Article pubs.acs.org/ac
Amplified Surface Plasmon Resonance and Electrochemical Detection of Pb2+ Ions Using the Pb2+-Dependent DNAzyme and Hemin/G-Quadruplex as a Label Gilad Pelossof, Ran Tel-Vered, and Itamar Willner* Institute of Chemistry, The Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel S Supporting Information *
ABSTRACT: The hemin/G-quadruplex nanostructure and the Pb2+-dependent DNAzyme are implemented to develop sensitive surface plasmon resonance (SPR) and electrochemical sensing platforms for Pb2+ ions. A complex consisting of the Pb2+-dependent DNAzyme sequence and a ribonuclease-containing nucleic acid sequence (corresponding to the substrate of the DNAzyme) linked to a G-rich domain, which is “caged” in the complex structure, is assembled on Au-coated glass surfaces or Au electrodes. In the presence of Pb2+ ions, the Pb2+-dependent DNAzyme cleaves the substrate, leading to the separation of the complex and to the self-assembly of the hemin/G-quadruplex on the Au support. In one sensing platform, the Pb2+ ions are analyzed by following the dielectric changes at the surface as a result of the formation of the hemin/G-quadruplex label using SPR. This sensing platform is further amplified by the immobilization of the sensing complex on Au NPs (13 nm) and using the electronic coupling between the NPs and the surface plasmon wave as an amplification mechanism. This method enables the sensing of Pb2+ ions with a detection limit that corresponds to 5 fM. The second sensing platform implements the resulting hemin/G-quadruplex as an electrocatalytic label that catalyzes the electrochemical reduction of H2O2. This method enables the detection of Pb2+ with a detection limit of 1 pM. Both sensing platforms reveal selectivity toward the detection of Pb2+ ions.
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ment of on-site Pb2+ analysis tests is required. One of the interesting catalytic nucleic acids is the Pb2+-dependent DNAzyme that cleaves a ribonucleobase-containing substrate.17 Indeed, this DNAzyme has been implemented to develop colorimetric Pb2+ ion sensors based on the catalyzed deaggregation of Au nanoparticles (NPs),18 electrochemical sensors based on the catalyzed removal of redox-active groups,19 fluorescence sensors that lead to the generation of fluorescence upon the cleavage of a fluorophore/quencherfunctionalized substrate,20 and sensors that use the Pb2+dependent DNAzyme and apply dynamic light scattering21 or surface-enhanced Raman spectroscopy22 as transduction signals. Also, other DNAzymes, such as the hemin/Gquadruplex horseradish peroxidase-mimicking DNAzyme, have been used for the colorimetric and chemiluminescence detection of Pb2+ ions.23 Recently, we reported on the surface plasmon resonance (SPR)-amplified detection of DNA, aptamer−substrate complexes, and Hg2+ ions using the hemin/G-quadruplex as a label for the amplified readout of the sensing events via monitoring changes in the dielectric properties of the sensing interfaces.24 In the present study we introduce highly sensitive SPR and electrochemical sensing
he use of catalytic nucleic acids (DNAzymes) as labels for the development of amplified sensor systems is attracting growing interest,1 and different DNAzyme-based sensing platforms for the detection of DNA,2 aptamer−substrate complexes,3 enzyme activities,4 and ions5 have been developed. Among the different DNAzyme labels, the hemin/G-quadruplex horseradish peroxidase (HRP)-mimicking DNAzyme has been extensively used, and different colorimetric6 or chemiluminescence7 sensing schemes and amplified DNA machineries for the detection of DNA,8 aptamer complexes,9 and ions10 that are based on the HRP-mimicking DNAzyme have been developed. Also, hemin/G-quadruplexes have been used as electrocatalysts for amplified sensing11 and as electron transfer quencher units, participating in optical sensing which is based on the application of quantum dots.12 In addition, different sensing platforms have implemented caged, inactive, nanostructures of the HRP-mimicking DNAzyme (e.g., hairpin structures), where upon the recognition event, the DNAzyme structure is uncaged and provides a catalytic label for the detection of the respective analytes.13 Lead ions (Pb2+) are a common environmental pollutant accompanied by severe health risks,14 and thus, their ultrasensitive and quantitative detection is important. While different spectroscopic techniques, such as atomic absorption spectroscopy (AAS)15 or inductively coupled plasma atomic emission spectroscopy (ICP-AES), are available,16 the develop© 2012 American Chemical Society
Received: January 23, 2012 Accepted: March 15, 2012 Published: March 15, 2012 3703
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it corresponded to 1.4 × 10−12 mol·cm−2. Figure 1A shows the SPR spectrum of the 1/2-functionalized Au surface before the addition of Pb2+ (curve a) and after the addition of Pb2+, 50 nM (curve b). A clear shift in the spectrum is observed. As the spectral shift is evident only in the presence of added Pb2+, and only in the presence of hemin, the SPR changes are attributed to the DNAzyme-catalyzed cleavage of the substrate 2 and to the self-assembly of the hemin/G-quadruplex on the surface. The time-dependent reflectance changes at a fixed angle of illumination, θ = 62.5°, upon the buildup of the Pb2+− DNAzyme assembly on the Au surface, and upon the sensing of Pb2+ ions, 10 nM, in the absence and presence of hemin, are shown in Figure S-1 (Supporting Information). One may realize that the deposition of the DNAzyme complex 1/2 on the surface results in an increase in the reflectance. A further addition of Pb2+ ions results in the cleavage of a part of the nucleic acid nanostructure, leading to a decrease in the reflectance intensity. The relatively small change observed may be attributed to the new K+-stabilized G-quadruplex associated with the surface. The subsequent addition of hemin leads, however, to a substantial increase in the reflectance, consistent with the formation of the hemin/G-quadruplex on the surface. This high reflectance change may be attributed to the high extinction coefficient of hemin. Figure 1B, curve a′, shows the time-dependent reflectance changes at a fixed angle of illumination (sensogram) upon treatment of the surface with different concentrations of Pb2+. As the concentration of the Pb2+ ions increases, the reflectance changes are intensified, and they level off to a saturation value of Pb2+ at ca. 10 nM. The response time for analysis of Pb2+ is ca. 6 min (cf. Figure 1B, inset). Figure 1B, curve b′, depicts the results of a control system, where the sensogram corresponds to the reflectance changes observed upon addition of Pb2+ to the duplex generated between 2 and a nucleic acid (5). In this control experiment 5 yields two duplex domains with 2, but generates a loop region that is unable to bind Pb2+ ions and, thus, lacks the ability to cleave 2. Evidently, minute reflectance changes are observed. Also, no reflectance changes are observed upon treatment of the 1/2 nanostructure with Pb2+ ions, 50 nM, in the absence of hemin (see the Supporting Information, Figure S-2A). These results further confirm that the SPR changes originate from the Pb2+-induced cleavage of the substrate strand 2 and the self-assembly of the hemin/G-quadruplex on the surface. Figure 1C shows the derived calibration curve indicating that Pb2+ can be analyzed by the appropriately modified surface with a detection limit corresponding to 1 pM. The successful detection of Pb2+ by means of SPR originates from the refractive index changes occurring on the sensing interface upon the formation of the hemin/G-quadruplex. To further enhance the sensitivity for the detection of Pb2+ ions, we attempted to immobilize the active DNAzyme/substrate complexes on Au NPs assembled on the Au-coated glass support. Metal NPs have been used to amplify the refractive index changes occurring on metal/NP interfaces as a result of sensing events.26 The coupling between the localized plasmon of the Au NPs and the surface plasmon wave was found to increase the SPR shifts originating from dielectric changes at the sensing interface. This phenomenon was previously implemented to amplify, by means of metallic NPs, the formation of antigen−antibody complexes,27 DNA hybridization,28 and formation of aptamer−substrate complexes,29 to follow enzymatic transformations, 30 and to probe the association of low-molecular-weight substrates to imprinted
platforms that use the hemin/G-quadruplex as an amplifying dielectric or electrocatalytic label, respectively. We design Pb2+dependent DNAzyme nanostructures on Au surfaces that include the DNAzyme sequence (1) and the DNAzyme ribonucleobase-containing substrate (2), Scheme 1. The Scheme 1. Sensing of Pb2+ Ions by the Pb2+-Dependent DNAzyme-Catalyzed Cleavage of the RibonucleaseContaining Nucleic Acid Sequence (2) and the Formation of a Hemin/G-Quadruplex Structure Acting as a Label
substrate sequence in 2 is elongated by a G-base-rich sequence containing a caged, inactive configuration of the HRPmimicking unit. In the presence of Pb2+ ions, cleavage of the DNAzyme substrate proceeds, resulting in the self-assembly of the hemin/G-quadruplex dielectric/electrocatalytic label. We further amplify the SPR sensing platform by the immobilization of the sensing probes on Au NPs, and we implement the electronic coupling between the localized plasmon of the NPs and the surface plasmon wave as an amplifying mechanism.
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RESULTS AND DISCUSSION Scheme 1 outlines the principles for the SPR analysis of the Pb2+ ions on a Au support. The thiolated nucleic acid complex 1/2 is assembled on the Au surface. Nucleic acid 2 includes two domains: domain I that consists of a ribonucleobase-containing substrate, and domain II that consists of the G-quadruplex sequence in a duplex caged configuration. Furthermore, nucleic acid 1 forms duplex structures with the two parts of 2 and generates the sequence-specific loop for binding Pb2+ ions. In the presence of Pb2+ ions, cleavage of 1 at the ribonucleobase site proceeds. The resulting duplex domains lack stability, and the complex is dissociated to free 1 while releasing the fragmented nucleic acid (3). Under these conditions the uncaged nucleic sequence (4), linked to the Au surface, selfassembles into the hemin/G-quadruplex. Due to the high extinction coefficient of the hemin label, the resulting changes in the dielectric properties at the surface lead to a shift in the surface plasmon resonance wave. The surface coverage of 1/2 on the Au electrode was determined by Tarlov’s method,25 and 3704
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sites in Au NP composites.31 Thus, we decided to amplify the SPR reflectance changes generated by the hemin/G-quadruplex by the immobilization of the Pb2+-sensing matrix on Au NPs. Scheme 2 depicts the integrated Au NP composite for the Scheme 2. Schematic Presentation for the Nucleic Acid 1/2Functionalized Au NP Monolayer-Modified Surface for the SPR Detection of Pb2+ Ions by the Pb2+-Dependent DNAzyme and the Hemin/G-Quadruplex Label
amplified SPR detection of Pb2+. The Au surface was modified with a benzenedithiol monolayer to which the 1/2-functionalized Au NPs (diameter 13 nm) were linked. The loading of the Au NPs with the 1/2 duplex structure was determined spectroscopically, and it corresponded to ca. 3 units of 1/2 per particle. The loading of the 1/2-functionalized Au NPs on the Au surface was evaluated by the deposition of the 1/2-modified Au NPs on a Au/quartz piezoelectric crystal. From the frequency changes of the crystal, and knowing the size of the NPs, we estimated the coverage of the Au NPs on the Au support to be 2.9 × 10−5 g·cm−2. Figure 2A shows the timedependent reflectance changes upon analysis of different concentrations of Pb2+ on the 1/2-functionalized Au NPmodified Au surface, and Figure 2B, curve a, shows the derived calibration curve. In the presence of Pb2+ ions, the substrate 1 is hydrolytically cleaved, leading to the self-assembly of the hemin/G-quadruplex. The resulting refractive index changes at the sensing interface are, consequently, amplified by the coupling between the localized plasmon of the NPs and the surface plasmon wave, leading to enhanced shifts in the SPR spectra. Evidently, reflectance changes are observed at a Pb2+ concentration as low as 5 fM. For comparison, Figure 2B, curve b, shows the calibration curve corresponding to the analysis of Pb2+ by the 1/2 monolayer configuration according to Scheme 1. In the absence of the amplifying Au NPs, the detection limit for the SPR analysis of Pb2+ by the 1/2 monolayer associated with the Au surface corresponds to 1 pM, a 200-fold higher value in comparison to that of the 1/2-functionalized Au NP-
Figure 1. (A) SPR curves corresponding to the nucleic acid 1/2modified Au surface: (a) in the absence of Pb2+ ions, (b) in the presence Pb2+ ions, 50 nM. (B) Sensograms, at θ = 62.5°, corresponding to the analysis of variable concentrations of Pb2+ on (a′) the 1/2-modified Au surface and (b′) the 5/2-modified Au surface. Pb2+ concentrations: (a) 1 pM, (b) 5 pM, (c) 50 pM, (d) 500 pM, (e) 1 nM, (f) 5 nM, (g) 10 nM, (h) 50 nM. The inset shows a magnified region of the sensogram corresponding to one of the analyzed samples of Pb2+ to illuminate the time course of reflectance changes (for simplicity, the time of injection of the respective concentration of Pb2+ is considered as t = 0). (C) Calibration curves corresponding to the reflectance changes at different concentrations of Pb2+: (a) in the presence of 1/2 as a sensing interface, (b) in the presence of the 5/2modified surface. Error bars correspond to a set of N = 5 measurements. All measurements were performed in a MES buffer solution (10 mM, pH 6.5, including 0.1 M KNO3 and 0.1 M NaNO3) containing hemin, 0.5 μM. 3705
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Figure 2. continued functionalized Pt NP-modified Au surface, (b) on the 1/2functionalized Au NP-modified Au surface. Error bars correspond to a set of N = 5 measurements. All measurements were performed in a MES buffer solution (10 mM, pH 6.5, including 0.1 M KNO3 and 0.1 M NaNO3) containing hemin, 0.5 μM.
modified surface. The enhanced sensitivity might originate from the increase of the coverage of the 1/2 duplex on the sensing surface due to the increased surface area of the Au NPs and/or due to the amplifying effect resulting from the coupling between the localized plasmon and the surface plasmon wave. The surface coverage of the 1/2 complex associated with the Au NPs linked to the Au support was estimated by quartz crystal microbalance (QCM) to be ca. 7 × 10−12 mol·cm−2. This value is ca. 5-fold higher than the surface coverage of 1/2 in the monolayer configuration and consistent with the loading of the complex on the Au NPs. Thus, the 200-fold increase in the sensitivity toward the analysis of Pb2+ ion in the presence of the Au NPs is mainly attributed to the coupling between the localized plasmon and surface plasmon wave. That is, even minute hemin/G-quadruplex-induced dielectric changes at the surface are amplified through the coupling between the localized plasmon of the NPs and the surface plasmon wave, thus enabling the SPR detection of low concentrations of Pb2+ ions. Further support that this electronic coupling leads to enhanced sensitivity was obtained by substituting the Au NPs with Pt NPs (ca. 10 nm diameter) functionalized with the complex 1/2, Figure 2C. The Pt NPs yield a high-surface-area electrode, but lack a localized plasmon that couples with the surface plasmon wave. Figure 2C, curve a, shows the calibration curve corresponding to the reflectance changes observed upon analysis of different concentrations of Pb2+ ions by the 1/2modified Pt NPs deposited onto the Au-coated glass support. For comparison, Figure 2C, curve b, depicts the calibration curve corresponding to the analysis of different concentrations of Pb2+ ions by the 1/2-functionalized Au NPs associated with the Au-coated support. While the Au NP-modified surface exhibits an enhanced sensing performance, only poor performance is evident for the Pt NP-modified electrode, thus supporting the amplification mechanism provided by the plasmonic coupling. A further aspect to consider relates to the selectivity for the analysis of the Pb2+ ions by the 1/2 DNAzyme construct linked to the Au NP monolayer. As the nucleic acid loop in 1 is specific for the binding of Pb2+ ions into a catalytically active nanostructure, one may expect selectivity toward the analysis of these ions. Figure 3 depicts the reflectance changes of the 1/2modified surface upon its interaction with different ions at 10 pM. Evidently, only the Pb2+ ions lead to a significant reflectance change, and all other ions yield minute reflectance changes that overlap the background signal of the system, implying that the system reveals the expected selectivity. The Pb2+-dependent DNAzyme-stimulated cleavage of the 1/2 complex associated with the electrode and the selfassembly of the hemin/G-quadruplex were further implemented for the electrochemical detection of Pb2+ ions. The hemin/G-quadruplex electrocatalyzes the reduction of H2O2 to water11 and, hence, provides an electrocatalytic label for the detection of Pb2+ ions, Figure 4A. The electrocatalytic cathodic currents upon analysis of different concentrations of Pb2+ ions and the resulting calibration curve are shown in Figure 4B and
Figure 2. (A) SPR sensogram, at θ = 62.5°, corresponding to the analysis of variable concentrations of Pb2+ on the 1/2-functionalized Au NP-modified Au surface. Pb2+ concentrations: (a) 5 fM, (b) 10 fM, (c) 50 fM, (d) 100 fM, (e) 500 fM, (f) 1 pM. (B) Calibration curves corresponding to the analysis of variable concentrations of Pb2+ on (a) the 1/2-functionalized Au NP-modified Au surface and (b) the 1/2modified Au surface. The inset shows a magnification of the lower concentration range of the calibration curves. (C) Changes in the reflectance intensities upon analysis of Pb2+ within a broad range of concentrations (presented on a semilogarithmic scale): (a) on the 1/23706
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electrochemical method enabled the analysis of Pb2+ ions with a detection limit that corresponded to 100 pM. The selectivity for the electrochemical analysis of Pb2+ ions was further demonstrated, Figure 4C. Amperometric responses were observed only with Pb2+, and all other ions generated significantly lower current signals. The SPR-based sensing platform that implements the amplified detection of Pb2+ ions by means of Au NPs was used to detect trace amounts of Pb2+ in mineral water. For this purpose, mineral water, 200 μL, was injected into the background solution containing 0.1 M KNO3, 0.1 M NaNO3, and 0.5 μM hemin in deionized water. Evidently, no reflectance changes were observed, implying that the mineral water contains
