Effect of Microelectrode Structure on Electrocatalysis at Nucleic Acid

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Effect of Microelectrode Structure on Electrocatalysis at Nucleic Acid-Modified Sensors Yige Zhou, Ying Wan, Andrew Sage, Mahla Poudineh, and Shana O. Kelley Langmuir, Just Accepted Manuscript • DOI: 10.1021/la502990s • Publication Date (Web): 06 Nov 2014 Downloaded from http://pubs.acs.org on November 17, 2014

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Effect of Microelectrode Structure on Electrocatalysis at Nucleic Acid-Modified Sensors Yi-Ge Zhou,a Ying Wan,a Andrew T. Sage,a Mahla Poudineh,c Shana O. Kelleya,b a

Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, bDepartment of Biochemistry, Faculty of Medicine,

University of Toronto, cDepartment of Electrical and Computer Engineering, University of Toronto, Toronto, ON, Canada M5S 3M2, Canada

Keywords: Electrochemical biosensing, mass transport, electron transfer kinetics, electrocatalysis ABSTRACT The electrochemical detection of nucleic acids using an electrocatalytic reporter system and nanostructured microelectrodes is a powerful approach to ultrasensitive biosensing. In this report we systematically study for the first time the behavior of an electrocatalytic reporter system at nucleic acid-modified electrodes with varying structures and sizes. [Ru(NH3)6]3+ is used as a primary electron acceptor that is electrostatically attracted to nucleic acids-modified electrodes, and [Fe(CN)6]3- is introduced into the redox system as a secondary electron acceptor to regenerate Ru3+ after electrochemical reduction. We found that the electrode structure has strong impact on mass transport and electrontransfer kinetics, with structures of specific dimensions yielding much higher electrochemical signals and catalytic efficiencies. The electrocatalytic signals obtained when gold sensors were electrodeposited in both circular and linear apertures were studied and the smallest structures plated in linear apertures were found to exhibit the best performance with high current densities and turnover rates. This study provides important information for optimal assay performance, and insights for the future design and fabrication of high performance biomolecular assays.

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INTRODUCTION

Nanostructured metal electrodes are widely applied as electrochemical sensors because they possess advantages over bulk materials, including enhanced mass transport, higher active surface area, and enhanced surface and electronic properties.1-10 Nanostructured electrodes can be made with tethered nanomaterials,11 threedimensional composites12 and templating methods.13 Three-dimensional nanostructured electrode systems are of particular interest because of the central role diffusion and surface area plays in electrochemical processes.14-16 These types of structures have been shown to present highly effective solutions for the development of new battery materials,17 sensors,18 hydrogen fuel cells,19 and photoelectrochemical devices.20 The last two decades has seen significant integration of these structures into biodetection systems, since the increase in electroactive surface area allows for lower detection limits and higher sensitivity to analytes.21

Three-dimensional nanostructured materials are of particular interest for the optimization of electrocatalytic processes. Electrocatalytic reactions are often based on an electron acceptor/donor system, with a primary redox moiety that exchanges electron with an electrode and a secondary electron acceptor or donor to regenerate the primary redox center. The strong reliance of electrocatalysis on high rates of diffusion makes threedimensional nanostructured electrodes with large surface areas ideal systems to for the pursuit of enhanced signals.

The advantages of this approach has been successfully

applied to the development of new sensing strategies22 and energy-delivering technologies19. We have applied three-dimensional nanostructured microelectrodes (NMEs) to the electrocatalytic detection of a variety of biomolecular analytes.23-28 This is an advantageous approach because the large footprint of NMEs facilitates high levels of collisions with analytes, while the nanostructuring further increases surface area and promotes the display of probe molecules in an active conformation. The NME sensors exhibit excellent sensitivity with attomolar detection limits and excellent levels of specificity.28 This approach is one of many strategies exploiting electrocatalysis for detection of biologically-relevant analytes, with examples of other systems including the catalytic oxidation of DNA guanines29, the catalytic oxidation of glucose,30 and the


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electrocatalytic detection of sulfide and arsenic (III).31 A unique electrochemical DNA sensor for the detection of DNA base was also developed using an electrocatalytic reaction through a DNA-mediated charge transport mechanism.32 In our electrocatalytic reporter strategy33, the presence of a target nucleic acid is read out using an electron acceptor/donor system (Figure 1). [Ru(NH3)6]3+ acts as a redox label and is electrostatically attracted to the electrode surface in the presence of negatively charged nucleic acids. [Fe(CN)6]3- is introduced into to the assay as a secondary electron acceptor and chemically turns Ru(II) over to make the redox cycle electrocatalytic.15 The use of NMEs and electrocatalysis provides superior sensitivity when used in conjunction with peptide nucleic acid (PNA) probes covalently attached to NMEs by a thiol linkage,25 which are charge neutral and therefore yield low background signals before the hybridization of negatively charged DNA targets. Using the chip-based NMEs and electrocatalysis, ultrasensitive detection of oligonucleotides,18 mRNAs,19,

20

and microRNAs,21 can be achieved. Recently the accurate identification and classification of circulating tumor cells (CTCs) was also enabled using this assay platform.22 While many applications have been pursued for this detection platform, little work has been done to characterize the features of the electrochemical reaction that has been used for readout.

Several studies have sought to characterize the interfacial electrochemistry occurring at conventional sensors functionalized with nucleic acids. Studies of interactions between cations and immobilized DNA,33-37 electrochemical quantification of DNA-modified electrodes,38,39 electrochemical studies of mediated redox reactions at DNA-modified electrodes,40 and the electrochemical detection of DNA by electrostatic repulsion41 or attraction42,43 of redox molecules have all provided important characterization of interfacial systems that can serve as powerful sensors for biomolecular analytes. Studying related phenomena occurring at the NME sensors has an additional level of complexity introduced by the three-dimensional surfaces of these sensors (Figure 1). The size and morphology of NMEs can be tuned by using different metal electrodeposition conditions to produce the sensors, and the shape of NMEs can also be tuned by varying the dimensions and shape of the aperture that is used as a template for the electrode on the surface of a silicon or glass chip. In the studies reported here, we


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explore how electrode structure impacts mass transport and electron transfer kinetics of the bound and catalytic redox molecules in order to improve the electrocatalytic efficiency and the performance of the assay. MATERIALS AND METHODS: Reagents

and materials: Hexaamine ruthenium chloride (99.9%), potassium

ferricyanide (99%), sodium chloride (≧99.5%), ethylenediaminetetraacetic acid (EDTA, ≧99%), Tris(2-carboxyethyl)phosphine (TCEP, ≧98%), and 6-mercapto-1-hexanol (MCH, 97%) were purchased from Sigma–Aldrich (Oakville, ON); sulphuric acid (70%), acetone (ACS grade), and isopropyl alcohol (IPA, ACS grade) were obtained from EMD (Gibbstown, NJ); 1 M Tris-HCl (pH 7.5) buffer solution and ultra pure distilled water were purchased from Invitrogen (Burlington, ON); and thiolated PNA probes (PNA Bio, Thousand Oaks, CA) and DNA oligomers (Integrated DNA Technologies (IDT), Coralville, IA) were purchased. The following probe and target sequences were used (N to C): PNA probe: Cys-O-CGC-CGT-AGC-CTC-AGC-C (where O represents an ethylene glycol linker and Cys represents the amino acid cysteine), complementary target DNA (5’ to 3’): GGC-TGA-GGC-TAC-GGC-G. Fabrication of nanostructured microelectrodes (NMEs): Microchips were fabricated in house from glass substrates (Telic Company, Valencia, CA). The glass substrates were precoated with 5 nm Cr, 50 nm Au, and a positive photoresist (AZ1600). The working electrodes and apertures were patterned on the glass substrates using standard contact lithography and etching techniques (Circular apertures were 5 µm in diameter and linear apertures were 5 x 100 µm). Microchips were washed with acetone and IPA, then O2 plasma etched. Electrodeposition of gold was performed at room temperature using a Bioanalytical Systems Epsilon potentiostat with a three-electrode system featuring an Ag/AgCl reference electrode and a platinum wire auxiliary electrode. Both linear and circular apertures electroplated in 50 mM HAuCl4 solution (Sigma–Aldrich, Oakville, ON) using DC potential amperometry at an applied potential of 0 mV. PNA-DNA hybridization and immobilization onto NMEs: When double-stranded hybrids were immobilized on electrodes, PNA-DNA hybridization was performed in 10 mM Tris-HCl, pH 7.5 containing 1 µM PNA probe and 1 µM DNA target, 1 mM TCEP,


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100 mM NaCl, and 1 mM EDTA. The hybridization solution was mixed thoroughly and incubated at 95°C for 5 minutes followed by incubation at 2°C. The immobilization solution was placed on the freshly prepared gold electrodes in a humidity chamber and the deposition was allowed to occur overnight at room temperature. The electrodes were then rinsed thoroughly using 10 mM Tris-HCl, pH 7.5 and backfilled with 1 mM MCH for 2 hours. Electrodes were washed and prepared for electrochemical measurements. For the two-step, in situ hybridization experiments, the electrodes were treated in a similar manner except that the hybridization step described above was performed on the sensors as a separate process. Electrochemical measurements: Electrochemical measurements were carried out using an Bioanalytical Systems Epsilon potentiostat in 10 mM Tris-HCl pH 7.5 and either 0 or 2 mM [Fe(CN)6]3-. Modified electrodes were incubated in 100 µM [Ru(NH3)6]3+ for 60 seconds then in rinsed in 10 mM Tris-HCl pH 7.5. A three-electrode system was used featuring an Ag/AgCl reference electrode and a platinum-wire auxiliary electrode. NMEs fabricated on the microchips were used as the working electrode and were contacted by using the exposed bond pads. A gold disk electrode (area = 0.02 cm2) (BASi, West Lafayette, IN)) was also used as the working electrode to study the electron transfer kinetics for NMEs coated with nucleic acids monolayers. RESULTS AND DISCUSSION: Electrochemical signals obtained at bare NMEs with varied sizes and structures. NMEs are fabricated within a template provided by a layer of photoresist on top of gold electrodes patterned on a glass chip (Figure 1). In order to obtain differently shaped and sized NMEs and compare the electrochemical processes occurring as a function of these parameters, we varied the deposition time and used different aperture shapes to template sensor electrodeposition. Figure 2 shows scanning electron micrographs of gold NMEs obtained at both circular and linear apertures as a function of plating time. For both circular and linear apertures, electroplated gold first fills in the apertures at short plating times and then forms nucleation sites for further growth. At short plating times, a sparse collection of gold structures are observed and the substructures are relatively isolated, while at longer plating times, the structures become much denser. For circular apertures, the gold structures have different submorphologies that are a mixture 5 ACS Paragon Plus Environment

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of leaf-like structures and needles, while for linear apertures, the gold structures are primarily needles that become decorated with other smaller needles. The surface areas of these structures were measured electrochemically by cyclic voltammetry (CV) in 0.5 M H2SO4 and subsequent integration of the cathodic peak (Figure 2c).44 The electrochemical measurements indicated that the surface area of NMEs grown with linear apertures increases faster with plating time than those made with circular apertures. The SEM images shown in Figure 2 support this observation, the structures produced from linear apertures grows with more branching and more active surface area exposed. previously,

45

It is noteworthy that these structures, like those reported

are highly reproducible with surface areas that differ by less than 10%

(Figure S2). To examine the diffusional profiles of electrochemical reactions occurring at these structures, we used cyclic voltammetry to monitor the reduction of [Fe(CN)6]3-. Figure 2d shows the CVs obtained with NMEs of increasing size plated with linear apertures. For short plating times (15 seconds) the voltammetry displays a steady-state current, implying that the diffusion of [Fe(CN)6]3- to NMEs is radial. With plating times of 30 seconds and 60 seconds, the voltammetric signal loses its steady-state character, implying that the contribution of radial diffusion decreases. With plating times of 90 seconds and 120 seconds, the voltammograms have well-defined peak currents that then decrease, indicating that the diffusion becomes linear. Therefore, the diffusional mode of [Fe(CN)6]3- to NMEs ranges from radial to linear as a function of sensor size. Similar results were obtained with sensors made with circular apertures except that the structure generated with a 30 second plating time still exhibited steady-state behavior (data not shown). In order understand how different sensor sizes and structures affect mass transport we calculated current densities (current normalized by surface area) observed during the reduction of [Fe(CN)6]3-, and compared these values with plating time for NMEs grown with both circular and linear apertures (Figure 3). Higher current density is expected to arise from enhanced mass transport to the electrode surface as a result of convergent (radial) diffusion.46 Sensors deposited for 15 and 30 seconds using circular apertures show the highest current densities. As the sensors get larger, the current densities


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decrease. With linear apertures, the current density is highest for the sensor generated with a 15 second plating time, and the densities then decrease for larger sensors. From the SEM images shown in Figure 2, it is clear that the shortest plating times generate the sensors with larger surface to volume rations. As the structures grow larger, there are more areas within the sensors where the supply of redox active molecules is depleted quickly. At longer plating times, the structures are compact and the diffusion zones of the fractal features overlap or partially overlap, leading to reliance on linear or partially linear diffusion over the entire surface and producing less efficient mass transport. Electrochemical signals obtained at modified NMEs with varied sizes and structures. Understanding the factors that affect interactions between a capture probe monolayer and an electrochemical reporter system is a key component in the development of sensing platforms. In order to achieve ultrasensitive detection of various biological analytes, NMEs can be functionalized with capture probes complementary to specific target sequences. In this study, we were interested in better characterizing the electrocatalytic reaction that occurs when PNA probes are used to target complementary DNA sequences in solution. To generate a well-defined interfacial environment, thiolmodified PNAs were hybridized with their DNA complements and immobilized on the sensors. In order to limit non-specific binding, control probe density, and orient the probes away from the sensor surface, the probe monolayers are backfilled with mercaptohexanol.

To study the properties of probe-modified linear and circular hybrid-modified NMEs, we used a previously described [Ru(NH3)6]3+/[Fe(CN)6]3- redox system.33 [Ru(NH3)6]3+ molecules accumulate at the sensor surface through electrostatic interactions with the negatively charged phosphate backbone of nucleic acids. In a potential sweep, adsorbed [Ru(NH3)6]3+ is reduced at -200 mV, producing a measureable current that reports on the presence of the target DNA. [Fe(CN)6]3- is introduced into the system to chemically oxidize the reduced Ru2+, which creates multiple redox cycles for a single [Ru(NH3)6]3+ molecule and thus amplifies the observed current.

In order to obtain an optimal

electrocatalytic efficiency for the reporter system of interest there must be: (i) enhanced mass transport of [Fe(CN)6]3- to the sensor, (ii) a slow direct electron transfer rate for


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[Fe(CN)6]3-, and (iii) only a small degree of interfering current contributed from the electrochemical reduction of [Fe(CN)6]3- at the NMEs. The interfering current that could be produced by direct reduction of [Fe(CN)6]3- is inhibited by the PNA/DNA monolayer. The negative charge of the PNA/DNA hybrid inhibits the electron transfer of [Fe(CN)6]3-, and the electrochemical signal of a PNA/DNA-modified electrode is approximately half of that of a bare gold NME (Figure 4). Therefore, the monolayer directly affects the catalytic activity of [Fe(CN)6]3- by slowing the electron transfer rate and decreasing the current from [Fe(CN)6]3. The presence of the neutral PNA monolayer also slows the electron transfer, but to a lesser extent than the PNA/DNA monolayer. [Fe(CN)6]

3-

The presence of

in solution does contribute a small, interfering current; however, this

interference only contributes to the background current during a potential sweep of [Ru(NH3)6]3+ and thus the peak currents remain unaffected. We examined the electrochemistry of redox reporter, [Ru(NH3)6]3+, with and without the catalytic enhancement provided by [Fe(CN)6]3-. The accumulation of [Ru(NH3)6]3+ was measured by preadsorbing [Ru(NH3)6]3+ with PNA/DNA-modified electrodes for 60 seconds, an amount of time where the cation would have equilibrated.43 By transferring to an buffer solution that contained either 0 mM or 2 mM [Fe(CN)6]3-, we were able to directly study the effects of [Fe(CN)6]3- on adsorbed [Ru(NH3)6]3+. This transfer has been shown to result in a loss of less than 10% of the adsorbed material.34 Figure 5a shows the typical differential pulse voltammetry (DPV) measurements for the electrochemical reduction of [Ru(NH3)6]3+ with and without the catalysis of [Fe(CN)6]3- at linear apertures with short (15s) plating times. We observed that the catalytic current for [Ru(NH3)6]3+ is more than four times larger in the presence of [Fe(CN)6]3- than that without [Fe(CN)6]3-. Furthermore, the reduction potential for the catalytic current shifts to more positive potentials, which demonstrates that [Fe(CN)6]3- accelerates the electron transfer kinetics of the reaction. We then compared the catalytic currents of NMEs generated with circular and lineshaped apertures for PNA/DNA-modified electrodes (Figure 5). The catalytic current increases with increasing plating time, which is consistent with our previous results indicating that larger surface areas are obtained with longer plating times (Figure 2). These results indicate that the active surface area plays a key role in the catalytic current as larger electrodes allow more PNA/DNA hybrids to modify the electrode


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surface and thus recruit more [Ru(NH3)6]3+. As evidenced by the error analysis shown in Figure 5, these experiments were highly reproducible despite the fractal nature of the sensors under study. To better understand how differences in electrode size related to the efficiency of the electrochemical reporter system, we examined current densities for the PNA/DNAmodified electrodes (Figure 5c). For the electrodes grown from linear apertures, we observed a decreasing trend that was consistent with the measurement of current density for [Fe(CN)6]3- measured at bare NMEs (Figure 3). This was expected as the higher current density was the result of the enhanced mass transport of [Fe(CN)6]3- to the smaller electrodes which subsequently allowed more [Fe(CN)6]3- to be involved in the catalytic process - leading to higher catalytic efficiency. In addition, the decreased electron transfer rate at short plating times also contributes somewhat in the catalytic process. Surprisingly, the catalytic current density at circular apertures does not differ much as a function of plating time, likely because the substructures of the NMEs have better solution exposure. In comparing all of the NME structures, a plating time of 15 s for the linear apertures produced the highest catalytic current density. In order to evaluate the catalytic efficiencies achieved with the different electrodes, we compared the turnover rate Ru3+/Fe3+ electrocatalysis using sensors grown from circular and linear apertures (Figure 5d). The turnover rate for the line-templated electrodes decreased with plating time due to the less advantageous diffusional profiles of these structures. The turnover rate at the sensors grown from circular apertures followed a similar trend. Of all the electrodes tested, linear apertures plated for 15 s produced highest turnover rate of [Ru(NH3)6]3+. Electron-Transfer Kinetics. The surface structure of an electrode can have a dramatic effect on the electrochemical reactivity of a redox species, which will ultimately impact the performance of a biosensor.

We sought to investigate how different surface

composition can affect the electron transfer reactivity of [Ru(NH3)6]3+. There are two ways to immobilize PNA-DNA hybrids onto an electrode: (i) sequential (two-step) immobilization of PNA probes and target DNA on an electrode and (ii) prehybridization of PNA probes and target DNA with subsequent deposition of the hybrid product on the electrode (one-step immobilization). The two-step method will produce a combination of


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single-stranded unhybridzed probes and DNA/PNA hybrids, while the one-step method will produce a uniform coating of hybrids. Solution-phase hybridization rates are 20- to 40- fold faster than surface hybridization rates for identical sequences and conditions.47 Therefore, the densities of PNA-DNA hybrids on the electrode surface was expected to differ based on the immobilization approach. Double-stranded hybrids (one-step immobilization) tend to be extended from the surface of the electrode due to their rigidity, forming better aligned SAMs. The two-step immobilization process would yield a mixture of hybrids extended from the surface and more flexible unhybridized probe sequences. Due to the difference in surface density, orientation, and morphology of the PNA-DNA hybrids, it was expected that the electron transfer kinetics of the bound [Ru(NH3)6]3+ in the two types of monolayers would differ. The electron-transfer kinetics of the reduction of the bound electroactive species at PNADNA modified electrode can be determined based on the Laviron classical model:48,49

= ′ −

υ ln

where Epc is the cathodic peak potential, E0’ is the formal potential (V), n is the number of electrons transferred, υ is the scan rate (V / s), ks is the electron transfer rate (s-1), and α is the transfer coefficient. For a plot of (Epc - E0’) versus scan rate, υ, the values of ks and α can be derived from the linear range at high scan rates. For the one-step immobilization method, ks and α were found to be 1.04 and 0.48. In contrast, ks and α were 0.74 and 0.55, respectively for a two-step immobilization process. These results demonstrate that the one-step immobilization method provides faster electron-transfer kinetics over a two-step method. As suggested by Fan et al.,50 the hybrids obtained from the one-step immobilization process are better aligned with optimal surface densities at the electrode surface and adsorbed [Ru(NH3)6]3+ is more uniformly distributed within the surface monolayer. This results in the homogeneous and equally fast electron-transfer properties of a one-step immobilization process.

This difference in electron-transfer

efficiency enhances the detection efficiency of the electrocatalytic assay, with greater levels of current being derived from probes that have hybridized with target sequences. CONCLUSIONS


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We have systematically studied electrochemical processes occurring at the surfaces of both bare and DNA/PNA-modified three-dimensional nanostructured microelectrodes. Various aperture shapes (circular and linear) and sizes were evaluated and there was a correlation between the electrode structure and performance in terms of the absolute catalytic current, density, and turnover rate. The structure of electrodes obtained at linear apertures with a plating time of 15 s exhibits the best performance (i.e. larger signals) among all NMEs. Lastly, we studied a one-step and two-step immobilization process of PNA-DNA hybrids observed differences in behavior. Taken together, this study provides key information for optimizing the performance of NME-based biomolecular assays. AUTHOR INFORMATION: Corresponding Author * Email [email protected] ACKNOWLEDGMENTS: The authors wish to acknowledge the Natural Sciences and Engineering Research Council for their support of this work.


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a)

Circular Apertures

Target binding

Linear Apertures

b) Fe3+

Fe3+ Fe3+

Fe3+

Fe3+

Ru3+

Fe3+

Fe3+ Ru3+

Fe3+

Fe3+

Fe3+ Fe3+

Ru3+ Ru3+

Fe3+ e- acceptors Fe3+ Ru3+

Ru3+ Ru3+

Ru3+

Ru3+

Ru3+

Ru3+

Fe2+

Fe3+

Redox Interface Ru3+ e- acceptors Nucleic acids (DNA/PNA) monolayer

Ru3+

-0.20V

Ru3+

Ru3+

Au electrode

Figure 1: Schematic representation of nanostructured microelectrode (NME) growth and use for nucleic acid detection. a) Circular and linear apertures created within a photoresist layer on top of gold leads serve as templates for sensor electrodeposition. When used for sequence-specific DNA detection, electrodes are functionalized with PNA probes (grey) and hybridized with complementary DNA targets (green). b) Electrochemical detection of the bound target is achieved using a Ru3+/Fe3+ redox reporter system where Ru3+ (red) interfaces with Fe3+ (green) at the nucleic acid (PNA/DNA) boundary (dashed line). The two redox reagents are separated because the Ru3+ species is positively charged and interacts with the monolayer, while the Fe3+ is part of an anionic complex (Fe(CN)63-) and is repelled from the monolayer.

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c)

a)

15 µm 15 µm Plating Time (s): Surface Area (cm2):

15 6.9x10-6

30 1.5x10-5

60 5.3x10-5

90 1.1x10-4

120 2.1x10-4

d)

100

b) 0

I / nA

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µm 2030µm Plating Time (s): Surface Area (cm2):

15 2.4x10-5

plating time -100

30 7.6x10-5

60 1.2x10-4

90 2.3x10-4

120 8.8x10-4 0.0

0.2

0.4

E/V

Figure 2: NME sensor morphology and surface area as a function of aperture shape and plating time. Scanning electron microscopy images are shown for gold NMEs templated in circular (a) and linear (b) apertures. The electrodeposition time and corresponding surface area of the sensors are indicated below each image. The scare bar for each template is indicated in the first panel. Surface areas of the sensors were measured as described in (c). c) Integrated surface area of bare NMEs templated from circular (●) and linear (n) apertures for plating times of 0 to 120 seconds in measured by scanning sensors in 0.5 M H2SO4 and measuring current generated upon the reduction of gold oxide. See Supporting Information Figure S1C for electrochemical scans. d) Cyclic voltammetry (CV) scans at 100 mV/s of bare NMEs templated from linear apertures for increasing plating times in 2 mM [Fe(CN)6]3-. ACS Paragon Plus Environment

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Figure 3: Effects of sensor morphology on mass transport. Current densities obtained with 2 mM [Fe(CN)6]3and bare NMEs templated from circular (●) and linear (n) apertures for plating times of 0 to 120 seconds.


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Figure 4: Electrochemical behavior of [Fe(CN)6]3- at NMEs modified with PNA/DNA duplexes. Cyclic voltammetry (CV) scans at 100 mV/s of linear-templated NMEs obtained with a plating time of 15 s for a bare gold (solid), PNA-modified (dotted) and PNA/DNA-modified (dashed) surfaces in 2 mM [Fe(CN)6]3-.


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a)

b)

c)

d)

current density mA/cm2

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Figure 5: Electrochemical behavior of NMEs modified with PNA/DNA duplexes. a) Differential pulse voltammetry (DPV) scans of line-templated NMEs modified with PNA/DNA duplexes and incubated in 100 µM [Ru(NH3)6]3+ for 1 minute then measured in a solution of 10 mM Tris-HCl pH 7.5 with (solid) and without (dashed) the presence of 2 mM [Fe(CN)6]3- as a catalytic enhancer. The catalytic current (b), current density (c), and turnover rate (d) obtained from NMEs for circular (●) and linear (n) templates at various plating times. Data shown are mean +/- SEM (n=15-20). ACS Paragon Plus Environment

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Nanostructured microelectrodes

Electrocatalytic DNA detection

Ru 2+ Fe 3+

3+ Ru Fe2+

_

0..20 V

-0.20V

2+ Ru3+ Fe

3+ Fe2+ Ru

TOC graphic ACS Paragon Plus Environment

Effect of Microelectrode Structure on Electrocatalysis at Nucleic Acid

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