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Phosphorylation-Directed Assembly of Single Quantum Dot-Based Nanosensor for Protein Kinase Assay Li-juan Wang, Yong Yang, and Chun-yang Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac504358q • Publication Date (Web): 31 Mar 2015 Downloaded from http://pubs.acs.org on April 8, 2015

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Analytical Chemistry

Phosphorylation-Directed Assembly of Single Quantum Dot-Based Nanosensor for Protein Kinase Assay

Li-juan Wang,‡ Yong Yang,‡ and Chun-yang Zhang* Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Guangdong 518055, China E-mail: [email protected]. Fax: +86-755-8639 2299.

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ABSTRACT Protein kinases play crucial roles in intracellular signal transduction and metabolic pathways, and the monitoring of protein kinase activity is essential to the understanding of fundamental biochemical processes and the clinical diagnosis. Here, we demonstrate the phosphorylation-directed assembly of single quantum dot (QD)-based nanosensor for sensitive detection of cAMP-dependent protein kinase (PKA). This assay involves (1) the PKA-directed simultaneous phosphorylation and biotinylation of Cy5labeled substrate peptides, (2) the assembly of phosphorylated and biotinylated peptides onto the surface of QD, and (3) the illumination of Cy5 by means of fluorescence resonance energy transfer (FRET) between the QD and Cy5. With an ATP analogue, γ-biotin-ATP, as the phosphoryl donor, the PKAcatalyzed phosphorylation reaction incorporates the biotin-conjugated phosphate group into the substrate peptides to form the biotinylated peptides. The biotin entity subsequently drives the assembly of peptides onto the surface of streptavidin-functionalized QD to form the sandwiched Cy5-peptide-QD nanostructure, enabling the occurrence of FRET between the QD and Cy5. The FRET signal can be easily recorded by either the conventional fluorescence spectrometer or the total internal reflection fluorescence (TIRF) microscope. In contrast, the absence of PKA cannot lead to the formation of Cy5-peptide-QD complex and no Cy5 signal can be detected. This protein kinase-actuated FRET assay is straightforward without the involvement of either washing or separation steps, and has a significant advantage of high sensitivity with a detection limit of 9.3 × 10-6 U/µL. Moreover, this method can be used to estimate the half-maximal inhibitory concentration (IC50) value of PKA inhibitor H-89 and to monitor Forskolin (Fsk) /3-isobutyl-1methylxanthine (IBMX)-triggered activation of PKA in cell lysates, thus holding great potential for further applications in protein kinase-related biological researches and drug discovery.

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INTRODUCTION The human kinome is the largest single family of enzymes with more than 500 members1 and may modify nearly one third of all human proteins.2 By transferring one or more γ-phosphate groups to specific amino acids (i.e., serine, threonine and tyrosine residues) in target proteins, the protein kinases modulate the activity, stability, conformation, interaction and the subcellular localization of substrate proteins,2,3 and play pivotal roles in cellular signal transduction pathway,4 immune response,5 learning and memory,6 brain development,7 cell-cycle regulation,8 and the maintenance of energy homeostasis.9 Nevertheless, the deregulation of protein kinase activities and the mutations of kinase proteins may lead to a number of human

diseases

including

cancers,8

diabetic

complications,10

neurological

disorders

and

neurodegenerative diseases7 as well as heart dysfunctions.11 The protein kinases are currently the second most important pharmaceutical drug targets,12 and several protein kinase inhibitors have been approved by FDA for the treatment of cancers.13 In addition, a large number of small-molecule inhibitors are undergoing clinical trials.13, 14 Given the crucial roles of kinases in normal physiology and drug discovery, the development of a universal and sensitive method for protein kinase assay is essential to the unraveling of complex signaling cascades and the screening of kinase-targeted inhibitors. So far, a variety of approaches have been developed to determine the activities of protein kinases. The radiometric assay relies on the transfer of radioactive phosphate group from [γ-32P]ATP to substrate proteins/peptides15 and is the standard method for protein kinase assay. This method is suitable for all kinds of substrates and can evaluate the enzyme activity directly, but it must be performed with caution because the radioactive isotopes may pose hazard to human health.16 The immunoassay employs phosphorylation-specific antibodies to sense phosphosubstrates generated from a kinase reaction,17 and can be accomplished by various analytical techniques such as fluorescence polarization,18 chemifluorescence,19 peptide array,20 and enzyme-linked immunosorbent assay (ELISA),21 but it does not work well for protein serine/threonine kinases.16 The matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) is frequently used for mapping the phosphorylation sites. By calculating the ratio of peak areas between the phosphorylated and the nonphosphorylated peptides, the

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activity of individual protein kinase can be determined indirectly.22 However, the intrinsic properties of phosphorylation reaction, such as the low stoichiometry of phosphopeptides relative to the unphosphorylated counterparts,23 the incorporation of negatively charged phosphate moiety to the substrate peptides,22 and the presence of salts and detergents in kinase reaction,24 may cause the underestimation of the signals of phosphorylated peptides. The artificial modification of substrate peptides with stable isotope tags and MS-sensitive enhancement reagent25 or separating phosphopeptides prior to MS analysis23 may solve these issues. Nevertheless, these additional treatments may increase the total analysis time. In addition, the protein kinase activity can be measured by the immobilized metal affinity chromatography (IMAC)/metal oxide affinity chromatography (MOAC) method due to the high affinity of phosphoryl groups toward some metal ions and metal oxides (e.g., Fe3+,26 Ga3+,26 ZrO2,27 and TiO223). The combination of IMAC/MOAC method with other analytical techniques such as MS analysis,23 flow cytometry28 and magnetic separation strategy29 has been applied for the identification of specific phosphorylation sites, the evaluation of kinase activity, and the screening of protein kinase inhibitors. However, the nonspecific absorption of acidic residue-rich nonphosphorylated peptides and the poor binding affinity of basophilic phosphopeptides may yield some biased or even false results.16,30 To improve the detection specificity, the chemical modification strategy with the involvement of βelimination and Michael addition have been developed to detect phospho-Ser/Thr residues.31 Even though the converted phosphopeptides can be selectively enriched and isolated from the complex mixtures,32 this strategy is not suitable for protein tyrosine kinases. Moreover, the protein kinase activity in living cells can be spatially and temporally mapped by genetically encoded FRET-based reporters,33 and the real-time monitoring of protein kinase activity can be achieved by the fluorescent peptide-based chemosensors.34 However, the resultant modest fluorescence changes may lead to poor sensitivity.28,29 Therefore, the development of sensitive, easy-manipulated and versatile methods for protein kinase assay still remains a great challenge. Recently, the semiconductor quantum dots (QDs) have attracted more and more attention in the biomedical fields owing to their unique optical and electronic characteristics, such as high quantum yields

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(QYs), narrow and size-tunable emission spectra along with broad excitation band, long fluorescence lifetime, and large surface area for chemical modification.35 The QDs are also considerably brighter and more resistant to photobleaching than the conventional fluorophores (e.g., fluorescent protein and organic dyes), and have been applied for multiplex analysis,36 optical barcoding,37 and multicolor imaging.38 In addition, single QD may act as a multifunctional nanoscaffold to conjugate various sensing molecules, enabling the development of QD-based biosensors with different sensing mechanisms (e.g., FRET and electron transfer).39-42 Here, we take advantage of the prominent features of QDs to develop a single QDbased sensing platform for protein kinase assay. With an ATP analogue, γ-biotin-ATP, as the phosphoryl donor,43 the protein kinase-catalyzed phosphorylation reaction leads to the biotinylation of Cy5-labed substrate peptides. The resultant substrate peptide may assemble onto the QD surface through specific biotin-streptavidin interaction to form a sandwiched QD-peptide-Cy5 nanostructure, enabling the occurrence of FRET between the QD and Cy5. By measuring the variance of Cy5 counts, the PKA activity can be quantitatively evaluated with high sensitivity and excellent selectivity. Moreover, this method can be further applied to evaluate the IC50 value of PKA inhibitor and to detect the cellular PKA activity.

EXPERIMENTAL SECTION Chemicals and Materials. The cAMP-dependent protein kinase (PKA) was purchased from New England Biolabs, Inc. (MA, USA). The RAC alpha serine/threonine protein kinase (AKT1), forskolin (Fsk),

3-isobutyl-1-methylxanthine

isoquinolinesulfonamide

dihydrochloride

(IBMX), (H-89),

N-[2-(p-Bromocinnamylamino)ethyl]-5magnesium

chloride

(MgCl2),

ethylenediaminetetraacetic acid (EDTA), trizma hydrochloride (pH 7.5 and pH 8.0, respectively), sodium chloride (NaCl), DL-Dithiothreitol (DTT), glycerol, ammonium sulfate (NH4)2SO4), glycine, potassium hydroxide (KOH), bovine serum albumin (BSA), trolox, glucose oxidase, D-glucose, and catalase were obtained from Sigma-Aldrich Co. LLC. (USA). The γ-[6-Aminohexyl]-ATP-biotin (γ-biotin-ATP) was purchased from Jena Bioscience (Jena, Germany). The Cy5-modified substrate peptide of PKA (Cy5-

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CLRRASLG) and the Cy5/biotin-modified peptide (Cy5-CLRRASLG-biotin) were synthesized and purified by Chinese Peptide Company (Hangzhou, China). The streptavidin-coated CdSe/ZnS QDs with the maximum emission at 605 nm (Qdot 605 ITK) were obtained from Invitrogen Corporation (CA, USA). Other reagents were of analytical grade and used just as received without further purification. The ultrapure water used in the experiments was prepared by a Millipore filtration system (Millipore, Milford, MA, USA). In vitro Kinase Assay, Assembly of QD-Peptide-Cy5 Nanostructure and Fluorescence Measurement. The PKA-catalyzed phosphorylation reaction involved three consecutive steps. Firstly, the stock solution of PKA was diluted with the dilution buffer (20 mM Tris–HCl, 50 mM NaCl, 2 mM DTT, and 1 mM EDTA, 50% glycerol, pH 7.5) to prepare the diluted PKA. Secondly, the diluted PKA at different concentrations was added into 100 µL of reaction buffer containing 50 mM Tris-HCl (pH 7.5), 1.5 µM Cy5-CLRRASLG, 3 µM γ-biotin-ATP, and 20 mM MgCl2 to prepare the phosphorylation reaction mixture. Thirdly, the mixture was incubated at 30 °C for 1 h to actuate the phosphorylation reaction. Then 20 µL of reaction mixture and certain amount of 0.2 µM streptavidin-coated CdSe/ZnS QDs were added into 100 µL of incubation buffer (1 M Tris-HCl, 100 mM (NH4)2SO4, and 30 mM MgCl2, pH 8.0), and incubated at room temperature for 10 min to allow the biotinylated substrate peptides binding to the streptavidin-coated CdSe/ZnS QDs. The fluorescence signals of the mixture were measured by an F-4600 spectrometer (Hitachi, Japan) with an excitation wavelength of 488 nm. The emission spectra were scanned from 550 nm to 750 nm and the emission intensities at 605 nm (the maximum emission of 605QDs) and 670 nm (the maximum emission of Cy5) were used for data analysis. TIRF Imaging and Data Analysis. For TIRF imaging, the imaging buffer (67 mM glycine-KOH (pH 9.4), 50 µg/mL BSA, 2.5 mM MgCl2, 1 mg/mL Trolox) and the oxygen-scavenging buffer (1 mg/mL glucose oxidase, 0.4 % (w/v) D-glucose, and 0.04 % mg/mL catalase) were freshly prepared before use. The mixture of QDs and the phosphorylation products was diluted 1200-fold with the imaging buffer prior to the fluorescence imaging. The oxygen-scavenging buffer was also added to minimize the photobleaching of Cy5. The QDs were excited by a sapphire 488 nm laser (50 mW, Coherent, USA) via

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the total internal reflection, and the fluorescence signals of QDs and Cy5 were collected by a UAPON 100× OTIRF objective (1.49 NA, Olympus) that coupled to an inverted microscope (IX71, Olympus, Japan). The fluorescence signals of QDs and Cy5 were separated by Optosplit II image splitter (Cairn Research) before imaging onto two halves of an Andor iXon DU897D EMCCD. The fluorescence images were obtained by the Micro-manager 1.4 software with an exposure time of 100 ms, and 7 frame of images derived from 7 different locations were acquired for every sample. The image J software (version 1.46) was used for data analysis. Generally, a central region of 200 × 400 pixels in the EMCCD image was used for fluorescence molecule counting. The number of Cy5 was obtained by using “analyze particles” function of image J with the particle size set at 2-12 pixels. At the same time, the numbers of QD were measured and used as an internal control to correct the number of Cy5. The number of Cy5 counts was calculated according to eq. 1. Nnet = NPKA− Nnegative

(1)

Where Nnet is the net number of Cy5, Nnegative is the corrected number of Cy5 in the absence of PKA, and NPKA is the corrected number of Cy5 in the presence of PKA. Inhibition of PKA Activity by H-89. For PKA inhibition assay, different concentrations of H-89 were added into the reaction mixture with a fixed concentration of PKA (0.1 U/µL). The corresponding Cy5 counts were measured as described above. The relative activity (RA) of PKA was calculated according to eq. 2. RA =

Ni − No ×100% Nt − No

(2)

Where No, Nt, and Ni represent the corrected number of Cy5 in the absence of PKA, in the presence of 0.1 U/µL PKA, and in the presence of 0.1 U/µL PKA and H-89, respectively. The RA was plotted against the concentrations of H-89, and the IC50 value of H-89 was calculated from the fitting curve. Cell Culture and Drug Treatment. HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum in a humidified chamber containing 5% CO2 at 37 °C. For the experiments with the treatment of forskolin (Fsk)/IBMX, the cells were cultured in serum-free

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medium for additional 4 h before the addition of Fsk and IBMX, and then the cells were incubated with Fsk and IBMX for 30 min to activate the cellular PKA. The cells treated with equal volume of DMSO were used as the negative control (the unstimulated sample). For H-89-induced inhibition assay, Fsk, IBMX, and H-89 were added to the cultured cells simultaneously, followed by the incubation for 30 min. Then the cells were washed with PBS buffer (pH 7.4) for three times before collection. The proteins were extracted with a commercial protein extraction kit (BSP022, Sangon Biotech, Shanghai, China), and the protein concentration was quantified by a modified Lowry protein assay kit (SK4041, Sangon Biotech, Shanghai, China). For PKA activity assay, the concentration of cell lysate was fixed at 10 µg/mL.

RESULTS AND DISCUSSION Principle of PKA Assay. The proposed method for PKA assay involves three consecutive steps including (1) the phosphorylation of Cy5-labeled substrate peptides by biotinylated γ-ATP (γ-biotin-ATP), (2) the formation of QD-peptide-Cy5 nanostructure by the self-assembly of phosphorylated substrate peptides onto the surface of QDs, and (3) the detection of kinase activity by the means of FRET between the QD and Cy5 (Scheme 1). In the presence of protein kinase, the protein kinase can catalyze the transfer of biotinylated γ-phosphate from γ-biotin-ATP to serine hydroxyl group of the substrate peptides,44 generating the biotinylated peptides. Then the biotin entity may drive the self-assembly of Cy5conjugated peptides onto the surface of QDs through specific biotin-streptavidin interaction to form the QD-peptide-Cy5 nanostructures, bringing QD and Cy5 in spatial proximity for the occurrence of fluorescence resonance energy transfer. The activity of PKA can be quantitatively measured by evaluating the Cy5 counts. In contrast, the substrate peptides can be neither phosphorylated nor biotinylated in the absence of protein kinase. Consequently, the Cy5-labeled peptides cannot assemble onto the surface of QDs and no FRET can happen. It should be noted that a single QD can assemble multiple Cy5-labeled substrate peptides to form the single-donor/multiple-acceptor configuration with an improved FRET efficiency.45 As a proof of concept, we used the cAMP-dependent protein kinase as a model and the Cy5labelled peptide (CLRRASLG) as the substrate (Scheme 1).

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Scheme 1. Schematic illustration of protein kinase assay. With γ-biotin-ATP as the phosphoryl donor, the PKA-catalyzed phosphorylation reaction incorporates the biotin entity into Cy5-labeled peptide substrate, which can assemble onto the surface of QDs through specific biotin-streptavidin interaction to form the sandwiched QD-peptide-Cy5 nanostructure. The subsequent FRET between the QD and Cy5 enables the detection of Cy5 signal. The protein kinase activity can be evaluated by the measurement of Cy5 signal. Inset shows the structure of γ-biotin-ATP.

The efficient FRET require two key factors to be satisfied simultaneously, i.e., (1) the spectral overlap between the acceptor excitation and the donor emission, and (2) the effective separation distance between the donor and the acceptor.45 In this research, we chose the 605 nm-emitting QD (605QD) as the energy donor and cyan dye Cy5 as the energy acceptor. This 605QD/Cy5 pair shows the negligible emission cross-talk but harbors significant spectral overlap between the QD emission and the Cy5 excitation.46 To verify the FRET between the 605QD and Cy5, we employed the 605QDs and the product of phosphorylation reaction (i.e., the biotinylated Cy5-labelled substrate peptides) to fabricate the sandwiched QD-peptide-Cy5 nanostructures. As shown in Figure 1A, with the increase of PKA concentration, the 605QD fluorescence intensity decreases, accompanied by the corresponding increase of Cy5 fluorescence intensity. Based on Scheme 1, the more the PKA concentration, the more the biotinylated Cy5-labelled substrate peptides assembled onto the single QD, and consequently the more the ratio of Cy5-peptide to QD. As a result, the FRET efficiency improves with the increasing ratio of Cy5-

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peptide to QD (see Supporting Information, Figure S1). Notably, the Cy5 fluorescence intensity improves gradually with the increasing ratio of Cy5-peptide to QD, but decreases beyond the ratio of 24 (see Supporting Information, Figure S1) due to the inner-filter effect caused by the high local concentration of Cy5.47 In addition, the average QD-Cy5 separation distance is calculated to be 102.7 ± 4.5 Å, within the efficient range of FRET (2R0 = 138.8 Å).46 These results demonstrate that efficient FRET can occur between the 605QD and Cy5 in the QD-peptide-Cy5 nanostructure formed by PKA-triggered phosphorylation reaction. It should be noted that the obtained Cy5 fluorescence signal results from FRET rather than the direct excitation of Cy5 acceptor by 488 nm laser (Figure 1A, Cy5 only). Moreover, the Cy5 fluorescence intensity shows a linear correlation with the PKA concentration in the range from 0.0001 U/µL to 0.1 U/µL (R2 = 0.9838) (Figure 1B), suggesting that the proposed method can be used for kinase assay.

Figure 1. (A) Variance of 605QD and Cy5 fluorescence as a function of PKA concentration (λex = 488 nm). (B) Calibration curve between Cy5 fluorescence intensity and the PKA concentration. Error bars show the standard deviation of three independent experiments.

Measurement of PKA Activity with TIRF Microscope. For TIRF-based fluorescence imaging, only the fluorescent molecules within the evanescent wave field (500), 1 making the specific measurement of kinase activity remain a great challenge. To demonstrate the specificity of the proposed method for PKA assay, we measured the fluorescence of QD and Cy5 in the presence of PKA, and compared them with those obtained in the absence of protein kinase (the control) or in the presence of a different kinase Akt1.29 As shown in Figure 4, in the presence of Akt1 (Figure 4A, blue color), neither distinct Cy5 fluorescence signal nor significant quenching of 605QD fluorescence is observed, suggesting no efficient FRET between 605QD and Cy5. In contrast, in the presence of PKA (Figure 4A, red color), both remarkable Cy5 fluorescence signal and significant quenching of 605QD fluorescence are observed, indicating the efficient FRET between 605QD and Cy5. These results are consistent with those obtained by TIRF-based counting measurement (see Supporting Information, Figure S6). As shown in Figure 4B, the Cy5 counts deriving from PKA (Figure 4B, red color) is much more than those obtained from Akt1 (Figure 4B, blue color). The failure of Akt1 to produce Cy5 signal may attribute to the fact that Akt1 cannot recognize the PKA-specific substrate.29 These results clearly demonstrate the high specificity of the proposed method for PKA assay.

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Figure 4. Specificity of PKA assay. (A) Emission spectra of 605QD and Cy5 in response to 0.1 U/µL Akt1 (blue color) and 0.1 U/µL PKA (red color). The reaction without protein kinase was used as the control (black color). (B) The Cy5 counts obtained in the presence of 0.1 U/µL Akt1 (blue color), 0.1 U/µL PKA (red color), and in the absence of protein kinase (black color). Error bars show the standard deviation of three independent experiments.

PKA Inhibitor Assay. Since PKA may serve as an extracellular tumor biomarker,52 the identification of effective PKA inhibitors is of great importance for PKA-targeted cancer therapy.53 To demonstrate the feasibility of the proposed method for PKA inhibitor assay, we employed H-89 as the model inhibitor (Figure 5A). The H-89 is a kind of H-series inhibitor that can block PKA activity by competitively inhibiting the ATP site of catalytic subunit.54 We measured the variance of Cy5 counts in response to different-concentration H-89 with a fixed amount of PKA (see Supporting Information, Figure S7). As shown in Figure 5B, the relative activity of PKA decreases gradually with the increasing concentration of H-89. The IC50 value of H-89 is calculated to be 75.26 nM, consistent with the reported IC50 value of 98 nM.55 This result demonstrates that the proposed method can be used for kinase inhibitor assay.

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Figure 5. (A) Chemical structure of H-89. (B) Relationship between the relative activity of PKA and the concentration of H-89. Error bars show the standard deviation of three experiments.

Measurement of PKA Activity in Cell Lysates. The activity of protein kinases inside the cells can be modulated by various internal and external stimuli.3 The treatment of cells with Fsk (a specific activator of adenylyl cyclase) and IBMX (an effective inhibitor of phosphodiesterase) can increase the intracellular cAMP level, which may in turn activate the cAMP-dependent protein kinases.29 To verify the feasibility of the proposed method for real sample analysis, we measured the cellular PKA activity induced by Fsk and IBMX using both fluorescence spectrometer (Figure 6A) and TIRF microscope (see Supporting Information, Figure S8). For the fluorescence spectrometer-based ensemble measurement, even though the treatment of Fsk and IBMX can induce significant QD quenching, the corresponding increase of Cy5 fluorescence signal as a result of FRET is too small to be distinguished among different conditions (Figure 6A). Therefore, in the ensemble measurement we used the quenching of 605QD fluorescence signal for analysis. For the unstimulated HeLa cells, no significant QD quenching (Figure 6A, green color) is observed relative to the control group without cell lysate (Figure 6A, olive color), implying the low PKA activity under normal physiological condition.28 In contrast, the treatment of HeLa cells with Fsk and IBMX induces remarkable quenching of 605QD fluorescence signal (Figure 6A, blue color) as compared with unstimulated HeLa cells (Figure 6A, green color), indicating the activation of PKA in

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Fsk/IBMX-stimulated cells.28 This conclusion is further confirmed by the continuous quenching of 605QD fluorescence signal with the increasing concentration of Fsk/IBMX (Figure 6A, red color) and the recovery of 605QD fluorescence signal by the addition of H-89 inhibitor (Figure 6A, black color). The results obtained by fluorescence spectroscopy (Figure 6A) are consistent with those obtained by the TIRF-based counting measurement (Figure 6B). The Cy5 counts obtained from the unstimulated HeLa cells (Figure 6B, green color) is a little more than that obtained from the control group (Figure 6B, olive color), consistent with low level of PKA activity under normal physiological condition.28 In contrast, the treatment of HeLa cells with Fsk/IBMX induces significant increase of Cy5 counts (Figure 6B, blue color and red color) due to the activation of PKA.28 The higher the concentration of Fsk/IBMX, the more the Cy5 counts obtained (Figure 6B, red color versus blue color). Moreover, the addition of H-89 inhibitor induces the decrease of Cy5 counts due to the inhibition of PKA activity by H-89 (Figure 6B, black color).54 For QD-based FRET assay with the QDs as the donor, the analysis of the quenching of QD fluorescence signal is a signal-off measurement which tends to induce false positivity, whereas the analysis of the increase of Cy5 fluorescence signal is a signal-on measurement with more accuracy. Our results demonstrate that the measurement of Cy5 counts by TIRF microscope (Figure 6B) is more suitable for cellular PKA assay than the ensemble measurement by fluorescence spectrometer. In addition, the TIRF-based counting measurement has significant advantages of high signal-to-noise ratio, high sensitivity, and low sample consumption as compared with the ensemble measurement.48,51,56

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Figure 6. Detection of endogenous PKA activity. (A) Emission spectra of 605QD and Cy5 in response to the control group without cell lysate (olive color), the unstimulated cell lysate (green color), 10 µM Fsk/20 µM IBMX-stimulated cell lysate (blue color), 50 µM Fsk/100 µM IBMX-stimulated cell lysate (red color), 50 µM Fsk/100 µM IBMX-stimulated cell lysate with the addition of 10 µM H-89 (black color). (B) The Cy5 counts obtained via the TIRF-based counting measurement as shown in A. The total protein concentration of HeLa cell lysates used in each experiment is 10 µg/mL. Error bars show the standard deviation of three experiments.

CONCLUSIONS In summary, we have developed a single QD-based FRET sensor for sensitive detection of protein kinase activity. The phosphorylation-directed assembly of QD-peptide-Cy5 nanostructure and the subsequent FRET from QD to Cy5 can translate the PKA activity into the visible fluorescence signal, which can be easily detected by TIRF imaging. The assembly of multiple Cy5-labeled peptides onto a single QD in combination with the high signal-to-noise ratio of TIRF imaging enables the measurement of PKA activity with an extremely low detection limit of 9.3 × 10-6 U/µL. Notably, this single QD-based FRET sensor has several distinct advantages: (1) the detection of FRET signal enables the assay to be carried out in a homogenous format without the involvement of washing and separation steps, making the assay

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much simple and easy manipulated; (2) the detection of Cy5 signal is a signal-on measurement, which can avoid the false positivity and increase the accuracy; (3) the TIRF-based imaging and particle counting techniques have a high signal-to-noise ratio, making the assay extremely sensitive; (4) this assay does not require the expensive antibodies for detection and consumes only a small amount of samples for imaging. Importantly, the proposed method can be used to characterize PKA inhibitors, and can be extended to detect other protein kinases by simply adopting appropriate substrate peptides, thus holding great potential for further applications in biological researches, clinic diagnosis and drug development.

ASSOCIATED CONTENT Supporting Information Supplementary Figures S1-S8. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author Tel.: +86 755 86392211. Fax: +86 755 86392299. E-mail: [email protected]. Author Contributions ‡

These authors contributed equally.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We gratefully acknowledge the financial supports from the National Basic Research Program 973 (Grant No. 2011CB933600), the Natural Science Foundation of China (Grant No. 21325523), the Award for the Hundred Talent Program of the Chinese Academy of Sciences, and the Funds for Shenzhen Engineering Laboratory of Single-molecule Detection and Instrument Development (Grant No. (2012) 433).

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Phosphorylation-directed assembly of a single quantum dot based nanosensor for protein kinase assay.

Protein kinases play crucial roles in intracellular signal transduction and metabolic pathways, and the monitoring of protein kinase activity is essen...
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