Article pubs.acs.org/ac
Upconversion Nanophosphor: An Efficient PhosphopeptidesRecognizing Matrix and Luminescence Resonance Energy Transfer Donor for Robust Detection of Protein Kinase Activity Chenghui Liu,*,†,‡ Lijuan Chang,‡ Honghong Wang,† Jie Bai,‡ Wei Ren,‡ and Zhengping Li†,‡ †
Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, Shaanxi Province P. R. China ‡ College of Chemistry and Environmental Science, Hebei University, Baoding 071002, Hebei Province P. R. China S Supporting Information *
ABSTRACT: Protein kinases play important regulatory roles in intracellular signal transduction pathways. The aberrant activities of protein kinases are closely associated with the development of various diseases, which necessitates the development of practical and sensitive assays for monitoring protein kinase activities as well as for screening of potential kinase-targeted drugs. We demonstrate here a robust luminescence resonance energy transfer (LRET)-based protein kinase assay by using NaYF4:Yb,Er, one of the most efficient upconversion nanophosphors (UCNPs), as an autofluorescence-free LRET donor and a tetramethylrhodamine (TAMRA)-labeled substrate peptide as the acceptor. Fascinatingly, besides acting as the LRET donor, NaYF4:Yb,Er UCNPs also serve as the phosphopeptide-recognizing matrix because the intrinsic rare earth ions of UCNPs can specifically capture the fluorescent phosphopeptides catalyzed by protein kinases over the unphosphorylated ones. Therefore, a sensitive and generic protein kinase assay is developed in an extremely simple mix-and-read format without any requirement of surface modification, substrate immobilization, separation, or washing steps, showing great potential in protein kinases-related clinical diagnosis and drug discovery. To the best of our knowledge, this is the first report by use of rare earth-doped UCNPs as both the phosphorecognizing and signal reporting elements for protein kinase analysis.
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harmful radioactive labels, specialized/sophisticated instrumentation, complicated sample handling, or tedious operations. For high throughput screening of potential PKs inhibitors, the fluorescence technique is highly preferred because of its high sensitivity and design flexibility and being compatible with various automatic equipment which allows for high-throughput signal readout.19 Fluorescence kinase assays typically use fluorescently labeled substrate peptides or phospho-recognizing antibodies to monitor the fluorescence changes upon kinaseinduced phosphorylation, which require labor-intense purification procedures to separate the phosphopeptides from unphosphorylated ones.20,21 In this regard, homogeneous fluorescence resonance energy transfer (FRET) assays show particular advantages because of their separation-free detection characteristic and robustness against matrix interferences.22−24 Notably, integrating fluorescent quantum dots (QDs) with the FRET technique offers an elegant way for detection of PKs activity. I. Willner’s group and M. Stevens’s group have separately established kinase assays by using QDs as the FRET
hosphorylation of cellular proteins catalyzed by protein kinases (PKs) plays vital roles in signal transduction pathways, which regulates many fundamental cellular processes including cell proliferation, growth, and apoptosis.1 It has been recognized that the dysfunction (hyperactivity in most cases) of some kinases is profoundly associated with the development of various human diseases including cancers.2−4 As such, PKs have emerged as promising molecular targets for the pharmaceutical intervention of several diseases by using kinase inhibitors to down-regulate the activities of related PKs. As a result, the screening of effective protein kinase inhibitors as potential anticancer drugs is undergoing intense study.5−7 In this regard, highly sensitive assays for accurate determination of the activity of specific PKs with low cost and simple operation are highly desired for PKs-related clinical diagnosis, drug discovery, as well as for further understanding the intracellular signal transduction pathways. Up until now, various approaches for the detection of PKs activities have been established, such as radioactive methods,8,9 electrochemical methods,10−13 nanomaterial-based assays,14,15 mass spectrometry-based assays,16,17 and magnetic resonance imaging (MRI).18 Although these methods all have their own advantages, most of them suffer from the drawbacks such as © 2014 American Chemical Society
Received: April 6, 2014 Accepted: May 16, 2014 Published: May 28, 2014 6095
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Figure 1. (a) Representative TEM image of the NaYF4:Yb,Er UCNPs; (b) XRD pattern of the UCNPs; (c) stability evaluation of NaYF4:Yb,Er colloidal solution (2 mg/mL) through time-course measurement of 545 nm emission. The inset is a photograph of 2 mg/mL NaYF4:Yb,Er colloidal solution; (d) investigation of the specific interaction between UCNPs and phosphopeptides. Unphosphorylated peptide (TAMRA-LRRASLG) (10 μM) and its phosphorylated counterpart (TAMRA-LRRApSLG) were, respectively, incubated with 2 mg/mL of UCNPs for 30 min, and then the UCNPs were centrifuged and the fluorescence signals of TAMRA remaining in the supernatants were recorded.
donors and organic fluorophore-labeled antiphosphoamino acid antibodies as the acceptors.25−27 Although these designs are smart, however, they heavily rely on the use of high-cost phosphoamino acid-recognition antibodies. Furthermore, these methods also show the potential drawbacks of inherent toxicity of heavy metal-containing QDs and specialized bioconjugation steps because substrate peptides should be preimmobilized on the surface of QDs, which may lead to undesired optical property changes. To overcome these limitations, very recently, we described an antibody-free PKs assay based on the metal ion-mediated FRET by using a fluorescent conjugated polymer as the donor, which enables highly sensitive detection of PKs activity with simple operations.28 However, it should be noted that the excitation of commonly used QDs, fluorescent conjugated polymer, as well as conventional dyes typically need the illumination of UV or short-wavelength visible light. The fluorescently labeled substrate peptides or antibodies as well as many other biological species can also be excited and strongly increase background fluorescence, which will greatly reduce the signal-to-background ratio and the detection sensitivity. Fortunately, the inherent shortcomings of QDs and organic dyes can be elegantly avoided by using rare earth (RE)-doped near-infrared (NIR)-to-visible upconversion nanophosphors (UCNPs).29−31 UCNPs are capable of emitting intense visible light by absorbing two or more NIR photons. They show distinct advantages over the traditional organic fluorophores and QDs, including large anti-Stokes shifts, high resistance to photobleaching and blinking, as well as low toxicity. More
fascinatingly, excitation of UCNPs via multiphoton absorption processes allows the use of NIR light, which would not be absorbed by conventional fluorescent dyes or biological species and thus effectively reduces autofluorescence and light scattering background. As a result, the signal-to-background ratio can be greatly enhanced in UCNPs-based biosensing systems, which allow high detection sensitivity. These advantages make UCNPs an ideal choice as the luminescence donor in designing luminescence resonance energy transfer (LRET) systems for biosensing and bioimaging.32−37 In light of these unique and excellent properties of RE-doped UCNPs, herein, we wish to report a robust LRET assay for the detection of PKs activity by using NaYF4:Yb,Er, one of the most efficient UCNPs known to date, as both the LRET donor and phosphopeptide-recognizing matrix because the intrinsic RE3+ ions of UCNPs can specifically capture the fluorescent phosphopeptides catalyzed by PKs, resulting in efficient upconversion LRET. Therefore, a sensitive and generic protein kinase assay is developed in an extremely simple mix-and-read manner without any requirement of surface modification of UCNPs, substrate immobilization, separation, or washing steps.
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EXPERIMENTAL SECTION Chemicals and Materials. All the peptides used in this study were synthesized by GL Biochem (Shanghai, China). cAMP-dependent protein kinase A (catalytic subunit, abbreviated as PKA) was purchased from New England Biolabs and AKT1 was obtained from Sigma-Aldrich. H-89 was supplied by EMD Biosciences and ATP was purchased from Sangon 6096
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Figure 2. Design principle of the UCNPs-based LRET assay for the detection of PKA activity.
(Shanghai, China). Ethylene imine polymer (EIP, Mw ∼1800) was obtained from Aladdin. Rare earth chlorides (RECl3·6H2O) were prepared by dissolving the corresponding rare earth oxides in hydrochloric acid and then evaporating the solvent at elevated temperature. All other reagents were of A.R. grade and used as received without further purification. Synthesis and Characterization of the EIP-Capped NaYF4:Yb,Er UCNPs. EIP-capped NaYF4:Yb,Er UCNPs were prepared by a modified solvothermal method.38 In a typical experiment, 1.2 mmol of RECl3 (Y3+/Yb3+/Er3+ = 80/18/2), 2.4 mmol of NaCl, and 0.3 g EIP were dissolved in 20 mL of ethylene glycol (EG) under agitation. Then 15 mL of EG solution containing 5 mmol of NH4F was added to the above mixture under vigorous stirring. The mixture was transferred into a 50 mL Teflon-linked autoclave and agitated thoroughly for another 5 min. Afterward, the autoclave was sealed and heated to 200 °C for 5 h. After cooling down to room temperature, the EIP-capped NaYF4:Yb,Er UCNPs were collected by centrifugation, washed with ethanol and distilled water, respectively, for several times, and dried at 60 °C. The morphology and size of NaYF4:Yb,Er UCNPs were characterized by transmission electron microscopy (TEM) using a JEM-2010 electron microscope (JEOL, Japan). Powder X-ray diffraction (XRD) patterns were recorded on a D8 Advance X-ray diffractometer (Bruker, Germany) with Cu Kα radiation. Upconversion photoluminescence (PL) spectra were recorded using a Fluorolog 3-211 fluorescence spectrophotometer (Horiba Jobin-Yivon, France) with an external 980 nm laser diode (Viasho, China) as the excitation source. All measurements were carried out at room temperature. Standard Procedures for Detection of PKA Activity and Inhibition Study. Typically, the PKA-catalyzed phosphorylation reaction was carried out in a 100-μL volume of Tris-HCl buffer (pH 7.5) containing 10 μM TAMRA-labeled PKA-specific peptide (TAMRA-LRRASLG), 100 μM ATP, 10 mM MgCl2, and a certain concentration of PKA at 30 °C for 1 h. Afterward, the reaction system was immediately mixed with an equal volume of NaYF4:Yb,Er colloidal solution (final concentration of 2 mg/mL) and incubated for 10 min at room temperature. Then the mixture was directly subject to upconversion luminescence measurement without any further treatment.
For the PKA inhibition study, the experimental procedures were similar to those stated above for detection of PKA activity, except for the preincubation of a fixed PKA concentration with varied concentrations of H-89 in the reaction system. In this study, the culture, drug stimulation and lysis of MCF7 breast carcinoma cells as well as the determination of total protein concentrations in the cell lysates were all performed according to the literature method.39 PKA activities in different MCF-7 cell lysates (10 μg/mL total protein concentration for each cell lysate sample) were detected according to the standard protocol described above.
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RESULTS AND DISCUSSION Characterization of the NaYF 4 :Yb,Er UCNPs. NaYF4:18%Yb,2%Er, one of the most efficient NIR-to-visible UCNPs, were synthesized by using a simple solvothermal method and the products were characterized by TEM. From the typical TEM image shown in Figure 1a, one can see that the as-prepared NaYF4:Yb,Er UCNPs display spherical shapes with an average diameter of ∼32 nm. X-ray powder diffraction (XRD) pattern of the UCNPs is shown in Figure 1b, where the peak positions and intensities can be well indexed to be cubic phase NaYF4:Yb,Er (JCPDS PDF 77-2042). Furthermore, the ethylene imine polymer (EIP)-capped NaYF4:Yb,Er UCNPs possess excellent dispersibility in water and aqueous buffers to form stable colloidal solutions (inset of Figure 1c), which is suitable for bioapplications. The NaYF4:Yb,Er UCNPs show intense upconversion photoluminescence (PL) at ∼545 nm (see the spectra below in Figure 3b) under the excitation of a 980 nm laser, which is assigned to the 4S3/2 → 4I15/2 transition of Er3+. Time course measurement at 545 nm emission of NaYF4:Yb,Er aqueous solution was also performed to further testify the stability of the UCNPs. The results shown in Figure 1c indicate that the upconversion PL intensity is stable for at least 8 h, indicating that the UCNPs which are highly dispersible in water are beneficial for fabricating homogeneous kinase assays. On the other hand, how to selectively recognize PKs-induced phosphopeptides by the UCNPs is the most critical issue to fabricate a UCNPs-based PKs assay. Inspired by the facts that some metal ions such as Zr4+ or Ti4+,40,41 particularly the rare earth ion La3+,42 can be applied to capture phosphorylated 6097
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Figure 3. (a) Overlap between the upconversion PL spectrum of NaYF4:Yb,Er UCNPs and the absorbance spectrum of TAMRA used in the LRET design; (b) upconversion PL spectra of the proposed LRET system under 980 nm excitation before (black line) and after the introduction of 0.05 U/ μL PKA (green line). The dashed red line is a negative control result of the LRET system in the presence of 0.05 U/μL PKA but without ATP. Experimental conditions: TAMRA-peptides, 10 μM; ATP, 100 μM; UCNPs, 2 mg/mL.
TAMRA-LRRASLG. After incubation with the NaYF4:Yb,Er, the phosphorylated TAMRA-peptides would be rapidly captured on the UCNPs through the high binding affinity between the RE3+ ions of UCNPs and the phosphate group. As a result, numerous TAMRA molecules would be accumulated on the very close proximity of NaYF4:Yb,Er UCNPs, resulting in background-free LRET from the UCNPs to TAMRA under the NIR light illumination (980 nm). By measuring the LRET signal, rapid and quantitative analysis of the TAMRA-peptides anchored on the UCNPs can be realized as a function of PKA activity. The feasibility of the proposed design is verified by the results shown in Figure 3b. It is well-known that FRET or LRET is a critically distance-dependent process which requires the donor and acceptor in very close proximity. So in the absence of PKA, although unphosphorylated TAMRA-peptides exist in the mixture with high concentration (10 μM), they are far away from the UCNPs and thus no LRET signal is observed (black line in Figure 3b). On the contrary, in the presence of PKA, a portion of TAMRA-peptides will be phosphorylated and then accumulated on the nearby surface of UCNPs, leading to the LRET from UCNPs to TAMRA accompanied by an intensity decrease at 545 nm but signal increase at 582 nm (characteristic emission of TAMRA), as shown in Figure 3b (green line). It is worth noting that only the emitter ions (Er3+) located near the surface of the UCNPs can transfer their energy at excited states to the captured TAMRA,43 so the decrease of upconversion luminescence at 545 nm is not so obvious compared with the fluorescence increase at 582 nm because a large portion of Er3+ are within the NaYF4 nanospheres and their emission do not contribute to the LRET. Furthermore, LRET phenomenon is not observed in the negative control experiment which is conducted in the presence of PKA but without ATP (red dashed line in Figure 3b), clearly indicating that the observed efficient LRET is indeed results from the PKA-actuated phosphorylation of TAMRA-peptides. In this proposed approach, separation of unbound TAMRApeptides as well as other biospecies involved in the system is not required because their excitation by NIR light does not lead to any background and only TAMRA-phosphopeptides anchored on the surface of UCNPs can be excited. Therefore, this design enables extremely simple kinase detection in a mix-
peptides/proteins from biological samples, we envision whether the RE-doped NaYF4:Yb,Er UCNPs can also act as an affinity material for recognizing phosphopeptides since a large portion of RE3+ ions are contained in the UCNPs. To verify this point, a protein kinase A (PKA)-specific substrate peptide (TAMRALRRASLG) and its corresponding phosphorylated counterpart (TAMRA-LRRApSLG) were, respectively, incubated with the NaYF4:Yb,Er UCNPs for 30 min, and then the UCNPs were centrifuged and the fluorescence signals of TAMRA-peptides remaining in the supernatants were recorded. As shown in Figure 1d, there is almost no fluorescence change for the unphosphorylated peptides before and after incubation with the UCNPs, indicating that the interaction between the UCNPs and the PKA-specific substrate peptides is negligible. However, a striking signal decrease is observed for the phosphorylated peptides, indicating that the phosphorylated peptides are tightly captured on the UCNPs and thus separated from the supernatant. These results clearly demonstrate that the NaYF4:Yb,Er UCNPs can specifically capturing the fluorescent phosphorylated peptides on their surfaces over the unphosphorylated ones through the strong interaction between RE3+ ions and phosphate groups, which will greatly simplify the fabrication of LRET-based PKs assay by using suitable fluorophore-labeled peptides as the acceptor and UCNPs as the donor without any requirement of functionalization of UCNPs with phospho-affinity antibodies, substrate immobilization, or separation procedures. Design Principle of the Mix-and-Read Assay for Detection of Protein Kinase Activity. On the basis of the facts that NaYF4:Yb,Er can serve as both a promising LRET donor and an efficient phosphopeptide-recognizing material, a robust LRET-based protein kinase assay can be facilely fabricated. The design principle is illustrated in Figure 2 by using PKA, a protein kinase critical to memory formation and an active target of drug development, as a proof-of-concept target. In this proposed LRET system, TAMRA-LRRASLG is used as the PKA-specific substrate peptide and the LRET acceptor because the emission spectrum of the UCNPs perfectly overlaps with the absorption spectrum of TAMRApeptide (Figure 3a), which is favorable for an efficient LRET process. Under the catalysis of PKA, the γ-phosphoryl of ATP will be transferred to the hydroxyl group of serine in the 6098
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Figure 4. (a) Normalized upconversion PL spectra of the proposed LRET system in the presence of different concentrations of PKA; (b) magnified spectra of (a) around the emission wavelength of 582 nm; (c) relationship between I582 nm/I545 nm ratios and PKA activities; (d) calibration curve between I582 nm/I545 nm ratios and PKA activities in the range from 0.0001 to 0.01 U/μL, which shows a good linear relationship. The error bars represent standard deviation of three replicates for each data point. Experimental conditions: TAMRA-peptides, 10 μM; ATP, 100 μM; UCNPs, 2 mg/mL. Other conditions are the same as those described in the Experimental Section.
(mU/μL) (correlation coefficient R = 0.9996) and the corresponding detection limit (3 s, n = 11) for PKA is estimated to be 0.000 05 U/μL, suggesting a high sensitivity. To the best of our knowledge, the most sensitive PKA assays reported recently are developed by using electrochemical or fluorometric techniques.41,44−46 They typically need sophisticated procedures and the detection limit for PKA are in the range of 0.0001−0.0005 U/μL, indicating that the sensitivity of the proposed method is much higher in spite of our extremely simple mix-and-read detection. In addition, the sensitivity of this method is also superior to that of our previous work for PKA detection by using fluorescent conjugated polymer as the FRET donor,28 clearly demonstrating the advantage of UCNPs as the luminescence donor. In this study, the zeta potential of EIP-capped NaYF4:Yb,Er UCNPs in PKA reaction buffer is tested to be 13.81 mV, indicating that the UCNPs are positively charged due to the EIP layer on their surface. On the other hand, the PKA-specific peptide is also positively charged but will become negatively charged after phosphorylation. So besides for our proposed mechanism that RE3+ ions are the source for the selective capture of phosphopeptides, it is also possible that the PKAinduced LRET may result from the electrostatic attraction between the positive charged EIP on the UCNPs surface and
and-read way, which only needs to mix the kinase reaction solution with the NaYF4:Yb,Er UCNPs for minutes and then directly record the upconversion LRET signal. Analytical Performance of the LRET Assay for PKA Detection. Under the systematically optimized experimental conditions (see the Supporting Information), the quantitative assessment of PKA activities using the LRET-based assay is investigated by monitoring the LRET ratios of I582 nm/I545 nm. Simultaneous measurement of emission changes at 582 and 545 nm can effectively preclude environmental or operation factors affecting PL measurements. To make the results more clear and easy to understand, the PL intensities of UCNPs at 545 nm are all normalized to 1. As can be seen from Figure 4a,b, the PL intensities at 582 nm are gradually increased with increasing concentrations of PKA, indicating that increasing PKA activity would bring more phosphorylated TAMRA-peptides close to UCNPs and hence enhance LRET efficiency. The calibration curves between the LRET ratios (I582 nm/I545 nm) and the PKA activities are plotted in Figure 4c, which shows that PKA activity can be detected over a wide concentration range from 0.0001 U/μL to 0.1 U/μL. In addition, the I582 nm/I545 nm ratios are found to vary linearly with the PKA activities in the measured range of 0.0001−0.01 U/μL (Figure 4d). The correlation equation is I582 nm/I545 nm = 0.027 + 0.011CPKA 6099
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activation of cAMP-dependent PKA.39 Therefore, in this study, MCF-7 cells are stimulated with different doses of forskolin/ IBMX, and the PKA activities in these cell lysates are examined by this UCNPs-based LRET assay. The results shown in Figure 5a manifest that the PKA activity in MCF-7 cell lysate without
the negatively charged phosphopeptides. If so, one would imagine that when negatively charged NaYF4:Yb,Er UCNPs were used for PKA analysis, no LRET signal would be observed with the increase of PKA activity. However, as can be seen from Figure S-4 in the Supporting Information, PKA-actuated LRET responses by using negatively charged NaYF4:Yb,Er UCNPs (zeta potential of −28.96 mV) are quite similar to those by using the positively charged EIP-NaYF4:Yb,Er UCNPs. Furthermore, Najam-ul-Haq et al. have recently demonstrated that lanthanum oxide, another kind of rare earth compound, can be applied to coprecipitate phosphorylated proteins from biological samples through the interaction between La3+ and phosphate groups.42 Therefore, it is reasonable to conclude that the RE3+ ions of UCNPs rather than the electrostatic interactions are the driving force for selective capture of phosphorylated peptides on UCNPs. To test whether this design is universally applicable, the activity of AKT1, another randomly selected protein kinase, is detected by using AKT1-specific peptide (TAMRACKRPRAASFAE) as the LRET acceptor. As can be seen from Figure S-5 in the Supporting Information, the I582 nm/ I545 nm ratios also increase gradually with increasing concentrations of AKT1, indicating that the proposed method can be used for the detection of other PKs. It should be noted that the peptide substrate of PKA has weakly positive charges in the reaction system, so the electrostatic interaction between the peptide and the UCNPs is fairly weak compared to the dominant RE3+-phosphate group interaction. However, if peptide substrates with much larger positive or negative charges were used for the detection of some other protein kinases, the electrostatic interaction might interfere with the RE3+-phosphate interaction and would affect the LRET result. According to previous work of Katayama’s group,47 the charge of a peptide substrate can be exactly tuned by adding (+)- or (−)-charged amino acid residues to a kinase-recognizing core peptide motif. Therefore, in such cases, the net charges of peptide substrates can be rationally controlled to be similar to that of PKA substrate used in this work in order to avoid the possible influence of electrostatic factor and make the proposed LRET assay more universally applicable for PKs analysis. To testify this viewpoint, two peptide substrates for kinases of PKC and Src are designed, respectively, with weakly positive charges (Supporting Information). As can be seen from Figure S-6 in the Supporting Information, no LRET phenomena are observed when only unphosphorylated peptide substrates are incubated with UCNPs, indicating that the electrostatic interaction between the UCNPs and the unphosphorylated peptides are negligible. As a contrast, the LRET signals increase gradually with the increasing concentration of phosphorylated peptide substrates, suggesting that the proposed method is undoubtedly feasible for monitoring the activity of PKC or Src. All of these results clearly suggest that with rationally designed peptide substrates, the proposed UCNPs-based LRET strategy can be easily extended to the detection of various types of PKs. Detection of Drug-Stimulated Activation of PKA in Cell Lysates. Protein kinase activities are highly regulated in cells in intracellular signaling pathways, so a practical kinase assay should be applicable for kinase analysis in real biological samples particularly in cell lysates. We further testify whether the UCNPs-based protein kinase assay could work in MCF-7 breast carcinoma cell lysates. It is well-known that stimulation of MCF-7 cells by the combination of forskolin/IBMX can greatly increase the intracellular level of cAMP, causing the
Figure 5. (a) Normalized upconversion PL spectra of the proposed LRET system for the detection of PKA activities in different types of MCF-7 cell lysates. Experimental conditions: TAMRA-peptides, 10 μM; ATP, 100 μM; UCNPs, 2 mg/mL; (b) detection of PKA activities in different types of MCF-7 cell lysates by using the Kinase-Glo Luminescent Kinase Assay kit. Sample a, blank without any cell lysate; b, unstimulated cell lysate; c, 10 μM forskolin/20 μM IBMXstimulated cell lysate; d, 50 μM forskolin/100 μM IBMX-stimulated cell lysate; e, 50 μM forskolin/100 μM IBMX-stimulated cell lysate premixed with 10 μM H-89. Experimental conditions: substrate peptides (LRRASLG), 10 μM; ATP, 20 μM; the total protein concentration of each type of MCF-7 cell lysate is fixed at 10 μg/mL and other conditions are all performed according to the standard technical bulletin of the commercial kit.
drug stimulation may be too low to be detectable. In contrast, the forskolin/IBMX-stimulated cell lysates all arouse obvious LRET responses, which increase gradually with increasing concentration of stimulants. If the drug-stimulated cell lysate is premixed with H-89, a well-recognized PKA inhibitor, the LRET signal will decrease again, clearly manifesting that the cell lysates-actuated LRET responses indeed result from the successful activation of PKA by drug stimulation. Meanwhile, PKA activities in these cell lysates are also evaluated by using a commercial assay kit (Kinase-Glo Luminescent Kinase Assay kit 6100
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from Promega). This commercial kit detects the protein kinase activity by quantitating the amount of ATP remaining in solution after a kinase reaction through an ATP-dependent bioluminescence pathway. Briefly, a proprietary luciferase accompanied by luciferin will generate a stable luminescence in the presence of ATP. So the luminescent signal is correlated with ATP concentration and would be inversely proportional to the kinase activity which would assume ATP during the phosphorylation reaction. As shown in Figure 5b, compared to the blank control without any cell lysate, the luminescence signal is almost unaffected for the unstimulated MCF-7 cell lysate, indicating a rather low PKA activity. However, with the increase of stimulants, more PKA are activated and then the phosphorylation reaction will consume more ATP, resulting in decreased luminescence signals. In addition, if the drugstimulated cell lysate is preincubated with H-89, no obvious luminescence decrease is observed. These results agree well with those obtained by our proposed LRET assay, indicating that the UCNPs-based LRET assay is feasible and reliable for in vitro detection of cell kinase activities in complex biological samples. Inhibition Study. It is well-known that the screening of potential small-molecule inhibitors of PKs is of great significance in drug discovery. To test whether this UCNPsbased assay can be employed to quantitatively assess kinase inhibitor, a proof-of-concept inhibition study for PKA is performed in this study. The experiments were conducted by preincubating a fixed PKA concentration with series dilutions of H-89, a potent inhibitor of PKA, then initiating the phosphorylation reaction and finally recording the LRET signals. Figure 6a shows the inhibitory results of H-89. As expected, the I582 nm/I545 nm ratios decrease gradually with increasing concentrations of H-89 because of the inhibition of PKA activity and thus low levels of peptide phosphorylation. The relationship between the I582 nm/I545 nm ratios and H-89 concentrations on the logarithm scale is plotted in Figure 6b, from which the IC 50 value (half maximal inhibitory concentration of the inhibitor) is determined to be 116 nM, which is well consistent with the literature values.28,48 These results clearly suggest that the UCNPs-based LRET assay is suitable for screening potential inhibitors of PKs and is of great potential in protein kinases-targeted drug development.
Figure 6. (a) Normalized upconversion PL spectra of the LRET system in the presence of different concentrations of H-89 (0−10 μM) by fixing PKA activity at 0.02 U/μL. The black line at the bottom refers to the blank control without PKA and H-89; (b) the relationship between the I582 nm/I545 nm ratios and H-89 concentrations on the logarithm scale.
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CONCLUSIONS In conclusion, by integrating the outstanding features of REdoped UCNPs for highly selective recognition of PKs-induced phosphopeptides and the inherent low-background upconversion luminescence property, a mix-and-read LRET assay is developed for highly sensitive detection of PKs activity. This approach shows distinct advantages. On one hand, the use of UCNPs as the LRET donor effectively reduces the autofluorescence and light scattering background, which allows separation-free detection with greatly improved signal-tobackground ratios. On the other hand, for the first time we demonstrate that UCNPs themselves can act as excellent affinity materials to selectively capture phosphopeptides catalyzed by PKs, which also greatly simplifies the detection procedures because complicated functionalization of UCNPs with additional phospho-recognizing elements (for example, antibodies) is no longer required. Therefore, this proposed strategy provides a practical and robust platform to detect PKs activity with extremely simple mix-and-read detection, absence of background interference, and high sensitivity, which may
serve as a powerful tool for PKs analysis in signal transduction pathways and kinase-related clinical diagnosis as well as drug discovery.
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ASSOCIATED CONTENT
S Supporting Information *
Detailed optimization of experimental conditions, PKA analysis by using negatively charged UCNPs, detection results of AKT1 activities by using the UCNPs-based LRET approach, and evaluation of the interaction between the UCNPs and peptide substrates for PKC and Src. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Fax: +86 29 81530859. Notes
The authors declare no competing financial interest. 6101
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(32) Wang, L.; Yan, R.; Huo, Z.; Wang, L.; Zeng, J.; Bao, J.; Wang, X.; Peng, Q.; Li, Y. Angew. Chem., Int. Ed. 2005, 44, 6054−6057. (33) Chen, Z.; Chen, H.; Hu, H.; Yu, M.; Li, F.; Zhang, Q.; Zhou, Z.; Yi, T.; Huang, C. J. Am. Chem. Soc. 2008, 130, 3023−3029. (34) Deng, R.; Xie, X.; Vendrell, M.; Chang, Y.-T.; Liu, X. J. Am. Chem. Soc. 2011, 133, 20168−20171. (35) Liu, C.; Wang, Z.; Jia, H.; Li, Z. Chem. Commun. 2011, 47, 4661−4663. (36) Liu, Q.; Peng, J.; Sun, L.; Li, F. ACS Nano 2011, 5, 8040−8048. (37) Liu, J.; Liu, Y.; Liu, Q.; Li, C.; Sun, L.; Li, F. J. Am. Chem. Soc. 2011, 133, 15276−15279. (38) Wang, F.; Liu, X. J. Am. Chem. Soc. 2008, 130, 5642−5643. (39) Xu, X.; Liu, X.; Nie, Z.; Pan, Y.; Guo, M.; Yao, S. Anal. Chem. 2011, 83, 52−59. (40) Ren, W.; Liu, C.; Lian, S.; Li, Z. Anal. Chem. 2013, 85, 10956− 10961. (41) Bai, J.; Zhao, Y.; Wang, Z.; Liu, C.; Wang, Y.; Li, Z. Anal. Chem. 2013, 85, 4813−4821. (42) Jabeen, F.; Hussaun, D.; Fatima, B.; Musharraf, G.; Huck, C. W.; Bonn, G. K.; Najam-ul-Haq, M. Anal. Chem. 2012, 84, 10180−10185. (43) Rantanen, T.; Järvenpäa,̈ M.-L.; Vuojola, J.; Kuningas, K.; Soukka, T. Angew. Chem., Int. Ed. 2008, 47, 3811−3813. (44) Xu, X.; Nie, Z.; Chen, J.; Fu, Y.; Li, W.; Shen, Q.; Yao, S. Chem. Commun. 2009, 6946−6948. (45) Miao, P.; Ning, L.; Li, X.; Li, P.; Li, G. Bioconjugate Chem. 2012, 23, 141−145. (46) Ji, J.; Yang, H.; Liu, Y.; Chen, H.; Kong, J.; Liu, B. Chem. Commun. 2009, 1508−1510. (47) Oishi, J.; Asami, Y.; Mori, T.; Kang, J.-H.; Tanabe, M.; Niidome, T.; Katayama, Y. ChemBioChem 2007, 8, 875−879. (48) Shiosaki, S.; Nobori, T.; Mori, T.; Toita, R.; Nakamura, Y.; Kim, C. W.; Yamamoto, T.; Niidome, T.; Katayama, Y. Chem. Commun. 2013, 49, 5592−5594.
ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grants 21335005 and 91127035), Program for Changjiang Scholars and Innovative Research Team in University (Grant IRT 1124), and the Fundamental Research Funds for the Central Universities (Grants GK201402051 and GK201303003).
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Upconversion Nanophosphor: An Efficient PhosphopeptidesRecognizing Matrix and Luminescence Resonance Energy Transfer Donor for Robust Detection of Protein Kinase Activity Chenghui Liu,*,†,‡ Lijuan Chang,‡ Honghong Wang,† Jie Bai,‡ Wei Ren,‡ and Zhengping Li†,‡ †
Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, Shaanxi Province P. R. China ‡ College of Chemistry and Environmental Science, Hebei University, Baoding 071002, Hebei Province P. R. China S Supporting Information *
ABSTRACT: Protein kinases play important regulatory roles in intracellular signal transduction pathways. The aberrant activities of protein kinases are closely associated with the development of various diseases, which necessitates the development of practical and sensitive assays for monitoring protein kinase activities as well as for screening of potential kinase-targeted drugs. We demonstrate here a robust luminescence resonance energy transfer (LRET)-based protein kinase assay by using NaYF4:Yb,Er, one of the most efficient upconversion nanophosphors (UCNPs), as an autofluorescence-free LRET donor and a tetramethylrhodamine (TAMRA)-labeled substrate peptide as the acceptor. Fascinatingly, besides acting as the LRET donor, NaYF4:Yb,Er UCNPs also serve as the phosphopeptide-recognizing matrix because the intrinsic rare earth ions of UCNPs can specifically capture the fluorescent phosphopeptides catalyzed by protein kinases over the unphosphorylated ones. Therefore, a sensitive and generic protein kinase assay is developed in an extremely simple mix-and-read format without any requirement of surface modification, substrate immobilization, separation, or washing steps, showing great potential in protein kinases-related clinical diagnosis and drug discovery. To the best of our knowledge, this is the first report by use of rare earth-doped UCNPs as both the phosphorecognizing and signal reporting elements for protein kinase analysis.
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harmful radioactive labels, specialized/sophisticated instrumentation, complicated sample handling, or tedious operations. For high throughput screening of potential PKs inhibitors, the fluorescence technique is highly preferred because of its high sensitivity and design flexibility and being compatible with various automatic equipment which allows for high-throughput signal readout.19 Fluorescence kinase assays typically use fluorescently labeled substrate peptides or phospho-recognizing antibodies to monitor the fluorescence changes upon kinaseinduced phosphorylation, which require labor-intense purification procedures to separate the phosphopeptides from unphosphorylated ones.20,21 In this regard, homogeneous fluorescence resonance energy transfer (FRET) assays show particular advantages because of their separation-free detection characteristic and robustness against matrix interferences.22−24 Notably, integrating fluorescent quantum dots (QDs) with the FRET technique offers an elegant way for detection of PKs activity. I. Willner’s group and M. Stevens’s group have separately established kinase assays by using QDs as the FRET
hosphorylation of cellular proteins catalyzed by protein kinases (PKs) plays vital roles in signal transduction pathways, which regulates many fundamental cellular processes including cell proliferation, growth, and apoptosis.1 It has been recognized that the dysfunction (hyperactivity in most cases) of some kinases is profoundly associated with the development of various human diseases including cancers.2−4 As such, PKs have emerged as promising molecular targets for the pharmaceutical intervention of several diseases by using kinase inhibitors to down-regulate the activities of related PKs. As a result, the screening of effective protein kinase inhibitors as potential anticancer drugs is undergoing intense study.5−7 In this regard, highly sensitive assays for accurate determination of the activity of specific PKs with low cost and simple operation are highly desired for PKs-related clinical diagnosis, drug discovery, as well as for further understanding the intracellular signal transduction pathways. Up until now, various approaches for the detection of PKs activities have been established, such as radioactive methods,8,9 electrochemical methods,10−13 nanomaterial-based assays,14,15 mass spectrometry-based assays,16,17 and magnetic resonance imaging (MRI).18 Although these methods all have their own advantages, most of them suffer from the drawbacks such as © 2014 American Chemical Society
Received: April 6, 2014 Accepted: May 16, 2014 Published: May 28, 2014 6095
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Figure 1. (a) Representative TEM image of the NaYF4:Yb,Er UCNPs; (b) XRD pattern of the UCNPs; (c) stability evaluation of NaYF4:Yb,Er colloidal solution (2 mg/mL) through time-course measurement of 545 nm emission. The inset is a photograph of 2 mg/mL NaYF4:Yb,Er colloidal solution; (d) investigation of the specific interaction between UCNPs and phosphopeptides. Unphosphorylated peptide (TAMRA-LRRASLG) (10 μM) and its phosphorylated counterpart (TAMRA-LRRApSLG) were, respectively, incubated with 2 mg/mL of UCNPs for 30 min, and then the UCNPs were centrifuged and the fluorescence signals of TAMRA remaining in the supernatants were recorded.
donors and organic fluorophore-labeled antiphosphoamino acid antibodies as the acceptors.25−27 Although these designs are smart, however, they heavily rely on the use of high-cost phosphoamino acid-recognition antibodies. Furthermore, these methods also show the potential drawbacks of inherent toxicity of heavy metal-containing QDs and specialized bioconjugation steps because substrate peptides should be preimmobilized on the surface of QDs, which may lead to undesired optical property changes. To overcome these limitations, very recently, we described an antibody-free PKs assay based on the metal ion-mediated FRET by using a fluorescent conjugated polymer as the donor, which enables highly sensitive detection of PKs activity with simple operations.28 However, it should be noted that the excitation of commonly used QDs, fluorescent conjugated polymer, as well as conventional dyes typically need the illumination of UV or short-wavelength visible light. The fluorescently labeled substrate peptides or antibodies as well as many other biological species can also be excited and strongly increase background fluorescence, which will greatly reduce the signal-to-background ratio and the detection sensitivity. Fortunately, the inherent shortcomings of QDs and organic dyes can be elegantly avoided by using rare earth (RE)-doped near-infrared (NIR)-to-visible upconversion nanophosphors (UCNPs).29−31 UCNPs are capable of emitting intense visible light by absorbing two or more NIR photons. They show distinct advantages over the traditional organic fluorophores and QDs, including large anti-Stokes shifts, high resistance to photobleaching and blinking, as well as low toxicity. More
fascinatingly, excitation of UCNPs via multiphoton absorption processes allows the use of NIR light, which would not be absorbed by conventional fluorescent dyes or biological species and thus effectively reduces autofluorescence and light scattering background. As a result, the signal-to-background ratio can be greatly enhanced in UCNPs-based biosensing systems, which allow high detection sensitivity. These advantages make UCNPs an ideal choice as the luminescence donor in designing luminescence resonance energy transfer (LRET) systems for biosensing and bioimaging.32−37 In light of these unique and excellent properties of RE-doped UCNPs, herein, we wish to report a robust LRET assay for the detection of PKs activity by using NaYF4:Yb,Er, one of the most efficient UCNPs known to date, as both the LRET donor and phosphopeptide-recognizing matrix because the intrinsic RE3+ ions of UCNPs can specifically capture the fluorescent phosphopeptides catalyzed by PKs, resulting in efficient upconversion LRET. Therefore, a sensitive and generic protein kinase assay is developed in an extremely simple mix-and-read manner without any requirement of surface modification of UCNPs, substrate immobilization, separation, or washing steps.
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EXPERIMENTAL SECTION Chemicals and Materials. All the peptides used in this study were synthesized by GL Biochem (Shanghai, China). cAMP-dependent protein kinase A (catalytic subunit, abbreviated as PKA) was purchased from New England Biolabs and AKT1 was obtained from Sigma-Aldrich. H-89 was supplied by EMD Biosciences and ATP was purchased from Sangon 6096
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Figure 2. Design principle of the UCNPs-based LRET assay for the detection of PKA activity.
(Shanghai, China). Ethylene imine polymer (EIP, Mw ∼1800) was obtained from Aladdin. Rare earth chlorides (RECl3·6H2O) were prepared by dissolving the corresponding rare earth oxides in hydrochloric acid and then evaporating the solvent at elevated temperature. All other reagents were of A.R. grade and used as received without further purification. Synthesis and Characterization of the EIP-Capped NaYF4:Yb,Er UCNPs. EIP-capped NaYF4:Yb,Er UCNPs were prepared by a modified solvothermal method.38 In a typical experiment, 1.2 mmol of RECl3 (Y3+/Yb3+/Er3+ = 80/18/2), 2.4 mmol of NaCl, and 0.3 g EIP were dissolved in 20 mL of ethylene glycol (EG) under agitation. Then 15 mL of EG solution containing 5 mmol of NH4F was added to the above mixture under vigorous stirring. The mixture was transferred into a 50 mL Teflon-linked autoclave and agitated thoroughly for another 5 min. Afterward, the autoclave was sealed and heated to 200 °C for 5 h. After cooling down to room temperature, the EIP-capped NaYF4:Yb,Er UCNPs were collected by centrifugation, washed with ethanol and distilled water, respectively, for several times, and dried at 60 °C. The morphology and size of NaYF4:Yb,Er UCNPs were characterized by transmission electron microscopy (TEM) using a JEM-2010 electron microscope (JEOL, Japan). Powder X-ray diffraction (XRD) patterns were recorded on a D8 Advance X-ray diffractometer (Bruker, Germany) with Cu Kα radiation. Upconversion photoluminescence (PL) spectra were recorded using a Fluorolog 3-211 fluorescence spectrophotometer (Horiba Jobin-Yivon, France) with an external 980 nm laser diode (Viasho, China) as the excitation source. All measurements were carried out at room temperature. Standard Procedures for Detection of PKA Activity and Inhibition Study. Typically, the PKA-catalyzed phosphorylation reaction was carried out in a 100-μL volume of Tris-HCl buffer (pH 7.5) containing 10 μM TAMRA-labeled PKA-specific peptide (TAMRA-LRRASLG), 100 μM ATP, 10 mM MgCl2, and a certain concentration of PKA at 30 °C for 1 h. Afterward, the reaction system was immediately mixed with an equal volume of NaYF4:Yb,Er colloidal solution (final concentration of 2 mg/mL) and incubated for 10 min at room temperature. Then the mixture was directly subject to upconversion luminescence measurement without any further treatment.
For the PKA inhibition study, the experimental procedures were similar to those stated above for detection of PKA activity, except for the preincubation of a fixed PKA concentration with varied concentrations of H-89 in the reaction system. In this study, the culture, drug stimulation and lysis of MCF7 breast carcinoma cells as well as the determination of total protein concentrations in the cell lysates were all performed according to the literature method.39 PKA activities in different MCF-7 cell lysates (10 μg/mL total protein concentration for each cell lysate sample) were detected according to the standard protocol described above.
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RESULTS AND DISCUSSION Characterization of the NaYF 4 :Yb,Er UCNPs. NaYF4:18%Yb,2%Er, one of the most efficient NIR-to-visible UCNPs, were synthesized by using a simple solvothermal method and the products were characterized by TEM. From the typical TEM image shown in Figure 1a, one can see that the as-prepared NaYF4:Yb,Er UCNPs display spherical shapes with an average diameter of ∼32 nm. X-ray powder diffraction (XRD) pattern of the UCNPs is shown in Figure 1b, where the peak positions and intensities can be well indexed to be cubic phase NaYF4:Yb,Er (JCPDS PDF 77-2042). Furthermore, the ethylene imine polymer (EIP)-capped NaYF4:Yb,Er UCNPs possess excellent dispersibility in water and aqueous buffers to form stable colloidal solutions (inset of Figure 1c), which is suitable for bioapplications. The NaYF4:Yb,Er UCNPs show intense upconversion photoluminescence (PL) at ∼545 nm (see the spectra below in Figure 3b) under the excitation of a 980 nm laser, which is assigned to the 4S3/2 → 4I15/2 transition of Er3+. Time course measurement at 545 nm emission of NaYF4:Yb,Er aqueous solution was also performed to further testify the stability of the UCNPs. The results shown in Figure 1c indicate that the upconversion PL intensity is stable for at least 8 h, indicating that the UCNPs which are highly dispersible in water are beneficial for fabricating homogeneous kinase assays. On the other hand, how to selectively recognize PKs-induced phosphopeptides by the UCNPs is the most critical issue to fabricate a UCNPs-based PKs assay. Inspired by the facts that some metal ions such as Zr4+ or Ti4+,40,41 particularly the rare earth ion La3+,42 can be applied to capture phosphorylated 6097
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Figure 3. (a) Overlap between the upconversion PL spectrum of NaYF4:Yb,Er UCNPs and the absorbance spectrum of TAMRA used in the LRET design; (b) upconversion PL spectra of the proposed LRET system under 980 nm excitation before (black line) and after the introduction of 0.05 U/ μL PKA (green line). The dashed red line is a negative control result of the LRET system in the presence of 0.05 U/μL PKA but without ATP. Experimental conditions: TAMRA-peptides, 10 μM; ATP, 100 μM; UCNPs, 2 mg/mL.
TAMRA-LRRASLG. After incubation with the NaYF4:Yb,Er, the phosphorylated TAMRA-peptides would be rapidly captured on the UCNPs through the high binding affinity between the RE3+ ions of UCNPs and the phosphate group. As a result, numerous TAMRA molecules would be accumulated on the very close proximity of NaYF4:Yb,Er UCNPs, resulting in background-free LRET from the UCNPs to TAMRA under the NIR light illumination (980 nm). By measuring the LRET signal, rapid and quantitative analysis of the TAMRA-peptides anchored on the UCNPs can be realized as a function of PKA activity. The feasibility of the proposed design is verified by the results shown in Figure 3b. It is well-known that FRET or LRET is a critically distance-dependent process which requires the donor and acceptor in very close proximity. So in the absence of PKA, although unphosphorylated TAMRA-peptides exist in the mixture with high concentration (10 μM), they are far away from the UCNPs and thus no LRET signal is observed (black line in Figure 3b). On the contrary, in the presence of PKA, a portion of TAMRA-peptides will be phosphorylated and then accumulated on the nearby surface of UCNPs, leading to the LRET from UCNPs to TAMRA accompanied by an intensity decrease at 545 nm but signal increase at 582 nm (characteristic emission of TAMRA), as shown in Figure 3b (green line). It is worth noting that only the emitter ions (Er3+) located near the surface of the UCNPs can transfer their energy at excited states to the captured TAMRA,43 so the decrease of upconversion luminescence at 545 nm is not so obvious compared with the fluorescence increase at 582 nm because a large portion of Er3+ are within the NaYF4 nanospheres and their emission do not contribute to the LRET. Furthermore, LRET phenomenon is not observed in the negative control experiment which is conducted in the presence of PKA but without ATP (red dashed line in Figure 3b), clearly indicating that the observed efficient LRET is indeed results from the PKA-actuated phosphorylation of TAMRA-peptides. In this proposed approach, separation of unbound TAMRApeptides as well as other biospecies involved in the system is not required because their excitation by NIR light does not lead to any background and only TAMRA-phosphopeptides anchored on the surface of UCNPs can be excited. Therefore, this design enables extremely simple kinase detection in a mix-
peptides/proteins from biological samples, we envision whether the RE-doped NaYF4:Yb,Er UCNPs can also act as an affinity material for recognizing phosphopeptides since a large portion of RE3+ ions are contained in the UCNPs. To verify this point, a protein kinase A (PKA)-specific substrate peptide (TAMRALRRASLG) and its corresponding phosphorylated counterpart (TAMRA-LRRApSLG) were, respectively, incubated with the NaYF4:Yb,Er UCNPs for 30 min, and then the UCNPs were centrifuged and the fluorescence signals of TAMRA-peptides remaining in the supernatants were recorded. As shown in Figure 1d, there is almost no fluorescence change for the unphosphorylated peptides before and after incubation with the UCNPs, indicating that the interaction between the UCNPs and the PKA-specific substrate peptides is negligible. However, a striking signal decrease is observed for the phosphorylated peptides, indicating that the phosphorylated peptides are tightly captured on the UCNPs and thus separated from the supernatant. These results clearly demonstrate that the NaYF4:Yb,Er UCNPs can specifically capturing the fluorescent phosphorylated peptides on their surfaces over the unphosphorylated ones through the strong interaction between RE3+ ions and phosphate groups, which will greatly simplify the fabrication of LRET-based PKs assay by using suitable fluorophore-labeled peptides as the acceptor and UCNPs as the donor without any requirement of functionalization of UCNPs with phospho-affinity antibodies, substrate immobilization, or separation procedures. Design Principle of the Mix-and-Read Assay for Detection of Protein Kinase Activity. On the basis of the facts that NaYF4:Yb,Er can serve as both a promising LRET donor and an efficient phosphopeptide-recognizing material, a robust LRET-based protein kinase assay can be facilely fabricated. The design principle is illustrated in Figure 2 by using PKA, a protein kinase critical to memory formation and an active target of drug development, as a proof-of-concept target. In this proposed LRET system, TAMRA-LRRASLG is used as the PKA-specific substrate peptide and the LRET acceptor because the emission spectrum of the UCNPs perfectly overlaps with the absorption spectrum of TAMRApeptide (Figure 3a), which is favorable for an efficient LRET process. Under the catalysis of PKA, the γ-phosphoryl of ATP will be transferred to the hydroxyl group of serine in the 6098
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Figure 4. (a) Normalized upconversion PL spectra of the proposed LRET system in the presence of different concentrations of PKA; (b) magnified spectra of (a) around the emission wavelength of 582 nm; (c) relationship between I582 nm/I545 nm ratios and PKA activities; (d) calibration curve between I582 nm/I545 nm ratios and PKA activities in the range from 0.0001 to 0.01 U/μL, which shows a good linear relationship. The error bars represent standard deviation of three replicates for each data point. Experimental conditions: TAMRA-peptides, 10 μM; ATP, 100 μM; UCNPs, 2 mg/mL. Other conditions are the same as those described in the Experimental Section.
(mU/μL) (correlation coefficient R = 0.9996) and the corresponding detection limit (3 s, n = 11) for PKA is estimated to be 0.000 05 U/μL, suggesting a high sensitivity. To the best of our knowledge, the most sensitive PKA assays reported recently are developed by using electrochemical or fluorometric techniques.41,44−46 They typically need sophisticated procedures and the detection limit for PKA are in the range of 0.0001−0.0005 U/μL, indicating that the sensitivity of the proposed method is much higher in spite of our extremely simple mix-and-read detection. In addition, the sensitivity of this method is also superior to that of our previous work for PKA detection by using fluorescent conjugated polymer as the FRET donor,28 clearly demonstrating the advantage of UCNPs as the luminescence donor. In this study, the zeta potential of EIP-capped NaYF4:Yb,Er UCNPs in PKA reaction buffer is tested to be 13.81 mV, indicating that the UCNPs are positively charged due to the EIP layer on their surface. On the other hand, the PKA-specific peptide is also positively charged but will become negatively charged after phosphorylation. So besides for our proposed mechanism that RE3+ ions are the source for the selective capture of phosphopeptides, it is also possible that the PKAinduced LRET may result from the electrostatic attraction between the positive charged EIP on the UCNPs surface and
and-read way, which only needs to mix the kinase reaction solution with the NaYF4:Yb,Er UCNPs for minutes and then directly record the upconversion LRET signal. Analytical Performance of the LRET Assay for PKA Detection. Under the systematically optimized experimental conditions (see the Supporting Information), the quantitative assessment of PKA activities using the LRET-based assay is investigated by monitoring the LRET ratios of I582 nm/I545 nm. Simultaneous measurement of emission changes at 582 and 545 nm can effectively preclude environmental or operation factors affecting PL measurements. To make the results more clear and easy to understand, the PL intensities of UCNPs at 545 nm are all normalized to 1. As can be seen from Figure 4a,b, the PL intensities at 582 nm are gradually increased with increasing concentrations of PKA, indicating that increasing PKA activity would bring more phosphorylated TAMRA-peptides close to UCNPs and hence enhance LRET efficiency. The calibration curves between the LRET ratios (I582 nm/I545 nm) and the PKA activities are plotted in Figure 4c, which shows that PKA activity can be detected over a wide concentration range from 0.0001 U/μL to 0.1 U/μL. In addition, the I582 nm/I545 nm ratios are found to vary linearly with the PKA activities in the measured range of 0.0001−0.01 U/μL (Figure 4d). The correlation equation is I582 nm/I545 nm = 0.027 + 0.011CPKA 6099
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activation of cAMP-dependent PKA.39 Therefore, in this study, MCF-7 cells are stimulated with different doses of forskolin/ IBMX, and the PKA activities in these cell lysates are examined by this UCNPs-based LRET assay. The results shown in Figure 5a manifest that the PKA activity in MCF-7 cell lysate without
the negatively charged phosphopeptides. If so, one would imagine that when negatively charged NaYF4:Yb,Er UCNPs were used for PKA analysis, no LRET signal would be observed with the increase of PKA activity. However, as can be seen from Figure S-4 in the Supporting Information, PKA-actuated LRET responses by using negatively charged NaYF4:Yb,Er UCNPs (zeta potential of −28.96 mV) are quite similar to those by using the positively charged EIP-NaYF4:Yb,Er UCNPs. Furthermore, Najam-ul-Haq et al. have recently demonstrated that lanthanum oxide, another kind of rare earth compound, can be applied to coprecipitate phosphorylated proteins from biological samples through the interaction between La3+ and phosphate groups.42 Therefore, it is reasonable to conclude that the RE3+ ions of UCNPs rather than the electrostatic interactions are the driving force for selective capture of phosphorylated peptides on UCNPs. To test whether this design is universally applicable, the activity of AKT1, another randomly selected protein kinase, is detected by using AKT1-specific peptide (TAMRACKRPRAASFAE) as the LRET acceptor. As can be seen from Figure S-5 in the Supporting Information, the I582 nm/ I545 nm ratios also increase gradually with increasing concentrations of AKT1, indicating that the proposed method can be used for the detection of other PKs. It should be noted that the peptide substrate of PKA has weakly positive charges in the reaction system, so the electrostatic interaction between the peptide and the UCNPs is fairly weak compared to the dominant RE3+-phosphate group interaction. However, if peptide substrates with much larger positive or negative charges were used for the detection of some other protein kinases, the electrostatic interaction might interfere with the RE3+-phosphate interaction and would affect the LRET result. According to previous work of Katayama’s group,47 the charge of a peptide substrate can be exactly tuned by adding (+)- or (−)-charged amino acid residues to a kinase-recognizing core peptide motif. Therefore, in such cases, the net charges of peptide substrates can be rationally controlled to be similar to that of PKA substrate used in this work in order to avoid the possible influence of electrostatic factor and make the proposed LRET assay more universally applicable for PKs analysis. To testify this viewpoint, two peptide substrates for kinases of PKC and Src are designed, respectively, with weakly positive charges (Supporting Information). As can be seen from Figure S-6 in the Supporting Information, no LRET phenomena are observed when only unphosphorylated peptide substrates are incubated with UCNPs, indicating that the electrostatic interaction between the UCNPs and the unphosphorylated peptides are negligible. As a contrast, the LRET signals increase gradually with the increasing concentration of phosphorylated peptide substrates, suggesting that the proposed method is undoubtedly feasible for monitoring the activity of PKC or Src. All of these results clearly suggest that with rationally designed peptide substrates, the proposed UCNPs-based LRET strategy can be easily extended to the detection of various types of PKs. Detection of Drug-Stimulated Activation of PKA in Cell Lysates. Protein kinase activities are highly regulated in cells in intracellular signaling pathways, so a practical kinase assay should be applicable for kinase analysis in real biological samples particularly in cell lysates. We further testify whether the UCNPs-based protein kinase assay could work in MCF-7 breast carcinoma cell lysates. It is well-known that stimulation of MCF-7 cells by the combination of forskolin/IBMX can greatly increase the intracellular level of cAMP, causing the
Figure 5. (a) Normalized upconversion PL spectra of the proposed LRET system for the detection of PKA activities in different types of MCF-7 cell lysates. Experimental conditions: TAMRA-peptides, 10 μM; ATP, 100 μM; UCNPs, 2 mg/mL; (b) detection of PKA activities in different types of MCF-7 cell lysates by using the Kinase-Glo Luminescent Kinase Assay kit. Sample a, blank without any cell lysate; b, unstimulated cell lysate; c, 10 μM forskolin/20 μM IBMXstimulated cell lysate; d, 50 μM forskolin/100 μM IBMX-stimulated cell lysate; e, 50 μM forskolin/100 μM IBMX-stimulated cell lysate premixed with 10 μM H-89. Experimental conditions: substrate peptides (LRRASLG), 10 μM; ATP, 20 μM; the total protein concentration of each type of MCF-7 cell lysate is fixed at 10 μg/mL and other conditions are all performed according to the standard technical bulletin of the commercial kit.
drug stimulation may be too low to be detectable. In contrast, the forskolin/IBMX-stimulated cell lysates all arouse obvious LRET responses, which increase gradually with increasing concentration of stimulants. If the drug-stimulated cell lysate is premixed with H-89, a well-recognized PKA inhibitor, the LRET signal will decrease again, clearly manifesting that the cell lysates-actuated LRET responses indeed result from the successful activation of PKA by drug stimulation. Meanwhile, PKA activities in these cell lysates are also evaluated by using a commercial assay kit (Kinase-Glo Luminescent Kinase Assay kit 6100
dx.doi.org/10.1021/ac501247t | Anal. Chem. 2014, 86, 6095−6102
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from Promega). This commercial kit detects the protein kinase activity by quantitating the amount of ATP remaining in solution after a kinase reaction through an ATP-dependent bioluminescence pathway. Briefly, a proprietary luciferase accompanied by luciferin will generate a stable luminescence in the presence of ATP. So the luminescent signal is correlated with ATP concentration and would be inversely proportional to the kinase activity which would assume ATP during the phosphorylation reaction. As shown in Figure 5b, compared to the blank control without any cell lysate, the luminescence signal is almost unaffected for the unstimulated MCF-7 cell lysate, indicating a rather low PKA activity. However, with the increase of stimulants, more PKA are activated and then the phosphorylation reaction will consume more ATP, resulting in decreased luminescence signals. In addition, if the drugstimulated cell lysate is preincubated with H-89, no obvious luminescence decrease is observed. These results agree well with those obtained by our proposed LRET assay, indicating that the UCNPs-based LRET assay is feasible and reliable for in vitro detection of cell kinase activities in complex biological samples. Inhibition Study. It is well-known that the screening of potential small-molecule inhibitors of PKs is of great significance in drug discovery. To test whether this UCNPsbased assay can be employed to quantitatively assess kinase inhibitor, a proof-of-concept inhibition study for PKA is performed in this study. The experiments were conducted by preincubating a fixed PKA concentration with series dilutions of H-89, a potent inhibitor of PKA, then initiating the phosphorylation reaction and finally recording the LRET signals. Figure 6a shows the inhibitory results of H-89. As expected, the I582 nm/I545 nm ratios decrease gradually with increasing concentrations of H-89 because of the inhibition of PKA activity and thus low levels of peptide phosphorylation. The relationship between the I582 nm/I545 nm ratios and H-89 concentrations on the logarithm scale is plotted in Figure 6b, from which the IC 50 value (half maximal inhibitory concentration of the inhibitor) is determined to be 116 nM, which is well consistent with the literature values.28,48 These results clearly suggest that the UCNPs-based LRET assay is suitable for screening potential inhibitors of PKs and is of great potential in protein kinases-targeted drug development.
Figure 6. (a) Normalized upconversion PL spectra of the LRET system in the presence of different concentrations of H-89 (0−10 μM) by fixing PKA activity at 0.02 U/μL. The black line at the bottom refers to the blank control without PKA and H-89; (b) the relationship between the I582 nm/I545 nm ratios and H-89 concentrations on the logarithm scale.
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CONCLUSIONS In conclusion, by integrating the outstanding features of REdoped UCNPs for highly selective recognition of PKs-induced phosphopeptides and the inherent low-background upconversion luminescence property, a mix-and-read LRET assay is developed for highly sensitive detection of PKs activity. This approach shows distinct advantages. On one hand, the use of UCNPs as the LRET donor effectively reduces the autofluorescence and light scattering background, which allows separation-free detection with greatly improved signal-tobackground ratios. On the other hand, for the first time we demonstrate that UCNPs themselves can act as excellent affinity materials to selectively capture phosphopeptides catalyzed by PKs, which also greatly simplifies the detection procedures because complicated functionalization of UCNPs with additional phospho-recognizing elements (for example, antibodies) is no longer required. Therefore, this proposed strategy provides a practical and robust platform to detect PKs activity with extremely simple mix-and-read detection, absence of background interference, and high sensitivity, which may
serve as a powerful tool for PKs analysis in signal transduction pathways and kinase-related clinical diagnosis as well as drug discovery.
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ASSOCIATED CONTENT
S Supporting Information *
Detailed optimization of experimental conditions, PKA analysis by using negatively charged UCNPs, detection results of AKT1 activities by using the UCNPs-based LRET approach, and evaluation of the interaction between the UCNPs and peptide substrates for PKC and Src. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Fax: +86 29 81530859. Notes
The authors declare no competing financial interest. 6101
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grants 21335005 and 91127035), Program for Changjiang Scholars and Innovative Research Team in University (Grant IRT 1124), and the Fundamental Research Funds for the Central Universities (Grants GK201402051 and GK201303003).
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