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Application of the Hybridization Chain Reaction on Electrodes for the Amplified and Parallel Electrochemical Analysis of DNA Alexander Trifonov, Etery Sharon, Ran Tel-Vered, Jason S. Kahn, and Itamar Willner* Institute of Chemistry, The Minerva Center for Biohybrid Complex Systems, The Hebrew University of Jerusalem, Jerusalem 91904, Israel S Supporting Information *

ABSTRACT: The hybridization chain reaction (HCR) is implemented for the development of amplified electrochemical DNA sensing platforms. The target analyte hybridizes with a probe oligonucleotide-functionalized electrode and triggers on the HCR process in the presence of the hairpins HA and HB. The formation of the analyte-triggered HCR chains is followed by Faradaic impedance spectroscopy or chronocoulometry using Fe(CN) 6 3−/4− or Ru(NH 3 ) 6 3+ as redox labels, respectively. By using two different probe-functionalized electrodes and a mixture of four hairpins, HA:HB and HC:HD, the parallel analysis of two analytes is demonstrated. Through the structural design of the hairpin structures to include caged G-quadruplex subunits, the analyte/probe hybrid associated with the electrode triggers on the HCR process, leading to G-quadruplex-functionalized HCR chains. The association of hemin to the matrix yields electrocatalytic hemin/Gquadruplex units that provide a secondary amplification path for the detection of DNA through an electrocatalyzed reduction of H2O2. The system allows the detection of the analyte DNA with a detection limit corresponding to 0.2 nM.

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INTRODUCTION The development of DNA sensors has attracted substantial research efforts in the past three decades.1−4 The significance of DNA sensors rests on their broad applications in medical diagnosis (detection of genetic disorders, tissue matching, detection of pathogens), environmental analysis (detection of pathogens in food or water), homeland security, forensic applications, and more. Different DNA sensing platforms were developed during the years, and these included a variety of optical,5−11 electrochemical,12−19 microgravimetric,20,21 photoelectrochemical,22−27 or magnetic sensors.28−32 For example, optical DNA detection systems were based on readout signals such as color,33,34 fluorescence,35−37 chemiluminescence,38,39 or surface plasmon resonance (SPR).40,41 Specifically, nanomaterials, such as metallic nanoparticles,42−48 semiconductor quantum dots,49,50 and luminescent metallic nanoclusters51−53 were used as optical labels. Furthermore, a variety of electrochemical DNA sensing platforms were developed, and these included systems based on specific redox-active complexes54 or intercalator units that bind to DNA,55,56 and the use of Faradaic impedance spectroscopy57,58 and conductivity measurements,59−64 employing metal nanoparticlefunctionalized duplex DNA sequences. Specific efforts were directed to enhance the sensitivity of the DNA sensing platforms. This has been accomplished by the coupling of catalytic or biocatalytic labels to the DNA recognition events, by implementing biocatalytic regeneration cycles of the analyte,65 or by applying plasmonic metal nanoparticles66 (e.g., enhancing surface plasmon resonance shifts by coupling © XXXX American Chemical Society

between the localized plasmon of nanoparticle labels and the surface plasmon wave). For example, different enzymes coupled to DNA detection events were used to amplify the sensing processes by intensifying the voltammetric,67,68 interfacial electron transfer resistances,69 or microgravimetric70,71 readout signals. The discovery of catalytic nucleic acids, DNAzymes, introduced many new optically- and electrochemicallyamplified sensing platforms for detection of DNA and, specifically, for the development of ultrasensitive detection paths involving several catalytic cascades. For example, the hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme has been applied for the color,72 electrochemical,73 chemiluminescence,74−76 or surface plasmon resonance transduction of DNA sensing systems,77 and Mg2+- or Pb2+dependent DNAzymes were applied for the fluorescence78 or surface plasmon resonance (SPR)77 detection of DNA, respectively. Furthermore, multistep catalytic cascades triggered by recognition events were applied to amplify DNA sensing processes. For example, by using a functional DNA scaffold, the recognition complex between the analyte gene and the scaffold triggered a cyclic polymerization/nicking process on the DNA scaffold that synthesized hemin/G-quadruplex79,80 or Mg2+/ DNAzyme sequences,33 acting as catalytic labels for the color, Special Issue: Kohei Uosaki Festschrift Received: November 18, 2015 Revised: December 30, 2015

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DOI: 10.1021/acs.jpcc.5b11308 J. Phys. Chem. C XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry C chemiluminescence, or fluorescence readout of the DNA recognition events. Similarly, the analyte-induced activation of the rolling circle amplification process on a functional circular DNA template was used to synthesize hemin/G-quadruplex chains as catalytic labels for the color or chemiluminescence readout of the recognition event between the analyte and the circular template.81,82 Also, the hybridization chain reaction stimulated by the target-triggered cross-opening of two functional hairpin structures was used for the synthesis of polymeric chains consisting of the hemin/G-quadruplex DNAzyme83 or Mg2+-dependent DNAzyme,84 for the color, chemiluminescence, or fluorescence detection of analyte genes. In the present study, we report on the development of a series of electrochemical DNA sensing platforms, which are based on the primary hybridization chain reaction (HCR) as the target amplification path.85 Specifically, we demonstrate that Faradaic impedance spectroscopy provides an effective means to follow the HCR process and that the derived interfacial electron transfer resistances yield a quantitative measure for probing the DNA analyte concentration. Furthermore, we show that the coulometric response of Ru(NH3)63+, associated with the HCR products generated on the electrodes, allows the quantitative analysis of the target DNA. We also demonstrate that by the appropriate design of the hairpin units involved in the HCR, the target-induced activation of the process yields hemin/G-quadruplex electrocatalytic chains, thus providing a secondary electrocatalytic amplification path to the primary HCR reaction. The study further demonstrates selective analysis and single-base mismatch discrimination and the possibility of performing parallel analysis of analytes. Recently, a tetrahedral DNA scaffold associated with an electrode was implemented for the amplified electrochemical detection of microRNA via the initiation of the hybridization chain reaction on the scaffold.86 In this study, the HCR products consisted of a biotin-labeled chain to which avidin-HRP was conjugated. The electrochemical regeneration of 3,3′,5,5′-tetramethylbenzidine (TMB) upon the HRPmediated oxidation of TMB by H2O2 provided an amplification path. In contrast to this study that required the enzyme as an amplification catalyst, our systems include enzyme-free amplification.

(2c) 5′-ACT TCA TTA GCC CAT AAG ACT ACT AAT TGA-3′ (3) 5′-GAT ATC AGC GAT CTT CTA ATT GAA AGT TAT TAA TCA ATT AGA AGT CTT ATG AAG CAC CCA TGT TAC TCT-3′ (4) 5′-GAT ATC AGC GAT CTT TTA ATA ACT TTC AAT TAG CAT AAG ACT TCT AAT TGA AAG CAC CCA TGT TAC TCT-3′ (5) 5′-CCT AGT CCA CCG AAA A- (CH2)6SH-3′ (6) 5′-CGG TGG ACT AGG AGA AGA AGG TGT TTA AGT A-3′ (7) 5′-AGG GCG GGT GGG TGT TTA AGT TGG AGA ATT GTA CTT AAA CAC CTT CTT CTT GGG T-3′ (8) 5′-TGG GTC AAT TCT CCA ACT TAA ACT AGA AGA AGG TGT TTA AGT TGG GTA GGG CGG G-3′ Modification of the Electrodes. Au electrodes (0.5 mm diameter, geometrical area ∼0.18 cm2) were cleaned by treating them with piranha solution, followed by rinsing with hot ethanol and sonication for 15 min. Prior to the modification, the electrodes were rinsed with water. The thiol functionality on the anchors (1) and (5) was deprotected by soaking the oligonucleotides for 2 h in a 0.1 M phosphate buffer, pH = 8.0, containing 0.1 M dithiothreitol. Subsequently, aliquots of the deprotected DNA solution were purified by G-25 microspin columns. The clean Au electrodes were reacted for 24 h with 10 mM HEPES buffer solution, pH = 7.4, containing (1) or (5), 1 × 10−6 M. The electrodes were, then, reacted for 20 min with an ethanolic solution of mercaptohexanol, 1 mM, followed by their washing in a HEPES buffer solution (10 mM, pH = 7.2, containing 50 mM NaCl, 20 mM MgCl2, and 20 mM KNO3) to remove any nonspecifically-adsorbed DNA. The (1)- or (5)modified electrodes were reacted for 5 min with different concentrations of the analyte sequences (2) or (6) in the HEPES buffer solution. The HCR process was carried out in the presence of 1 μM of the DNA hairpins (3)/(4) or (7)/(8), in the HEPES buffer solution. Before their use, the functional hairpin structures (1 μM) were heated for 5 min to 95 °C and were, then, allowed to cool down to room temperature for at least 2 h. It should be noted that an additional polishing of the electrodes with alumina slurry of different sizes had a minute effect on the electrochemical responses of the electrode (Figure S1). Methods and Instrumentation. Electrochemical measurements were performed using an Autolab potentiostat (ECO Chemie, The Netherlands) driven by FRA (impedance measurements) or GPES (chronocoulometry, voltammetry measurements) software. A KCl-saturated calomel electrode (SCE) and a carbon rod (5 mm diameter) were used as the reference and the counter electrodes, respectively. Chronocoulometric analyses were performed according to Tarlov’s method.39 Prior to the application of the potential pulse, from E = −0.5 V to E = 0.1 V vs SCE, the DNA-modified electrodes were allowed to react for 60 s with a HEPES buffer solution (10 mM, pH = 7.2, containing 50 mM NaCl, 20 mM MgCl2, and 20 mM KNO3) that contained Ru(NH3)6, 300 μM. The surface coverage of the DNA units, as well as the calibration curves corresponding to Figures 2(B) and 5(A), were derived by extrapolating the tangent of the chronocoulometric responses of the charge vs the square root of pulse duration, to time zero. Also, prior to the electrocatalysis

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EXPERIMENTAL SECTION Materials and Reagents. Ultrapure water from NANOpure Diamond (Barnstead Int., Dubuque, IA) source was used throughout the experiments. Hemin was purchased from Frontier Scientific and was used without any further purification. All other chemicals were obtained from SigmaAldrich and were used as supplied. The DNA strands were purchased from Integrated DNA Technologies Inc. (IDT). All oligonucleotides were HPLC-purified and freeze-dried by the supplier. The oligonucleotides were diluted in a 10 mM phosphate buffer solution, pH = 7.4, to yield 100 μM stock solutions. The sequences of the oligomers which were used in this study are: (1) 5′-GGC TAA TGA AGT AAA A-(CH2)6SH-3′ (2) 5′-ACT TCA TTA GCC CAT AAG ACT TCT AAT TGA-3′ (2a) 5′-ACT TCA TTA GCC CAT AAG ACA ACT AAT TGA-3′ (2b) 5′-ACT TAT TTA GCC CAT AAG ACT TCT AAT TGA-3′ B


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The Journal of Physical Chemistry C

Figure 1. (A) Schematic presentation of the amplified detection of nucleic acid sequence (2) following hybridization chain reaction on an electrode surface. (B) Faradaic impedance spectra (in the form of Nyquist plots) corresponding to (a) the (1)-modified Au surface and (b) the (1)/(2)modified Au surface. Curves (c)−(g) correspond to the impedance spectra obtained upon subjecting the (1)/(2)-modified Au electrode to the HCR process for variable time intervals: (c) 15, (d) 25, (e) 35, (f) 45, and (g) 55 min. Measurements were performed at a (2) concentration of 500 nM. Inset: The equivalent circuit simulated for the impedance spectra. (C) Faradaic impedance spectra obtained upon subjecting the (1)-modified Au electrode to variable concentrations of the analyte (2): (a) 0, (b) 1, (c) 20, (d) 100, and (e) 500 nM, for 5 min, and allowing the HCR process to operate for 45 min. Inset: Calibration curve corresponding to the dependence of the interfacial electron transfer resistance values on the various concentrations of the analyte (2). Error bars correspond to a set of N = 3 measurements. All measurements were performed in the presence of a HEPES buffer (10 mM, pH = 7.2) containing NaCl, 50 mM, KNO3, 20 mM, MgCl2, 20 mM, and 2 mM of K3Fe(CN)6 and K4Fe(CN)6. The HCR experiments were performed in the presence of 1 μM of the hairpins (3) and (4). Data were recorded in the frequency range of 10 kHz to 100 mHz at E = 0.17 V vs SCE.

with the electrode. It is well established that negatively charged interfaces repel negatively charged redox species, thus leading to an increased interfacial electron transfer resistance, Ret, which is monitored by Faradaic impedance spectroscopy.38 That is, the negative charge associated with the electrode surface upon the initiation of the HCR process is anticipated to be controlled by two factors: (i) The concentration of the analyte (2) that initiates the HCR process and (ii) the time interval that is allowed to operate the HCR process. Figure 1(B) depicts the Faradaic impedance spectra (in the form of Nyquist plots) corresponding to the monolayer of (1) and formation of the recognition complex (1)/(2) on the electrode, in the presence of (2), 500 nM, curves (a) and (b), respectively, and upon allowing the progress of the HCR reaction for different time intervals, curves (c)−(g). In these experiments Fe(CN)63−/4− is used as a negatively charged redox label, and its repulsion from the surface is anticipated to be controlled by the buildup of the negative charge associated with the surface. The assembly of the probe nucleic acid (1) on the electrode and the further hybridization of the analyte (2) increase the interfacial electron transfer resistance of the bare electrode by ΔRet = 320 ± 10 Ohm. These results are consistent with the initial formation of the negatively charged probe monolayer (1) and the increase of the negative charge associated with the electrode upon hybridization with (2). Figure 1(B), curves (c)−(g), shows the impedance spectra observed upon the (1)/(2)-triggered activation of the HCR reaction in the presence of HA and HB for different time intervals. As the HCR process is prolonged,

experiments, the modified electrodes were equilibrated in a HEPES buffer solution (10 mM, pH = 7.2, containing 50 mM NaCl, 20 mM MgCl2, and 20 mM KNO3) that contained hemin, 1 mM, for 15 min. The electrodes were, then, washed off with a HEPES buffer that contained 1 μM hemin. Detection limits were calculated according to the IUPAC recommendations, where the detection limit (CL) is “the lowest detectable concentration that can be detected with reasonable certainty” and is given by CL = Cn̅ + t(Sn/√n) where Cn̅ is the average value of the lowest detectable concentration and t the student’s factor chosen according to a 99% confidence level and using n = 3 or 4 measurements. Sn is the measured standard deviation for the separate measurements.

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RESULTS AND DISCUSSION The general scheme for analyzing a target DNA by the HCR amplification path is shown in Figure 1(A). The electrode is modified with the thiolated probe (1) that is complementary to part of the analyte (2). In the presence of the analyte (2), hybridization with the probe proceeds, leading to the formation of a (1)/(2)-modified electrode that includes a single-stranded toehold sequence. Interaction of the modified electrode with a mixture of the hairpins HA, (3), and HB, (4), initiates the HCR process. The single-stranded tether (2) opens hairpin HA, leading to the free tether “W” that opens hairpin HB, resulting in the sequence “X” that opens hairpin HA, etc. That is, the cross-opening of hairpins HA and HB leads to polymer chains consisting of the cross-interacting hairpins, which are associated C


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Figure 2. (A) Chronocoulometric analysis corresponding to (a) the (1)/(2)-modified Au electrode in the absence of Ru(NH3)63+ redox label. Curves (b) and (c) correspond, respectively, to the (1)-modified Au electrode and to the (1)/(2)-modified Au electrode in the presence of the Ru(NH3)63+, 300 μM. Curves (d)−(h) correspond to the chronocoulometric transients obtained upon subjecting the (1)/(2)-modified Au surface to the HCR process for variable time intervals: (d) 15, (e) 25, (f) 35, (g) 45, and (h) 55 min. Measurements were performed at a (2) concentration of 500 nM. (B) Chronocoulometric transients obtained upon subjecting the (1)-modified Au electrode to variable concentrations of the analyte (2): (a) 0, (b) 0.5, (c) 1.0, (d) 20, (e) 100, and (f) 500 nM, for 5 min, and performing the HCR process for 45 min. Inset: Calibration curve showing the dependence of the extrapolated charge on the various concentrations of the analyte (2). Unless specified, all measurements were performed in the presence of a HEPES buffer (10 mM, pH = 7.2) containing NaCl, 50 mM, KNO3, 20 mM, MgCl2, 20 mM, and 300 μM of Ru(NH3)63+. The HCR experiments were performed in the presence of 1 μM of the hairpins (3) and (4). In all experiments a potential pulse from E = −0.5 V to E = 0.1 V vs SCE was employed, and the error bars correspond to a set of N = 3 measurements.

the interfacial electron transfer resistances, Ret, increase, consistent with the buildup of the negatively charged HCR polymer chains. The equivalent circuit corresponding to the buildup of the HCR product on the electrodes is shown in Figure 1(B) (inset). The values Ret = 560 kΩ, Cdl = 200 μF, and Rs = 18 Ω were derived for the spectrum generated following the operation of the HCR process for 45 min. Figure 1(C) depicts the Faradaic impedance spectra observed upon activation of the HCR on the (1)/(2)-modified electrode in the presence of variable concentrations of (2), for a fixed time interval of 45 min. As the concentration of (2) increases, the interfacial electron transfer resistances are intensified, consistent with the higher content of negatively charged polymer chains. Figure 1(C) (inset) shows the derived calibration curve corresponding to the interfacial electron transfer resistance in the presence of the various concentrations of the analyte. The method enabled the analysis of the analyte with a detection limit that corresponded to 1.2 nM. The binding of Ru(NH3)63+ to DNA monolayers associated with electrodes and the chronocoulometric analysis of the charge associated with the redox label linked to the nucleic acid-modified interface is the “gold-label” procedure to elucidate the surface coverage of the nucleic acids on surfaces, e.g., Tarlov’s method.87 We have implemented this method not only to evaluate the surface coverage of (1) and of the (1)/(2) hybrid on the electrode, but also to follow the time-dependent and concentration-dependent buildup of the HCR chains. Figure 2(A) depicts the chronocoulometric curves corresponding to the (1)/(2) hybrid in the absence of the Ru(NH3)63+

label, curve (a), and the (1)-modified or (1)/(2)-modified electrodes in the presence of Ru(NH3)63+, curves (b) and (c), respectively (concentration of (2) 500 nM). The derived surface coverage of (1) on the electrode is 9.8 × 10−11 mol· cm−2, whereas the surface coverage of (2) hybridized with the probe layer is estimated as 3.2 × 10−11 mol·cm−2. Figure 2(A), curves (d)−(h), depicts the chronocoulometric transients corresponding to the Ru(NH3)63+ associated with the HCR product generated at different time intervals of the HCR. As the HCR process is prolonged, the charge associated with the bound Ru(NH3)63+ increases, consistent with the buildup of the DNA chains. For example, we estimate that after 45 min of the HCR process, 6.6 × 10−9 mol·cm−2 of negatively charged phosphate groups are associated with the electrode surface. Figure 2(B) shows the chronoamperometric transients corresponding to the Ru(NH3)63+ associated with the HCR products generated by the (1)-modified electrode that was subjected to different concentrations of the target (2) and to the subsequent operation of the HCR process for a fixed time interval of 45 min. As the concentration of the analyte increases, the charge associated with the DNA-bound Ru(NH3)63+ is higher, consistent with the elevated content of the DNA chains formed by the HCR process. Figure 2(B) (inset) presents the derived calibration curve corresponding to the charge associated with the Ru(NH3)63+ units linked to the DNA chains generated by the HCR process for a fixed time interval of 45 min in the presence of variable concentrations of the analyte. This method enabled the analysis of the analyte (2) with a detection limit of 1.4 nM. Further support for the D


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unchanged, ΔRet = 190 Ohm, implying that the mutants failed to activate the HCR process since the hairpin HA was not opened due to insufficient stability of the resulting duplexes. It should be noted that similar selectivity results were observed upon analyzing the mutants in comparison to the target (2), using Ru(NH3)63+ as a redox label and chronocoulometry as a readout signal. The amplified electrochemical detection of a target nucleic acid by the HCR reaction was further improved by coupling a secondary amplification path that involves the HCR generation of electrocatalytic DNAzyme units (Figure 4(A)). It was reported that hemin/G-quadruplexes immobilized onto electrodes electrocatalyze the reduction of H2O2.39 The thiolated nucleic acid, (5), was immobilized on a Au electrode, and it acted as an anchor for the hybridization of the target analyte (6), which initiated the HCR process, leading to the formation of catalytic hemin/G-quadruplex units electrocatalyzing the reduction of H2O2 to water. The system is composed of a solution that includes hairpins HC, (7), and HD, (8), which are programmed to stimulate the HCR process and assemble the catalytic hemin/G-quadruplex HCR products. The analyte (6) hybridizes with the probe (5) to yield a duplex, which includes a single-strand tether toehold of the analyte. The (5)/(6)functionalized electrode is then introduced into the HC/HD solution to trigger the hybridization chain reaction. The toehold tether of (6) opens hairpin HC, and the resulting released domain “Y” opens the hairpin HD. The latter process releases the sequence “Z” that interacts to open HC. That is, the duplex (5)/(6) initiates the opening of HC with the subsequent cross-opening of the hairpins HD and HC to yield the HCR chains. The 5′ end of the stem region of hairpin HC includes an encoded sequence that consists of three-fourths of the Gquadruplex sequence, domain I. The 3′ end of the hairpin consists of sequence II, comprising one-fourth of the Gquadruplex sequence. Hairpin HD is functionalized at its 5′ and 3′ ends with one-fourth and three-fourths of the G-quadruplex sequences, II and I, respectively. It should be noted that the Gquadruplex subunit tethers associated with the HC and HD are partially caged in the stem regions of the respective hairpins, and thus the formation of a G-quadruplex structure is eliminated in the absence of the target DNA. Accordingly, the initiation of the HCR process by the analyte (6) triggers the cross-opening of HC and HD, leading to an HCR chain carrying as tethers the G-rich subunits, and these self-assemble, in the presence of the K+ ions present in the electrolyte, into Gquadruplex chains. In the presence of added hemin, the electrocatalytic hemin/G-quadruplex chains are formed, leading to the amplified electrocatalyzed reduction of H2O2. Figure 4(B) depicts the time-dependent Faradaic impedance spectra (in the form of Nyquist plots) upon the triggered operation of the HCR process on the electrode surface. As the HCR process is prolonged, the interfacial electron transfer resistance (in the presence of Fe(CN)63−/4− as a redox label) increases, consistent with the higher content of the negatively charged chains on the electrode. Figure 4(C) shows the Faradaic impedance spectra observed upon sensing different concentrations of the analyte (6) and allowing the HCR process to operate for a fixed time interval of 45 min. As the concentration of (6) increases, the interfacial electron transfer resistance is higher due to the elevated contents of the negatively charged HCR products associated with the electrode. From the respective calibration curve, Figure 4(D), the detection limit for analyzing (6) using

analyte-induced formation of duplex DNA chains via the HCR process was obtained by the interaction of SYBRgold with the HCR-generated chains (Figure S2). Also, gel electrophoresis experiments supported the (2)-stimulated activation of the hybridization chain reaction, in the presence of the hairpins (3) and (4), leading to the HCR-generated polymer chains (Figure S3). It should be noted that Faradaic impedance spectroscopy and chronocoulometry are superior means to other electrochemical methods when probing the hybridization chain reaction occurring on the electrodes. Figure S4 depicts the application of different voltammetry techniques to detect the formation of the HCR product. Evidently, even at the highest concentration of the analyte, 500 nM, small changes in the cyclic voltammogram (CV), differential pulse voltammogram (DPV), and square wave voltammogram (SWV) of the Fe(CN)63−/4− redox label are observed, implying the low sensitivities of these transduction means. The HCR-mediated, amplified electrochemical detection of the target (2) by Faradaic impedance spectroscopy or chronocoulometry reveals impressive selectivity. Figure 3,

Figure 3. Faradaic impedance spectra obtained: (a) before and (b) after subjecting the (1)/(2)-modified Au electrode to the HCR process for 45 min and in the presence of the analyte (2) 500 nM. Curves (c), (d), and (e) correspond, respectively, to the impedance spectra recorded for the two base mismatches (2a) and (2b) and the single base mismatch (2c), all at 500 nM, prior to the operation of the HCR. Curves (c′), (d′), and (e′) correspond, respectively, to the impedance spectra recorded for the two base mismatches (2a) and (2b) and the single base mismatch (2c), all at 500 nM, after subjecting the (1)/(2)-modified Au electrode to the HCR process for 45 min. All measurements were performed in the presence of a HEPES buffer (10 mM, pH = 7.2) containing NaCl, 50 mM, KNO3, 20 mM, MgCl2, 20 mM, and 2 mM of K3Fe(CN)6 and K4Fe(CN)6. The HCR experiments were performed in the presence of 1 μM of the hairpins (3) and (4). Data were recorded in the frequency range of 10 kHz to 100 mHz at E = 0.17 V vs SCE.

curves (a) and (b), show the impedance spectra of the (1)/ (2)-modified electrode (analyzing (2), 500 nM), prior to and following the operation of the HCR process for 45 min, respectively. The interfacial electron transfer resistance increases by ΔRet = 2870 Ohm. Figure 3, curves (c), (d), and (e), depicts the impedance spectra generated upon challenging the (2) sensing interface with the two base mismatch nucleic acids (2a) or (2b), or the one base mismatch (2c) (concentration of the mutants 500 nM). Figure 3, curves (c′), (d′), and (e′), shows the impedance spectra generated by the mutant-treated, (1)-modified electrode after allowing the HCR process for 45 min in the presence of hairpins HA and HB. The interfacial electron transfer resistances are almost E


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Figure 4. (A) Schematic presentation of the double-amplified detection of nucleic acid sequence (6) following hybridization chain reaction on an electrode surface. The HCR process generates G-quadruplex units, which, combined with hemin, electrocatalyze the reduction of H2O2 to H2O. (B) Faradaic impedance spectra obtained upon subjecting the (5)/(6)-modified Au electrode to the HCR process for variable time intervals: (a) 0, (b) 15, (c) 25, (d) 35, (e) 45, and (f) 55 min. Measurements were performed at a (6) concentration of 500 nM. (C) Faradaic impedance spectra obtained upon subjecting the (5)-modified Au electrode to variable concentrations of the analyte (6): (a) 0, (b) 0.5, (c) 1.0, (d) 10 (e) 20, (f) 100, and (g) 500 nM, for 5 min, and allowing the HCR process to operate for 45 min. (D) Calibration curve showing the dependence of the interfacial electron transfer resistance values on the various concentrations of the analyte (6). Error bars correspond to a set of N = 3 measurements. For clarity, the lower concentration region of the calibration curve was further magnified. All measurements were performed in the presence of a HEPES buffer (10 mM, pH = 7.2) containing NaCl, 50 mM, KNO3, 20 mM, MgCl2, 20 mM, and 2 mM of K3Fe(CN)6 and K4Fe(CN)6. The HCR experiments were performed in the presence of 1 μM of the hairpins (7) and (8). Data were recorded in the frequency range of 10 kHz to 100 mHz at E = 0.17 V vs SCE.

upon analyzing variable concentrations of (6). A further control experiment (Figure S5) confirmed the functions of the hemin/ K+ ion-stabilized G-quadruplex as an electrocatalyst for the amplified detection of (6). In this experiment, the cyclic voltammograms corresponding to the detection of (6), 500 nM, by the HCR product in the presence of added hemin were recorded in the presence and absence of added K+ ions. While in the presence of K+ ions, the saturated, ca. 70 μA, electrocatalytic cathodic current was observed, and in the absence of the K+ ions only the background current of the electrolyte solution was recorded. This control experiment reveals that K+ ions, indeed, stabilize the formation of the hemin/G-quadruplex complex. The detection limit for analyzing (6) by the HCR-induced hemin/K+ ion-stabilized G-quadruplex sensing platform is 0.2 nM. This value is nearly an order of magnitude lower than the methods involving Faradaic impedance spectroscopy (measuring interfacial electron transfer resistance) or the coulometric analysis of the surface-bound Ru(NH3)63+. That is, the application of a dual amplification cascade that involves the HCR process and the hemin/G-quadruplex-catalyzed reduction of H2O2 leads to the improved sensitivities. The successful separate analyses of the two sequences (2) and (6) suggest the parallel analysis of both the analytes using

the HCR process and Faradaic impedance as a transduction signal was estimated to be ca. 1.5 nM. Figure 5(A) shows the chronocoulometric transients corresponding to the Ru(NH3)63+ redox label associated with the HCR products formed on the electrode surface in the presence of variable concentrations of the analyte (6) and upon operating the HCR process for a fixed time interval of 45 min. As the concentration of (6) increases, the charge of Ru(NH3)63+ associated with the DNA is higher, consistent with the elevated contents of DNA generated by the HCR process. Figure 5(A) (inset) presents the derived calibration curve. This method enabled the sensing of (6) with detection limit of 1.2 nM. Figure 5(B) depicts the cyclic voltammograms of the hemin/G-quadruplex-functionalized HCR chains, generated upon challenging the (5)-modified electrode with variable concentrations of (6) and operating the HCR process for a fixed time interval of 45 min. The cyclic voltammograms correspond to the electrocatalyzed reduction of H2O2 by the hemin/G-quadruplex-functionalized chains. The electrocatalytic cathodic currents are intensified as the concentration of (6) increases, consistent with the higher content of the hemin/Gquadruplex electrocatalytic units generated by the HCR process. Figure 5(B) (inset) depicts the derived calibration curve corresponding to the resulting cathodic currents observed F


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Figure 5. (A) Chronocoulometric transients obtained upon subjecting the (5)-modified Au electrode to variable concentrations of the analyte (6): (a) 0.5, (b) 1.0, (c) 10, (d) 20, (e) 100, and (f) 500 nM, for 5 min, and allowing the operation of the HCR process for 45 min. Inset: Calibration curve showing the dependence of the extrapolated charge on the various concentrations of the analyte (6). For clarity, the lower concentration region of the calibration curve was further magnified. All measurements were performed in the presence of a HEPES buffer (10 mM, pH = 7.2) containing NaCl, 50 mM, KNO3, 20 mM, MgCl2, 20 mM, and 300 μM of Ru(NH3)63+. In all experiments a potential pulse from E = −0.5 V to E = 0.1 V vs SCE was employed, and the error bars correspond to a set of N = 3 measurements. (B) Cyclic voltammograms, corresponding to the electrocatalytic reduction of hydrogen peroxide by the HCR-assembled hemin/G-quadruplex electrocatalytic units, formed upon the interaction of the (5)-modified electrode with variable concentration of the analyte (6): (a) 0, (b) 0.1, (c) 0.5, (d) 1.0, (e) 5.0, (f) 20, (g) 100, and (h) 500 nM. Scan rate: 10 mV·s−1. During the measurements, the concentrations of hemin and H2O2 were 1 μM and 5 mM, respectively. Inset: Calibration curve showing the dependence of the electrocatalytic current, at E = −0.6 V vs SCE, on the various concentrations of the analyte (6). The error bars correspond to a set of N = 4 measurements. For clarity, the lower concentration region of the calibration curve was further magnified. In all measurements the HCR experiments were performed in the presence of 1 μM of the hairpins (7) and (8).

HCR processes were activated on the two electrodes by the analytes (2) and (6).

the amplified HCR paradigm. Figure 6(A) depicts the Faradaic impedance spectra of the (1)- and (5)-modified electrodes upon their interaction with the analyte (2), 500 nM, and the operation of the HCR process in the presence of all hairpins, HA, HB, HC, and HD, for a time interval of 45 min. Treatment of the two electrodes with the analyte (2) in the presence of all four hairpins resulted in an increase in the interfacial resistance, ΔRet = 2300 ± 60 Ohm, consistent with the selective activation of the HCR process by hairpins HA and HB. Similarly, Figure 6(B) shows the Faradaic impedance spectra observed upon challenging of the two electrodes with the analyte (6), 500 nM, and operating the HCR process in the presence of all four hairpins. Evidently, only the (5)-modified electrode reveals an increase in the interfacial electron transfer resistance, ΔRet = 2100 ± 50 Ohm, consistent with the selective (6)-triggered operation of the HCR process that opens the hairpins HC and HD. The simultaneous analysis of the two analytes is presented in Figure 6(C). Subjecting the (1)- and (5)-modified electrodes to both of the analytes (2) and (6), at 500 nM, followed by the operation of the HCR process in the presence of all four hairpins HA, HB, HC, and HD, for a time interval of 45 min, leads to an increase in the electron transfer resistances of the (1)-modified electrode and the (5)-functionalized electrode, ΔRet = 2200 and 2100 Ohm, respectively, implying that the

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CONCLUSIONS The present study has implemented the hybridization chain reaction (HCR) as the primary amplification path for the electrochemical sensing of target DNA sequences. Faradaic impedance spectroscopy (interfacial electron transfer resistance) and chronocoulometry of a redox label, Ru(NH3)63+, associated with the HCR products provided readout signals for the sensing events. By the engineering of the hairpins involved in the hybridization process to include “caged” G-quadruplex subunits, a secondary amplification path to the primary HCR amplification process was introduced. The triggered HCRstimulated opening of the hairpins that included the Gquadruplex subunits led to G-quadruplex structures that were tethered to the HCR product. The binding of hemin to the Gquadruplexes associated with the electrode yielded hemin/Gquadruplex units acting as electrocatalysts for the electrochemical reduction of H2O2 to water. The electrocatalytic process provided a secondary amplification step for analyzing the DNA. The different sensing platforms revealed selectivity, and single-base mismatches could be discriminated. Furthermore, the simultaneous analysis of two analytes using the G



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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.5b11308. Interfacial electron-transfer resistance of the sensing platform, where the electrode is cleaned by the experimental protocol with and without terminal polishing with alumina slurries; following the HCR process by SYBRgold label; gel electrophoresis performed for the HCR process; results corresponding to the electrochemical transduction of the sensing process by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and square-wave voltammetry (SWV); the cyclic voltammograms corresponding the G-rich HCR products associated with the respective electrodes in the presence, and absence, of K+ ions and added hemin and H2O2 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +972-2-6585272. Fax: +972-2-6527715. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported in part by the Israel Science Foundation and by the EU FET Open MICREAgents Project #318671.

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REFERENCES

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Figure 6. (A) Faradaic impedance spectra corresponding to the (a) (1)-modified electrode, (b) (5)-modified electrode, and (c) (5)/(2)modified electrode following the application of the HCR process for 45 min, and (d) the (1)/(2)-modified electrode following the application of the HCR process for 45 min. (B) Faradaic impedance spectra corresponding to the (a) (1)-modified electrode, (b) (5)modified electrode, and (c) (1)/(6)-modified electrode following the application of the HCR process for 45 min and (d) the (5)/(6)modified electrode following the application of the HCR process for 45 min. (C) Faradaic impedance spectra corresponding to the (a) (1)modified electrode, (b) (5)-modified electrode, (c) (1)/(2)-modified electrode, (d) (5)/(6)-modified electrode, and (e) (1)/(2)-modified electrode following the application of the HCR process for 45 min and (f) the (5)/(6)-modified electrode following the application of the HCR process for 45 min. All measurements were performed in the presence of a HEPES buffer (10 mM, pH = 7.2) containing NaCl, 50 mM, KNO3, 20 mM, MgCl2, 20 mM, and 2 mM of K3Fe(CN)6 and K4Fe(CN)6. The HCR experiments were performed in the presence of the respective hairpins (3) and (4) or (7) and (8). Data were recorded in the frequency range of 10 kHz to 100 mHz at E = 0.17 V vs SCE.

Faradaic impedance responses of the electrodes was demonstrated. H


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