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Feb 17, 2013 - 651 Hamilton Hall, University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0304, United States. •S Supporting Information. ABSTRACT: ...
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Effect of Signaling Probe Conformation on Sensor Performance of a Displacement-Based Electrochemical DNA Sensor Zhi-gang Yu†,‡ and Rebecca Y. Lai*,‡ †

Key Laboratory of Green Chemical Engineering and Technology of College of Heilongjiang Province, College of Chemical and Environmental Engineering, Harbin University of Science and Technology, Harbin 150040, P. R. China ‡ 651 Hamilton Hall, University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0304, United States S Supporting Information *

ABSTRACT: Here we report the effect of the signaling probe conformation on sensor performance of a “signal-on” folding-based electrochemical DNA sensor. The sensor is comprised of a methylene blue (MB)-modified signaling probe and an unlabeled capture probe that partially hybridize to each other at the distal end. In presence of the full-complement target which binds to the unlabeled capture probe, the labeled signaling probe is released. Two different signaling probes were used in this study, in which one is capable of assuming a stem-loop conformation (SLP-MB), whereas the other probe adopts a flexible linear conformation (LP-MB). In the presence of the full complement target DNA, both sensors showed a large increase in MB current when interrogated using alternating current (ac) voltammetry, verifying the release of the signaling probe. Overall, the SLP-MB sensor showed higher % signal enhancement; the LP-MB sensor, however, showed distinctly faster binding kinetics when interrogated under the same experimental conditions. The SLP-MB sensor displayed a wider usable ac frequency range when compared to the LP-MB sensor. Despite these differences, the detection limit and dynamic range were found to be similar among the two sensors. In addition to 6-mercapto-1-hexanol, longer chain hydroxylterminated alkanethiols were used to construct these sensors. Our results showed that sensors fabricated with longer chain diluents, independent of the sensor architecture, were not only functional, the signaling capability was significantly enhanced.

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that is in a linear rather than a stem-loop conformation.9,10 The signaling of this linear probe sensor originates from the binding-induced changes in the dynamics of the redox-labeled probe, which is dampened due to the decrease in the flexibility of the probe-target duplex, leading to a significant reduction in the redox current.11 Both stem-loop and linear probe-based E-DNA sensors have been proven to be highly sensitive and selective enough to be employed directly in whole blood and other realistically complex biological samples.7,9,10,12 However, both sensors are inherently “signal-off” sensors, which suffer from limited signaling capacity, in which only a maximum of 100% signal suppression can be attained under any experimental conditions.6−12 To circumvent this limitation, several “signal-on” E-DNA sensors have been developed in the past few years.13−16 While most sensors of this class utilize one type of surface-immobilized probe, we recently published a short communication on the design and fabrication of a “signalon” E-DNA sensor that features two surface-attached DNA probes.17 In this sensor architecture, an unlabeled capture probe and a MB-modified signaling probe are coimmobilized onto the interrogating electrode. The two probes are designed

rowing demands for point-of-care medical diagnostics and rapid methods for the specific detection of nucleic acids has resulted in the development of a number of DNA and RNA sensing methods in the past few decades.1−3 While many of the sensing approaches feature impressive sensitivity and generalizability, a DNA sensing technology that is highly sensitive and selective does not use exogenous reagents, requires relatively low quantities of mass/power/volume, and is operationally convenient has yet to be realized. One of the more promising sensing approaches developed to date is the electrochemical DNA (E-DNA) sensing platform.4,5 The first E-DNA sensor reported in 2003 is the electrochemical equivalent of the optical “molecular beacon” detection system.6 The signaling of this sensor originates from the binding-induced changes in the conformation of the stem-loop probe and the efficiency with which the attached redox label transfers electrons to the electrode. In the absence of the target, the stemloop structure holds the redox label, methylene blue (MB), in proximity to the electrode, enabling efficient electron transfer. Upon hybridization with the perfect match target DNA, the double-stranded conformation of the probe DNA forces the redox label away from the electrode, impeding electron transfer, resulting in a detectable reduction in the MB redox current.6−8 An alternate version of this E-DNA sensor was reported in 2005; this sensor design utilizes a redox-labeled DNA probe © 2013 American Chemical Society

Received: December 30, 2012 Accepted: February 15, 2013 Published: February 17, 2013 3340

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evidence to support the proposed signaling mechanisms. While defining experimental conditions in which the two sensors can achieve optimal sensor performance is essential for applicational purposes, fundamental understanding of the signaling mechanism of these sensors could be beneficial in designing future generations of E-DNA sensors for real-world applications.

to be partially complementary to each other, enabling the formation of a short DNA duplex at the distal end of the probes. In the absence of the target DNA, the short duplex prevents the MB labels on the signaling probe from accessing the electrode surface for efficient electron transfer; formation of the target/ capture probe duplex, however, disrupts the short interprobe duplex, liberating the signaling probe and allowing it to assume a stem-loop conformation. The formation of the stem-loop structure results in an increase in MB current, owing to the proximity of the redox labels to the electrode. This dual-probe sensor design has been demonstrated to be sensitive, specific, and works well under a wide range of experimental conditions.17 Similar to the original E-DNA sensor, this “signal-on” sensor architecture is equally versatile. It is conceivable that the signaling probe can be redesigned to assume a linear conformation once it has been released from the short duplex upon target hybridization. The change in the probe dynamics is likely to result in an increase in MB current. This equally advantageous linear probe-based design, however, has not been explored previously. Here we systematically characterized both the stem-loop and linear probe-based “signal-on” E-DNA sensors that are fabricated using close-to-identical 25-mer signaling probe sequences (Scheme 1). We utilized alternating current voltammetry

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EXPERIMENTAL AND METHODS Materials. A thiolated and MB-modified stem-loop (SLPMB) or linear DNA probe (LP-MB) was used as the surfaceimmobilized signaling probe (Figure S1, Supporting Information). A 25-base thiolated DNA probe was used as the target capturing probe (T8-P). All three probes were purchased from Biosearch Technologies Inc. (Novato, CA). Five DNA targets obtained from Integrated DNA Technologies (Coralville, IA) were used as received. The sequence information of the probes and targets are shown in the following. The single underlined portion of SLP-MB and LP-MB forms a 10-base duplex with the italicized portion of the unlabeled capture probe (T8-P), whereas the double underlined portion of T8-P hybridizes to the target DNA. The mismatches in the targets are highlighted in bold:

Scheme 1. Design and Signaling Mechanism of Stem-Loop (SLP-MB) (A) and Linear Probe-Based (LP-MB) (B) E-DNA Sensors

6-Mercapto-1-hexanol (C6-OH), 8-mercapto-1-octanol (C8-OH), 9-mercapto-1-nonanol (C9-OH), tris-(2-carboxyethyl) phosphine hydrochloride (TCEP), guanidine hydrochloride (GHCl), and iron fortified bovine calf serum were used as received (Sigma-Aldrich, St. Louis, MO). All other chemicals were of analytical grade. All the solutions were made with deionized (DI) water purified through a Millipore system (18.2 MΩ cm, Millipore, Billerica, MA). All sensors were interrogated in either a phosphate buffer containing 10 mM sodium phosphate and 300 mM sodium chloride at pH 7.4 (PBS) or 50% bovine calf serum (1:1 bovine calf serum−PBS). E-DNA Sensor Preparation. Prior to sensor fabrication, gold disk electrodes with a geometric area of 0.0314 cm2 (CH Instruments, Austin, TX) were polished with a 0.1 μm diamond slurry (Buehler, Lake Bluff, IL), rinsed with deionized water, and sonicated in a low power sonicator for 5 min to remove bound particulates. They were then electrochemically cleaned by a series of oxidation and reduction cycles in 0.5 M H2SO4. The real surface area of each electrode was estimated based on the amount of charge consumed during the reduction of the gold surface oxide monolayer in 0.05 M H2SO4 and the reported value of 400 μC cm−2 was used for the calculation.18 The average surface roughness factor, which is the ratio between the real surface area and the geometric surface area, was found to be 1.13 ± 0.11 (n = 3). Fabrication of the E-DNA sensor involved several steps. First, 1.5 μL of 200 μM T8-P was added to 1 μL of 200 μM SLP-MB or LP-MB, and 2.5 μL of 10 mM TCEP was subsequently added to the solution to reduce the disulfide bond. The solution was then diluted with Phys2 buffer (20 mM Tris, 140 mM NaCl, 5 mM KCl, 1 mM

(ACV) to investigate the sensors’ hybridization efficiency and binding kinetics, with the goal of obtaining a set of individually optimized experimental conditions for the two sensors. Cyclic voltammetry (CV) was used to generate concrete experimental 3341

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by incubating in 4 M GHCl for 4 min, followed by rinsing with DI water for 30 s for sensors interrogated in 50% serum. Mechanical stirring was not used in any of the hybridization experiments. All experiments were performed at room temperature (∼23 °C). Unless mentioned otherwise, all experimental results presented in this study are averaged from three different sensors.

MgCl2, and 1 mM CaCl2, pH 7.4) to achieve a probe concentration of 5 μM (3 μM T8-P and 2 μM SLP-MB or LP-MB) . Both T8-P and SLP-MB or LP-MB were coimmobilized onto the gold electrode surface by incubating the clean electrode in this probe DNA solution for 1 h. The modified electrode was then rinsed with PBS and further passivated with 2 mM C6-OH (C8-OH or C9-OH) in PBS overnight to displace nonspecifically bound oligonucleotides. The density of electroactive DNA probes on the electrode surface was determined by integration of charges under the MB reduction peak in the CV scans collected at low scan rates (20, 50, and 100 mV/s).19 To calculate the electron transfer rate constant of the MB, ks, for both SLP-MB and LP-MB modified electrodes, a series of CV scans were collected. Increasing values of CV peak separation (ΔEp = Ep,a − Ep,c) as a function of increasing scan rate (v) reflects control of the voltammetry by the rate of heterogeneous electron transfer reactions of the MB labels in the monolayer. When ΔEp > 200/n mV, a graph of ΔEp versus log v yields a straight line that is in accordance with the Laviron equation (eq 1).20

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RESULTS AND DISCUSSION Sensor Design and Characterization Using ACV. Sensor architecture and signaling mechanism of these two reagentless and reusable sensors are shown in Scheme 1. Fabrication of the sensors involves coimmobilization of a redox-labeled signaling probe and an unlabeled capture probe onto a gold electrode using thiol-gold chemistry. SLP-MB has a 15-base loop that is flanked by two 5-base self-complementary regions. LP-MB has the same 15-base core sequence as SLP-MB; the 5-base sequences at the two termini are identical and thus incapable of forming a double stranded stem. T8-P, the unlabeled capture probe used in the fabrication of both sensors, possesses a 17-base sequence that binds specifically to the target DNA. Eight additional thymine bases are located at the 3′-end of T8-P to improve probe flexibility. T8-P is designed to be partially complementary to both SLP-MB and LP-MB, which allows the formation of a 10-base DNA duplex at the distal end of the probes. In absence of the target, the short DNA duplex prevents the MB labels on the signaling probe from accessing the electrode surface for efficient electron transfer; formation of the target/capture probe duplex, however, disrupts the short duplex, liberating the signaling probe and allowing it to assume either a stem-loop or linear conformation. The change in the probe conformation and/or dynamics will result in an enhancement in electron transfer, which will be reflected in the increase in MB current. Since ACV is one of the more commonly used sensor interrogation techniques, it was first used to characterize the sensors. In the absence of the target DNA, a well-defined MB peak was observed at ∼−0.29 V (vs Ag/AgCl) for both SLPMB and LP-MB sensors (Figure 1). The low MB current could be attributed to the formation of the 10-base duplex between T8-P and SLP-MB or LP-MB.17 Using the optimized sensor fabrication protocol, a probe coverage of 1.2 × 1012 ± 1.2 × 1011 molecules cm−2 was obtained for the SLP-MB sensor; whereas a slightly higher probe coverage of 1.5 × 1012 ± 2.1 × 1011 molecules cm−2 was evident for the LP-MB sensor. These values are lower than those obtained when only the signaling probes were used in the sensor fabrication. Such difference is not unexpected since more signaling probes can be immobilized on the electrode in the absence of T8-P. However, in the presence of 1.0 μM perfect match target (PM-17), a large signal increase was registered. For this class of sensors, the change in probe flexibility and/or conformation often parallels the change in the rate of electron transport and the resultant redox current.4,5,9 The large increase in ac current post hybridization is attributed to the liberation of the signaling probe. For the SLP-MB sensor, the MB label is confined near the electrode surface owing to the formation of the stem-loop structure, resulting in a large increase in MB current.17 The increase in the probe flexibility, however, is the origin of the increase in MB current for the LP-MB sensor.9,21 In addition to PM-17, two longer DNA targets with additional adenine bases (i.e., PM-21, PM-25) were used in this study, aiming at understanding the effect of target length on

log ks = α log(1 − α) + (1 − α)log α − log(RT /nFv) − α(1 − α)nF ΔEp/2.3RT

(1) −1

where ks is the electron transfer rate constant (s ), α is the electron transfer coefficient, v is the CV potential scan rate (V/s), and ΔEp is the difference between the anodic potential and cathodic potential (V). α can be determined from the slope of the straight line, and ks can be calculated with the help of the intercept. Electrochemical Measurements. Electrochemical measurements were performed at room temperature using a CHI 1040A Electrochemical Workstation (CH Instruments, Austin, TX). The E-DNA sensors were characterized by ACV over a wide range of frequencies (1−5000 Hz) using an amplitude of 25 mV. Higher ac frequencies were used with sensors interrogated in pure buffer. Owing to the small MB peak current observed at high frequencies, only low frequencies were used with sensors interrogated in 50% serum. CV was also used to determine the surface probe coverage and electron transfer rate. DNA probe-modified gold disk electrodes were used as working electrodes. A platinum wire electrode was used as the counter electrode, and a Ag/AgCl (3.0 M KCl) electrode served as the reference electrode (CH Instruments, Austin, TX). Prior to sensor interrogation, the modified electrode was allowed to equilibrate in PBS or 50% fetal bovine serum. Target DNA (1 μM) was then added to the electrochemical cell for sensor interrogation. Alternating current voltammograms were collected at different time intervals until a stable peak current was obtained. The ratio between the MB peak current in the presence and absence of the target DNA was used to calculate the % signal enhancement (eq 2). signal enhancement (%) = [(I − I0)/ I0] ∗100

(2)

where I is the baseline-subtracted peak current obtained in the presence of the target, and I0 is the baseline-subtracted peak current in the target-free solution. Calibration curve results were obtained by sequential addition of the target DNA at an interval of 60 min. Concentrations of the target DNA used to obtain the calibration curves were 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, and 3 μM. Sensor regeneration was achieved by rinsing with room temperature DI water for 30 s for sensors interrogated in PBS or 3342

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The increase in current for the LP-MB sensor, however, does not rely on the formation of a defined probe conformation. The probe becomes highly flexible immediately upon release from the 10-base duplex. Similarly fast binding kinetics was observed for the two longer DNA targets. For this class of sensors, the MB peak current obtained both before and after target hybridization is highly dependent on the applied ac frequency.9,10,22 The peak current should be proportional to frequency for a surface-confined reversible redox system where the applied frequency is much lower than the electron-transfer rate. However, the peak current often decreases at higher frequencies. This phenomenon can be explained in terms of the relative time scale of interfacial electron transfer and the potential fluctuation, as the applied frequency approaches a critical value above which electron transfer can no longer keep up with the rapidly oscillating potential, the peak current diminishes relative to the background current.23−25 This “threshold” frequency is an intrinsic characteristic of a specific sensor design. For these two sensors, in the absence of PM-17, the MB current was very low; the current gradually decreased at frequencies beyond 5 Hz. In contrast, upon hybridization with PM-17, a large increase in MB current with increasing frequency was evident prior to the “threshold” frequency that is unique for each sensor (Figure S3, Supporting Information).10 The “threshold” frequency was found to be 2000 and 200 Hz for the SLP-MB and LP-MB sensors, respectively. These values provides evidence in support of the signaling mechanism, in which the SLP-MB sensor, owing to the formation of the stem-loop structure in the hybridized state, can support efficient electron transfer at extremely high frequencies; the dynamics-based LP-MB sensor, however, cannot.9,10 Since the % SE is calculated using the two MB currents, it is equally dependent on the applied ac frequency.9,10 For example, in the presence of PM-17 a steady increase in % SE from 31 ± 4% to 8972 ± 347% was observed for the SLP-MB sensor between 1 and 1000 Hz (Figure 2A). The same trend can be seen with the other two targets. The % SE for PM-17 and PM-21 was very similar at all frequencies, whereas a consistently lower % SE was detected for PM-25. Unlike the SLP-MB sensor, the dynamics-based LP-MB sensor exhibited a different frequencydependent profile, as demonstrated in this study as well as our previous studies.9,10 As shown in Figure 2B, a sharp increase in % SE from 26 ± 1% to 787 ± 126% was observed as the frequency increased from 1 to 100 Hz. A rapid decrease in % SE was evident at frequencies beyond 100 Hz. The % SE at an extreme frequency such as 2000 Hz was low for all three targets. The results highlight one practical difference between the two sensors; this wide usable frequency range is undoubtedly a unique feature of the SLP-MB sensor.10 Sensor Characterization Using CV. While ACV has been the method of choice for E-DNA sensor characterization, CV has proven to be an equally useful means to elucidate a sensor’s signaling mechanism.10 Shown in Figure S4 (Supporting Information) are the CV scans of the two sensors before, after hybridization with PM-17, and after sensor regeneration at a scan rate of 1 V/s. The voltammetric hysteresis (ΔE) was found to be 95 mV for SLP-MB and 99 mV for LP-MB, further confirming that both sensors are in a similar duplex-state prior to target hybridization. The 10-base duplex positions the MB redox label away from the electrode, thereby impeding efficient electron transfer, which results in the large hysteresis observed in CV as well as the low MB current in ACV. Upon hybridization to PM-17, a large change in the hysteresis was observed

Figure 1. Alternating current voltammograms of the SLP-MB (A) and LP-MB (B) sensors before and after hybridization with 1 μM PM-17, PM-21, and PM-25. For both systems, all three curves were obtained using the same sensor. Sensor regeneration curves are not shown since they essentially overlapped with the “Without Target” curve.

sensor performance. When interrogated using ACV at 10 Hz, the SLP-MB sensor showed signal enhancement (SE) of 904 ± 56%, 871 ± 29%, 626 ± 21% for PM-17, PM-21, and PM-25, respectively. Slightly lower % SE was recorded for the LP-MB sensor for all three targets (437 ± 19%, 468 ± 28%, and 350 ± 14% for PM-17, PM-21, and PM-25, respectively) (Table S1, Supporting Information). The difference in % SE between the two sensor systems can be ascribed to the difference in the released signaling probe conformation. Formation of the stem-loop conformation produces a larger change in the electron transfer rate between the “off” and “on” states, which is reflected by the larger % SE. For both sensors, PM-17 and PM-21 showed higher % SE when compared to PM-25, the target sequence capable of hybridizing to the entire capture probe sequence. The difference in % SE between PM-17 and PM-25 was more pronounced for the SLP-MB sensor, presumably due to steric hindrance, in which complete hybridization to T8-P could impede formation of the stem-loop structure. The LP-MB sensor performance appears to be less dependent on target chain length. It is worth noting that all three interrogations were performed using the same sensor since this sensor, like other E-DNA sensors, is regenerable and reusable. In addition to quantifying the extent of signal increase, the response time, which is another important indicator of sensor performance, was also evaluated. Our results suggest that the binding kinetics of the two sensors is different (Figure S2, Supporting Information).10 For example, when challenged with PM-17, only ∼75% of the total % SE was observed within 12 min for the SLP-MB sensor, whereas ∼92% of the total % SE was evident within the same time period for the LP-MB sensor. This result is not unexpected since the change in MB current for the SLP-MB sensor originates from the formation of the stem-loop structure, which is not an instantaneous event. 3343

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Sensor Sensitivity and Specificity. Sensor sensitivity and dynamic range are two other important indicators of sensor performance. Sensors with high sensitivity and a large dynamic range are sought after for real-world DNA analysis. Since PM-17 and PM-21 showed relatively similar sensor performance for both sensors, we chose PM-17 as the model target for this study. As shown in Figure 3, both sensors showed a change

Figure 2. Effect of ac frequency on signal enhancement upon hybridization to 1 μM PM-17, PM-21, and PM-25 for the SLP-MB (A) and LP-MB (B) sensors.

for both sensors (ΔE = 3 mV for SLP-MB and ΔE = 38 mV for LP-MB). A small posthybridization hysteresis is anticipated for the SLP-MB sensor since the signaling probe assumes the stem-loop conformation, which positions the MB label close to the electrode. The posthybridization hysteresis for the LP-MB sensor is expected to be larger than that observed with the SLPMB sensor since target hybridization induces a morphological change rather than an actual conformational change.9,10 These results agree well with the ACV results, in which higher % SE was observed for the SLP-MB sensor in the presence of the target DNA. It is worth mentioning that the CV scans obtained post sensor regeneration completely overlapped with the scans recorded prior to target hybridization, suggesting that the probes in both sensors are capable of resuming the original partially hybridized structure. Electron transfer kinetics of MB was determined by fitting the aforementioned CV results using the Laviron equation. A ks value of 2.8 ± 0.1 was obtained for both the SLP-MB and LPMB sensors in the unhybridized state (Table S2, Supporting Information). The electron transfer rate constant of the hybridized SLP-MB sensor, ks′, was found to be significantly larger when compared to the prehybridization value (ks′ = 137 ± 3 s−1 vs ks = 2.8 ± 0.1 s−1). Since electron transfer kinetics varies exponentially with the distance between the redox label and the electrode surface, the results here again indicate the formation of the stem-loop structure in the presence of the target. For the LP-MB sensor, the increase was less substantial (ks′ = 13 ± 1 s−1 vs ks = 2.8 ± 0.1 s−1), which is consistent with the signaling mechanism. ks′ values clearly reflect the differences in electronic coupling distance between MB and the electrode for the two sensors in the hybridized state. It is noteworthy that the ks′ values in the presence of different targets agree well with the % SE observed in ACV for both sensors (Tables S1 and S2, Supporting Information).

Figure 3. Dose−response curves for the SLP-MB (A) and LP-MB (B) sensors in the presence of 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, and 3 μM PM-17. The inset figures show the dose− response curves for the lower target concentrations. The data were collected at an ac frequency of 10 Hz.

in % SE that is concentration-dependent. A detection limit of 1 pM (S/N = 3) was achieved for both sensors; this detection limit is lower than that determined for the original “signal-off” E-DNA sensor.6 When compared to the LP-MB sensor, the SLP-MB sensor exhibited significantly higher % SE at most target concentrations. The linear dynamic range, however, was found to be similar for both sensors. Negligible signal change was observed at concentrations beyond 1 μM; this is not unexpected since most binding sites available on the sensor surface are likely to be occupied at these concentrations. Unfortunately, since the dose−response data could not be fitted to a one-site binding model, Kd values could not be accurately determined. Nonetheless, it is worth noting that the MB current is rather stable in the absence of the target, thereby allowing clear distinction between target-induced current change and baseline drift. Shown in Figure S5 in the Supporting Information are the ACVs obtained before and after addition of 10 pM PM-17; an increase in the MB peak current was clearly observable. Overall, sensor performance is reproducible for both sensors, as evidenced by the small error bars shown in the dose−response curves (Figure 3). In addition to determining the sensors’ sensitivity and limit of detection, we also evaluated the sensors’ specificity. Both sensors are specific and are capable of discriminating between 3344

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Figure 5. Hybridization-regeneration curves for the SLP-MB (A) and LP-MB (B) sensors in the presence of 1 μM PM-17. The results presented here were collected at an ac frequency of 10 Hz. Figure 4. Response curves for the SLP-MB (A) and LP-MB (B) sensors in presence of 1 μM of PM-17, 17-1M, and 17-2M. The results presented here were collected at an ac frequency of 10 Hz.

effects of reduction in ionic strength and increase in solvent viscosity (Figure 1 and Figure S6, Supporting Information).9,17 Lowering the ionic strength is likely to affect the formation of interprobe duplexes prior to target hybridization, which is the key feature of this sensor design and vital to optimal sensor signaling. Hybridization efficiency between the target and the capture probe could be also affected by the change in the ionic strength. However, despite the difference in % SE, both sensors can be readily regenerated via a 4-min incubation in GHCl. While both sensors showed good regenerability, a slight decrease in % SE was observed after each regeneration cycle (Figure 5). The SLP-MB sensor showed more consistent sensor performance than the LP-MB sensor, as indicated by the lower % SE loss over the course of six hybridizations (SE loss of ∼11% for SLP-MB vs SE loss of ∼20% for LP-MB). One possible reason behind the signal loss with extensive sensor usage is DNA probe degradation by the nucleases present in the blood serum. To determine the sensors’ specificity in this complex medium, we challenged the sensors against all three targets (PM-17, 17-1M, and 17-2M). Shown in Figure S6 in the Supporting Information are the ac voltammograms of the two sensors collected in 50% serum in the presence of the three targets. For the SLP-MB sensor, Fs was found to be 2.6 and 101 for 17-1M and 17-2M, respectively. For the LP-MB sensor, Fs was determined to be 2.3 for 17-1M and 34 for 17-2M. Independent of the sensor architecture, Fs for 17-1M was slightly lower than that determined in a pure buffer; however, Fs for 17-2M was significantly higher, in particular, for the SLP-MB sensor (Fs = 101 vs 17.4). While both sensors are capable of retaining sensor specificity, the SLP-MB sensor operates more optimally in this medium. Characterization of Sensors Fabricated with Longer Diluents. On the basis of our previous study on the “signal-off” E-DNA sensors, the use of longer diluents allows the sensors to be interrogated at a higher temperature, which contributes to the enhanced mismatch discrimination.26 The use of longer diluents such as C8-OH and C9-OH is likewise advantageous

the perfect match and mismatch targets (Figure 4). Specifically, our results showed large differences in % SE between PM-17 and the mismatch targets, 17-1M and 17-2M (Table S1, Supporting Information). The discrimination factor (Fs = % SEPM‑17/% SE17‑1M or 17‑2M) was found to be 3.5 and 17.4 for 17-1M and 17-2M, respectively, for the SLP-MB sensor. The LP-MB sensor displayed lower Fs values for both targets (Fs = 3.0 for 17-1M and 9.3 for 17-2M). Independent of the sensor architecture, slightly faster hybridization kinetics were observed for the mismatch targets (Figure 4). Overall, the sensors displayed improved specificity when compared to the original “signal-off” E-DNA sensors, which are only capable of differentiating between the perfect match and single-base mismatch targets at elevated temperature and with the help of longer passivating diluents.26 This significant enhancement in sensor specificity is presumably due to the displacement-replacement sensing mechanism. It could be, in part, due to the preferential binding of the mismatch target DNA to the excess T8-P capture probes. The sensor surface is likely to have excess T8-P probes since the probe ratio of T8-P to SLP-MB (or LP-MB) used in the immobilization step was 3:2. Hybridization to the unlabeled capture probes that are not part of a duplex does not contribute to the signal enhancement, but it could affect the sensor’s mismatch discrimination capability. The change in the probe ratio could have a significant effect on general sensor performance. A comprehensive study that focuses on elucidating the effect of excess capture probes on sensor performance, in particular, sensor specificity, is currently under investigation in the lab and the findings will be disseminated in a future publication. Sensor Selectivity and Reusability. Similar to previously developed E-DNA sensors; both sensors are fully functional in a realistically complex medium such as 50% blood serum (Figure 5).7,12,17 The % SE, however, was lower than that recorded in a pure buffer, presumably due to the combined 3345

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for these two “signal-on” sensors, in particular, for improvement in signaling efficiency. The initial MB current was lower for these sensors, presumably due to the lower surface probe coverage (Table S3, Supporting Information).26 Additionally, in the presence of the longer diluent the interprobe duplex is more sterically constricted and thus impedes efficient electron transfer to the electrode, resulting in the lower MB current. However, upon hybridization to PM-17, the % SE was significantly larger when compared to sensors passivated with C6-OH (Figure S7, Supporting Information). It is worth noting that this diluent chain length-dependent effect is more pronounced for the SLP-MB system. For example, % SE for sensors passivated with C9-OH was about 2.6 times higher than those with C6-OH for the SLP-MB system; whereas only a 1.9 times increase was evident for the LP-MB system. The sensors passivated with C8-OH and C9-OH showed close to identical % SE upon target hybridization for the LP-MB sensor. The SLP-MB sensor, however, showed the largest % SE for sensors passivated with C9-OH. The use of longer diluents also results in the systematic decrease in both ks and ks′, indicating the increase in electronic coupling distance between the MB redox label and the electrode with increasing diluent chain length.26 The decrease in ks′, however, was less substantial for the SLP-MB system; formation of the stem-loop structure does not appear to be adversely affected by the longer diluents. Furthermore, the hybridization kinetics was slightly improved, which could be attributed to the lower probe coverage (Figure S7 and Table S3, Supporting Information).26 The double layer capacitance calculated from the CV scans (data not shown) decreased with increasing diluent chain length, suggesting a decrease in the electric double layer, which has been shown to speed up diffusion of the negatively charged target DNA to the electrode surface, thus shortening the hybridization time.26−28 Overall, the results presented here highlight the advantages of using longer passivating diluents in sensor fabrication, in particular, for the SLP-MB sensor.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by Nebraska EPSCoR (Grant EPS-1004094) and the NSF Career Award (Grant CMI0955439). The authors would like to thank Anita J. Zaitouna for the helpful discussions.

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CONCLUSION In this study, we designed and fabricated two new “signal-on” E-DNA sensors that utilize a displacement-replacement sensing mechanism. For the SLP-MB sensor, target hybridization leads to the release of the MB-labeled signaling probe that is capable of forming a stem-loop structure. In contrast, the signaling probe used in the LP-MB system is only capable of assuming a linear structure post hybridization. We systematically evaluated the sensors’ performance, including the limit of detection, specificity, selectivity, and reusability. We also studied the effect of longer chain diluents on sensor performance. While both sensors are sensitive, specific, and selective, our results also suggest that the SLP-MB sensor is, in general, the more advantageous design, as indicated by its ability to perform optimally under a wider range of experimental conditions.

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REFERENCES

(1) Sassolas, A.; Leca-Bouvier, B. D.; Blum, L. J. Chem. Rev. 2008, 108, 109−139. (2) Vercoutere, W.; Akeson, M. Curr. Opin. Chem. Biol. 2002, 6, 816−822. (3) Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2003, 21, 1192−1199. (4) Ricci, F.; Plaxco, K. W. Microchim. Acta 2008, 163, 149−155. (5) Lubin, A. A.; Plaxco, K. W. Acc. Chem. Res. 2010, 43, 496−505. (6) Fan, C.; Plaxco, K. W.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 9134−9137. (7) Lubin, A. A.; Lai, R. Y.; Baker, B. R.; Heeger, A. J.; Plaxco, K. W. Anal. Chem. 2006, 78, 5671−5677. (8) Mao, Y.; Luo, C.; Ouyang, Q. Nucleic Acids Res. 2003, 31, e108. (9) Ricci, F.; Lai, R. Y.; Plaxco, K. W. Chem. Commun. 2007, 3768− 3770. (10) Yang, W.; Lai, R. Y. Langmuir 2011, 27, 14669−14677. (11) Anne, A.; Bouchardon, A.; Moiroux, J. J. Am. Chem. Soc. 2003, 125, 1112−1113. (12) Lai, R. Y.; Lagally, E. T.; Lee, S.-H.; Soh, H. T.; Plaxco, K. W.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 4017−4021. (13) Immoos, C. E.; Lee, S. J.; Grinstaff, M. W. J. Am. Chem. Soc. 2004, 126, 10814−10815. (14) Xiao, Y.; Qu, X.; Plaxco, K. W.; Heeger, A. J. J. Am. Chem. Soc. 2007, 129, 11896−11897. (15) Xiao, Y.; Lubin, A. A.; Baker, B. R.; Plaxco, K. W.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 16677−17780. (16) March, G.; Noël, V.; Piro, B.; Reisberg, S.; Pham, M.-C. J. Am. Chem. Soc. 2008, 130, 15752−15753. (17) Yu, Z.; Lai, R. Y. Chem. Commun. 2012, 48, 10523−10525. (18) Angerstein-Kozlowska, H.; Conway, B. E.; Hamelin, A.; Stoicoviciu, L. J. Electroanal. Chem. 1987, 228, 429−453. (19) Dong, S.; Che, G.; Xie, Y. Chemically Modified Electrodes, 1st ed.; Science Press: Beijing, 1995; p 123. (20) Laviron, E. J. Electroanal. Chem. 1979, 101, 19−28. (21) Yang, W.; Lai, R. Y. Chem. Commun. 2012, 48, 8703−8705. (22) Ricci, F.; Lai, R. Y.; Heeger, A. J.; Plaxco, K. W.; Sumner, J. J. Langmuir 2007, 23, 6827−6834. (23) Sumner, J. J.; Weber, K. S.; Hockett, L. A.; Creager, S. E. J. Phys. Chem. B 2000, 104, 7449−7454. (24) Sumner, J. J.; Creager, S. E. J. Phys. Chem. B 2001, 105, 8739− 8745. (25) Creager, S. E.; Wooster, T. T. Anal. Chem. 1998, 70, 4257−4263. (26) Yang, W.; Lai, R. Y. Analyst 2011, 136, 134−139. (27) Ricci, F.; Zari, N.; Caprio, F.; Recine, S.; Amine, A.; Moscone, D.; Palleschi, G.; Plaxco, K. W. Bioelectrochemistry 2009, 76, 208−213. (28) Rentsch, S.; Siegenthaler, H.; Papastavrou, G. Langmuir 2007, 23, 9083−9091.

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dx.doi.org/10.1021/ac3037987 | Anal. Chem. 2013, 85, 3340−3346