Amplified Chemiluminescence Surface Detection of DNA and

Feb 25, 2004 - The DNA is analyzed with a detection limit of ∼1 × 10-9 M. In the second system, telomerase from HeLa cancer cells induces telomeriz...
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Anal. Chem. 2004, 76, 2152-2156

Amplified Chemiluminescence Surface Detection of DNA and Telomerase Activity Using Catalytic Nucleic Acid Labels Valeri Pavlov,† Yi Xiao,† Ron Gill,† Arnon Dishon,‡ Moshe Kotler,‡ and Itamar Willner*,†

The Institute of Chemistry, The Farkas Center for Light-Induced Processes, The Hebrew University of Jerusalem, Jerusalem 91904, Israel, and Department of Pathology, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel

A G-rich nucleic acid sequence binds hemin and yields a biocatalytic complex (DNAzyme) of peroxidase activity, namely, the biocatalyzed generation of chemiluminescence in the presence of H2O2 and luminol. The DNAzyme is used as a label for the amplified detection of DNA, or for the analysis of telomerase activity in cancer cells, using chemiluminescence as an output signal. In one configuration, the analyzed DNA is hybridized with a primer nucleic acid that is associated with a Au surface, and the DNAzyme label is hybridized with the surface-confined analyte DNA. The DNA is analyzed with a detection limit of ∼1 × 10-9 M. In the second system, telomerase from HeLa cancer cells induces telomerization of a primer associated with a Au surface and the complementary DNAzyme units are hybridized with the telomere to yield the chemiluminescence. The detection limit of the system corresponds to 1000 HeLa cells in the analyzed sample. The preparation of DNA-based enzymes attracts substantial research efforts directed to the development of novel biocatalysts.1,2 Many different nucleic acids were employed as catalysts for different chemical transformations such as cleavage of RNA or DNA phosphoesters,3 porphyrin metalation,4 and DNA ligation.5 An interesting example of a catalytic DNA is a single-stranded guanine-rich nucleic acid (aptamer) that upon complexation with hemin revealed peroxidase activity6 (the H2O2-mediated oxidation of 2,2′-azinobis(3-ethylbenzothiozoline)-6-sulfonic acid, ABTS). It was suggested that the intercalation of the hemin into the guanine * To whom correspondence should be addressed. Tel: 972-2-6585272. Fax: 972-2-6527715. E-mail: [email protected]. † The Hebrew University of Jerusalem. ‡ The Hebrew University-Hadassah Medical School. (1) (a) Breaker, R. R. Nat. Biotechnol. 1997, 15, 427-431. (b) Breaker, R. R. Nat. Biotechnol. 1999, 17, 422-423. (2) Emilsson, G. M.; Breaker, R. R. Cell Mol. Life Sci. 2002, 59, 596-607. (3) (a) Breaker, R. R.; Joyce, G. F. Chem. Biol. 1995, 2, 655-660. (b) Carmi, N.; Shultz, L. A.; Breaker, R. R. Chem. Biol. 1996, 3, 1039-1046. (4) Li, Y. F.; Sen, D. Nat. Struct. Biol. 1996, 3, 743-747. (5) (a) Sugimoto, N.; Wakizaka, N. Nucleosides Nucleotides 1998, 17, 565574. (b) Levy, M.; Ellington, A. D. Bioorg. Med. Chem. 2001, 9, 25812587. (c) Cuenoud, B.; Szostak, J. W. Nature 1995, 375, 611-613. (6) (a) Travascio, P.; Bennet, A. J.; Wang, D. Y.; Sen, D. Chem. Biol. 1999, 6, 779-787. (b) Travascio, P.; Witting, P. K.; Mauk, A. G.; Sen, D. J. Am. Chem. Soc. 2001, 123, 1337-1348. (c) Witting, P. K.; Travascio, P.; Sen, D.; Mauk, A. G. Inorg. Chem. 2001, 40, 5017-5023.

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quadruplex docked layers of the nucleic acid leads to the biocatalytic structure. Enzymes are often employed as biocatalysts for the amplified detection of DNA. Horseradish peroxidase (HRP) was used as biocatalytic label for the amperometric detection of DNA.7 The biocatalyzed precipitation of an insoluble product on electrode surfaces was reported as a means for the amplified electrochemical sensing of DNA,8,9 and the generation of redox-active DNA replica and the secondary activation of bioelectrocatalytic processes was used for the amplified amperometric analysis of DNA.10 Recently, we reported on the analysis of DNA by the HRP-mediated generation of chemiluminescence.11 In this system, doxorubicin was intercalated into double-stranded DNA, and the doxorubicinmediated electrocatalyzed generation of H2O2 allowed the generation of chemiluminescence in the presence of luminol and HRP. The use of nucleic acids as catalytic labels (DNAzymes) for the amplified analysis of DNA may reveal several advantages: (i) The use of nucleic acids as biocatalysts might reduce the nonspecific adsorption caused by proteins to the nucleic acid interfaces or the electrode surfaces. This problem of nonspecific adsorption is often encountered in biosensing, and the use of noncomplementary negatively charged, nucleic acid labels may reduce this limitation. (ii) The base sequence in the nucleic acid label may be tailored to include the catalytic DNAzyme domain and a nucleic acid domain that is complementary and hybridizes with the analyzed DNA. Thus, the easy synthesis of the DNA label may eliminate the need for biocatalytic conjugates (e.g., avidinenzyme conjugates) or the need for intermediate labeled nucleic acids for the association of the protein-based conjugates (e.g., biotin-labeled nucleic acids). This may reduce the number of analysis steps involved in the sensing of DNA. Here we report on (7) (a) Caruana, D. J.; Heller, A. J. Am. Chem. Soc. 1999, 121, 769-774. (b) de Lumley-Woodyear, T.; Caruana, D. J.; Campbell, C. N.; Heller, A. Anal. Chem. 1999, 71, 394-398. (8) (a) Patolsky, F.; Lichtenstein, A.; Kotler, M.; Willner, I. Angew. Chem., Int. Ed. 2001, 40, 2261-2265. (b) Patolsky, F.; Katz, E.; Bardea, A.; Willner, I. Langmuir 1999, 15, 3703-3706. (9) (a) Patolsky, F.; Lichtenstein, A.; Willner, I. Nat. Biotechnol. 2001, 19, 253-257. (b) Alfonta, L.; Singh, A. K.; Willner, I. Anal. Chem. 2001, 73, 91-102. (10) Patolsky, F.; Weizmann, Y.; Willner, I. J. Am. Chem. Soc. 2002, 124, 770772. (11) (a) Patolsky, F.; Katz, E.; Willner, I., Angew. Chem., Int. Ed. 2002, 41, 3398-3402. (b) Patolsky, F.; Weizmann, Y.; Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2003, 42, 2372-2376. 10.1021/ac035219l CCC: $27.50

© 2004 American Chemical Society Published on Web 02/25/2004

the use of a catalytic nucleic acid (DNAzyme) as a label for the amplified chemiluminescence surface detection of DNA. We also apply the method for the amplified detection of telomerase activity in HeLa cancer cells. EXPERIMENTAL SECTION Materials. Hemin was purchased from Porphyrin Products (Logan, UT) and used without further purification. The concentration of diluted hemin solutions was determined using standard spectroscopic methods.12 A hemin stock solution was prepared in DMSO and stored in the dark at -20 °C. Luminol and other chemicals were obtained from Sigma and used as supplied. All buffer solutions used in the different measurements contained the nonionic detergent Triton X-100 (0.05% w/v) and DMSO (1%, v/v). DNA oligonucleotides were synthesized by Sigma Genosys. They were purified using the PAGE method. The sequences of the oligomers are as follows: 5′-HS(CH2)6CGATTCGGTACTGG3′ (1); 5′-TTGAGCATGCGCATTATCTGAGCCAGTACCGAATCG3′ (2); 5′-ATGCGCATGCTCAATTTGGGTAGGGCGGGTTGGG3′ (3); 5′-HS(CH2)6TTTTTTAATCCGTCGAGCAGAGTT-3′ (5); 5′CTAACCCTAACCTTTGGGTAGGGCGGGTTGGG-3′ (7). Immobilization of the Thiolated DNA Primer and Hybridization of the DNAzyme Label. The Au-coated (50-nm gold layer) glass plate (22 mm × 11 mm) was prepurified by treatment with a piranha solution (consisting of 70% concentrated sulfuric acid and 30% hydrogen peroxide) for 20 min and then thoroughly rinsed with pure water. The plate was then soaked in concentrated nitric acid for 5 min and rinsed again with water. The plate was interacted with a solution of 1, 6 µM in 0.4 M phosphate buffer, pH 7.4, for 12 h. The resulting plate was washed with the phosphate buffer and then the 1-functionalized Au surface was treated with 1-mercaptohexanol, 1 mM in 0.1 M phosphate buffer, pH 7.4, for 1 h. The resulting monolayer-functionalized surface was treated with different concentrations of the complementary analyte DNA 2 in a solution composed of 0.1 M phosphate buffer and the Perfect Hyb Plus hybrization buffer (Sigma) (1:1), for 5 h to yield the ds-DNA assembly on the surface. A solution of 25 µM DNAzyme 3 was heated at 95 °C for 9 min in 0.01 M Tris buffer, pH 7.4, to dissociate any intermolecular quadruplex, and allowed to cool to room temperature. An identical volume of a buffer solution consisting of 50 mM HEPES, 40 mM KCl, 400 mM NaCl, 0.1% Triton X-100, and 2% DMSO, pH 7.4, was added to the solution of 3 to allow appropriate folding. Hemin, 12 µM, was added to the nucleic acid solution, and the system was allowed to form the supramolecular complex for 3 h. The surface was then allowed to hybridize with a 2.5 µM solution of the DNAzyme in a 0.1 M phosphate buffer that included 25 mM HEPES, 20 mM KCl, 200 mM NaCl, 0.05% Triton X-100, and 1% DMSO, for 12 h. Preparation of Telomerase Extracts. HeLa cells were removed from the substrate by trypsinization, washed twice with PBS, and pelleted at 2000 rpm for 10 min at 4 °C. The cells were resuspended in a cold CHAPS lysis buffer (10 mM Tris-HCl, pH 7.4, 1 mM MgCl2, 1 mM EGTA, 0.1 mM PMSF, 0.5% CHAPS (Sigma), and 10% glycerol) at a concentration of 5 × 106 cells/ mL, incubated for 30 min in ice, and then centrifuged for 20 min (12) Lavallee, D. K. The Chemistry and Biochemistry of N-substituted Pophyrins; VCH Publications: New York, 1987.

(12 000 rpm, 4 °C). The supernatant was flash frozen and stored at -70 °C. Immobilization of 5 and Telomerization on a Au-Coated Glass Plate. The telomerase extract from the respective number of cells was introduced into a 50-µL mixture of 20 mM Tris-HCl, pH 8.3, 4 mM MgCl2, 1 mM EGTA, 63 mM KCl, 0.05% Tween 20, 2 mM dATP, 2 mM dGTP, and 2 mM dTTP. The reaction mixture, 50 µL, was placed on the Au-coated glass plate modified with 5. Modification of the plate with 5 was performed as described for 1. The plate was covered, and the telomerization was allowed to proceed for 12 h at 37 °C. The resulting plate was rinsed with a phosphate buffer solution and allowed to hybridize with the DNAzyme solution 7, 2.5 µM, that was prepared as described for the analysis of 2. For the control experiments utilizing heat-treated HeLa cells, the cell extract was heated for 10 min at 85 °C. Light Emission Measurements. Light emission was measured using a photon counting spectrometer (Edinburgh Instruments, FLS 920) equipped with a cooled photomultiplier detection system, connected to a computer (F900 v. 6.3 software). Before the sample analyses, a background run without sample was done, and all spectral results were corrected from the background and integrated. Measurements were made after the plates were placed in a cuvette that included 3.3 mL of a buffer solution consisting of 25 mM HEPES, 20 mM KCl, and 200 mM NaCl, pH 9.0, and included 0.5 mM luminol and 30 mM H2O2. The light emission intensity was measured at 1-nm intervals in the region of 300600 nm, while reading at each wavelength the photons for a time interval of 0.2 s. The integrated light signal is accumulated within 1 min for each experiment. RESULTS AND DISCUSSION Previous studies have indicated that a guanine-rich nucleic acid, with the base sequence depicted in Figure 1, structure I, is capable of forming a supramolecular G-quadruplex structure with hemin.6 The resulting complex exhibited peroxidase-like catalytic activity, and it catalyzed the oxidation of ABTS by H2O2. We find that this nucleic acid-hemin complex reveals also peroxidase-like functions toward the oxidation of luminol by H2O2 and the generation of chemiluminescence. We use this property to develop predesigned DNAzyme label for the amplified detection of DNA. Figure 1 outlines the method for the nucleic acid-based detection of DNA. The thiolated nucleic acid 1 is assembled on a Au surface and it is complementary to a part of the analyzed DNA, 2. The modified surface is hybridized with 2 on the surface. The catalytic nucleic acid label, 3, is predesigned by introducing into the label the nucleic acid sequence that produces with hemin the catalytic DNAzyme and linking to it a nucleic acid residue that can hybridize with the free 5′-end of the analyzed DNA, 2, that is associated with the surface. The hybridization of the DNA label with the resulting interface, and the formation of the supramolecular complex with hemin, yield the catalytic unit on the surface, and in the presence of H2O2 and luminol, 4, the system generates chemiluminescence. Note that chemiluminescence occurs only if the analyte DNA 2 is hybridized to the interface, provided that nonspecific adsorption is eliminated. Microgravimetric quartz crystal microbalance measurements indicate that the surface coverage of 1 on the Au surface is ∼3.1 × 10-11 mol‚cm-2. Upon treatment of the surface with 2, 0.5 µM, the surface coverage of the hybridized DNA is 7.3 × 10-12 mol‚cm-2, and the hybridization Analytical Chemistry, Vol. 76, No. 7, April 1, 2004

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Figure 1. Scheme for the analysis of a DNA using a DNAzyme label and chemiluminescence as a detection signal.

of the hemin/3 complex, 2.5 µM, with the interface yields a surface coverage of the catalytic DNA label that corresponds to ∼1.5 × 10-12 mol‚cm-2. Figure 2, curve a, shows the integrated light intensity emitted by the system upon analyzing 2, 0.5 µM. Control experiments reveal that the 1/2 hybridized interface does not emit light in the presence of hemin and H2O2/luminol, Figure 2, curve b. Also, the direct treatment of the 1-modified surface with the 3/hemin complex does not lead to any biochemiluminescence. These control experiments indicate that the 3/hemin complex acts as a catalyst for the generation of chemiluminescence, and the light emission occurs only by the intermediate hybridization of the analyte DNA with the sensing interface. The surface coverage of the hybridized analyzed DNA is controlled by the bulk concentration of 2. Hence, the emitted light is anticipated to be controlled by the concentration of 2, Figure 2, curves c-h. Figure 2, inset, shows the derived calibration curve 2154

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that corresponds to the emitted light intensity upon analyzing variable concentrations of 2. The analyzed DNA is detected with a detection limit of 1 nM. It should be noted that the control experiment shown in Figure 2, curve b represents the background light intensity originating from the system and may be considered as the noise level of nonspecific binding of hemin to the surface, This intensity is ∼1.8% of the light intensity emitted by the system that analyses 2, 0.5 µM. and the light emitted by the system analyzing 2, 1.0 nM reveals a S/N of ∼2.5. The chromosomes are protected by nucleic acids of constant repeats termed telomeres.13 The gradual erosion of the telomere units during cell proliferation provides a cellular signal for terminating the cell cycle. In certain cells, there is accumulation (13) (a) Harley, C. B.; Futcher, A. B.; Greider, C. W. Nature 1990, 345, 458460. (b) Hastie, N. D.; Dempster, M.; Dunlop, M. G.; Thompson, A. M.; Green, D. K.; Allshire, R. C. Nature 1990, 346, 866-867.

Figure 2. Integrated light intensities corresponding to (a) the analysis of 2, 0.5 µM, using the DNAzyme label 3, 2.5 µM and (b) The analysis of 2 without added DNAzyme label but upon treatment with hemin 2.5 µM. (c-h) Analyzing 2: 0.1, 0.07, 0.04, 0.01, 0.005, and 0.001 µM, respectively. Inset: Calibration curve corresponding to the analysis of 2.

of the ribonucleoprotein telomerase that incorporates the telomere units into the chromosome ends, and this turns the cells into immortal entities.14 Indeed, in over 95% of the different cancer or

malignant cells, elevated amounts of telomerase were detected,15 and the monitoring of telomerase activity in cells is promising for cancer diagnostics.16 Several analytical procedures for the determination of telomerase activity were developed, and these include the telomeric repeat amplification protocol (TRAP) method,17 the fluorescence detection of telomerase activity,18 or the recently reported19 optical detection of telomerase using CdSe/ZnS quantum dots. We have applied the catalytic DNAzyme as a label for the amplified chemiluminescence detection of telomerase activity on a functionalized surface. Figure 3 depicts the method for the amplified analysis of telomerase activity. The primer 5 is assembled on a Au surface, and the functionalized surface is interacted with the HeLa cancer cell extract in the presence of the nucleotide mixture dNTPs. Since the telomerization leads to a long nucleic acid with constant repeat units (6), the interface may be hybridized with a complementary catalytic label. The nucleic acid 7 is predesigned to include the G-rich sequence that forms the catalytic complex with hemin and a nucleic acid domain that is complementary to the telomere repeat units. The hybridization of the catalytic DNAzyme label with the telomere associated with the surface then enables the chemiluminescence detection of telomerase activity by the biocatalytic oxidation of luminol by H2O2 and the concomitant light emission. The analysis of telomerase involves two consecutive amplification steps. The first step

Figure 3. Scheme for the analysis of telomerase activity using DNAzyme labels and chemiluminescence as a detection signal.

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Figure 4. Integrated light intensities corresponding to (a) the analysis of 10 000 HeLa cells using the DNAzyme 7, 2.5 µM, (b) the analysis of heat-treated HeLa cells (10 000) in the presence of the DNAzyme, 2.5 µM, and (c) the analysis of HeLa cells (10 000) without the DNAzyme but upon interaction with hemin, 2.5 µM. (d-f) The analysis of 5000, 2500, and 1000 HeLa cells, respectively. Inset: Calibration curve corresponding to the analysis of variable numbers of cells.

involves the hybridization of several catalytic entities to the telomere and the second includes the catalytic DNAzyme that generates numerous photons as a result of a single telomere formation. The system assembled on the gold surface was characterized by quartz crystal microbalance experiments. The immobilization of 5 on the Au/quartz surface resulted in a frequency change of -40 Hz that translates to a surface coverage of 7.9 × 10-12 mol‚cm-2 of 5. The telomerization occurring upon the treatment of the functionalized surface with a cell lysate (10 000 cells) in the presence of dNTPs leads to a frequency decrease of 52 Hz, and this translates to a coverage of 6.9 × 10-11 telomere units‚cm-2. That is, an average ∼9 telomere units are linked to each primer associated with the surface (this frequency change corresponds to the incorporation of 54 bases into each primer linked to the electrode). The association of the catalytic 7/hemin label with the surface further decreases the crystal frequency by 50 Hz, indicating a surface coverage of ∼1.3 × 10-11 mol‚cm-2 or the binding of ∼2 DNAzyme units to each telomeric primer. Figure 4, curve a, shows the integrated light intensity emitted from the system upon analyzing the telomerase activity originated (14) Harley, C. B.; Villeponteau, B. Curr. Opin. Genet. 1995, 5, 249-255. (15) Wright, W. E.; Piatyszek, M. A.; Rainey, W. E.; Byrd, W.; Shay, J. W. Rev. Genet. 1996, 18, 173-179. (16) (a) Shay, J. W.; Wright, W. E. Curr. Opin. Oncol. 1996, 8, 66-71. (b) Shay, J. W.; Bacchetti, S. Eur. J. Cancer 1997, 33, 787-791. (17) Kim, N. W.; Piatyszek, M. A.; Prowse, K. R.; Harley, C. B.; West, M. D.; Ho, P. C. H.; Coviello, G. M.; Wright, W. E.; Weinricht, S. L.; Shay, J. W. Science 1994, 266, 2011-2015. (18) Schmidt, P. M.; Lehmann, C.; Matthes, E.; Bier, F. F. Biosens. Bioelectron. 2002, 17, 1081-1087. (19) Patolsky, F.; Gill, R.; Weizmann, Y.; Mokari, T.; Banin, U.; Willner, I. J. Am. Chem. Soc., in press.

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from 10 000 cells. A control experiment revealed that, upon analysis of heat-treated HeLa cells (85 °C for 10 min) according to Figure 3, no light emission is observed, Figure 4, curve b. That is, the thermal deactivation of the telomerase in the HeLa cells prevents telomerization, and the subsequent hybridization of 7 and the biocatalyzed generation of chemiluminescence is inhibited. Furthermore, this control experiment demonstrates the advantages and utility of the DNAzyme as a label for the amplified detection of DNA. The fact that no chemiluminescence is generated by the heat-treated cells implies that no nonspecific interference takes place in the system. Thus, even if cell ingredients bind nonspecifically to the surface, their affinity to the catalytic DNAzyme is negligible. Also, the treatment of the telomere units on the surface with hemin leads to only a negligible generation of chemiluminescence, Figure 4, curve c. Thus, although the telomere units includes G-bases, no biocatalytic complex is generated with hemin, and only the base sequence of 7 is specific to generate with hemin the DNAzyme of peroxidase activity. As the telomerization is controlled by the content of telomerase in the cell lysate samples, the amount of hybridized DNAzyme label, and the intensity of emitted light, should relate to the concentration of cancer cells. Figure 4 shows the integrated light intensity emitted from the system analyzing variable numbers of HeLa cells (curves d-f). As expected, the chemiluminescence decreases as the content of HeLa cells in the sample is lower. Figure 4, inset, shows the calibration curve that corresponds to the emitted light intensity as a function of the number of cells that are analyzed. The detection limit in this experiment corresponds to ∼1000 HeLa cells in the analyzed sample. CONCLUSION The present study has demonstrated a new nucleic acid-based catalytic label for the analysis of DNA. We formulated a general assay for analyzing DNA based on the hybridization of the DNAzyme to the analyte DNA. It should be noted that, although a real comparison is difficult, the present DNAzyme-based assay of DNA is ∼2 orders of magnitude lower in sensitivity as compared to the analogous analysis using horseradish peroxidase as a biocatalyst. Realizing, however, that our system is not optimized and that nonspecific adsorption processes are eliminated, one may anticipate the advantages of the present assay. Further analytical progress was achieved by the development of a telomerase activity assay based on the catalytic DNA. We believe that the reported nucleic acid/hemin biocatalyst will provide a versatile tool in different assays of nucleic acid analysis. Experiments directed to the analysis of the telomerase activity in different kinds of cancer cells are in progress. ACKNOWLEDGMENT This research is supported by The Prostate Cancer Charitable Trust (PCCT, U.K.). Received for review October 15, 2003. Accepted February 3, 2004. AC035219L