Optical, Electrical and Surface Plasmon Resonance Methods for

Sep 17, 2010 - The hybridization of Au NPs functionalized with nucleic acids complementary to the telomere repeat units with the telomeres amplifies t...
1 downloads 10 Views 285KB Size
Download PDF
Anal. Chem. 2010, 82, 8390–8397

Letters to Analytical Chemistry Optical, Electrical and Surface Plasmon Resonance Methods for Detecting Telomerase Activity Etery Sharon,† Ronit Freeman,† Michael Riskin,† Noa Gil,‡ Yehuda Tzfati,‡ and Itamar Willner*,† Institute of Chemistry, The Center for Nanoscience and Nanotechnology, and Department of Genetics, The Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel Three different sensing platforms for the analysis of telomerase activity in human cells are described. One sensing platform involves the label-free analysis of the telomerase activity by a field-effect-transistor (FET) device. The telomerase-induced extension of a primer associated with the gate of the FET device, in the presence of the nucleotide mixture dNTPs, alters the gate potential, and this allows the detection of telomerase extracted from 65 ( 10 293T (transformed human embryonic kidney) cells/ µL. The second sensing platform involves the optical detection of telomerase using CdSe/ZnS quantum dots (QDs). The telomerase-stimulated telomerization of the primer-functionalized QDs in the presence of the nucleotide mixture dNTPs results in the synthesis of the G-rich telomeres. The stacking of hemin on the self-organized G-quadruplexes found on the telomers results in the electron transfer quenching of the QDs, thus providing an optical readout signal. This method enables the detection of telomerase originating from 270 ( 20 293T cells/ µL. The third sensing method involves the amplified surface plasmon resonance (SPR) detection of telomerase activity. The telomerization of a primer associated with Au film-coated glass slides, in the presence of telomerase and the nucleotide mixture (dNTPs), results in the formation of telomeres on the surface, and these alter the dielectric properties of the surface resulting in a shift in the SPR spectrum. The hybridization of Au NPs functionalized with nucleic acids complementary to the telomere repeat units with the telomeres amplifies the SPR shifts due to the coupling between the local plasmon of the NPs and the surface plasmon wave. This method enables the detection of telomerase extracted from 18 ( 3 293T cells/µL. Telomeres cap and protect the eukaryotic chromosome ends from undesired degradation, recombination, or end-to-end fusion. * To whom correspondence should be addressed. Phone: 972-2-6585272. Fax: 972-2-6527715. E-mail: [email protected]. † Institute of Chemistry, The Center for Nanoscience and Nanotechnology. ‡ Department of Genetics.

8390

Analytical Chemistry, Vol. 82, No. 20, October 15, 2010

They are composed of short tandem DNA repeats (TTAGGG in vertebrates) covered with specialized proteins. In human somatic cells, telomeres are progressively shortened during cell proliferation and, at a certain length, signal the cell to arrest its life cycle.1-4 Telomerase is a ribonucleoprotein reverse transcriptase that binds to the telomere ends and elongates them by copying telomeric repeats from its endogenous RNA template.5 The elongation of the telomeres by telomerase compensates for their natural shortening, thus it extends the cellular lifespan and may lead to malignant transformation. Indeed, in over 85% of the different cancer types, elevated amounts of telomerase are detected.6-10 Accordingly, the rapid, affordable, and sensitive analysis of telomerase activity is important for diagnosis and development of anticancer therapeutic treatments. Different analytical methods to assay telomerase activity were developed. The most frequently used method is the telomeric repeat amplification protocol (TRAP).11 However, TRAP is limited since it is based on polymerase chain reaction (PCR) amplification, which is time-consuming, susceptible to inhibition by cell-extract,12 and requires expensive equipment and reagents. Furthermore, the exponential amplification of the telomerase products increases the risk of false positive and makes accurate quantification very difficult. Other reported methods for detecting telomerase activity include optical means, such as imaging of the extended telomerase products by fluorophore-labeled hybridization probes, and fluorescence reso(1) Moyzis, R. K.; Buckingham, J. M.; Cram, L. S.; Dani, M.; Deaven, L. L.; Jones, M. D.; Meyne, J.; Ratcliff, R. L.; Wu, J. R. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 6622–6626. (2) Bryan, T. M.; Cech, T. R. Curr. Opin. Cell. Biol. 1999, 11, 318–324. (3) Hayflick, L.; Moorhead, P. S. Exp. Cell. Res. 1961, 25, 585–621. (4) Olovnikov, A. M. Exp. Gerontol. 1996, 31, 443–448. (5) Moyzis, R. K.; Buckingham, J. M.; Cram, L. S.; Dani, M.; Deaven, L. L.; Jones, M. D.; Meyne, J.; Ratliff, R. L.; Wu, J. R. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 6622–6626. (6) Wright, W. E.; Piatyszek, M. A.; Rainey, W. E.; Shgy, J. W. Dev. Genetics 1996, 181, 73–18179. (7) Shay, J. W.; Bacchetti, S. Eur. J. Cancer. 1997, 33, 787–789. (8) Kim, N. W. Eur. J. Cancer. 1997, 33, 781–786. (9) Morin, G. B. Cell 1989, 59, 521–529. (10) Rhyu, M. S. Natl. J. Cancer Int. 1995, 87, 884–894. (11) Kim, N. W.; Piatyszek, M. A.; Prowse, K. R.; Harley, C. B.; West, M. D.; Ho, P. L.; Coviello, G. M.; Wright, W. E.; Weinrich, S. L.; Shay, J. W. Science 1994, 266, 2011–2015. (12) Kim, N. W.; Wu, F. Nucleic Acids Res. 1997, 25, 2595–2597. 10.1021/ac101976t  2010 American Chemical Society Published on Web 09/17/2010

nance energy transfer (FRET) stimulated between quantum dots and a dye incorporated into the telomeres.13 Also, the colorimetric detection of the extended telomeric sequences based on their ability to form a quadruplex hairpin with Hoogsteen-type base pairing and a catalytic DNAzyme activity,14 the labeling of the extended telomeric sequences attached to Au nanoparticles by horseradish peroxidase mimicking DNAzyme to generate chemiluminescence,15 or the amplified fluorescence analysis by an isothermal circular displacement polymerization process.16 Different electrochemical methods to detect telomerase activity were also reported, including the use of the ferrocenyl naphthalene diimide binder to the telomeric tetraplex structure,17 and the coulometric analysis of Ru(NH3)63+ that binds to the duplex DNA regions of telomeres hybridized with complementary labels.18 Other reported telomerase assays involved the surface plasmon resonance (SPR) detection of the elongated telomeres on Au chips19 or the magnetic beads-induced magnetic separation of the telomeres and their chemiluminescence-amplified detection.20 In addition, different nanotechnology-based methods to detect telomerase activity were reported, including the magnetomechanical deflection of cantilevers resulting in the association of modified magnetic nanoparticles to the telomeres generated on the cantilevers,21,22 and the catalytic enlargement of Au nanoparticle labels that bind to the telomeric products, with gold.23 The sensitivity of the different optical or electronic methods are, however, usually insufficient, requiring a preceding-PCR amplification step, which is by itself problematic, as described above. In the present study we describe three new methods to analyze the activity of telomerase. The first method involves the incorporation of hemin into the G-quadruplex structure generated by the extended G-rich telomeric sequences associated with CdSe-ZnS quantum dots (QDs) and the resulting electron transfer quenching of the QDs by the hemin units. The second method involves the electronic detection of the extended telomerase substrate on a field-effect-transistor device. The third method includes the surface plasmon resonance (SPR) detection of the telomerization process and the amplification of the sensing platform by the binding of Au nanoparticles labels to the telomeres. We discuss the sensitivities of the different methods and reveal that the amplified SPR detection of the telomerization process can detect telomerase activity that originates from 18 ± 3 293T cells/µL. (13) Patolsky, F.; Gill, R.; Weizmann, Y.; Mokari, T.; Banin, U.; Willner, I. J. Am. Chem. Soc. 2003, 125, 13918–13919. (14) Xiao, Y.; Pavlov, V.; Niazov, T.; Dishon, A.; Kotler, M.; Willner, I. J. Am. Chem. Soc. 2004, 126, 7430–7431. (15) Niazov, T.; Pavlov, V.; Xiao, Y.; Gill, R.; Willner, I. Nano Lett. 2004, 4, 1683–1687. (16) Ding, C.; Li, X.; Ge, Y.; Zhang, S. Anal. Chem. 2010, 82, 2850–2855. (17) Sato, S.; Kondo, H.; Nojima, T.; Takenaka, S. Anal. Chem. 2005, 77, 7304– 7309. (18) Li, Y.; Liu, B.; Li, X.; Wei, Q. Biosens. Bioelectron. 2010, 25, 2543–2547. (19) Maesawa, C.; Inaba, T.; Sato, H.; Lijima, S.; Ishida, K.; Terashima, M.; Sato, R.; Suzuki, M.; Yashima, A.; Ogasawara, S.; Oikawa, H.; Sato, N.; Saito, K.; Masuda, T. Nucleic Acids Res. 2003, 31, e4. (20) Zhou, X.; Xing, D.; Zhu, D. Anal. Chem. 2009, 81, 255–261. (21) Grimm, J.; Perez, J. M.; Josephson, L.; Weissleder, R. Cancer Res. 2004, 64, 639–643. (22) Weizmann, Y.; Patolsky, F.; Liubashevski, O.; Willner, I. J. Am. Chem. Soc. 2004, 126, 1073–1080. (23) Weizmann, Y.; Patolsky, F.; Popov, I.; Willner, I. Nano Lett. 2004, 4, 787– 792.

EXPERIMENTAL SECTION Materials and Reagents. Ultrapure water from NANOpure Diamond (Barnstead Int., Dubuque, IA) source was used throughout the experiments. Hemin was purchased from Porphyrin Products (Logan, Utah) and used without further purification. The concentration of diluted hemin solutions was determined using the standard spectroscopic method.24 Hemin stock solution was prepared in DMSO and stored in the dark at -20 °C. Hops Yellow Core Shell EviDots, CdSe/ZnS Quantum dots in toluene were purchased from Evident Technologies. N-[e-Maleimidocaproyloxy]succinimide ester (EMCS) was purchased from Pierce Biotechnologies. The DNA constructs were purchased from Sigma Life Science (U.K.). All oligonucleotides were HPLC-purified and freeze-dried by the supplier. The oligonucleotides were used as provided and diluted in 10 mM phosphate buffer solution, pH 7.4, to give stock solutions of 100 µM. The deoxynucleotide solution set, 25 µM each, was purchased from New England BioLabs. The sequences of the oligomers are as follows: (1) 5′-HS(CH2)6 TTTTAATCCGTCGAGCAGAGTT-3′; (2) 5′-CCCTAACCCTAAAAAA (CH2)3SH-3′. Preparation of GSH-Capped QDs. QDs were precipitated from the toluene solution by addition of 2 mL of methanol to 0.5 mL of the QDs in toluene, followed by centrifugation for 5 min at 3000 rpm. The resulting precipitate was dissolved in 1 mL of chloroform, to which was added 200 µL of a glutathione, GSH, solution (containing 0.142 g of GSH and 40 mg of KOH in 2 mL of methanol), and the resulting mixture was shaken. After the addition of 1.5 mL of 1 mM NaOH solution in water, all particles were transferred to the water phase. The QDs solution was separated from the chloroform by centrifugation for 1 min. The excess of GSH was removed by two successive precipitation steps of QDs, using NaCl and methanol followed by centrifugation. The resulting QDs were dissolved in 200 µL of a 10 mM HEPES buffer, pH ) 7.4. It should be noted that the GSH-capped QDs exhibit high stabilities, and they can be stored for several months without precipitation while retaining their photophysical properties. Also, the particles are stable in the pH range of 6.3-8.8, and are not affected by the change of the buffer solution. Preparation of Nucleic Acid-Capped QDs. To the GSHcapped QDs (1 nmol) in HEPES buffer, 200 µL, were added 100 µL of an EMCS stock solution (1 mg mL-1), and the mixture was shaken for 15 min. The QDs were purified by precipitation by the addition of 1 mL of methanol and 3 mg of NaCl to remove excess of EMCS, and the QDs were redissolved in 10 mM HEPES buffer (pH 7.4). The DNA stock solution 1 × 10-4 M was added, and then the resulting solution was shaken for 2.5 h. Finally, the excess of DNA was removed by precipitation of the QDs, and the purified particles were dissolved in phosphate buffer solution (100 µL, pH 7.4, 10 mM). The resulting QDs were stable for at least 4 weeks with no noticeable precipitation or change in their luminescence properties. (24) Lavallee, D. K. The Chemistry and Biochemistry of N-Substituted Porphyrins; VCH Publications: New York, 1987.

Analytical Chemistry, Vol. 82, No. 20, October 15, 2010

8391

Preparation and Functionalization of Au-NPs. The 13 nm AuNPs were prepared using a standard citrate method.25 The concentration of the 13 nm Au-NPs was determined by following the absorbance spectra, λ ) 519 nm, and by using the appropriate extinction coefficient. Before DNA loading, the thiol functionality on nucleic acid 1 was deprotected by soaking the oligonucleotide in 0.1 M phosphate buffer (pH ) 8) containing 0.1 M dithiothreitol for at least 2 h. Then, aliquots of the deprotected DNA solution were purified through G-25 microspin columns. AuNPs were functionalized by derivatizing aqueous Au colloids with deprotected thiol-oligonucleotides (final concentration, 10 mM for oligonucleotides and 10 nM for AuNPs). The resulting solution was then incubated at room temperature for 24 h. Afterwards, the NaCl concentration of the solution was increased to 100 mM, and the resulting solution was incubated for an additional 24 h at room temperature. The resulting DNA-AuNPs were purified by three consecutive precipitation/resuspension steps by separating the precipitate by centrifugation (30 000g for 45 min), and the resuspension of the Au-NPs in a 10 mM phosphate buffer (pH ) 7.0). Preparation of the 1-Modified SPR Gold Surfaces. Aucoated semitransparent glass slides (0.5 mm thickness, Militec GmbH, Analytical µ-Systems, Germany) were used for the SPR measurements. Prior to modification, the Au electrode was cleaned in a hot ethanol for 5 min, followed by a gentle rinse with water, and subsequently dried under nitrogen. The clean Au SPR slides were reacted with 1 × 10-4 M of 1 for 24 h in a 10 mM phosphate buffer solution (pH ) 7.4), at room temperature. The slides were then rinsed with the same buffer solution and dried under argon. The surface coverage of 1 on the SPR gold surfaces was determined to be ∼2.1 × 10-12 mol cm-2 by chronocoulometry,26 using Ru(NH3)63+ as the redox label. Preparation of the 1-Modified Ion-Sensitive Field-Effect Transistor (ISFET) Devices. The primary modification of the Al2O3 gate of an ISFET device was achieved by the treatment of the ISFET with 3-aminopropyltriethoxysilane (0.2 mL, 10% (v/v) solution in toluene) at room temperature for 12 h. The silylated chips were thoroughly rinsed with toluene and then with water. Nucleic acid 1 was covalently linked to the aminosiloxane-functionalized gate interface by treatment of the gate with EMCS (1 mg/mL, Hepes 10 mM, pH ) 7.4) at room temperature for 30 min. The chips were rinsed with water, then with a HEPES buffer solution (10 mM, pH 7.4), and then treated with 1 (0.2 mL) for 2 h. Knowing the surface area of the gate surface (0.2 cm2) and using transconductance measurements,27 the surface coverage of 1 was estimated to be ∼1.2 × 10-12 mol cm-2. Telomerase Extract Preparation. 293T, a transformed human embryonic kidney (HEK) cell line, was used for telomerase positive cells. Primary human foreskin fibroblasts were used as a telomerase negative control. Cells were grown in DMEM supplemented with 10% fetal calf serum and 1% nonessential amino acids (for the fibroblasts only), collected by trypsinization and centrifugation, counted, washed twice in PBS, aliquotted, and stored as

cell pellets at -80 °C until extraction by the CHAPS method.28 Cells were collected in the exponential phase of growth. Typically, an aliquot containing 40 × 106 cells was resuspended in 200 µL of ice cold lysis buffer (0.5% CHAPS, 10 mM Tris-HC1, pH 7.5, 1 mM MgC12, 1 mM EGTA, 5 mM β-mercaptoethanol, 0.1 mM PMSF, 10% glycerol) by pipetting at least three times and incubated in ice for 30 min to thoroughly lyse the cells. The lysate was then centrifuged at 14 000 rpm for 30 min at 4 °C, and the supernatant was collected carefully and aliquotted into several 1.5 mL Eppendorf tubes (about 25 µL per tube). The resulting extract was used immediately or was flash-frozen in liquid nitrogen and stored at -80 °C. Telomerization on the 1-Modified QDs/ISFET Electrode/ SPR Au Surface. The telomerization reaction was performed by using telomerase primer 1-modified QDs/ISFET electrode/SPR Au surface, in the presence of dATP, dTTP, dGTP, dCTP (2 mM each), and telomerase solution (20 mM Tris-HCl buffer, pH 8.3, 1.5 mM MgCl2, 63 mM KCl, 0.005% Tween 20, 1 mM EGTA, BSA 0.1 mg/mL) at 38 °C for 1 h. (Telomerase solution consisted of telomerase originating from the specified number of 293T cells). For control experiments, telomerase extracts were heat treated (90 °C for 4 min). Amplification of Telomerization Reaction. Telomerase extension products were hybridized with 30 µL (1 nM) of AuNPs functionalized with nucleic acids complementary to the constant repeat units of the telomeres (2). Experimental Setup. ISFET Instrumentation. ISFET experiments were performed using a home-built system that included a Keithley 617 programmable electrometer, Keithley 238 high current source measurement unit, and Keithley 237 high voltage measurement unit. ISFET devices (IMT, Neuch_tel, Switzerland) with Al2O3 gates (20 µm × 700 µm) were used in all of the experiments. A conventional Ag/AgCl electrode was used as a reference electrode. The total solution volume in the cell was 800 µL. SPR Instrumentation. A surface plasmon resonance (SPR) Kretschmann type spectrometer NanoSPR 321 (NanoSPR devices), with a LED light source, λ ) 650 nm, and a prism, refraction index of n ) 1.61, was used in this work. The in situ measurements were conducted using a home-built fluid cell (0.2 cm2 area Au Surface exposed to the solution, total volume of 300 µL of solution). Optical Instrumentation. Real-time fluorescence measurements were carried out using a Cary Eclipse fluorometer (Varian Inc.); solution volume for fluorescence measurements was 200 µL.

(25) Turkevich, J.; Stevenson, P. C.; Hillier, J. Discuss. Faraday Soc. 1951, 11, 55–75. (26) Steel, A. B.; Herne, T. M.; Tarlov, M. J. Anal. Chem. 1998, 70, 4670–4677. (27) Kharitonov, A. B.; Wasserman, J.; Katz, E.; Willner, I. J. Phys. Chem. B 2001, 105, 4205–4213.

(28) Piatyszek, M. A.; Kim, N. W.; Weinrich, S. L.; Hiyama, K.; Hiyama, E.; Wright, W. E.; Shay, J. W. Methods Cell Sci. 1995, 17, 1–15. (29) Volotovsky, V.; Kim, N. Sens. Actuators, B 1998, 49, 253–257. (30) Park, K. Y.; Choi, S. B.; Lee, M.; Sohn, B. K.; Choi, S. Y. Sens. Actuators, B 2002, 83, 90–97.

8392

Analytical Chemistry, Vol. 82, No. 20, October 15, 2010

RESULTS AND DISCUSSION Detection of Telomerase on Field-Effect Transistors. Fieldeffect transistor (FET) devices find growing interest as electronic transducers that follow biocatalytic transformations and biorecognition events. The control of the gate potential by enzyme-stimulated reactions that change the pH of the reaction medium and, thus, the degree of ionization of the oxide layer of the device,29,30 or by altering

Figure 1. (A) ISFET configuration and gate modification for the analysis of telomerase activity. (B) Time-dependent gate-to-source potential changes upon telomerization of the primers associated with the gate using a 293T cell extract of 67 ( 5 cells/µL. (C) Gate-tosource potential changes of the device upon the telomerization of the primer 1 associated with the gate for a fixed time-interval of 60 min, using different concentrations of 293T cells.

the redox state of a modifier of the gate,31 was used to follow biocatalyts, such as nitrate reductase and their substrates. Similarly, the control of the electrical charge associated with the gate surface of the FET devices through the hybridization of DNA or the formation of nucleic acid complexes,32-34 such as aptamer-substrate complexes,35 were used for the electronic detection of DNA, substrates of aptamer, or for the probing of biocatalytic processes occurring on DNA. The telomerase-induced elongation of a telomerase primer linked to the gate of the ISFET device, Figure 1A, could then alter the charge on the gate, thus enabling the sensing of telomerase activity. Accordingly, the Al2O3 gate surface of the FET device was modified with aminoethyl trialkoxysilane, and the nucleic acid 1, acting as a primer for telomerase, was covalently linked to the surface, using N-[e-maleimidocaproy(31) Zayats, M.; Kharitonov, A. B.; Katz, E.; Willner, I. Analyst 2001, 126, 652– 657.

loxy]succinimide ester as a covalent binding unit. The functionalized device was then interacted with telomerase and a mixture of nucleotides (dNTPs) that stimulated the telomerization process on the gate. As the time-dependent telomerization proceeds on the ISFET device, it stimulates a potential change that enables the analysis of telomerase activity. Figure 1B shows the gate-to-source potential changes, ∆VGS, observed upon the telomerase-induced telomerization of 1 on the gate surface. In this experiment, a constant source-to-drain potential of ∆VSD ) -0.5 V is applied, and a constant source-to-drain current, ISD ) -6.7 × 10-5A, is driven through the device. As a result of telomerization, the charge on the gate alters, and in order to retain the constant current passing through the device, the changes in the gate-to-source potential compensate for the charging of the gate by the telomeres, thus providing the electronic readout for the telomerase activity. The ∆VGS values change with time, consistent with the telomerization process, and they reach a saturation value after ∼60 min. The saturation effect is attributed to the deactivation of telomerase as telomerization is prolonged. Control experiments show that only minute changes in ∆VGS values (±5 mV) are observed upon using the thermally treated telomerase from a similar number of 293T cells (cells heated at 90 °C for 4 min) in the presence of dNTPs or the telomerase-negative cell extract (1010 ± 50 cells/µL) and dNTPs, confirming that the potential changes ∆VGS observed in the presence of telomerase originate, indeed, from the telomerization process that alters the charge of the gate interface. Accordingly, the ∆VGS values of the 1-functionalized ISFET devices treated with telomerase extracted from a variable number of 293T cells and dNTPs for a fixed time interval of 60 min are recorded. Figure 1C shows the resulting calibration curve that corresponds to the analysis of a different number of 293T cells by the FET device. The method enabled the detection of telomerase originating from 65 ± 10 293T cells/µL. Optical Analysis of Telomerase through Quantum DotsHemin/G-Quadruplex Hybrids. In a recent study, we demonstrated that hemin/G-quadruplex nanostructures linked to CdSe/ ZnS quantum dots (QDs) lead to the electron transfer quenching of the luminescence of the QDs.36 This process was implemented to sense DNA or the formation of aptamer-substrate complexes by using hairpin-functionalized QDs that include, in their stem region, the protected G-rich sequences. The opening of the hairpin structures by the DNA, or the aptamer substrate, released the G-rich sequence that self-assembled into the hemin/G-quadruplex structure that quenched the luminescence of the QDs. The G-rich telomere units are known to self-assemble into G-quadruplex structures.37 Accordingly, we assumed that hemin could stack into G-quadruplexes generated on CdSe/ZnS QDs, thus leading to the electron transfer quenching of the fluorescence of the QDs. This process would, then, enable the optical analysis of telomerase. (32) Uslu, F.; Ingebrandt, S.; Mayer, D.; Bo ¨ker-Meffert, S.; Odenthal, M.; Offenha¨usser, A. Biosens. Bioelectron. 2004, 19, 1723–1731. (33) Fritz, J.; Cooper, E. B.; Gaudet, S.; Sorger, P. K.; Manalis, S. R. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 14142–14146. (34) Shin, J.-K.; Kim, D.-S.; Park, H.-J.; Lim, G. Electroanalysis 2004, 16, 1912– 1918. (35) Sharon, E.; Freeman, R.; Tel-vered, R.; Willner, I. Electroanalysis 2009, 21, 1291–1296. (36) Sharon, E.; Freeman, R.; Willner, I. Anal. Chem. 2010, 82, 7073–7077. (37) Parkinson, G. N.; Lee, M. P. H.; Neidle, S. Nature 2002, 417, 876–880.

Analytical Chemistry, Vol. 82, No. 20, October 15, 2010

8393

Figure 2. Optical analysis of telomerase activity by telomerization of the primer associated with CdSe/ZnS QDs and following the luminescence quenching of the QDs by the hemin/G-quadruplexes formed within the telomerase chains.

CdSe/ZnS QDs (diameter, 5.8 nm, λem ) 614 nm) were functionalized with a glutathione capping stabilizing monolayer, and the primer 1 was covalently linked to the capping layer, Figure 2. The loading of the QDs with 1 was determined spectroscopically to be 8 units of 1 per particle. The telomerization of 1 was then stimulated in the presence of telomerase and the dNTPs mixture for different time-intervals or in the presence of variable numbers of 293T cells. The resulting hemin/G-quadruplex and the quenching of the luminescence of the QDs provided then the optical readout for the telomerase activity. It should be noted that telomerase is inhibited by hemin. Hence, all telomerization processes were performed in the absence of hemin, and hemin was added to the system to follow the luminescence quenching of the QDs, only at the end of the telomerization. Figure 3A shows the time-dependent luminescence spectra of the QDs, on which the telomerization was activated by an extract of 2700 ± 200 293T cells/µL for different timeintervals. As the telomerization is prolonged, the quenching of the QDs is enhanced, consistent with the formation of a higher content of the hemin/G-quadruplex quencher units on the telomere chains. For comparison, Figure 3B depicts the time-dependent luminescence spectra of the 1-functionalized QDs upon interaction with a thermally treated 293T cell extract (6700 ± 300 cells/µL) and dNTPs. Only a minute decrease in the luminescence intensities of the QDs is observed, implying that the thermally deactivated telomerase eliminates the telomerization process. Also, the treatment of the 1-functionalized QDs with the fiber blast cell extract (6700 ± 300 cells/ µL), which lacks telomerase, in the presence of dNTPs, did not lead to any noticeable quenching of the luminescence of the QDs (see the Supporting Information, Figure 1S). These control experiments imply that the quenching of the luminescence of the QDs is specific to the system where telomerization proceeded, resulting in the formation of the hemin/G-quadruplex quencher units. The results also indicate that hemin in solution does not quench the luminescence of the QDs and that hemin does not bind and quench the luminescence of QDs through a nonspecific adsorption process. The 1-functionalized QDs were then applied to follow the activities of telomerase extracted from different numbers of 293T cells. In these experiments the 1-modified QDs were subjected to telomerase extracted from different numbers of 293T cells in the presence of dNTPs, and the telomerization was conducted for a fixed time-interval corresponding to 60 min. Figure 3C shows the resulting calibration curve corresponding to the fluorescence intensity changes of the 1-functionalized QDs upon analyzing telomerase activity in different numbers of 293T cells. The method 8394

Analytical Chemistry, Vol. 82, No. 20, October 15, 2010

enables the detection of telomerase activity originating from an extract of 270 ± 20 cells/µL. Surface Plasmon Resonance Detection of Telomerase Activity. Surface Plasmon Resonance (SPR) is a versatile tool to follow biosensing or biocatalytic events occurring on thin Au layers.38-41 The dielectric changes occurring on the metal thin films as a result of the recognition events shift the SPR spectra, thus providing a readout signal for the analytical processes. At low concentrations of the analyte, the SPR changes are, however, small, leading to minute shifts in the SPR spectra. Amplified SPR analysis of biorecognition or biocatalytic processes was accomplished by the coupling of Au (or Ag) nanoparticles as amplifying labels to the sensing layers. The electronic coupling between the local NPs plasmon and the surface plasmon wave was found to induce an enhanced large shift in the SPR spectra, and thus, minute changes in the dielectric properties of the sensing interface are amplified.42 Indeed, Au NPs provided a versatile means for amplified SPR biosensing or sensing. For example, the coupling of Au NPs to antigen-antibody43,44 complexes or nucleic acid-functionalized Au NPs to duplex DNA structures45 formed on Au surfaces were used for amplified biosensing. Also, the conjugation of Au NPs as labels for the amplified probing of biocatalytic transformations was reported.46 Similarly, π-donor-functionalized Au NPs aggregates generated on Au surfaces were used for the amplified SPR detection of explosives, such as TNT47 or RDX,48 through the formation of donor-acceptor complexes on the sensing interface. The telomerase-induced telomerization of telomerase primers associated with a Au surface is anticipated to alter the dielectric properties of the surface, and thus, the telomerase activity could be, in principle, monitored by SPR. Furthermore, as the telomeres (38) Homola, J. Chem. Rev. 2008, 108, 462–493. (39) Phillips, K. S.; Cheng, Q. Anal. Bioanal. Chem. 2007, 387, 1831–1840. (40) Heyse, S.; Ernst, O. P.; Dienes, Z.; Hofmann, K. P.; Vogel, H. Biochemistry 1998, 37, 507–522. (41) Berger, C. E. H.; Beumer, T. A. M.; Kooyman, R. P. H.; Greve, J. Anal. Chem. 1998, 70, 703–706. (42) Agarwal, G. S.; Dutta Gupta, S. Phys. Rev. B 1985, 32, 3607–3611. (43) Lyon, L. A.; Musick, M. D.; Natan, M. J. Anal. Chem. 1998, 70, 5177– 5183. (44) Mauriz, E.; Calle, A.; Lechuga, L. M.; Quintana, J.; Montoya, A.; Manclus, J. Anal. Chim. Acta 2006, 561, 40–47. (45) He, L.; Musick, M. D.; Nicewarner, S. R.; Sallinas, F. G.; Benkovic, S. J.; Natan, M. J.; Keating, C. D. J. Am. Chem. Soc. 2000, 122, 9071–9077. (46) Zayats, M.; Pogorelova, S. P.; Kharitonov, A. B.; Lioubashevski, O.; Katz, E.; Willner, I. Chem.sEur. J. 2003, 9, 6108–6114. (47) Riskin, M.; Tel-vered, R.; Lioubashevski, O.; Willner, I. J. Am. Chem. Soc. 2009, 131, 7368–7378. (48) Riskin, M.; Tel-vered, R.; Willner, I. Adv. Mater. 2010, 22, 1387–1391.

Figure 3. (A) Time-dependent luminescence changes of the 1-functionalized CdSe/ZnS QDs that were subjected to telomerization in the presence of 293T cell extract, 2700 ( 200 cells/µL, and 2 mM dNTPs for 1 h, and subsequently treated with hemin (changes represent the time-dependent assembly of the G-quadruplex formation). Curve (a) in the absence of hemin and curves (b)-(i) after addition of hemin, 1 × 10-7 M. (B) Time-dependent luminescence changes of the 1-modified CdSe/ZnS QDs that were subjected to telomerization by thermally treated 293T cell extract 6700 ( 300 cells/ µL and dNTPs 2 mM, for 1 h, and followed by the treatment with hemin. Curve (a) in the absence of hemin and curves (b)-(e) after addition of hemin, 1 × 10-7 M. (C) Calibration curve corresponding to the luminescence quenching changes upon treatment of the 1-functionalized CdSe/ZnS QDs with variable concentrations of 293T cell extracts and dNTPs 2 mM for 1 h and subsequently treated with hemin 1 × 10-7 M for a fixed time-interval of 70 min.

consist of more constant repeat units, the hybridization of Au NPs functionalized with nucleic acids complementary to the constant repeat units, 2, of the telomeres would amplify the formation of the telomers. Accordingly, the sensitivity toward the detection of telomerase activity could be enhanced. A Au surface was modified

with the thiolated primer 1, and the surface was subjected to telomerase, extracted from 293T cells, in the presence of dNTPs. Figure 4A shows the SPR spectra of the 1-modified Au surface before telomerization, curve (a), and after the telomerization of the primer in the presence of telomerase, extracted from 110 ± 10 293T cells/µL, and in the presence of the dNTPs, for a time interval of 30 min, curve (b). Evidently, the telomerization of the primer units leads to a shift in the SPR spectrum, presumably, due to the changes in the dielectric properties of the modified surface as a result of the formation of the telomeres. Furthermore, the hybridization of the 2-functionalized Au NPs with the telomeres yields a substantial shift in the SPR spectrum, Figure 4A, curve (b′). As the interaction of the 2-modified NPs with the primer-functionalized surface does not yield any change in the SPR spectrum curve (a′), we conclude that the Au NPs-induced shift in the presence of the telomeres is due to the hybridization of the 2-modified Au NPs with the telomeres. The coupling between the localized plasmon of the NPs with the surface plasmon wave result in an enhanced shift in the SPR spectrum of the surface, leading to the amplified detection of telomerase activity. In view of these results, we examined in detail the SPR readout of the telomerase activities in the absence and presence of Au NPs labels. Figure 4B, Panel A, shows the sensogram corresponding to the reflectance changes upon interacting the 1-functionalized surface with telomerase extracted from different numbers of 293T cells. As the content of telomerase increases, the reflectance changes are intensified, Panel A, curve (1). The lowest detectable telomerase activity corresponds to telomerase extracted from 180 ± 15 293T cells/µL. Control experiments revealed that telomerase-negative cells did not cause any reflectance changes, Figure 4B, Panel A, curve (2). Thermally denaturated telomerase (90 °C for 4 min) did not lead to any detectable reflectance changes upon the treatment of the 1-modified surface with denaturated telomerase in the presence of dNTPs. Also, the treatment of the 1-modified surface with the fiber blast cell extract (200 cell/µL), which lacks telomerase, in the presence of dNTPs and 2-modified Au NPs leads to much lower reflectance changes (see the Supporting Information, Figure 2S). Figure 4B, panel B, depicts the sensogram corresponding to the reflectance changes upon analyzing the telomerase activity from extracts that include different numbers of 293T cells using the 2-modified Au NPs as amplifying labels, Figure 4B, Panel B, curve (1). The method enables the detection of telomerase extracted from 18 ± 3 cells/ µL. Control experiments reveal that thermally treated 293T cells did not affect the telomerization, and the interaction of the 1-functionalized with the thermally treated 293T cell extract in the presence of dNTPs and the 2-functionalized Au NPs yielded only a minute reflectance changes, Figure 4B, Panel B, curve (2). The time-dependent reflectance changes upon analyzing the telomerase activity originating from different numbers of cells after treatment with the 2-modified Au NPs is presented in Figure 4C. As the number of cells increases, the reflectance changes are intensified, consistent with the higher content of telomerase in the analyzed samples. Also, the intensities of the reflectance changes increase with time, and after ∼2 h they tend to level off to saturation values. The leveling-off effect is attributed to the gradual denaturation of telomerase in the non-native environments Analytical Chemistry, Vol. 82, No. 20, October 15, 2010

8395

Figure 4. (A) Surface plasmon resonance spectra corresponding to: Panel I - The 1-functionalized Au surface before and after telomerization, curve (a) and (b), respectively. Panel II - The 1-functionalized Au surface before telomerization, curve (a′), and after telomerization, and the binding of the 2-functionalized AuNPs, 1 nM, curve (b′). Telomerization was performed by telomerase extracted from 293T cells, 110 ( 10 cells/µL, in the presence of dNTPs, 2 mM, for 50 minutes. (B) Sensograms corresponding to the reflectance changes observed upon analyzing different numbers of 293T cell extracts. Panel A (1) corresponds to the reflectance changes observed upon telomerization of the 1-functionalized interface in the presence of different numbers of cell extracts (telomerization was performed for 40 min in the presence of dNTPs 2 mM). Cell concentrations (cells/µL): (a) 180 ( 15, (b) 270 ( 20, (c) 350 ( 30, (d) 450 ( 35, (e) 540 ( 45, (f) 630 ( 50, (g) 810 ( 65. Panel A (2) corresponds to the reflectance changes observed upon analyzing telomerase-negative cells. Panel B (1) corresponds to the reflectance changes observed upon telomerization of the 1-modified surface in the presence of different numbers of 293T cells extracts in the presence of 2-functionalized Au NPs, 1 nM (telomerization was performed for 40 min in the presence of dNTPs, 2 mM). Cell concentrations (cells/µL): (a) 18 ( 3, (b) 110 ( 10, (c) 200 ( 15, (d) 290 ( 25, (e) 380 ( 30, (f) 465 ( 40, (g) 555 ( 45, (h) 645 ( 55, (i) 825 ( 65. Panel B (2) corresponds to the reflectance changes observed upon interaction of the 1-modified surface with thermally treated 293T cell extract, 290 ( 25 cells/µL (90 °C for 4 min), in the presence of dNTPs, 2 mM, and the 2-functionalized Au NPs, 1 nM. (C) Time-dependent reflectance changes of different 1-functionalized Au surfaces treated with variable concentrations of 293T cell extracts and dNTPs, 2 mM, in the presence of 2-modified Au NPs, 1 nM. Concentration of cells (cells/µL) (a) 18 ( 3, (b) 110 ( 10, (c) 290 ( 25, (d) 420 ( 35, (e) 550 ( 45. (D) Calibration curves corresponding to the reflectance changes of the 1-functionalized Au surface upon analyzing variable numbers of 293T cells extracts: (a) reflectance changes as a result of telomerization for 40 min and (b) reflectance changes as a result of telomerization for 40 min and amplification by the 2-modified Au NPs labels.

of the analytical samples. In fact, the telomerase extracted from the 293T cells lost ∼80% of its activity after storage for 2 h, at room temperature, in the reaction medium. Figure 4D depicts the resulting calibration curves corresponding to the SPR detection of telomerase originating from different numbers of 293T cells by the direct monitoring of the reflectance changes as a result of telomerization, curve (a), and by the amplified detection of telomerase using the 2-functionalized Au NPs labels, curve (b). The readout signals using the Au NPs are ∼3-fold higher, allowing the detection of telomerase originating from ∼18 ± 3 293T cells/ µL with a time interval of ∼60 min. In conclusion, the present study has introduced three new analytical platforms for the analysis of telomerase extracted from 293T cells. These included two optical methods and one electronic sensing platform. One optical method has included the luminescence quenching of CdSe/ZnS QDs by hemin 8396

Analytical Chemistry, Vol. 82, No. 20, October 15, 2010

stacked into the G-quadruplex of the telomerase-synthesized telomeres. This method is the least sensitive analytical procedure, among the three methods, enabling the detection of telomerase from 270 ± 20 cells/µL. The other sensing platform involved the electronic detection of telomerase activity by using an ion-sensitive field-effect transistor device. This method led to a detection limit corresponding to 65 ± 10 cells/µL. While the advantage of this method is the fact that it is label-free, the method requires prefabricated transistor devices and adequate instruments to follow the potential changes occurring on the gate, as a result of telomerization. The third sensing platform has involved the amplified SPR detection of telomerase using Au NPs as amplifying labels. This method led to an impressive sensitivity that enabled the detection of telomerase originating from 18 ± 3 293T cells/µL with an analysis timeinterval of ∼60 min. Furthermore this method revealed a broad

linear detection range of 18-600 cells/µL. Realizing that the amplifying effect of the NPs labels is controlled by the dimensions of the NPs, one may anticipate that by further optimization this sensitivity could be further improved. An attempt to compare the detection limits of the three analytical platforms reported in the present paper to other available methods is quite difficult since many of the reports lack information on the volume of the cell samples. A comparison of the different methods for analyzing telomerase20 indicates, as expected, that the PCR based assays reveal the highest sensitivities that enable the detection of telomerase originating from ∼10 cells. Realizing that the Au NPs-amplified platform for detecting telomerase enabled the detection of ∼18 cells/ µL and the fact that SPR sensing chips of low volume are

available suggest that this method could be competitive with the PCR assays. ACKNOWLEDGMENT This research was supported by the Israel Science Foundation, Israel. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review July 26, 2010. Accepted September 3, 2010. AC101976T

Analytical Chemistry, Vol. 82, No. 20, October 15, 2010

8397