Dual Signaling DNA Electrochemistry: An Approach To Understand

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Dual Signaling DNA Electrochemistry: An Approach To Understand DNA Interfaces Saimon Moraes Silva, Roya Tavallaie, Vinicius R. Gonçales, Robert H. Utama, Mehran B. Kashi, D. Brynn Hibbert, Richard D. Tilley,* and J. Justin Gooding* School of Chemistry, Australian Centre for NanoMedicine, and ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, The University of New South Wales, Sydney 2052, Australia S Supporting Information *

ABSTRACT: Electrochemical DNA biosensors composed of a redox marker modified nucleic acid probe tethered to a solid electrode is a common experimental construct for detecting DNA and RNA targets, proteins, inorganic ions, and even small molecules. This class of biosensors generally relies on the binding-induced conformational changes in the distance of the redox marker relative to the electrode surface such that the charge transfer is altered. The conventional design is to attach the redox species to the distal end of a surface-bound nucleic acid strand. Here we show the impact of the position of the redox marker, whether on the distal or proximal end of the DNA monolayer, on the DNA interface electrochemistry. Somewhat unexpectedly, greater currents were obtained when the redox molecules were located on the distal end of the surface-bound DNA monolayer, notionally furthest away from the electrode, compared with currents when the redox species were located on the proximal end, close to the electrode. Our results suggest that a limitation in ion accessibility is the reason why smaller currents were obtained for the redox markers located at the bottom of the DNA monolayer. This understanding shows that to allow the quantification of the amount of redox labeled target DNA strand that hybridizes to probe DNA immobilized on the electrode surface, the redox species must be on the distal end of the surface-bound duplex.

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INTRODUCTION Surface DNA hybridization is the foundation of modern technologies broadly used in applied genomics for genotyping, gene expression profiling, drug discovery, and biosensing.1,2 Electrochemical DNA biosensors have become increasingly attractive because they benefit from developments of microfluidics, microsystems technology, and nanotechnology, which is promising for miniaturization and utilization in point-of-care diagnostics.3−6 Typically, a DNA biosensor comprises a singlestranded probe sequence tethered to a surface, and as a result of base-pairing interactions, the DNA target binds to the interface.7 For successful hybridization, the probe and target should be free to coil around each other.4 The common strategy used to obtain maximum configurational freedom is immobilizing the tethered DNA probe at the end of the strand.8,9 There have been two main approaches to electrochemically explore DNA hybridization, and they are usually referred to as label-free and labeled methods. The labeled approaches, whether the interfaces uses long-range charge transfer through the DNA duplex or elastic bending/rotational motions of © XXXX American Chemical Society

tethered DNA, use redox-active molecules that bind to DNA. The most commonly used redox probes are daunomycin, anthraquinone, methylene blue, and ferrocene.7,9−12 The advantage of this type of DNA interfaces is high selectivity, being able to distinguish not only complementary sequences from noncomplementary targets but also single mismatches.13 Contradictory results are reported for redox-labeled DNA interfaces regarding whether hybridization increases or decreases faradaic electron transfer. Heeger and co-workers14 reported a strategy using a ferrocene-labeled DNA stem loop tethered to a gold electrode to monitor DNA hybridization. Physical changes in the DNA upon hybridization cause the redox label to move away from the electrode surface, increasing the electron transfer distance. In this study, a greater electrochemical response was obtained for the ferrocene positioned close to the electrode surface. However, weaker responses were obtained by King and co-workers for Received: August 10, 2017 Revised: November 18, 2017

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DOI: 10.1021/acs.langmuir.7b02787 Langmuir XXXX, XXX, XXX−XXX

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Scheme 1. Schematic Representation of Ferrocene Labeled DNA Probe Modified Gold Electrode before and after Hybridization with Methylene Blue Labeled DNA Targeta

a

Formation of a rigid duplex after hybridization results in an increase in the average distance of the ferrocene redox label in the DNA probe from the surface while the methylene blue redox label in the DNA target is located close to the electrode surface.

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comparable systems.15 This inconsistency may be a consequence of the complex nature of DNA interfaces. That is, DNA hybridization causes a change in a number of properties at the electrode surface as a result of the immobilized single-stranded DNA probe hybridizing with a complementary target sequence.16 The changes caused by hybridization include (1) changes in physical properties of the interface as a flexible single-strand DNA becomes a rigid double strand,17 (2) changes in electronic properties as a DNA duplex can transfer electrons more efficiently than a single strand of DNA,18,19 and (3) the electrode interface becoming more open as a consequence of the duplex DNA being in an upright position relative to the electrode surface. In the case of an electrochemical interfaces, the electrode interface becoming more open on hybridization means an increase in ion accessibility of the surface and a decrease in charge transfer resistance.8 Because of these many changes, the impact of the position of the redox label in the tethered DNA on the electron transfer between the redox label and electrode surface it is still not well understood. In this work we investigate how the position of the redox labels in the DNA film affects the electrochemical behavior. The preparation of the interface is based on the Tarlov interface, where a thiol-modified single-stranded DNA probe is chemisorbed on gold surface.20−22 A dual signaling approach was adopted by using redox labels on the probe strand and on the target strand. There are two potential advantages of using such configuration: it seeks to avoid any ambiguous observation caused by the DNA dynamics, and the system can be configured to determine the extent of hybridization in a direct manner. Square-wave voltammetry (SWV) was used as measurement technique. The initial DNA interface, and a depiction of the expected change in the interface upon hydridization, is illustrated in Scheme 1. The impact of the dynamic properties of the surface-tethered duplex DNA (electron transfer in a nonrigid system) was investigated by changing the viscosity of the electrolyte solution. Furthermore, the effect of changing the accessibility of the counterion was investigated by changing the position of the redox label in the DNA interface.

EXPERIMENTAL SECTION

Materials and Reagents. All the DNA sequences were purchased from Biosearch Technologies (Petaluma, Canada). The motivation of using these DNA probe sequences is due to the possibility to translate the approach presented here to the direct detection of micro-RNAs, which have shown a great potential to be used as biomarker to many disease, mainly cancer. Probe DNA strands (22-mer): 5′-SH-(CH 2 ) 6 -p-TCAACATCAGTCTGATAAGCTA-MB-3′ and 5′-SH-(CH2)6-p-TCAACATCAGTCTGATAAGCTA-Fc-3′. The motivation of using these DNA probe sequences is due to the possibility of translating the approach presented here to the direct detection of micro-RNAs, which have shown a great potential to be used as biomarker to many diseases, mainly cancer. Target DNA strands: 5′-TAGCTTATCAGACTGATGTTGA-Fc3′, 5′-TAGCTTATCAGACTGATGTTGA-MB-3′, 5′-MB-TAGCTTATCAGACTGATGTTGA, and 5′-Fc-TAGCTTATCAGACTGATGTTGA. Ethanol, Dulbecco’s phosphate buffered saline modified without calcium chloride and magnesium chloride, H2SO4, 6-mercaptohexan-1ol (MCH), and glycerol were purchased from Sigma (Sydney, Australia). All reagents were of analytical grade and used as received. Preparation of Electrodes and DNA Immobilization. Polycrystalline gold electrodes (CH Instruments, Austin, TX) were polished with wet alumina slurries with particles sizes of 1.0, 0.3, and 0.05 μm on polishing cloths (Thermo Fisher Scientific, Sydney). Subsequently, the electrodes were rinsed with Milli-Q water electrochemically cleaned in 0.05 M H2SO4 by cycling in the potential range from −0.1 to 1.5 V vs Ag|AgCl (KCl, 3 M) at 100 mV s−1 until a reproducible voltammogram was obtained. After cleaning procedure, the electroactive areas were evaluated by integrating the cathodic peak in the gold electropolishing voltammograms and converting them into the real surface area by using a factor of 390 μC cm−2.23 The clean electrodes were used to tether the DNA capture probes. Immobilization of DNA was carried out by incubating the clean electrodes for 2 h in 4 μM DNA probes solution containing the redox label in PBS, pH 7.4, at room temperature. Afterward, the electrodes were thoroughly rinsed with PBS to remove any excess of reagents or any weakly attached DNA strands. To finalize the electrode preparation, the single-strand DNA-modified electrodes were immersed into a 2 mM mercaptohexan-1-ol (MCH) solution in ethanol for 30 min to produce the binary monolayer of the DNA capture probe and MCH, followed by rinsing with PBS. As reported in the literature, the efficiency of DNA hybridization depends on the surface coverage of the probe (Γmax).24,25 According to Levicky and co-workers, the pseudo-Langmuir regime, where B

DOI: 10.1021/acs.langmuir.7b02787 Langmuir XXXX, XXX, XXX−XXX

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Langmuir maximum DNA hybridization efficiency is achieved, occurs at low DNA probe coverage, around Γmax ≤ 1 × 1012 molecules cm−2.26 Tagging DNA probes with redox probes allowed the determination of DNA probe concentration, Γp. Hence, in this work to ensure maximum efficiency of DNA hybridization the DNA probe surface coverage was kept ≈1 × 1012 molecules cm−2. Hybridization of DNA Target. Hybridization of target nucleic acid was performed at room temperature by immersing the DNA capture probe/MCH modified electrode in the desired concentration solutions of target in PBS for 2 h, followed by rinsing with PBS. Complementary DNA molecules used in this present work were tagged at 3′ or 5′ end with the redox marker ferrocene or methylene blue. After hybridization, the redox markers at 3′ end in the complementary DNA were located at the bottom of the binary DNA/MCH monolayer, while the redox markers at the 5′ end were located at the top of the DNA/MCH film. Tagging DNA target with a redox label allowed quantification of complementary DNA hybridized to the prepared electrodes. Electrochemistry. Cyclic and square-wave voltammetry were performed with a μAutolab PGSTAT 128N potentiostat/galvanostat (Metrohm Autolab B.V., Netherlands) connected to a computer, and GPES 4.9 software was used for data acquisition and analysis. The experiments were carried out in a conventional three-electrode cell, with an Ag|AgCl (KCl, 3 M) electrode as reference electrode and platinum electrode as counter electrode. Dulbecco’s phosphate buffered saline (PBS) modified without calcium chloride and magnesium chloride was used as electrolyte due to the fact that Cl− ions exhibit high affinity to gold surfaces. Square-wave voltammetry of the strongly adsorbed quasi-reversible redox molecules gave information on kinetics and surface coverage.27 The square-wave current was fitted to a model of Butler−Volmer charge transfer kinetics for the absorbed redox molecules, ignoring any contributions from solution-resident species, by an in-house program written in the MATLAB (Mathworks, USA, R2016a) environment. Viscosity Measurements. Viscosity measurements were conducted using an AntonPaar rheometer equipped with a cup and spindle system (CC17/S). Briefly, 5 mL of the sample solution was transferred into the cup, and the spindle was lowered until it was fully immersed. First, an increasing (logarithmic ramp) shear rate from 10 to 1000 s−1 was applied to confirm that the viscosity of solution was constant at all shear rates. Subsequently, a constant shear rate of 50 s−1 was applied over 5 min, with a viscosity measurement recorded at every 5 s, giving 60 data points. The presented viscosity values were the average of the 60 measurement points.

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Figure 1. (a) Square-wave voltammograms of single-stranded DNA labeled with ferrocene in its 3′ end before hybridization (black line) and after hybridization with the DNA target labeled with methylene blue in its 3′ end in different concentrations: 0.5 μM (red line), 1 μM (green line), 2.5 μM (blue line), and 5 μM (dark yellow line). (b) Plot of the concentration of DNA target versus the number of electroactive molecules of DNA target calculated from methylene blue redox peak (blue y-axis) and plot of the concentration of DNA target versus the number of electroactive ferrocene molecules in the DNA probe (red yaxis). Experimental conditions: DNA probe immobilization: clean gold electrodes were incubated for 2 h in 4 μM PBS solution of DNA probe labeled with ferrocene; MCH immobilization: 30 min incubation in 2 mM MCH ethanolic solution, followed by rinsing with PBS; DNA hybridization: the modified electrodes were exposed to different concentrations of the methylene blue labeled DNA target in PBS for 2 h at room temperature, followed by rinsing with PBS; SWV were recorded in PBS using a frequency of 10 Hz.

concentrations of DNA target tagged with methylene blue. Before the hybridization, a high current peak at a potential of +0.27 V vs Ag|AgCl(3 M KCl) was observed related to the ferrocene electrochemical oxidation. After hybridization, the associated ferrocene peak current decreases. The diminution in current is proportional to the increase of DNA target concentration. Moreover, after the formation of the DNA duplex, a peak around −0.25 V vs Ag|AgCl(3 M KCl) appears due to the methylene blue electrochemical oxidation, and this peak increases with the DNA target concentration. However, it can be observed from Figure 1a that the magnitudes of the changes in peak current for the ferrocene and methylene blue redox labels are very different. The amount of ferrocene switching off is significantly greater than the amount of methylene blue switching on. This was unexpected considering that methylene blue is allocated closer to the surface, and its electrochemical oxidation involves two electrons. Hence, the changes in the methylene blue peaks were expected to be much larger than our observations. Figure 1b shows a plot of the estimated number of DNA target molecules that bind to the electrode surface (blue y-axis) versus the concentration of the target DNA hybridized. The number of DNA target molecules was calculated by fitting the entire square-wave voltammograms assuming the condition of strongly adsorbed quasi-reversible redox molecules (Oxads + ne− = Redads) and based on the Butler−Volmer charge transfer kinetics.27 The red y-axis shows the relationship between the number of electrochemically active ferrocene molecules as a function of the DNA target concentration. The number of ferrocene molecules in the DNA probe was calculated using a similar procedure employed for the methylene blue attached to the DNA target. Thus, with the increasing of the DNA target concentration, a decreasing of the number of ferrocene molecules reaching the electrode surface is observed. The relation between the ferrocene signal suppression and DNA target concentration follows a similar trend of the number of methylene blue molecules versus DNA target concentration. However, the decrease in charge transferred from the ferrocene

RESULTS AND DISCUSSION

The DNA recognition interface is shown in Scheme 1. The DNA probe strand possesses a ferrocene moiety at its distal end which can easily access the electrode due to the low persistence length of the DNA. Upon hybridization, the redox marker in the DNA probe, ferrocene, is positioned further away from the electrode surface as the persistence length of the DNA duplex is approximately 50 nm16 while the 20-base-pair single-stranded DNA is only 12−13 nm long. In this position the ferrocene charge transfer could occur either by (1) the elastic bending and free rotational motions of the duplex that brings the ferrocene group close to electrode surface and/or (2) directly through the DNA base stacking mechanisms,11 whereas the redox marker in the DNA target, methylene blue, is located at the bottom of the DNA/MCH monolayer. In this position, it is expected that methylene blue electron transfer can proceed directly to the electrode due to the redox species’ close proximity to the electrode surface.7,11,28 At first, the quantification of the amount of DNA target hybridized was attempted. Figure 1a presents the square-wave voltammograms for the DNA probe labeled with ferrocene before (black line) and after hybridization with the different C

DOI: 10.1021/acs.langmuir.7b02787 Langmuir XXXX, XXX, XXX−XXX

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Langmuir molecules is not a simple 1:1 relationship with the increase in the number of methylene blue molecules interrogated electrochemically. In addition, for a given change in DNA target concentration, the observed current from ferrocene in the DNA probe, that is, the redox species distal from the electrode surface, was always higher than the current arising from methylene blue, which is the species closer to the surface. Hence, it is apparent from these results that the magnitude of the current from the ferrocene and methylene blue labels cannot be directly correlated with the amount of target and probe DNA on the surface with this system. This raises the question, what other factors are influencing the electrochemically measured amount of probe or target DNA? Answering this question, and understanding under what conditions the changes in probe and target electrochemistry directly correlate, may then allow redox labels on the target DNA to be used to quantify the extent of hybridization. Inspection of the literature shows that our results are consistent with the observations of others. A lower current for the redox label closer to electrode surface when compared with the redox label located far of the electrode was also reported by Kraatz29 and co-workers and King15 and co-workers. Those studies investigated the impact of the position of ferrocene molecule in the DNA duplexes on the electron transfer process. A strong electrochemical response was observed when the ferrocene was located on top of DNA duplex, while weaker responses were obtained for the systems with the redox labels placed closer to the interface. Kraatz and co-workers hypothesized that this phenomenon is caused by the elastic bending and free rotation motions of the of the DNA duplex, which would bring the ferrocene close to the electrode surface when located on top of the film.29 However, this hypothesis has remained untested. Motivated by these previous reports, and the results obtained, three hypotheses were investigated in order to explain the electrochemical results obtained here. The first hypothesis to be tested was if the electrochemical behavior of the proposed system is to a large extent dictated by the dynamics properties of the surface tethered double-stranded DNA. To test this, the viscosity of the supporting electrolyte was altered. Changing the viscosity of the environment was expected to limit the mobility of the DNA duplex and thus diminish its elastic bending and free rotation motions. Figure 2a shows a square-wave voltammogram of the DNA duplex surface where the probe DNA is labeled with ferrocene and the DNA target labeled with methylene blue in different electrolyte viscosities. The viscosity of the electrolyte was varied by the addition of glycerol. Glycerol was chosen because it does not present electrochemical activity in the potential window studied (control experiments with MCH modified gold electrode are presented in Figure SI2). The changes in electrolyte viscosity did not cause significant modification of the electrochemistry of methylene blue, which is located at the bottom of the DNA/ MCH monolayer. In contrast, the increasing in the electrolyte solution viscosity led to an increase of the ferrocene oxidation current. This finding suggests two things. First, the mobility of the DNA decreases as the viscosity increases. Second, under these experimental conditions, DNA duplexes bend toward the electrode surface such that the ferrocene on the distal end of the DNA spends the majority of its time near the electrode surface. As a result, a greater electrochemical response is observed as the viscosity increases and the mobility of the duplex decreases.

Figure 2. SWV recorded in different viscosities of electrolyte solution of (a) duplex DNA where the probe DNA is labeled with ferrocene and the target DNA is labeled with methylene blue. (b) Plot of the ferrocene standard heterogeneous electron transfer rate constant versus viscosity of electrolyte support. Experimental conditions: SWV were recorded at 10 Hz in PBS plus different concentrations of glycerol to change the solution viscosity. The concentrations of glycerol used were 0 g/L (1.00 mPa·s), 200 g/L (1.61 mPa·s), 400 g/L (2.14 mPa·s), 600 g/L (3.48 mPa·s), and 1000 g/L (5.06 mPa·s). Concentration of DNA target: 5 μM of methylene blue labeled DNA.

Figure 2b presents the relationship between the standard heterogeneous electron transfer rate constant and solution viscosity for the ferrocene redox marker in the DNA probe of the duplex. As the standard heterogeneous electron transfer constant is independent of the viscosity, this suggests that although more ferrocene redox-active species are in close proximity to the electrode surface as the viscosity is increased, the mechanism of electron transfer remains the same. This corroborates that the elastic bending/rotational motions play an important role in the electrochemistry of the DNA duplex. However, this observation does not explain why greater charge transfer is obtained for the redox species on top of the DNA/ MCH film compared with the redox species located at the bottom of the film and closer to the electrode surface (as observed in Figure 1). The second hypothesis to be investigated was if the unexpected behavior is related to electrostatic interactions between the surface and the redox markers of different natures. Methylene blue is a positively charged molecule, and ferrocene is a neutral molecule. As such, the position of the ferrocene was swapped to the target sequence and located at the proximal end of the duplex while the methylene blue was on the distal end of the duplex attached to the probe strand (Figure 3). In both surface constructs the current arising from the distal redox label on the DNA probe was again higher than the current from the redox species in the proximal end, independently of the squarewave frequency. This confirms that the unexpected behavior is not an artifact of the redox marker. From these results, a question arises: is the lesser response from the proximal species due to poor accessibility of the counterion during the electrochemical reaction? When the redox label is located in the bottom of the DNA/MCH film, it may be harder for the ions to access this redox species, while the redox reporter located at the top of the film would be more accessible. The question regarding ion accessibility can be answered with a final experiment. For this purpose, the redox labels for D

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Figure 3. SWV recorded in PBS at 10 Hz (black lines) and 50 Hz (red lines) of (a) DNA duplex where the DNA probe is labeled with ferrocene and the DNA target with methylene blue; (b) DNA duplex where the DNA probe is labeled with methylene blue and the DNA target with ferrocene. Probe DNA immobilization: gold electrodes were incubated for 2 h in 4 μM PBS solution of labeled DNA probe; MCH: 30 min incubation in 2 mM MCH ethanolic solution; the modified electrodes were exposed to 2.5 μM labeled DNA target solution in PBS for 2 h.

Figure 4. SWV of DNA duplex interface as a function of pulse frequency where (a) the DNA probe carries the redox label ferrocene and the DNA target carries the methylene blue label at its 5′ end and (b) the DNA probe carries the redox label methylene blue and the DNA target carries the ferrocene at its 5′ end. (c) Plot of the surface coverage versus pulse frequency for the surface presented in (a); the red circles represent the surface coverage for the ferrocene redox process, and the blue triangles represent the surface coverage for the methylene blue redox process. (d) Plot of the surface coverage versus pulse frequency for the surface presented in (b); the red circles represent the surface coverage for the ferrocene redox process, and the blue triangles represent the surface coverage for the methylene blue redox process. Experimental conditions: DNA probe immobilization: clean gold electrodes were incubated for 2 h in 4 μM PBS solution of labeled probe DNA; MCH immobilization: 30 min incubation in 2 mM MCH ethanolic solution, followed by rinsing with PBS; DNA hybridization: the modified electrodes were exposed to 2.5 μM solution of the DNA target in PBS for 2 h at room temperature, followed by rinsing with PBS; SWV were recorded in PBS using the frequencies of 10, 30, 50, 70, 100, and 150 Hz.

the DNA targets were placed at the 5′ end (in the previous experiments it was on the 3′ end). In such position, after the hybridization has occurred, both redox labels (in the probe and in the target) are located on the distal end of the DNA/MCH monolayer. In this position both redox probes have similar counterion accessibility. Figure 4 presents the SWV for the DNA duplex formed in this configuration for both systems recorded in PBS using different frequencies. Figure 4a presents the system where the DNA probe carries the ferrocene and the target strand carries the methylene blue. With an increase in the square-wave frequency there is an increase in the current monitored for both methylene blue and ferrocene redox labels. At low frequency, 10 Hz, the signal from the redox tag in the DNA probe, ferrocene, was significantly greater than the signal from the methylene blue allocated in the DNA target, but at higher frequencies, the responses of the two redox labels were similar in magnitude (Figure 4a). In a similar manner, the inverse system (Figure 4b) where the DNA probe carries the methylene blue and the DNA target carries the ferrocene, the current also increased with increasing frequency, and the charge transfers of the two redox labels were similar in magnitude. Figures 4c and 4d present the plot of the surface coverage for ferrocene and methylene blue as a function of frequency for the surfaces presented in Figures 4a and 4b, respectively. The surface coverage was calculated as described in the Experimental Section. For the surface where the DNA probe carries ferrocene and the DNA target carries methylene blue at the 5′ end, the surface coverage for ferrocene molecules (red circles, Figure 4c) and methylene blue (blue triangles, Figure 4c) decreases from 10 to 30 Hz; after 30 Hz the apparent surface coverage of both redox species does not change with further changes in frequency. In this frequency range, upon hybridization of the target with the surface bound probe strand, at high concentrations of target, the number of redox molecules on the probe strands directly correlates with the number of redox species from the target strands. For the surface at which the DNA probe carries methylene blue and the strand DNA target carries ferrocene (Figure 4d), the surface coverages from

ferrocene and methylene blue are approximately the same above 50 Hz. So, in the configuration where both redox species are located at the top of the DNA monolayer, for a given DNA target concentration, the number of target strands that hybridize to the probe strands can be directly quantified. In the case of the data presented in Figure 4 where the concentration of target DNA is high (2.5 μM), the hybridization efficiency is close to 100%. The results reported in Figure 4 suggest that counterion accessibility plays a key role in the DNA duplex electrochemistry. They also suggest that at optimized square-wave frequencies the charge involved in the redox process of the labels does not depend on which DNA strand carries that redox marker, probe or target strand. It is also important to note that in such configuration both redox labels are susceptible to elastic bending and rotational motions of the DNA duplex.

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CONCLUSIONS In this work a dual signaling strategy was used to investigate the effect of varying the position of redox moieties within the DNA/MCH monolayer. It was observed that the currents arising from redox labels located at the bottom of the film DNA E

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platform for functional genomics. Trends Biotechnol. 1998, 16 (7), 301−306. (3) Drummond, T. G.; Hill, M. G.; Barton, J. K. Electrochemical DNA sensors. Nat. Biotechnol. 2003, 21 (10), 1192−1199. (4) Du, Y.; Lim, B. J.; Li, B. L.; Jiang, Y. S.; Sessler, J. L.; Ellington, A. D. Reagentless, Ratiometric Electrochemical DNA Sensors with Improved Robustness and Reproducibility. Anal. Chem. 2014, 86 (15), 8010−8016. (5) Yang, W. W.; Lai, R. Y. Comparison of the Stem-Loop and Linear Probe-Based Electrochemical DNA Sensors by Alternating Current Voltammetry and Cyclic Voltammetry. Langmuir 2011, 27 (23), 14669−14677. (6) Wang, J. Electrochemical nucleic acid biosensors. Anal. Chim. Acta 2002, 469 (1), 63−71. (7) Anne, A.; Demaille, C.; Moiroux, J. Elastic bounded diffusion and electron propagation: Dynamics of the wiring of a self-assembly of immunoglobulins bearing terminally attached ferrocene poly(ethylene glycol) chains according to a spatially controlled organization. J. Am. Chem. Soc. 2001, 123 (20), 4817−4825. (8) Gooding, J. J.; Chou, A.; Mearns, F. J.; Wong, E.; Jericho, K. L. The ion gating effect: using a change in flexibility to allow label free electrochemical detection of DNA hybridisation. Chem. Commun. 2003, No. 15, 1938−1939. (9) Wong, E. L. S.; Chow, E.; Gooding, J. J. DNA recognition interfaces: The influence of interfacial design on the efficiency and kinetics of hybridization. Langmuir 2005, 21 (15), 6957−6965. (10) Wong, E. L. S.; Gooding, J. J. Electronic detection of target nucleic acids by a 2,6-disulfonic acid anthraquinone intercalator. Anal. Chem. 2003, 75 (15), 3845−3852. (11) Anne, A.; Demaille, C. Electron transport by molecular motion of redox-DNA strands: Unexpectedly slow rotational dynamics of 20mer ds-DNA chains end-grafted onto surfaces via C(6) linkers. J. Am. Chem. Soc. 2008, 130 (30), 9812−9823. (12) Li, H.; Arroyo-Curras, N.; Kang, D.; Ricci, F.; Plaxco, K. W. Dual-Reporter Drift Correction To Enhance the Performance of Electrochemical Aptamer-Based Sensors in Whole Blood. J. Am. Chem. Soc. 2016, 138 (49), 15809−15812. (13) Wong, E. L. S.; Gooding, J. J. Charge transfer through DNA: A selective electrochemical DNA biosensor. Anal. Chem. 2006, 78 (7), 2138−2144. (14) Fan, C. H.; Plaxco, K. W.; Heeger, A. J. Electrochemical interrogation of conformational changes as a reagentless method for the sequence-specific detection of DNA. Proc. Natl. Acad. Sci. U. S. A. 2003, 100 (16), 9134−9137. (15) Di Giusto, D. A.; Wlassoff, W. A.; Giesebrecht, S.; Gooding, J. J.; King, G. C. Multipotential electrochemical detection of primer extension reactions on DNA self-assembled monolayers. J. Am. Chem. Soc. 2004, 126 (13), 4120−4121. (16) Mearns, F. J.; Wong, E. L. S.; Short, K.; Hibbert, D. B.; Gooding, J. J. DNA biosensor concepts based on a change in the DNA persistence length upon hybridization. Electroanalysis 2006, 18 (19− 20), 1971−1981. (17) Anne, A.; Bouchardon, A.; Moiroux, J. 3′-ferrocene-labeled oligonucleotide chains end-tethered to gold electrode surfaces: Novel model systems for exploring flexibility of short DNA using cyclic voltammetry. J. Am. Chem. Soc. 2003, 125 (5), 1112−1113. (18) Pheeney, C. G.; Barton, J. K. DNA Electrochemistry with Tethered Methylene Blue. Langmuir 2012, 28 (17), 7063−7070. (19) Zwang, T. J.; Hurlimann, S.; Hill, M. G.; Barton, J. K. HelixDependent Spin Filtering through the DNA Duplex. J. Am. Chem. Soc. 2016, 138 (48), 15551−15554. (20) Herne, T. M.; Tarlov, M. J. Characterization of DNA probes immobilized on gold surfaces. J. Am. Chem. Soc. 1997, 119 (38), 8916−8920. (21) Petrovykh, D. Y.; Kimura-Suda, H.; Whitman, L. J.; Tarlov, M. J. Quantitative analysis and characterization of DNA immobilized on gold. J. Am. Chem. Soc. 2003, 125 (17), 5219−5226. (22) Peterlinz, K. A.; Georgiadis, R. M.; Herne, T. M.; Tarlov, M. J. Observation of hybridization and dehybridization of thiol-tethered

duplex (proximal end) are lower than the currents of the redox labels located at the top of the film. We conclude that the lower currents obtained for the redox species located on the proximal end close to the electrode surface are due to the limitations of ion accessibility. DNA is a highly charged polyelectrolyte that assembles at the surface and so may form a permselective coating. As in this configuration the redox-active molecule was located underneath the DNA duplex, the ion transport along the DNA duplexes influences the redox process. Because of the search for neutrality, the negatively charged DNA backbone attracts cations, resulting in formation of a compact layer of positive ions around the DNA duplexes.28,30 The results presented here are relevant to the signaling mechanisms of a variety of conformational-based biosensors, ranging from electrochemical DNA sensors to surface-bound molecular beacons. The results also suggest that careful consideration of the position of the redox label in the DNA film is relevant to obtain maximum performance across this broad range of sensors.

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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.langmuir.7b02787. Figures S1−S5 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail [email protected] (R.D.T.). *E-mail [email protected]; Tel +61-2 9385 5384 (J.J.G.). ORCID

D. Brynn Hibbert: 0000-0001-9210-2941 Richard D. Tilley: 0000-0003-2097-063X J. Justin Gooding: 0000-0002-5398-0597 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the generous financial support from the Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology (CE140100036) and the ARC Australian Laureate Fellowship (FL150100060). S.M.S. is a scholarship student from CNPq, Conselho Nacional ́ de Desenvolvimento Cientifico e Tecnológico and INCTBio, ́ Instituto Nacional de Ciência e Tecnologia em Bioanalitica − Brazil.

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ABBREVIATIONS PBS, Dulbecco’s phosphate buffered saline modified without calcium chloride and magnesium chloride; MCH, 6-mercaptohexan-1-ol; MB, methylene blue; Fc, ferrocene; SWV, squarewave voltammetry.

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DOI: 10.1021/acs.langmuir.7b02787 Langmuir XXXX, XXX, XXX−XXX