ZnS Quantum Dots

May 18, 2010 - the CK2-catalyzed phosphorylation of a serine-containing peptide (1), linked to CdSe/ZnS QDs, with Atto-590-functionalized. ATP. The se...
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Probing Protein Kinase (CK2) and Alkaline Phosphatase with CdSe/ZnS Quantum Dots Ronit Freeman, Tali Finder, Ron Gill, and Itamar Willner* Institute of Chemistry, The Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel ABSTRACT Semiconductor quantum dots (QDs) are used for the optical analysis of casein kinase (CK2) or the hydrolytic activity of alkaline phosphatase (ALP). Two schemes for the analysis of CK2 by a FRET-based mechanism are described. One approach involves the CK2-catalyzed phosphorylation of a serine-containing peptide (1), linked to CdSe/ZnS QDs, with Atto-590-functionalized ATP. The second analytical method involves the specific association of the Atto-590-functionalized antibody to the phosphorylated product. The hydrolytic activity of ALP is followed by the application of phosphotyrosine (4)-modified CdSe/ZnS QDs in the presence of tyrosinase as a secondary reporter biocatalyst. The hydrolysis of (4) yields the tyrosine units that are oxidized by O2/tyrosinase to the respective dopaquinone product. The latter quinone units quench the QDs via an electron transfer route, leading to the optical detection of the ALP activity. KEYWORDS Quantum dots, casein kinase, CdSe/ZnS, alkaline phosphatase, sensor

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phorylated product.8 Other methods included a fluorescence polarization assay,9 an optical method involving the kinaseinduced aggregation of Au nanoparticles,10 and an indirect method that probes the depletion of ATP as a result of the phosphorylation process.11 Also, label-free bioelectronic sensors for protein kinases were developed, and these included the monitoring of the phosphorylation process on a field-effect-transistor12 and an electrochemical method that applied impedance spectroscopy.13 Also, different methods to follow the activity of ALP were developed; these included the electrochemical detection of different redox-active products generated by ALP14 or a potentiometric assay.15 The use of semiconductor quantum dots (QDs) attracts growing interest in bioanalysis.16 Different QDs-based enzymatic assays were developed.17 For example, the replication of DNA in the presence of polymerase or the synthesis of telomers on nucleic acid-functionalized QDs were followed by a fluorescence resonance energy transfer process (FRET),18 the hydrolytic activities of different proteases were monitored by a FRET process involving CdSe QDs,19 the activities of NAD+-dependent enzymes were probed by a FRET reaction,20 and the activity of tyrosinase was followed by an electron-transfer quenching of CdSe/ZnS QDs.21 In the present study, we report on the analysis of casein kinase, CK2 (as a model for kinases), and on the detection of alkaline phosphatase, ALP, using CdSe/ZnS QDs as optical transducer. We implement fluorescence resonance energy transfer (FRET) and electron transfer (ET) quenching processes in the different bioanalytical platforms. Scheme 1A depicts one sensing configuration for analyzing casein kinase, CK2. The CdSe/ZnS QDs (λem ) 560 nm) were functionalized by the tethering of the peptide (1) to glutathione (GSH)-modified QDs (see Supporting Information). The peptide sequence (1) is recognized by CK2 and

rotein kinases represent a large family of enzymes that modulate the activity of proteins by their phosphorylation, and they play a key role in signal transduction and regulation of intracellular processes.1 One of the important protein kinases is casein kinase (CK2), a serine/ threonine selective protein kinase that was found to phosphorylate more than 160 different proteins. It was reported that CK2 is involved in signal transduction, transcription control, apoptosis, and more.2 Aberrant activity of CK2 was related to a number of diseases. For example, reduced activity of CK2 was found in neurons of patients with Alzheimer’s disease,3 while elevated amounts of CK2 were found in various types of cancer.4 Particular interest in analyzing kinase activities originates from the growing need to develop inhibitors toward kinases as a therapeutic means. For example, CK2 plays an important role in the cell cycle of HIV-1, and inhibitors of CK2 act as HIV-1 transcription inhibitors.5 The hydrolytic cleavage of phosphate esters by phosphatases represents the reverse reaction stimulated by kinases. A representative of this class of enzymes is alkaline phosphatase, ALP, which concentrates in the liver and bones, and monitoring its activity is an indicator for liver or bone diseases.6 Indeed, substantial efforts are directed to the development of rapid and sensitive analytical methods for following the activities of kinases (e.g., CK2) and phosphatases (e.g., ALP). Different methods to monitor the activities of protein kinases have been developed, such as, the radioactive labeling of the phosphorylated product with radioactive ATP7 or the use of immunoassays that involve fluorophore-labeled antibodies that are specific to the phos* To whom correspondence should be addressed. E-mail: [email protected]. Tel: 972-2-6585272. Fax: 972-2-6527715. Received for review: 03/25/2010 Published on Web: 05/18/2010 © 2010 American Chemical Society

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SCHEME 1. Processesa

Optical Analysis of Casein Kinase, CK2, via FRET

FIGURE 1. (A) Time-dependent luminescence spectra corresponding to the (1)-modified QDs upon treatment with γ-ATP-Atto-590 (2). (a) Before the addition of CK2, and (b-g) after the interaction with CK2, 1 unit. Spectra were recorded at time intervals of 7 min. (B) Calibration curve corresponding to the optical analysis of CK2. The calibration curve corresponds to the degree of the luminescence quenching of the (1)-modified QDs at variable concentrations of CK2. All experiments were performed after a fixed time-interval of 50 min in a Tris buffer (20 mM, pH 7.5) that included KCl (50 mM) and MgCl2 (10 mM) in the presence of the (1)-modified QDs, 10 nM; the fluorophore-labeled ATP (2), 50 nM, and the respective concentrations of CK2.

a (A) By the biocatalytic phosphorylation of the (1)-functinalized QDs with γ-ATP-Atto-590, (2). (B) By the binding of Atto-590-modified antiphosphoserine-antibody, (3), to the phosphorylated (1)-functionalized QDs.

includes the serine unit for phosphorylation. Reaction of the modified QDs with γ-adenosine triphosphate-Atto-590, γ-ATPAtto-590, (2), results in the phosphorylation of the serine unit with the fluorophore-labeled γ-phosphate, leading to the fluorescence resonance energy transfer, FRET, from the QDs to the fluorophore acceptor. Figure 1A shows the timedependent luminescence changes of the CdSe/ZnS QDs system upon treatment with γ-ATP-Atto-590 (2) and CK2, 1 unit. The luminescence of the QDs (λem ) 560 nm) decreases with time while the fluorescence of the Atto-590 label (λem ) 634 nm) is intensified with the progress of the reaction. Control experiments revealed that only minute change in the luminescence of the QDs was observed in the presence of γ-ATP-Atto-590 (2), but in the absence of CK2. These results imply that CK2 catalyzed the phosphorylation of the serine unit to yield the Atto-590-functionalized peptide. The spatial proximity between the QDs and the dye, and the appropriate spectral overlap between the luminescence of the QDs and the absorbance of Atto-590 dye, resulted in the FRET from the QDs to the acceptor dye. To further support the FRET mechanism, we estimated the Fo¨rster distance (R0) for energy transfer, and derived from it the approximated distance (R) separating the donor-acceptor pair. The spectral features of the Atto-590 are provided in the Supporting © 2010 American Chemical Society

Information. The extracted R0 value corresponded to 5.4 nm, and this translated to a value of R ) 4.8 nm, separating the donor-acceptor pair. This value is close to the estimated distance (by geometrical bond considerations) separating the acceptor dye from the modified QDs, ca. 4 nm. Realizing that the FRET process probes the phosphorylation reaction, we examined the quantitative assay of CK2 by probing the extent of phosphorylation of (1) by variable concentrations of CK2 using a fixed time-interval for the phosphorylation reaction that corresponded to 50 min. Figure 1B shows the resulting calibration curve corresponding to the FRET quenching degree of the luminescence of the modified QDs upon interaction with different concentrations of CK2. As the concentration of CK2 increases, the FRET process is intensified, consistent with a higher content of the fluorophore-labeled phosphorylated product. This method enabled us to detect CK2 with a sensitivity corresponding to 0.1 units. 2193

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the incorporation of the Atto-590-labeled phosphate units into the serine unit of (1) in the presence of CK2. This suggests that the rate-limiting step in the analysis of CK2 using the immuno-complex configuration, Scheme 1B, is the phosphorylation process, rather than the binding of the labeled antibody to the phosphorylated antigen. Control experiments revealed that the luminescence of the (1)modified QDs is not affected in the presence of CK2 and the labeled antibodies in the absence of ATP. This implies that the antibody is specific for the phosphorylated product, and that no nonspecific adsorption of the antibody to the QDs occurs. Figure 2B shows the calibration curve corresponding to the quenching degree of the luminescence of the modified QDs upon analysis of different concentrations of CK2 by the antibody platform. In these experiments, the phosphorylation process was allowed to proceed for a fixed time-interval of 50 min, and the respective FRET process from the QDs to the Atto-590 was recorded. As the concentration of CK2 is elevated, the FRET emission of the Atto-590 is intensified, consistent with the higher content of the phosphorylated product. This method enables the detection of ca. 0.1 units of the enzyme. We then applied the QDs to detect the activity of phosphatases, specifically, alkaline phosphatase, ALP. The analysis approach is schematically outlined in Scheme 2A that implement O-phospho-L-tyrosine (4)-modified QDs (λem ) 620 nm, average loading ca. 30 ( 10 units of (4) per particle, see Supporting Information). The analysis of ALP is based on our previous observation that tyrosinase hydroxylates tyrosine to the respective DOPA derivative, and it catalyzes the oxidation of the product to the dopaquinone. The latter quenches via electron transfer the luminescence of the QDs.21 Accordingly, the ALP-induced hydrolysis of the (4)modified QDs yields the tyrosine (5)-functionalized QDs that, in the presence of the coadded tyrosinase, acting as catalytic reporter, results in the catalytic oxidation that yields the dopaquinone (6)-modified QDs. Thus, the activity of ALP can be monitored via the quenching of the luminescence of the QDs. Figure 3A shows the time-dependent luminescence quenching of the CdSe/ZnS QDs upon treatment of the (4)functionalized QDs with ALP, 0.13 units, in the presence of tyrosinase, 25 units. As the hydrolytic cleavage of (4) is prolonged, the quenching of the QDs is enhanced, consistent with the increase in the content of the dopaquinone quencher units with time. Control experiments indicated that in the absence of tyrosinase or in the presence of tyrosinase under nitrogen, no quenching of the QDs occurred. This implies that the formation of the dopaquinone quencher proceeds only in the presence of tyrosinase, and that the supply of oxygen is essential for this reaction. Realizing that tyrosinase transforms the ALP-generated product into the dopaquinone quencher, we used this process to detect and follow ALP. Figure 3B shows the degree of quenching of the luminescence of the (4)-modified QDs upon interaction with different concentrations of ALP for a fixed time-interval of 90 min

FIGURE 2. (A) Time-dependent luminescence spectra of the (1)modified QDs upon the CK2 phosphorylation of the peptide by ATP in the presence of the Atto-590-modified-antiphosphoserine-antibody (3). (a) Before the addition of CK2 (b-f) after the interaction with CK2, 1 unit. Spectra were recorded at time intervals of 8 min. (B) Calibration curve corresponding to the optical analysis of CK2. The calibration curve corresponds to the degree of the luminescence quenching of the (1)-modified QDs at variable concentrations of CK2. All experiments were performed after a fixed time-interval of 50 min in Tris buffer (20 mM, pH 7.5), that included KCl (50 mM) and MgCl2 (10 mM) in the presence of the modified QDs, 10 nM; ATP, 100 µM; the atto-590-modified antibody, (3), 50 nM, and the respective concentrations of CK2.

Scheme 1B depicts a second QDs-based CK2 sensing configuration. The (1)-functionalized QDs were reacted with CK2 and ATP to yield the phosphorylated peptide. The resulting functionalized particles were then interacted with the Atto-590-modified antiphosphoserine-antibody (3). The association of (3) to the QDs resulted in the FRET process from the QDs to the fluorophore, thus providing the optical readout for the phosphorylation process. Figure 2A depicts the time-dependent luminescence spectra of the (1)-modified QDs upon reaction with ATP, 100 µM, and CK2, 1 unit, in the presence of (3), (average loading of ca. 3 Atto-590 units per antibody). As the time of interaction of the (1)-functionalized QDs with CK2 is prolonged, the FRET-stimulated luminescence of Atto-590 at 634 nm is intensified, consistent with the increase in the content of the phosphorylated peptide and, thus, a higher content of the resulting immunocomplex. The time-dependent increase in the FRET signal of Atto-590 is very similar to the kinetic profile observed for © 2010 American Chemical Society

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SCHEME 2. Optical Analysis of the Hydrolytic Activity of Alkaline Phosphatase, ALP, via an Electron-Transfer Quenching Path and Using Tyrosinase in the Presence of O2 As Biocatalytic Reportera

FIGURE 3. (A) Time-dependent luminescence spectra of the (4)modified QDs upon treatment with tyrosinase, 25 units, in the presence of O2. (a) Before the addition of ALP and (b-t) after the interaction with ALP, 0.13 units. Spectra were recorded at time intervals of 5 min. (B) Calibration curve corresponding to the optical analysis of ALP. The calibration curve corresponds to the degree of the luminescence quenching of the (4)-modified QDs at variable concentrations of ALP. All experiments were performed in a 10 mM phosphate buffer, pH ) 7.5, in the presence of the QDs, 10 nM; tyrosinase, 25 units and the respective concentrations of ALP that were interacted for 90 minutes, prior to the recording of the fluorescence spectra.

a (A) By the application of the phosphorylated tyrosine substrate, (4)modified QDs. (B) By using the phosphorylated (7)-modified QDs as substrate.

in the presence of tyrosinase. As the concentration of ALP increases, the quenching of the QDs is enhanced. The results indicate that ALP can be detected with a sensitivity that corresponds to ca. 0.01 units. Similar results were observed when the CdSe/ZnS QDs (λem ) 620 nm) were functionalized with the phosphorylated peptide, (7), Scheme 2B. (The average loading of (7) per particle corresponded to 8 ( 2 units, see Supporting Information.) In this system, the phosphorylated peptide, (7), was subjected to the two enzymes, ALP and tyrosinase. The hydrolytic cleavage of the phosphorylated peptide by ALP was followed by the oxidation of the resulting tyrosine to the dopaquinone residue that quenches the luminescence of the QDs. Figure 4A shows the time-dependent quenching of the QDs by the dopaquinoneformed peptide, as a result of the ALP hydrolysis of the respective phosphoester, and the tyrosinase-catalyzed oxidation of the tyrosine residue by O2 to the respective dopaquinone-functionalized peptide. Similarly, Figure 4(B) depicts the resulting calibration curve upon analyzing different concentrations of ALP. In conclusion, the present study has implemented QDs as an optical labels for the detection of the activities of casein © 2010 American Chemical Society

kinase, CK2, and of alkaline phosphatase, ALP, as a model for analyzing other kinases or phosphatases. We describe two methods for the detection of the activities of CK2 and two methods for analyzing ALP. While the two methods for analyzing CK2 yield comparable sensitivities, the two methods described for the detection of ALP demonstrate that the analysis of ALP by the tyrosine residue that is directly linked to the QDs, Scheme 2A, is ca. 10-fold more sensitive than the method using the tyrosine-containing peptide, Scheme 2B. This is reasonable because the resulting quinone quencher is closer to the QDs in the configuration shown in Scheme 2A, as compared to the structure shown in Scheme 2B. The major advantage of providing the two methods for analyzing the kinase and the two methods to follow the phosphatase rests, however, on the fact that these allow the multiplexed analysis of several kinases or several phosphatases. One way is to use different-sized QDs and combine the direct phosphorylation or the generation of immuno-complexes to follow simultaneously different enzymes. While the sensitivity for the detection of CK2 is comparable to other previous 2195

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(2) (3)

(4) (5) (6)

(7) (8) (9)

(10)

(11) (12) (13) FIGURE 4. (A) Time-dependent luminescence spectra of the (7)modified QDs upon treatment with tyrosinase, 25 units, in the presence of O2. (a) Before the addition of ALP and (b-w) after the interaction with ALP, 0.5 units. Spectra were recorded at time intervals of 4 min. (B) Calibration curve corresponding to the optical analysis of ALP. The calibration curve corresponds to the degree of the luminescence quenching of the (7)-modified QDs at variable concentrations of ALP. All experiments were performed in a 10 mM phosphate buffer, pH ) 7.5, in the presence of the QDs, 10 nM; tyrosinase, 25 units and the respective concentrations of ALP that were interacted for 90 min, prior to the recording of the fluorescence spectra.

(14) (15) (16)

(17)

studies7-11 and our approach to analyze ALP is better than the reported methods,14,15 the main advantage of the present study seems to be the possibility for multiplexed assay of the enzymes. (18)

Acknowledgment. This research is supported by the EC NANOGNOSTICS research project.

(19)

Supporting Information Available. Materials and methods. This material is available free of charge via the Internet at http://pubs.acs.org. REFERENCES AND NOTES (1)

(20)

(a) Nishizuka, Y. Nature. 1984, 308, 693–698. (b) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam, S. Science. 2002, 298, 1912–1934.

© 2010 American Chemical Society

(21)

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Wang, H.; Davis, A.; Yu, S.; Ahmed, K. Mol. Cell. Biochem. 2001, 227, 167–174. (a) Flajolet, M.; He, G.; Heiman, M.; Lin, A.; Nairn, A. C.; Greengard, P. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 4159–4164. (b) Hanger, D. P.; Byers, H. L.; Wray, S.; Leung, K.; Saxton, M. J.; Seereeram, A.; Reynolds, C. H.; Ward, M. A.; Anderton, B. H. J. Biol. Chem. 2007, 282, 23645–23654. Critchfield, J. W.; Coligan, J. E.; Folks, T. M.; Butera, S. T. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 6110–6115. Houseman, B. T.; Huh, J. H.; Kron, S. J.; Mrksich, M. Nat. Biotechnol. 2002, 20, 270–274. (a) Gomez, B., Jr.; Ardakani, S.; Ju, J.; Jenkins, D.; Cerelli, M. J.; Daniloff, G. Y.; Kung, V. T. Clin. Chem. 1995, 41, 1560–1566. (b) Moss, D. W. Clin. Chem. 1982, 28, 2007–2016. Lehel, C.; Daniel-Issakani, S.; Brasseur, M.; Strulovici, B. Anal. Biochem. 1997, 244, 340–346. Till, J. H.; Annan, R. S.; Carr, S. A.; Miller, W. T. J. Biol. Chem. 1994, 269, 7423–7428. (a) Fowler, A.; Swift, D.; Longman, E.; Acornley, A.; Hemsley, P.; Murray, D.; Unitt, J.; Dale, I.; Sullivan, E.; Coldwell, M. Anal. Biochem. 2002, 308, 223–231. (b) Turek-Etienne, T. C.; Lei, M.; Terracciano, J. S.; Langsdorf, E. F.; Bryant, R. W.; Hart, R. F.; Horan, A. C. J. Biomol. Screening. 2004, 9, 52–61. (a) Wang, Z.; Levy, R.; Fernig, D. G.; Brust, M. J. Am. Chem. Soc. 2006, 128, 2214–2215. (b) Oishi, J.; Asami, Y.; Mori, T.; Kang, J. H.; Tanabe, M.; Niidome, T.; Katayama, Y. ChemBioChem. 2007, 8, 875–879. Kupcho, K.; Somberg, R.; Bulleit, B.; Goueli, S. A. Anal. Biochem. 2003, 317, 210–217. Freeman, R.; Gill, R.; Willner, I. Chem. Commun. 2007, 3450–3452. Wilner, O. I.; Guidotti, C.; Wieckowska, A.; Gill, R.; Willner, I. Chem.sEur. J. 2008, 14, 7774–7781. Ito, S.; Yamazaki, S.; Kano, K.; Ikeda, T. Anal. Chim. Acta 2000, 424, 57–63. Hassan, S. S. M.; Sayour, H. E. M.; Kamel, A. H. Anal. Chim. Acta 2009, 640, 75–81. (a) Gill, R.; Zayats, M.; Willner, I. Angew. Chem., Int. Ed. 2008, 47, 7602–7625. (b) Algar, W. R.; Krull, U. J. Anal. Bioanal. Chem. 2008, 391, 1609–1618. (c) Frasco, M. F.; Chaniotakis, N. Anal. Bioanal. Chem. 2010, 396, 229–240. (a) Schubert, K.; Khalid, W.; Yue, Z.; Parak, W. J.; Lisdat, F. Langmuir. 2010, 26, 1395–1400. (b) Wang, Z.; Xu, Q.; Wang, H. Q.; Yang, Q.; Yu, J. H.; Zhao, Y. D. Sens. Actuators, B. 2009, 138, 278–282. (c) Cavaliere-Jaricot, S.; Darbandi, M.; Kuc¸ur, E.; Nann, T. Microchim. Acta. 2008, 160, 375–383. (d) Stoll, Ch.; Gehring, C.; Schubert, K.; Zanella, M.; Parak, W. J.; Lisdat, F. Biosens. Bioelectron. 2008, 24, 260–265. (e) Duong, H. D.; Rhee, J. I. Talanta 2007, 73, 899–905. Patolsky, F.; Gill, R.; Weizmann, Y.; Mokari, T.; Banin, U.; Willner, I. J. Am. Chem. Soc. 2003, 125, 13918–13919. (a) Xia, Z.; Xing, Y.; So, M.; Koh, A. L.; Sinclair, R.; Rao, J. Anal. Chem. 2008, 80, 8649–8655. (b) Medintz, I.; Clapp, A. R.; Brunel, F. M.; Tiefenbrunn, T.; Uyeda, H. T.; Chang, E. L.; Deschamps, J. R.; Dawson, P. E.; Mattoussi, H. Nat. Mater. 2006, 5, 581–589. (c) Shi, L.; De Paoli, V.; Rosenzweig, N.; Rosenzweig, Z. J. Am. Chem. Soc. 2006, 128, 10378–10379. Freeman, R.; Gill, R.; Shweky, I.; Kotler, M.; Banin, U.; Willner, I. Angew. Chem., Int. Ed. 2009, 48, 309–313. Gill, R.; Freeman, R.; Xu, J.; Willner, I.; Winograd, S.; Shweky, I.; Banin, U. J. Am. Chem. Soc. 2006, 128, 15376–15377.

DOI: 10.1021/nl101052f | Nano Lett. 2010, 10, 2192-–2196