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Nucleic Acid-Functionalized Pt Nanoparticles: Catalytic Labels for the Amplified Electrochemical Detection of Biomolecules Ronen Polsky, Ron Gill, Lubov Kaganovsky, and Itamar Willner*
Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
Nucleic acid-functionalized Pt nanoparticles (Pt-NPs) act as catalytic labels for the amplified electrochemical detection of DNA hybridization and aptamer/protein recognition. Hybridization of the nucleic acid-modified Pt-NPs with a sensing nucleic acid/analyte DNA complex associated with an electrode enables the amperometric, amplified, detection of the DNA by the Pt NP electrocatalyzed reduction of H2O2 (sensitivity limit, 1 × 10-11 M). Similarly, the association of aptamer-functionalized PtNPs to a thrombin aptamer/thrombin complex associated with an electrode allowed the amplified, electrocatalytic detection of thrombin with a sensitivity limit corresponding to 1 × 10-9 M. The amplified detection of biorecognition events, and specifically of DNA hybridization, is one of the challenges in bioanalytical chemistry.1,2 The coupling of enzymes as biocatalytic amplifying labels is a general paradigm in developing bioelectronic sensing devices. The biocatalytic generation of a redox product upon binding of the label to the recognition event,3 the incorporation of redox mediators into DNA assemblies that activate bioelectrocatalytic transformations,4 or the use of enzyme labels that yield an insoluble product on electrode surfaces5 was extensively used to amplify biorecognition events. Metal nanoparticles,6 microbeads,7 or functionalized liposomes8 were widely employed to amplify DNA hybridization. Metal nanoparticles were used as catalytic labels for the enlargement of the particles linked to the biorecognition events with metals and for the detection of the deposited metals by conductivity measurements,9 dissolution of the metals and their electrochemical detection by stripping voltammetry,10 and the analysis of the metal enlargement processes by microgravimetric quartz crystal microbalance measure* To whom correspondence should be addressed. E-mail: willnea@ vms.huji.ac.il. Tel: 972-2-6585272. Fax: 972-2-6527715. (1) Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2003, 21, 11921199. (2) Gooding, J. J. Electroanalysis 2002, 14, 1149-1156. (3) Rishpon, J.; Rosen, I. Biosensors 1989, 4, 61-74. (4) Patolsky, F.; Weizmann, Y.; Willner, I. J. Am. Chem. Soc. 2002, 124, 770772. (5) Patolsky, F.; Katz, E.; Bardea, A.; Willner I. Langmuir 1999, 15, 37033706. (6) Katz, E.; Willner I. Angew. Chem., Int. Ed. 2004, 43, 6042-6108. (7) Wang, J.; Polsky, R.; Merkoci, A.; Turner, K. L. Langmuir 2003, 19, 989991. (8) Patolsky, F.; Lichtenstein A.; Willner I. Angew. Chem., Int. Ed. 2000, 39, 940-943. (9) Park, S. J.; Taton, T. A.; Mirkin, C. A. Science 2002, 295, 1503-1506.
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ments.11 Here, we report on the application of nucleic acidfunctionalized Pt nanoparticles as catalytic labels for the amperometric detection of biomolecules and demonstrate as a proof of concept the electrical detection of DNA and of thrombin. In contrast with the more common methods of using metal nanoparticles as labels for electrochemical detection described above, the current method uses the metal nanoparticle as a catalyst analogous to the way enzymes are traditionally used as redox labels. Using metal nanoparticles might overcome some of the problems associated with the thermal and environmental instability inherent in biological materials such as enzymes. EXPERIMENTAL SECTION Materials and Reagents. Ultrapure water from NANOpure DIamond (Barnstead Int., Dubuque, IA) source was used throughout the experiments. Potassium phosphate monobasic, potassium phosphate dibasic, ethanol, hydrogen peroxide, sodium citrate, sodium chloride, potassium chloride, calcium chloride, bovine serum albumin (BSA), magnesium chloride, tris-HCl, nitric acid, sulfuric acid, potassium hexachloroplatinate, mercaptohexane, and mercaptohexanol were purchased from Sigma-Aldrich Inc. Hexanedithiol was purchased from Acros Organics (Geel, Belgium). DNA was purchased from Sigma-Genosys. Preparation of Platinum Nanoparticles (Pt-NPs). Platinum particles were prepared by heating 100 mL of a 1 mM PtCl6solution to reflux and adding 10 mL of 38.8 mM aqueous sodium citrate, followed by boiling of the mixture for an additional time interval of 30 min at which point the solution turned from clear to black. The heat was then turned off, and the solution was stirred for an additional 10 min. Finally, the solution was allowed to cool to room temperature, filtered through a 0.2-µm cellulose acetate filter (Schleicher and Schuell, Keene, NH), and washed two times through a 100 000 MW cutoff Centricon tube (Millipore Inc., Billerica, MA) with water. Preparation of Pt-NPs on Dithiol-Modified Gold Plates. For the analysis of the model test system, two gold slides (Aucoated glass microarray slides were purchased from Nalge Nunc International, Rochester, NY), cut to the size of 9 × 25 mm, placed in an ethanolic solution of 1 mM hexanethiol and 0.1 mM hexanedithiol overnight, washed with ethanol and then water, and dried under argon. One of the slides was then placed in a solution of the as-prepared Pt-NP solution for a time interval of 16 h, then (10) Wang, J.; Xu, D.; Kawde, A.-N.; Polsky, R. Anal. Chem. 2001, 73, 55765581. (11) Weizmann Y.; Patolsky F.; Willner I. Analyst 2001, 126, 1502-1504. 10.1021/ac0519864 CCC: $33.50
© 2006 American Chemical Society Published on Web 02/18/2006
washed with water, and dried under argon in order to bind the Pt-NPs to the gold slide. Preparation of DNA-Modified Pt-NPs. The DNA and DNAaptamer-modified Pt-NPs were prepared by mixing 2.5 mL of filtered and washed Pt-NP solution with 10 nmol of the appropriate thiol-functionalized ss-DNA overnight and then slowly brought to 0.1 M NaCl and 10 mM phosphate buffer and allowed to stir for 24 h. Subsequently, the solution was centrifuged for 30 min at 14 000 rpm. The DNA-modified Pt-NPs were resuspended in 1.5 mL of 10 mM phosphate buffer that included 0.3 M NaCl, while the DNA aptamer-modified Pt-NPs were brought to 1.5 mL of the binding buffer (20 mM Tris-HCl, pH 7.4, 140 mM NaCl, 5 mM KCl, 5 mM CaCl2, 1 mM MgCl2, and 5% w/v glycerol). DNA and Thrombin Experimental Protocol. DNA and thrombin analysis was accomplished by first cleaning gold slides with a piranha solution (70% sulfuric acid, 30% H2O2) (Caution: piranha solution reacts violently with many organic materials and should be handled with great care), followed by treatment of the electrodes with nitric acid, and then washing them with water and drying under argon. A 100-µL drop of 1-nmol solution of the appropriate DNA was allowed to interact with the electrode for 16 h, washed, dried, and placed in a solution of 1 mM mercaptohexanol for 1 h to create a DNA/mercaptohexanol mixed monolayer probe surface. For DNA detection, the analyte was captured by the hybridization of the nucleic acid-functionalized slides with the appropriate concentrations of the analyte DNA in 100 µL of 0.3 M NaCl, 10 mM phosphate buffer for 4 h, and, subsequently, hybridized with the Pt-NP (38 µL) labels for another 4 h in the same buffer. For thrombin detection, the analyte capture was accomplished by mixing the slides with the appropriate concentration of thrombin in 100 µL of binding buffer for 45 min; subsequently, the aptamer-thrombin-modified surface was mixed with the Pt-NP labels for another 45 min in the same buffer. Washing of the surface with 0.1 M phosphate buffer and drying under argon was performed between each of the steps. Experimental Setup. All electrochemical experiments were carried out using an Autolab electrochemical system (ECO Chemie, The Netherlands) driven by GPES software. Cyclic voltammograms and linear sweep voltammograms were recorded by submerging the prepared gold slides, area 36 mm2, into 5 mL of 0.1 M phosphate buffer solution and recording the voltammetric response using a saturated calomel electrode as a reference and a carbon counter electrode. Chronoamperometric experiments were carried out by measuring the change of the steady-state current 90 s after addition of 2 mM H2O2 in a stirred 10-mL 0.1 M phosphate buffer solution while holding the electrode at a potential of -0.28 V versus an Ag/AgCl reference and using platinum as a counter electrode. Transmission electron microscope (TEM) images were recorded on a Tecnai F20 G2 (FEI Co.) using an accelerating voltage of 200 kV. Samples were prepared by placing a 5-µL drop of the Pt-NPs solution on a 3-mm copper TEM grid and allowing the droplet to evaporate to dryness. Scanning electron microscope (SEM) images were taken on a Sirion high-resolution SEM (FEI Co.). Images were recorded at 200000× magnification. RESULTS AND DISCUSSION A model system was first employed to show the electrocatalytic properties of Pt-NPs toward the reduction of H2O2. Citrate-capped
Figure 1. (A) Cyclic voltammograms corresponding to a nonmodified Au-coated slide in (a) 0.1 M phosphate buffer and (b) 0.1 M phosphate buffer that included 10 mM H2O2. (B) Cyclic voltammograms of the Pt-NP-modified Au slide in (a) 0.1 M phosphate buffer and (b) 0.1 M phosphate buffer that included 10 mM H2O2.
Pt-NPs, 4.0 ( 0.8 nm, were tethered to a dithiol-modified Au-coated plate. Figure 1A shows the cyclic voltammograms of the unmodified Au surface in the absence, curve a, and presence of H2O2, curve b. Within the potential range of 0.4 to -0.6 V. no redox reaction is observed. Figure 1B shows the cyclic voltammograms of the Pt-NP-functionalized electrode in the absence of H2O2, curve a, and upon addition of H2O2, curve b. In the absence of H2O2, a low-intensity cathodic wave at -0.2 V is observed, corresponding to the electrocatalytic reduction of oxygen. In the presence of H2O2, an intense cathodic current, in the region -0.1 to -0.6 V is observed, and this corresponds to the Pt-NP-catalyzed reduction of H2O2. Also, at positive potentials, E > 0.2 V, an anodic current is observed, and this corresponds to the catalyzed oxidation of H2O2. Further experiments in a deoxygenated solution after bubbling argon showed that the reductive wave due to H2O2 reduction is largely unaffected by the presence of dissolved oxygen, and therefore, all further experiments were carried out without degassing. Furthermore, since the Pt-NP-catalyzed reduction of H2O2 reveals an intense voltammetric response, implying a high catalytic turnover (note that the catalytic current is in the mA region), the application of the Pt-NP as a catalytic label was employed in the potential region where the electrocatalytic reduction of H2O2 proceeds. Figure 2A depicts the analytical procedure for the amplified electrochemical analysis of the DNA analyte 1. The thiolated primer 2, which is complementary to a segment of the analyte DNA 1, is assembled on an Au electrode. Hybridization of the Analytical Chemistry, Vol. 78, No. 7, April 1, 2006
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Figure 3. SEM micrographs of the Au-covered slide. (A) Slide used in the DNA detection scheme with 10-8 M DNA analyte. Pt particles are seen as very small bright spots on the background of the Au grains (marked with arrows). (B) Slide used as control in the DNA detection scheme.
Figure 2. Scheme depicting the analytical procedure for using the Pt-NPs in the analysis of (A) DNA and (B) thrombin.
resulting duplex DNA with the nucleic acid, 3-functionalized PtNPs, results in the tricomponent Pt-NP labeled structure. The PtNP-electrocatalyzed reduction of H2O2 is then used as the amplifying reaction to detect the DNA. According to this detection scheme, the Pt-NPs were modified with the nucleic acid 3. The average coverage of the Pt-NP was estimated spectroscopically to be ∼20 units of 3 per particle. The surface coverage of 2 assembled on the electrode was determined by Tarlov’s method12 to be ∼5 × 1011 DNA strands/cm2. The 2-modified electrode was interacted with variable concentrations of 1, and the 3-functionalized Pt-NPs were then linked to the surface. Figure 3A shows the SEM image of the Pt-NPs after linkage to the double-stranded DNA. The Pt-NPs (seen as very small white spots and marked with arrows) are observed on the surface, which includes the analyte 1, while they are absent on the control surface that lacks 1 (Figure 3B). Figure 4A exemplifies the linear sweep voltammograms observed upon analyzing 1, 1 × 10-8 M, and those obtained in a series of control experiments. In the presence of 1, 1 × 10-8 M, an electrocatalytic cathodic wave is observed at -0.3 V indicating that the Pt-NP labels are bound to the duplex DNA and that the electrocatalyzed reduction of H2O2 proceeds, curve a. In the absence of 1 only, or upon the addition of the foreign, noncomplementary DNA 4, curves b and c, respectively, no electrocatalytic currents are observed, indicating that the Pt-NPs do not bind to the surface and that no nonspecific adsorption of the NPs takes place. In addition, the DNA 5 that includes a singlebase mismatch as compared to 1 was analyzed according to this detection scheme. As the melting temperatures of the 2/1 and (12) Steel, A. B.; Herne, T. M.; Tarlov, M. J. Anal. Chem. 1998, 70, 4670-4677.
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Figure 4. (A) Linear sweep voltammograms corresponding to different types of DNA according to Figure 2A. (a) Analysis of 1 × 10-8 M target sample 1. (b) Control experiment in the absence of a DNA analyte. (c) Analysis of 1 × 10-6 M noncomplementary analyte DNA (4). (d) Analysis of 1 × 10-8 M analyte DNA with a single-base mismatch compared to the target sample 5. (e) Analysis of the 1 × 10-8 M target sample in a buffer without H2O2. (B) Chronoamperometric results for different concentrations of DNA. All experiments except for (e) were performed in 0.1 M phosphate buffer (pH 7.4) that included 10 and 2 mM H2O2 for (A) and (B), respectively. scan rate ) 0.1 V/s.
2/5 duplexes are 51.1 and 35.6 °C, respectively, the hybridization between the 2-modified electrode with 5 was conducted at 48 °C. The resulting duplex was then hybridized with the 3-functionalized Pt-NPs. The electrocatalytic cathodic current as a result of the electrocatalyzed reduction of H2O2 is depicted in Figure 4A, curve d. Clearly, the resulting current is substantially lower, indicating that a lower content of 5 is hybridized with the sensing interface, resulting in a lower coverage of the catalytic Pt-NPs. This result implies that a nucleic acid with a single-base mismatch can be
Figure 5. (A) Linear sweep voltammograms corresponding to the analysis of different concentrations of thrombin according to Figure 2B. (a) 1 × 10-6, (b) 1 × 10-7, (c) 1 × 10-8, and (d) 1 × 10-9 M. (e) Control sample in the absence of thrombin. (B) Comparison of the currents at -0.15 V for different concentrations of thrombin. All experiments were performed in 0.1 M phosphate buffer (pH 7.4) that included 10 mM H2O2; scan rate, 0.02 V/s.
differentiated from the analyte DNA. Figure 4B shows the amperometric responses that correspond to the analysis of different concentrations of 1. The target DNA 1 is analyzed through the Pt-NP label and the catalyzed reduction of H2O2 with a sensitivity limit that corresponds to 1 × 10-11 M. Chronoamperometric experiments performed upon the analysis of the target DNA 1, 1 × 10-8 M, in the presence of H2O2, 2 × 10-3 M, allowed us to estimate the charge associated with the reduction of H2O2. The estimated surface coverage of the Pt-NPs associated with the 2/1/3 complex, from the SEM image, is ∼7 × 1010 particles cm-2. Thus, we estimate that ∼5 × 104 electrons s-1 are transported from each Pt-NP to H2O2 during the Pt-NP-catalyzed process. Aptamers are DNA or RNA sequences selected in vitro for their ability to bind specific molecular or macromolecular targets.13,14 In the last couple of years, intensive research efforts were placed in developing aptamer-based biosensors, using fluorescence15,16 or electrochemical17,18 signals as readout outputs. Recently, aptamers were linked to Au-NPs, and the catalytic deposition of Au on the NPs was used as an enhancement step in developing an optical biosensor.19 To show the versatility of the DNA-modified Pt-NPs, we further developed an aptamer-based detection scheme for (13) Ellington, A. D.; Szostak, J. W. Nature 1990, 346, 818-822. (14) Bock, L. C.; Griffin, L. C.; Latham, J. A.; Vermaas, E. H.; Toole, J. J. Nature 1992, 355, 564-566. (15) Vicens, M. C.; Sen, A.; Vanderlaan, A.; Drake, T. J.; Tan, W. H. ChemBioChem 2005, 6, 900-907. (16) Xiao, Y.; Pavlov, V.; Niazov, T.; Dishon, A.; Kotler, M.; Willner, I. J. Am. Chem. Soc. 2004, 126, 7430-7431. (17) Rodriguez, M. C.; Kawde, A. N.; Wang, J. Chem. Commun. 2005, 34, 42674269. (18) Xiao, Y.; Lubin, A. A.; Heeger, A. J.; Plaxco, K. W. Angew. Chem., Int. Ed. 2005, 44. 5456-5459.
thrombin in solution. Figure 2B shows the analytical procedure for the detection of thrombin using the Pt-NP labels. Au electrodes were modified with the thiolated nucleic acid 6, which included a thrombin aptamer section. The Au electrodes were subjected to solutions with different concentrations of thrombin. The modified electrodes were then reacted with the Pt-NPs that were modified with the thiolated nucleic acid 6. The average coverage of the Pt-NPs was estimated spectroscopically to be ∼15 units of 6/particle. As thrombin includes two binding sites for the aptamer 6, the nucleic acid-functionalized Pt-NPs bind to the thrombin complex associated with the surface. The Pt-NP labels associated with the thrombin were then used as sites for the electrocatalytic reduction of H2O2. Figure 5A shows the linear sweep voltammograms corresponding to the analysis. Preliminary experiments (data not shown), using a high scan rate (100 mV/s), similar to that used in the DNA analysis, did not show any observable difference between control and target samples in the same potential region observed in the DNA analysis. Catalytic currents could only be seen at extreme negative potentials. This is most likely due to increased electron-transfer resistance from the bound protein layer between the Pt-NP and the electrode surface. When the scan rate was decreased to 20 mV/s, a catalytic current was observed starting at ∼0.1 V and increasing steadily with a broad shoulder at ∼-0.15 V for a thrombin concentration of 1 µM (Figure 5A, curve a). No electrocatalytic current was observed in control samples without thrombin (Figure 5A, curve e) or a control sample that included 1 µM BSA protein instead of thrombin (curve not shown). A dependence of the transduced cathodic current on thrombin concentration was observed upon analyzing varying concentrations of thrombin corresponding to 100, 10, and 1 nM, (Figure 5A, curves b-d, respectively). Figure 5B shows the signal response for each thrombin concentration, and the control sample measured at -0.15 V. We can conclude that the detection limit for analyzing thrombin by the aptamers is at least 1 nM. In conclusion, the present study has introduced a new method to amplify DNA and protein biosensing by employing Pt-NP labels as catalysts for the reduction of H2O2. The sensitivity of the method for analyzing thrombin is ∼100-fold improved as compared to the present aptamer-based thrombin detection schemes. This suggests that other proteins could be detected at high sensitivities using appropriate aptamers. The analysis of DNA by the Pt-NPs is comparable, and eventually slightly lower, to the available procedures for the amplified detection of DNA. The elimination of enzymes or antibodies for the amplified detection of DNA or antigens and the use of an inorganic catalytic label is certainly an advantage of the present analytical procedure. As far as we are aware, this is the first demonstration of amplified biosensing by electrocatalytic nanoparticles. The application of other NPs and substrates could further improve the sensitivity of this method. ACKNOWLEDGMENT This research is supported by the Israel Ministry of Science as an Infrastructure project. Received for review November 8, 2005. Accepted January 17, 2006. AC0519864 (19) Pavlov, V.; Xiao, Y.; Shlyahovsky, B.; Willner, I. J. Am. Chem. Soc. 2004, 126, 11768-11769.
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