the absence of PKA (curve c). It was the negative control measurements. The results showed that the pDNA@DNAzyme-AuNPs could effectively quench the ECL intensity of GQDs. To further confirm the efficient double-quenching effect of the pDNA@DNAzyme-AuNPs, the phosphorylated peptide modified electrode treated with only G-quadruplex–hemin DNAzyme (curve d) and AuNPs just assembled with phosphorylated linker DNA (curve e) were also investigated, respectively. It was demonstrated that both the DNAzyme and AuNPs can quench the ECL intensity of GQDs. Clearly, with integrating the DNAzyme and AuNPs together, an enhanced quenched ECL signal was obtained (curve b), indicating that the ECL intensity of GQDs could be
quenched by pDNA@DNAzyme-AuNPs more effectively. In addition, some control experiments to test for false positive in the absence of Zr4 þ and pDNA@DNAzyme-AuNPs and in the absence of pDNA@DNAzyme-AuNPs have been applied in the supporting information. From the results of Fig. S5, We found that the ECL intensity has little decrease in these control experiments by comparing with peptide/GQDs modified electrode. These results further showed that the sensitive ECL assay for PKA activity and inhibition could be carried out. 3.4. PKA activity detection Under the optimized conditions (60 min of phosphorylation time, 1 mM Zr4 þ , 80 μM ATP and pH 8) (Fig. S6), the relationship between the ECL intensity and PKA activity were investigated. From the results of Fig. 3A, the ECL intensity of GQDs was sensitive to the change of the PKA activity with enzymatic unit from 0 to 20 U mL 1. As the enzymatic unit of PKA increased, the ECL intensity decreased sharply and then slowed down at higher enzymatic unit. The results showed that the ECL decrement ΔI (ΔI¼ I0 I, where I0 and I were ECL intensity of the peptide/GQDs modified ITO electrode before (I0) and after (I) phosphorylated by PKA and then treatment with pDNA@DNAzyme-AuNPs, respectively) was logarithmically related to the PKA enzymatic unit in the range from 0.05 U mL 1 to 5 U mL 1 with a correlation coefficient of 0.985 (the inset in Fig. 3B). The linear regression equation was calculated as ΔI¼ 534 log [PKA] þ789 and the detection limit (LOD) of PKA was as low as 0.04 U mL 1 at 3 times signal-to-noise. Compared with the previous reporters (Liu et al., 2014b; Xu et al., 2009), the ECL assay have a lower detection limit, indicating that the developed ECL assay can be used to highly sensitive PKA activity detection with a wide concentration range. 3.5. Influence of inhibitor on PKA activity Ellagic acid, as a cell-permeable and potent antioxidant (Cozza et al., 2006), was selected to further test the potential applications of the ECL assay in protein kinase inhibitor detection. Fig. 4A shows the inhibitory results of ellagic acid. As expected, the ΔI decreased gradually with increasing the concentration of ellagic acid owning to the inhibition of PKA activity and low levels of peptide phosphorylation. As a result, IC50 (the half maximal inhibitory concentration) was calculate to be 4.22 μM, which was
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Fig. 3. (A) ECL signals of the ECL assay incubated with different activity of PKA (0, 0.05, 0.1, 0.5, 1, 2, 5, 10, 20 U mL 1). (B) Current intensity as a function of PKA activities. The inset is the linear relationship between current intensity and the activity of PKA Linear relationship between ECL decrement ΔI (I0 I) and the logarithm of PKA activity, three measurements for each point.
well comparable with the previous literature (Xu et al., 2010). The result indicated that the developed ECL assay has an excellent potential in screening the activity of the kinase inhibitor. 3.6. Selectivity and reproducibility of the ECL assay In this ECL assay, the selectivity was evaluated by incubating the same concentration of PKA, CK2 and T4 PNK, respectively. As displayed in Fig. S7, the ECL assay showed a good selectivity for PKA over other kinases. These results indicated that the developed ECL assay exhibit good specificity. The reproducibility of the ECL assay was also used to examine PKA activity with intraassay and interassay. The relative standard deviation (RSD) of intraassay, examined by testing the same electrode of five dependent measurements, was 4.95%. While the interassay estimated for five reduplicate measurements on different electrodes was 9.38%. The result indicated that the ECL assay have an acceptable fabrication reproducibility in PKA activity detection. 3.7. PKA activity assay in complex biological samples To further investigate the proposed ECL assay in complex biological samples for clinic application, the phosphorylation reaction solution was prepared by adding a desired amount of PKA in FBS rather than buffer solution. As shown in Table S1, the relative
errors of two different enzymatic unit of PKA were 8.4% and 11.07%, respectively, suggesting that the as-proposed ECL assay was utilizable for the detection of PKA activity in FBS fluids. In addition, the cell lysates samples were still used to kinase activity assay. In this case, three cell lysates from HEK293 normal cell line and two cancer cell lines of lung (A549) and breast (MCF-7) were investigated in this work. As displayed in Fig. 4B, the MCF-7 cell line emerged the higher level of PKA than the other cell lines. Moreover, the PKA expression levels of cancer cells are higher than normal cell (HEK293), which was compared with the previous result (Wang et al., 2014).
4. Conclusion In conclusion, with the confluence of G-quadruplex–hemin DNAzyme and AuNPs, a highly sensitive ECL assay was developed for the PKA activity and inhibition detection. In this method, the pDNA@DNAzyme-AuNPs can highly quench the ECL intensity of GQDs as a joint result of the G-quadruplex–hemin DNAzyme catalytic reaction and ECL-RET analytical technique. Based on the efficient double-quenching effect, the proposed ECL assay showed high sensitivity and good selectivity for PKA activity assay with a LOD of 0.04 U mL 1. Furthermore, this ECL assay can also be taken as a general platform to detect different protein kinase by simply changing specific substrate peptide sequences. Moreover, the
Fig. 4. (A) Inhibition of the activity of PKA by ellagic acid. The concentrations of ellagic acid were 0, 1, 2, 4, 6, 10, and 15 μM. (B) Detection of PKA activities from BEK293, A549, and MCF-7 cell lysates. All reactions were carried out at optimized condition except for the addition of different concentrations of inhibitor or cell lysates.
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application of inhibition assay and kinase detection in serum samples and cell lysates were also indicated that the proposed ECL assay has the promise for potential applications in protein kinaserelated biochemical fundamental research and clinical diagnosis.
Acknowledgment This work was supported in part by the Key Project of National Natural Science Foundation of China (Grants 21175039, 21322509, 21305035, 21190044, and 21221003), Research Fund for the Doctoral Program of Higher Education of China (Grant 20110161110016) and the project supported by Hunan Provincial Natural Science Foundation of China (Grant 14JJ3066) and Hunan Provincial Science and Technology Plan of China (2012TT1003).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.03.026.
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A highly sensitive electrochemiluminescence assay for protein kinase based on double-quenching of graphene quantum dots by G-quadruplex–hemin and gold nanoparticles Jinquan Liu, Xiaoxiao He n, Kemin Wang n, Dinggeng He, Yonghong Wang, Yinfei Mao, Hui Shi, Li Wen State Key Laboratory of Chemo/Biosensing and Chemometrics, Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, College of Biology, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China
art ic l e i nf o
a b s t r a c t
Article history: Received 31 December 2014 Received in revised form 9 March 2015 Accepted 10 March 2015 Available online 11 March 2015
A highly sensitive electrochemiluminescence (ECL) strategy was developed for the protein kinase A (PKA) activity and inhibition assay based on double-quenching of graphene quantum dots (GQDs) ECL by G-quadruplex–hemin DNAzyme and gold nanoparticles (AuNPs). In this strategy, the GQDs were modified onto the indium-tin oxide (ITO) electrode and further assembled with substrate peptide of target protein kinase through covalent coupling, which can exhibit high and stable ECL signal. The AuNPs, functionalized with the phosphorylated DNA and G-quadruplex–hemin DNAzyme via Au–S chemistry, were selected as quenching probes. In the presence of PKA, the peptide on the electrode was phosphorylated and the AuNPs functionalized with the phosphorylated DNA and G-quadruplex–hemin DNAzyme were subsequently integrated onto the phosphorylated peptide by Zr4 þ . Owing to the reduction of coreactant H2O2 resulting from G-quadruplex–hemin DNAzyme catalytic reaction and the ECL energy transfer (ECL-RET) between AuNPs and GQDs, the ECL intensity of GQDs was significantly decreased. By taking advantage of the double-quenching effect, this assay can detect PKA with a linear range of 0.05 to 5 U mL 1 and a detection limit of 0.04 U mL 1. In addition, the PKA inhibition assay and interferences experiments of CK2 and T4 PNK have been studied respectively. This assay was also successfully applied to PKA assay in serum samples and cell lysates, indicating that the developed method have the potential applications in protein kinase-related biochemical fundamental research and clinical diagnosis. & 2015 Elsevier B.V. All rights reserved.
Keywords: Electrochemiluminescence Graphene quantum dots Protein kinase A Gold nanoparticles G-quadruplex–hemin DNAzyme
1. Introduction Phosphorylation of proteins, chemically adding phosphate groups to the proteins by kinase enzymes, will result in a functional change of the target protein (substrate) by changing enzyme activity, cellular location, or association with other proteins (Nestler and Greengard, 1983), which plays important roles in numerous biological systems, including metabolism regulation, signal transduction, genes expression, cell proliferation, differentiation and apoptosis (López-Otín and Hunter, 2010; Cohen, 2002). The human genome contains about 500 protein kinase genes and they constitute about 2% of all human genes. According to its action on specific amino acids, the protein kinases can be divided n
Corresponding authors. Fax: þ 86 731 88821566. E-mail addresses: [email protected] (X. He), [email protected] (K. Wang). http://dx.doi.org/10.1016/j.bios.2015.03.026 0956-5663/& 2015 Elsevier B.V. All rights reserved.
into the following kinds: serine/threonine-specific protein kinases, tyrosine-specific protein kinases, histidine-specific protein kinases, and so on (Adams, 2001). Protein kinases A (PKA), is a kind of serine/threonine-specific protein kinases that modulate the phosphorylation of proteins by transferring the ATP terminal phosphates to the serine, or threonine residues. The activation of the PKA could mediate diverse cellular responses to external signals such as proliferation, ion transport, regulation of metabolism and gene transcription (Sims et al., 2013; Dupré et al., 2014). It has been reported that the abnormal expression and changing of PKA might have implications for many human diseases such as the carney complex, dilated cardiomyopathy and Huntington's disease (Moujalled et al., 2010; Lin et al., 2013). In this case, the assay of the PKA activity and potential inhibitors screening are of immediate significance for biochemical fundamental research and clinical diagnostics. Up to now, various methods have been designed for assaying
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activity of protein kinases including 32P radioactive (Park et al., 2007), surface-enhanced Raman spectroscopy (SERS) (Siddhanta et al., 2013), resonance-light scattering (RLS) (Li et al., 2010), mass spectroscopy (MS) (Shigaki et al., 2006), colorimetric sensing (Kitazaki et al., 2012; Zhou et al., 2014), fluorescence (Bai et al., 2013; Freeman et al., 2010; Wang et al., 2014), quartz crystal microbalance (QCM) (Xu et al., 2012), electrochemical (Ji et al., 2009; Martić et al., 2012; Wang et al., 2014, 2010; Yin et al., 2015) and electrochemiluminescence (ECL) technique (Chen et al., 2013). In particular, the ECL technique, a light emission process in a redox reaction of electrogenerated reactants, has recently attracted increasing concern for protein kinase analysis owing to its intrinsic advantages of low cost, wide range of analytes, rapid response, high sensitivity, good stability, and low background (Richter, 2004). For example, Xu et al. (2010) developed a novel ECL biosensor for protein kinase activity and inhibition assay using gold (nanoparticles) AuNPs as signal transduction probes. The AuNPs were assembled onto the electrode based on the introduction of thiol group during the kinase catalyzed-phosphorylation process. Due to its good conductivity, large surface area, and excellent electroactivity to luminol oxidization, low detection limit of 0.07 U mL 1 PKA had been obtained. Zhao et al. (2012) reported an ECL biosensor for protein kinase activities and inhibition monitoring based on the magnetic beads (MB) technology and signal enhancement of AuNPs. In this work, the coupling of AuNPs carrying lots of ECL compounds resultstris-(2, 2′-bipyridyl) ruthenium (TBR) and the MB surfaces used for separation and reaction with unmodified ECL electrode detection greatly improved the quality of kinase activity assay. Indeed, these reported ECL methods provided simple and highly sensitive strategies for PKA kinase activity assay and inhibition monitoring. However, the luminescence reagents involved in the ECL based protein kinase biosensors are confined to molecular reagents, which unfortunately suffer from expensive and/or poisonous reagents or complex modification (Tang et al., 2014). In recent years, semiconductor quantum dots (QDs), as new arising ECL indicators, have received great attention (Gill et al., 2008; Zhang et al., 2012). The QDs could generate cathodic emission in the presence of different coreactants such as oxygen, hydrogen peroxide or peroxydisulfate. By taking advantages the
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peroxidase-like property of G-quadruplex–hemin DNAzyme to catalyze the reduction of H2O2 or dissolved oxygen, QDs-based ECL biosensors have been developed and used for the ECL detection of DNA, ATP, and α-fetoprotein (Zhou et al., 2013; Liu et al., 2014a; Lin et al., 2011). Although these QDs indicators involved ECL biosensors displayed sensitive and selective detection and have a promising application for ultrasensitive analysis in biomedicine, the serious health and environmental concerns raised from cadmium-based QDs is still a challenge. Graphene quantum dots (GQDs), as recently emerging carbon-based materials, are grapheme sheets smaller than 100 nm (Dong et al., 2012). Due to quantum confinement and edge effects, GQDs are attracting extensive attention in the fields of photovoltaic devices, bioimaging, biosensing and drug delivery as they are superior to traditional semiconductor QDs in terms of chemical inertness, low toxicity, good biocompatibility, excellent solubility and stable photoluminescence (Li et al., 2013). Apart from their photoluminescence properties, it's very exciting to see that GQDs also show some brilliant ECL properties with H2O2 as coreactant (Lu et al., 2013). In addition, it has been recently reported the ECL signal GQDs could be quenched by AuNPs due to the occurrence of an electrochemiluminescence resonance energy transfer (ECL-RET) between the GQDs and the AuNPs (Lu et al., 2014). Thus, it is very encouraged to develop GQDs indicator based ECL strategy for PKA activity and inhibition assay. Herein, for the first time, an effective ECL assay for high sensitive detection of PKA activity and inhibition was constructed based on double-quenching of GQDs ECL by G-quadruplex–hemin DNAzyme and AuNPs. As shown in Scheme 1, the assay platform was just developed by assembling the substrate peptide to the electrode which was functionalized with GQDs through covalent coupling. In this strategy, the peptide/GQDs modified electrode showed a strong and stable ECL emission in the presence of coreactant H2O2 at the moment. After the substrate peptide was phosphorylated by PKA using ATP as the co-substrate, AuNPs functionalized with the phosphorylated linker DNA and G-quadruplex–hemin DNAzyme were attached on the electrode surface by the Zr4 þ -mediated reaction between the phosphorylated peptide and the phosphorylated linker DNA. The G-quadruplex–hemin DNAzyme on the surface of AuNPs could catalyze the reduction of
Scheme 1. Schematic illustration of ECL assay for PKA based on double-quenching of GQDs by pDNA@DNAzyme-AuNPs.
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coreactant H2O2 and then reduced the ECL intensity of GQDs. Meanwhile, the ECL intensity was also quenched due to the presence of ECL-RET between the proximal AuNPs and GQDs at a relative short distance. As a joint result of the DNAzyme catalytic reaction and ECL-RET analytical technique, the present ECL assay opened up a new way and showed an excellent analytical performance for the high sensitive detection of PKA activity and inhibition. Moreover, the PKA assay in serum samples and cell lysates was also indicated that the proposed method have the promise for potential applications in protein kinase-related biochemical fundamental research and clinical diagnosis.
2. Experimental 2.1. Chemicals and materials Protein kinase A (PKA) was obtained from Ray Biotech, Inc. (Specific activity is 415,000,000 U/mg). Casein kinase II (CK2) and T4 polynucleotide kinase (T4 PNK) were obtained from New England Biolabs, Inc. Adenosin triphosphate (ATP), bovine serum albumin (BSA) and fetal bovine serum (FBS) were obtained from Dingguo Biological Products Company (China). Adenosine 3′, 5′cyclic monophosphate sodium salt monohydrate (cAMP), ZrOCl2, 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich. Hydrogen tetrachloroaurate trihydrate (HAuCl4 3H2O) was purchased from Beijing Chemicals (Beijing, China). Ellagic acid and tris (2-carboxyethyl) phosphine hydrochloride (TCEP) were purchased from TCI Development Co. Ltd. (Shanghai, China). Citric acid (CA) was obtained from Shanghai Reagent (Shanghai, China). Sodium dodecylsulfate (SDS) and hydrogen peroxide (H2O2) were obtained from Sinopharm Chemical Reagent Co. Ltd. All other chemicals were obtained from Reagent & Glass Apparatus Corporation of Changsha and were of analytical grade used without further purification. The washing buffer was 10 mM Tris–HCl containing 0.05% tween-20, pH 7.4. Ultrapure water (Milli-Q 18.2 MΩ, Millipore System Inc.) was used for all the experiment. The peptide and oligonucleotides were purchased and purified by Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. (China). The sequences of peptide and oligonucleotides used in this experimental were given below Substrate peptide: CLRRASIG. Phosphorylated linker DNA: 5′-SH-C6-AAAAAAGTGT GTGTGTGTGTGTGTG TG-P-3′. G-quadruplex: 5′-SH-C6-AAAAAAGGGTTGGGCGGGATGGGT-3′. Phosphorylated G-quadruplex: 5′AAAAAAGGGTTGGGCGGGATGGGT-3′. Indium tin oxide (ITO)-coated (thickness, 100 nm; resistance, 15 Ω/square) aluminosilicate glass slides were purchased from Zhuhai Kaivo Optolelectronic Technology Co. Ltd. (Zhuhai, China).
2.3. Preparation of DNA functionalized AuNPs The phosphorylated linker DNA and G-quadruplex modified AuNPs were prepared as literatures with little modification (Alhasan et al., 2012). Firstly, 50 μL 5′-terminal disulfide groups of the DNA strands were deprotected by 10 mM TCEP for 30 min, and then added to 450 μL citrate-capped 13 nm AuNPs solutions (the final phosphorylated linker DNA concentration was 2 mM and G-quadruplex concentration was 4 mM) for 2 h at room temperature. Next, the mixed solution was salted to 0.1 M NaCl in PBS (10 mM, 0.01% SDS, pH 7.4,) and incubated for an additional 2 h at room temperature. Two aliquots of NaCl were added with an incubation time of 1 h between each addition to achieve a final concentration of 0.3 M. After incubation overnight at room temperature with gentle shaking, the AuNPs solution was centrifuged (15,000 rpm, 30 min at 4 °C) in order to remove excessive DNA. The precipitate was washed with 0.1 M NaCl, 10 mM phosphate buffer (pH 7.4) solution, recentrifuged, and finally dispersed in 10 mM phosphate buffer (pH 7.4) containing 0.1 M KCl. The phosphorylated linker DNA and G-quadruplex modified AuNPs were characterized by UV–vis spectra (Fig. S1). Finally, a 50 μL portion of 5 μM hemin was added to the resulting DNA modified AuNPs solutions to form the phosphorylated linker DNA and G-quadruplex–hemin DNAzyme modified AuNPs (pDNA@DNAzyme-AuNPs) and then stored at 4 °C for further use. 2.4. Preparation of the ECL electrode and phosphorylation of substrate peptide The ITO electrode (1 cm 1.5 cm each) was ultrasonically cleaned with acetone, ethanol, and water subsequently and dried at room temperature. Then 20 μL GQDs solution was coated on the ITO electrode and dried in shade at room temperature. To further conjugate the substrate peptide onto the ECL electrode, the GQDs modified ITO electrode was dropped on 20 mL EDC/NHS mixture (200 mM EDC and 50 mM NHS) for 1 h at 4 °C to activate the terminal carboxylic acid groups of GQDs. The activated GQDs modified electrode was then soaked in a PBS (50 mM, pH 7.4) solution containing 0.3 μM amino terminated substrate peptide overnight at 4 °C. Finally, the electrode was dipped in 20 μL of 1% BSA solution at 4 °C for 1 h to block possible remaining active sites against nonspecific adsorption. The electrode surface was rinsed with blank buffer after each step to remove nonspecifically adsorbed species. A PKA-catalyzed phosphorylation reaction was then performed by adapting the manufacturer's protocol. Briefly, 20 μL reaction mixtures (40 mM Tris–HCl and 20 mM MgCl2, pH 7.4) containing the final assay concentration of 200 μM cAMP, 80 μM ATP and a desired amount of PKA were added onto the substrate peptide modified ITO electrode surface at 30 °C for 1 h. Then the electrode was washed with blank buffer to remove the excess reagents. For the PKA inhibitor assay, the same experiments were performed, except for different concentrations of ellagic acid were added in the PKA reaction mixture.
2.2. Preparation of GQDs The GQDs were prepared by directly pyrolyzing CA according to previous report (Dong et al., 2012). Briefly, 2 g CA was heated to liquated at 200 °C. After further heated, the color of the liquid was changed from colorless to pale yellow, and then orange, it indicated that the GQDs were formed. The resulted orange liquid was then added drop by drop into 100 mL of 10 mg/mL NaOH solution under vigorous stirring. After the pH value was adjusted to 7.0, the solution changed to greenish yellow. The final obtained GQDs solution was stored in the refrigerator (4 °C) and ready for use.
2.5. Linkage of phosphorylated peptide with pDNA@DNAzymeAuNPs by Zr4 þ The above mentioned phosphorylated peptide modified electrode was then treated with 20 μL 1 mM Zr4 þ solution for 1 h at room temperature. After incubation, the electrode was rinsed thoroughly with blank buffer solution, followed by dipping twice in water. After being dried with nitrogen, the electrode was coated with 20 μL pDNA@DNAzyme-AuNPs at room temperature for 1 h. The resulted electrode was washed with washing buffer and dried by nitrogen and then used for the ECL measurement.
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2.6. Preparation of cell lysates To demonstrate the application of this ECL kinase assay in cell lysates, normal cells HEK293, lung cells A549 and breast cells MCF7 were incubated in DMEM medium supplemented with 10% FBS under a humidified atmosphere containing 5% CO2 at 37 °C, respectively. Subsequently, the cells solution ( 1.5 106 cells) was centrifuged and washed three times with PBS buffer (10 mM, pH 7.4). After the supernatant was removed, the precipitated cells were treated with 200 μL lysis buffer at 4 °C for 30 min. Finally, the cells lysate were centrifuged (12,000 rpm, 20 min at 4 °C) and then the supernatant were stored at 20 °C for further use. For detection of the kinase activity in complex biological samples, the cell lysates rather than PKA were added into desired volumes phosphorylation reaction solutions. 2.7. ECL and electrochemical measurements ECL signals were measured with MPI-E ECL analyzer (Remax Electronic Instrument Limited Co., Xi’ an, China) with the voltage of the photomultiplier tube (PMT) set at 800 V. The ECL assay was carried out in 4 mL solution (0.1 M PBS, 0.1 M KCl, pH 8.0) with 1 mM H2O2 as coreactant at a potential from 0 V to 2.0 V and a scan rate of 0.1 V/s. The electrochemical impedance spectroscopy (EIS) was obtained on a CHI 660 A electrochemical workstation (Shanghai Chenhua Instrument Corporation, China) in PBS buffer (10 mM PBS, containing 5 mM [Fe(CN)6]3 /4 and 0.1 M KCl, pH ¼7.4) in the frequency range from 0.01 Hz to 100 kHz. Both ECL and EIS measurements were investigated on a three-electrode system with ITO electrode as the working electrode and a platinum wire and an Ag/AgCl (saturated KCl solution) as the counter and reference electrode, respectively. 2.8. Apparatus and characterization Transmission electron microscopy (TEM) images were performed on JEM-3010 (JEOL, Japan). Atomic force microscopy (AFM) images were obtained in ScanAsyst mode with MultiMode-8 (Bruker, Germany). Fluorescence measurement was performed using an F-7000 spectrofluorometer (Hitachi, Japan). The UV–vis absorption spectrum was collected on UV-2600 spectrophotometer (Shimadzu, Japan). The Fourier transform infrared (FTIR) spectrum was recorded with TENSOR27 FT-IR spectrometer
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(Bruker, Germany).
3. Results and discussion 3.1. Characterization of GQDs To observe the morphology and microstructure of the GQDs, the TEM and AFM images were acquired. TEM images showed that the diameters and uniformity of GQDs were not evenly distributed but mainly dispersed in the range of 6–12 nm with average diameter of 10 nm (Fig. 1A), which was similar to previous reports (Dong et al., 2012). The AFM results indicated that the topographic height of the GQDs were mostly between 1.2 and 3.6 nm (Fig. 1B), suggesting that most of GQDs were single layered or bilayered. The FTIR spectrum (Fig. S2A) exhibited characteristic absorption bands of hydroxyl functional group at 3423 cm 1, COO stretching vibrations at 1608 cm 1 and 1400 cm 1, indicating that the GQDs possess excellent solubility in water. The UV–vis and PL spectra were also used to characterize the formation of GQDs (Fig. S2B). The synthesized GQDs showed a broad UV–vis absorption band at 362 nm (curve a), and the PL spectrum of the GQDs solution showed a strong emission peak at 470 nm under the excitation at 320 nm (curve b) and the maximum emission wavelength was not changed with different batches of samples. 3.2. Characterization of the ITO Electrodes modification EIS, as an effective method for probing the interfacial properties of modified electrode, was employed to confirm the fabrication process of the ITO electrode modification using [Fe (CN) 6] 3 /4 as redox probe. Fig. 2A shows the nyquist plots of EIS observed upon the stepwise fabrication processes. The bare ITO electrode given a very small semicircular domain (curve a) and the resistances (Ret) value of bare ITO electrode was about 34.15 Ω. Because of the GQDs can promote electron-transfer processes, upon the GQDs assembled on electrode surface, the Ret value decrease to 26.42 Ω (curve b). After the substrate peptide and BSA were self-assembled onto the electrode, the Ret increased to 55.89 Ω (curve c). This is ascribed to the poor conductivity of substrate peptide and BSA. Due to the electrostatic repulsion between the negative charged phosphorylation sites and redox probe, the Ret was increased to 58.89 Ω after the phosphorylation by PKA (curve d). Subsequently,
Fig. 1. (A) The TEM image of GQDs. (B) AFM image and the corresponding height profiles of GQDs.
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Fig. 2. (A) The EIS of bare ITO electrode (a) GQDs modified electrode, (b) peptide and BSA modified electrode, (c) phosphorylated peptide modified electrode before, (d) and after, (e) the assembly of pDNA@DNAzyme-AuNPs in 0.01 M PBS solution containing 5 mM [Fe(CN)6]3 /4 electroactive probes within the range from 0.1 Hz to 100 KHz, the amplitude of the applied sine wave potential was 5 mV and quiet time was 2 s. (B) ECL-potential curves of peptide/GQDs modified ITO (a) peptide phosphorylated, (b) or unphosphorylated, (c) by PKA after treatment with pDNA@DNAzyme-AuNPs mediated by Zr4 þ and the Zr4 þ mediated phosphorylated DNAzyme, (d) or phosphorylated linker DNA modified AuNPs and (e) assembled phosphorylated peptide modified electrode. ECL conditions: the voltage of PMT was 800 V and amplifier gain was set at 4. Scan rate: 100 mV/s.
when the pDNA@DNAzyme-AuNPs was assembled onto the electrode by the linkage of Zr4 þ , the Ret emerged a further increase corresponding to the large amount of negatively charged DNA linked on the AuNPs (curve e), and the Ret value was 84.88 Ω. The EIS results proved that the modification process of the electrode was achieved successfully. The AFM image of the resulting pDNA@DNAzyme-AuNPs bound to the phosphorylated peptide modified electrode was shown in Fig. S3. It is noted that a number of pDNA@DNAzymeAuNPs with height of 15 nm were uniformly distributed on the surface of the electrode, and no aggregation was found (Fig. S3A). However, only little pDNA@DNAzyme-AuNPs were observed on the surface of the control electrode which was without PKA treatment (Fig. S3B). This is probably due to nonspecific absorption of pDNA@DNAzyme-AuNPs. As the result of AFM, the pDNA@DNAzyme-AuNPs could be successfully attached to the phosphorylated peptide modified electrode. The ECL behaviors of the GQDs and mechanism of the proposed ECL assay was shown in supporting information (Fig. S4). 3.3. Feasibility investigation of the ECL assay After confirmation the modification of the electrode and the conjugation of pDNA@DNAzyme-AuNPs onto the phosphorylated peptide, the feasibility of this ECL assay was then investigated by the ECL emission. As it is shown in Fig. 2B, the peptide/GQDs modified electrode exhibited a strong ECL signal with 1 mM H2O2 as coreactant at the applied potential range of 0 to 2.0 V (curve a). In the presence of PKA, the ECL intensity significantly decreased upon phosphorylation by PKA with the linkage of pDNA@DNAzyme-AuNPs by Zr4 þ (curve b). In comparison, a weak decrease in ECL intensity can be observed in the absence of PKA (curve c). It was the negative control measurements. The results showed that the pDNA@DNAzyme-AuNPs could effectively quench the ECL intensity of GQDs. To further confirm the efficient double-quenching effect of the pDNA@DNAzyme-AuNPs, the phosphorylated peptide modified electrode treated with only G-quadruplex–hemin DNAzyme (curve d) and AuNPs just assembled with phosphorylated linker DNA (curve e) were also investigated, respectively. It was demonstrated that both the DNAzyme and AuNPs can quench the ECL intensity of GQDs. Clearly, with integrating the DNAzyme and AuNPs together, an enhanced quenched ECL signal was obtained (curve b), indicating that the ECL intensity of GQDs could be
quenched by pDNA@DNAzyme-AuNPs more effectively. In addition, some control experiments to test for false positive in the absence of Zr4 þ and pDNA@DNAzyme-AuNPs and in the absence of pDNA@DNAzyme-AuNPs have been applied in the supporting information. From the results of Fig. S5, We found that the ECL intensity has little decrease in these control experiments by comparing with peptide/GQDs modified electrode. These results further showed that the sensitive ECL assay for PKA activity and inhibition could be carried out. 3.4. PKA activity detection Under the optimized conditions (60 min of phosphorylation time, 1 mM Zr4 þ , 80 μM ATP and pH 8) (Fig. S6), the relationship between the ECL intensity and PKA activity were investigated. From the results of Fig. 3A, the ECL intensity of GQDs was sensitive to the change of the PKA activity with enzymatic unit from 0 to 20 U mL 1. As the enzymatic unit of PKA increased, the ECL intensity decreased sharply and then slowed down at higher enzymatic unit. The results showed that the ECL decrement ΔI (ΔI¼ I0 I, where I0 and I were ECL intensity of the peptide/GQDs modified ITO electrode before (I0) and after (I) phosphorylated by PKA and then treatment with pDNA@DNAzyme-AuNPs, respectively) was logarithmically related to the PKA enzymatic unit in the range from 0.05 U mL 1 to 5 U mL 1 with a correlation coefficient of 0.985 (the inset in Fig. 3B). The linear regression equation was calculated as ΔI¼ 534 log [PKA] þ789 and the detection limit (LOD) of PKA was as low as 0.04 U mL 1 at 3 times signal-to-noise. Compared with the previous reporters (Liu et al., 2014b; Xu et al., 2009), the ECL assay have a lower detection limit, indicating that the developed ECL assay can be used to highly sensitive PKA activity detection with a wide concentration range. 3.5. Influence of inhibitor on PKA activity Ellagic acid, as a cell-permeable and potent antioxidant (Cozza et al., 2006), was selected to further test the potential applications of the ECL assay in protein kinase inhibitor detection. Fig. 4A shows the inhibitory results of ellagic acid. As expected, the ΔI decreased gradually with increasing the concentration of ellagic acid owning to the inhibition of PKA activity and low levels of peptide phosphorylation. As a result, IC50 (the half maximal inhibitory concentration) was calculate to be 4.22 μM, which was
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Fig. 3. (A) ECL signals of the ECL assay incubated with different activity of PKA (0, 0.05, 0.1, 0.5, 1, 2, 5, 10, 20 U mL 1). (B) Current intensity as a function of PKA activities. The inset is the linear relationship between current intensity and the activity of PKA Linear relationship between ECL decrement ΔI (I0 I) and the logarithm of PKA activity, three measurements for each point.
well comparable with the previous literature (Xu et al., 2010). The result indicated that the developed ECL assay has an excellent potential in screening the activity of the kinase inhibitor. 3.6. Selectivity and reproducibility of the ECL assay In this ECL assay, the selectivity was evaluated by incubating the same concentration of PKA, CK2 and T4 PNK, respectively. As displayed in Fig. S7, the ECL assay showed a good selectivity for PKA over other kinases. These results indicated that the developed ECL assay exhibit good specificity. The reproducibility of the ECL assay was also used to examine PKA activity with intraassay and interassay. The relative standard deviation (RSD) of intraassay, examined by testing the same electrode of five dependent measurements, was 4.95%. While the interassay estimated for five reduplicate measurements on different electrodes was 9.38%. The result indicated that the ECL assay have an acceptable fabrication reproducibility in PKA activity detection. 3.7. PKA activity assay in complex biological samples To further investigate the proposed ECL assay in complex biological samples for clinic application, the phosphorylation reaction solution was prepared by adding a desired amount of PKA in FBS rather than buffer solution. As shown in Table S1, the relative
errors of two different enzymatic unit of PKA were 8.4% and 11.07%, respectively, suggesting that the as-proposed ECL assay was utilizable for the detection of PKA activity in FBS fluids. In addition, the cell lysates samples were still used to kinase activity assay. In this case, three cell lysates from HEK293 normal cell line and two cancer cell lines of lung (A549) and breast (MCF-7) were investigated in this work. As displayed in Fig. 4B, the MCF-7 cell line emerged the higher level of PKA than the other cell lines. Moreover, the PKA expression levels of cancer cells are higher than normal cell (HEK293), which was compared with the previous result (Wang et al., 2014).
4. Conclusion In conclusion, with the confluence of G-quadruplex–hemin DNAzyme and AuNPs, a highly sensitive ECL assay was developed for the PKA activity and inhibition detection. In this method, the pDNA@DNAzyme-AuNPs can highly quench the ECL intensity of GQDs as a joint result of the G-quadruplex–hemin DNAzyme catalytic reaction and ECL-RET analytical technique. Based on the efficient double-quenching effect, the proposed ECL assay showed high sensitivity and good selectivity for PKA activity assay with a LOD of 0.04 U mL 1. Furthermore, this ECL assay can also be taken as a general platform to detect different protein kinase by simply changing specific substrate peptide sequences. Moreover, the
Fig. 4. (A) Inhibition of the activity of PKA by ellagic acid. The concentrations of ellagic acid were 0, 1, 2, 4, 6, 10, and 15 μM. (B) Detection of PKA activities from BEK293, A549, and MCF-7 cell lysates. All reactions were carried out at optimized condition except for the addition of different concentrations of inhibitor or cell lysates.
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application of inhibition assay and kinase detection in serum samples and cell lysates were also indicated that the proposed ECL assay has the promise for potential applications in protein kinaserelated biochemical fundamental research and clinical diagnosis.
Acknowledgment This work was supported in part by the Key Project of National Natural Science Foundation of China (Grants 21175039, 21322509, 21305035, 21190044, and 21221003), Research Fund for the Doctoral Program of Higher Education of China (Grant 20110161110016) and the project supported by Hunan Provincial Natural Science Foundation of China (Grant 14JJ3066) and Hunan Provincial Science and Technology Plan of China (2012TT1003).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.03.026.
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