nihms97838.pdf

Scanning Electrochemical Microscopy of DNA Monolayers

Modified with Nile Blue

Alon A. Gorodetsky

†,||

William J. Hammond

‡,||

Michael G. Hill

*,§

Krzysztof Slowinski

*,‡

and

Jacqueline K. Barton

*,†

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,

California 91125, Department of Chemistry and Biochemistry, California State University, Long

Beach, California 90840, and Department of Chemistry, Occidental College, Los Angeles, California

90041

Abstract

Scanning electrochemical microscopy (SECM) is used to probe long-range charge transport (CT)

through DNA monolayers containing the redox-active Nile Blue (NB) intercalator covalently affixed

at a specific location in the DNA film. At substrate potentials negative of the formal potential of

covalently attached NB, the electrocatalytic reduction of Fe(CN)

−

generated at the SECM tip is

observed only when NB is located at the DNA/solution interface; for DNA films containing NB in

close proximity to the DNA/electrode interface, the electrocatalytic effect is absent. This behavior

is consistent with both rapid DNA-mediated CT between the NB intercalator and the gold electrode

as well as a rate-limiting electron transfer between NB and the solution phase Fe(CN)

−

. The DNA-

mediated nature of the catalytic cycle is confirmed through sequence-specific and localized detection

of attomoles of TATA-binding protein, a transcription factor that severely distorts DNA upon

binding. Importantly, the strategy outlined here is general and allows for the local investigation of

the surface characteristics of DNA monolayers both in the absence and in the presence of DNA

binding proteins. These experiments highlight the utility of DNA-modified electrodes as versatile

platforms for SECM detection schemes that take advantage of CT mediated by the DNA base pair

stack.

Introduction

Numerous studies have shown that DNA efficiently mediates long-range charge transport both

in solution

1–5

and at DNA-modified surfaces.

6,7

The electrochemical response at DNA self-

assembled monolayers (SAMs) is remarkably sensitive to base stacking perturbations, with

the efficiency of CT to an electro-active probe reflecting the integrity of the base pair stack.

Consequently, DNA electrochemistry has been exploited successfully in the development of

simple assays for the rapid detection of single-nucleotide polymorphisms,

8–10

base lesions,

and DNA/protein binding.

12–18

*Corresponding authors. E-mail: jkbarton@caltech.edu (J.K.B.), mgh@oxy.edu (M.G.H.), kslowins@csulb.edu (K.S.).

†

California Institute of Technology.

‡

California State University at Long Beach.

Occidental College.

These authors contributed equally to this work.

Supporting Information Available:

Structure of the NB-modified uridine. Cyclic voltammetry and plots of peak current as a function

of scan rate for DNA monolayers modified with NB at both the top and the bottom. Cyclic voltammetry of ferricyanide at a bare and a

mercaptoundecylphosphoric acid-modified gold electrode. SECM approach curves for a DNA monolayer modified with NB at the bottom

in the presence and absence of MB. This material is available free of charge via the Internet at http://pubs.acs.org.

NIH Public Access

Author Manuscript

Langmuir

. Author manuscript; available in PMC 2009 December 16.

Published in final edited form as:

Langmuir

. 2008 December 16; 24(24): 14282–14288. doi:10.1021/1a8029243.

NIH-PA Author Manuscript

Controlling the orientation and packing of DNA helices within these SAMs is critical for many

analytical applications, yet assessing film morphology and homogeneity, particularly in

aqueous media, remains a difficult challenge. Previously, the morphology of DNA SAMs on

gold has been investigated by scanning tunneling microscopy (STM),

19–21

atomic force

microscopy (AFM),

22–24

and, most recently, scanning electrochemical microscopy (SECM).

25–28

SECM provides submicrometer visualization of DNA films through in situ imaging of

chemical reactivity at modified insulating or conducting substrates.

29,30

SECM is also

inherently compatible with aqueous environments, thereby allowing for the direct

electrochemical profiling of DNA arrays with micrometer resolution.

29,30

In addition, SECM

has been used successfully to monitor DNA hybridization and to detect DNA lesions.

29–34

One advantage of this technique over conventional bulk electrochemistry is that the

micrometer-level diameter of the SECM tip inherently minimizes contributions from

imperfections on large-sized electrodes by sampling a local area of the film at a specific

substrate potential.

Electrochemical assays based on DNA CT have frequently utilized noncovalent mediators to

collect global information,

8–11,35,36

but the electrochemistry of these mediators can be

significantly complicated by contributions from CT pathways that are not DNA-mediated.

The use of covalent probes at fixed positions within the film,

38–45

particularly in conjunction

with backfilling of the DNA monolayer with a suitable straight-chain diluent molecule,

minimizes such contributions from defects, pinholes, and other imperfections. Therefore,

SECM investigations of DNA monolayers can particularly benefit from the use of covalently

attached probes.

Here, we report the application of SECM in probing long-range CT across a DNA monolayer

containing the redox-active intercalator Nile Blue (NB) covalently attached at discrete sites

along the individual DNA helices. Nile Blue is particularly useful for such studies because it

can be easily incorporated into DNA on solid support and displays electrocatalytic activity.

Therefore, on the basis of our previous investigations of the catalytic reduction of

ferricyanide by methylene blue (MB) at DNA-modified electrodes,

8–11,46

we have replaced

MB with covalently bound NB to reduce ferricyanide, which is generated at the SECM tip in

our current experiments. Only Nile Blue attached at the solution-exposed periphery of the film

is capable of supporting catalytic regeneration of ferrocyanide present in solution. Furthermore,

this cycle can be interrupted in a sequence-specific manner by addition of TATA binding

protein (TBP), a transcription factor that bends DNA by ~90° upon binding.

12,13

Taken

together, these studies establish the upright orientation of individual helices within a relatively

homogeneous dilute monolayer and demonstrate the critical role of efficient long-range

electrochemical charge transport through the

-stack. They additionally suggest a sensitive and

highly selective SECM method for assaying specific DNA/protein interactions with even dilute

and imperfect assemblies of probe DNA.

Experimental Section

Materials

All reagents for DNA synthesis were purchased from Glen Research. Methylene blue,

ruthenium hexammine chloride, potassium ferricyanide, potassium ferrocyanide, and 11-

mercaptoundecylphosphoric acid were purchased from Sigma in the highest purity available

and used as received. Nile Blue perchlorate was purchased from Acros in laser grade purity.

TATA binding protein was custom ordered from Protein One, Inc. Bovine serum albumin

(BSA) was obtained from New England Biolabs, Inc. Platinum scanning electrochemical

microscopy tips were purchased from CH Instruments, Inc., and used as received. Au(111) on

mica substrates packaged under argon were purchased from Agilent, Inc., and utilized

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immediately. Sodium phosphate buffers were prepared with Milli-Q water, and pH adjustments

were made with sodium hydroxide, if necessary.

Synthesis of Thiol and Nile Blue-Modified DNA

Oligonucleotides were prepared on solid support using standard phosphoramidite chemistry

on an Applied Biosystems 394 DNA synthesizer. All of the prepared sequences were purified

by multiple rounds of reverse phased, high performance liquid chromatography (HPLC). The

pure oligonucleotides were characterized via matrix-assisted laser desorption (MALDI) mass

spectrometry and UV–visible (UV–vis) spectrophotometry.

Thiol-terminated oligonucleotides were synthesized according to established protocols from

Glen Research, Inc., using the C6 S–S thiol modifier. After deprotection and cleavage from

solid support with ammonium hydroxide (60 °C for 8 h), the disulfide containing DNA was

purified by HPLC. The disulfide was subsequently reduced with an excess of dithiothreitol in

ammonium acetate buffer at pH = 8, and the free thiol containing single-stranded DNA was

then purified with a second round of HPLC.

DNA modified with NB at the 5

′

terminus was prepared according to ultramild protocols (Glen

Research, Inc.) to avoid degradation of the NB moiety. Additionally, Pac-protected bases and

ultramild reagents were utilized during the synthesis to prevent undesirable capping of the

protecting groups. A 17mer sequence (either 5

′

-UGC GTG CTT TAT ATC TC-3

′

or 5

′

-UGC

GCG CCC GGC GCC TC-3

′

) was prepared on solid support with a 5-[3-acrylate NHS ester]-

deoxy uridine as the terminal 5

′

base. The beads were then removed from the synthesizer and

dried thoroughly. The solid support was reacted with a 10 mg/mL Nile Blue perchlorate

solution in either 9:1

-dimethylformamide/

-diisopropylethylamine or 9:1

dichloromethane/

-diisopropylethylamine for 12–48 h. The beads were subsequently

washed up to three times with dichloromethane or

-dimethylformamide, methanol, and

acetonitrile. Subsequently, the Nile Blue-containing sequence was simultaneously cleaved

from the support and deprotected with 0.05 M potassium carbonate in methanol at room

temperature for 12–14 h. The overall yields of the reaction ranged from 30% to 80%.

DNA modified with NB one base in from the 3

′

terminus was prepared according to ultramild

protocols in a similar fashion. A 2mer sequence containing a 5-[3-acrylate NHS ester]-deoxy

uridine as the terminal 5

′

base was prepared on solid support. The beads were removed from

the synthesizer and reacted with a 10 mg/mL Nile Blue perchlorate solution in either 9:1

-dimethylformamide/

-diisopropylethylamine or 9:1 dichloromethane/

diisopropylethylamine for 12–48 h. After thorough washing of the beads, the Nile Blue

containing 2mer was placed back on the synthesizer, and the remaning 15 bases were added

to the sequence, yielding the oligonucleotide 5

′

-TGC GTG CTT TAT ATC UC-3

′

. The

resulting sequence contained an internal Nile Blue and was deprotected with 0.05 M potassium

carbonate in methanol at room temperature for 12–14 h. The overall yields of the reaction

ranged from 30% to 80%.

Both thiol- and NB-modified DNA was quantified using the extinction coefficient of the single-

stranded DNA at 260 nm on a Beckman UV–vis spectrophotometer. Equimolar amounts of

each strand (50

M) were combined before the resulting solution was purged with argon.

Duplexes were formed by thermally annealing in deoxygenated buffer containing 5 mM

NaP

, pH 7.1, 50 mM NaCl to 90 °C, followed by cooling to ambient temperature.

Preparation of Backfilled DNA/11-Mercaptoundecylphosphoric Acid Monolayers

Electrode areas were defined with a Viton O-ring with estimated surface areas of either 0.1 or

0.2 cm

in either a scanning electrochemical microscope Teflon cell from CH Instruments,

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Inc., or a custom-built Teflon cell. To ensure successful experiments, great care was taken to

minimize mechanical damage to the gold substrate upon mounting in the cell. After cell

assembly, a fresh 25–50

M duplex NB-DNA solution was deposited onto the gold surface.

Monolayer formation was allowed to proceed in a humidified environment for a period of 24–

72 h. Incubation times outside of this window (either shorter or longer) were found to be

detrimental to proper monolayer formation. Upon completion of film formation, the cell was

rinsed thoroughly with phosphate buffer to remove residual DNA before the DNA-modified

surface was backfilled with 11-mercaptoundecylphosphoric acid for 10–20 min.

Mercaptoundecylphosphoric acid rapidly displaces DNA from the surface, so backfilling times

were short and the cell was rinsed thoroughly to eliminate any residual alkanethiols.

Electrochemistry/Scanning Electrochemical Microscopy Experiments

Cyclic voltammetry and scanning electrochemical microscopy experiments were performed

using a CH Instruments electrochemical analyzer (Austin, TX). Unless otherwise noted,

experiments were performed at ambient temperature in phosphate buffer at pH 7 containing

either 5 mM NaP

and 50 mM NaCl or 20 mM NaP

and 80 mM NaCl under an argon

atmosphere (the oxygen content was constantly monitored). The buffer was supplemented with

Fe(CN)

for SECM experiments. A Teflon electrochemical cell was used for all experiments

with a Pt auxiliary electrode, a gold working electrode, and a silver/silver chloride (Ag/AgCl)

reference electrode. Because we have observed some variation in the potential of covalently

attached NB depending on the type of gold surface and the buffer conditions, the voltammetric

potentials reported in this work have a maximum uncertainty of 50 mV.

In a typical SECM experiment, total or partial passivation of the substrate was confirmed

through cyclic voltammetry of the ferri/ferrocyanide couple at the substrate. Approach curves

were obtained with either 2 or 10

m diameter platinum tips, which were polarized between

600 mV and 1 V, and identical results were obtained regardless of the tip bias. To aid

interpretation of the results, substrates featuring a significant free NB signal or virtually no

passivation against ferricyanide were discarded. Initial approach curves to the substrate were

recorded at a 0 mV substrate bias to confirm the insulating behavior of the NB-DNA monolayer.

Subsequent approach curves were recorded with the substrate bias modulated between 0 and

−

400 mV. For imaging of the substrate, the SECM tip was moved to within less than one tip

radius of the substrate before being retracted by 2–5

m prior to scans. Great care was taken

during all experiments to avoid damaging the DNA monolayer through direct contact of the

tip with the surface.

TATA Binding Protein Experiments

In a typical TBP detection experiment by SECM, approach curves or substrate scans were

initially recorded with the bias modulated between 0 and

−

400 mV to confirm switching from

negative to positive feedback. Subsequently, TBP was added to the SECM cell from a

concentrated ~16

M stock in phosphate buffer at pH 7 containing 5 mM KP

, 50 mM NaCl,

4 mM MgCl

, 4 mM spermidine, 50

M EDTA, 10% glycerol. The solution was equilibrated

via gentle pipetting, and approach curves or substrate scans were again recorded with the bias

modulated between 0 and

−

400 mV.

Results

Electrochemistry of Backfilled DNA Monolayers Modified with NB

Figure 1 shows a schematic illustration of DNA monolayers modified with NB either at the

top or at the bottom (see Supporting Information for the chemical modification). DNA

monolayers modified with NB display a pH-dependent redox couple at ~

−

200 mV versus Ag/

AgCl due to the 2e

−

reduction of the bound probe.

13,48,49

Because modification of the

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exocyclic amine of NB perturbs its aromatic core with an accompanying shift in its redox

potential of ~200 mV relative to free NB,

50,51

DNA monolayer stability can be assessed in

situ with the appearance of a NB signal at ~

−

400 mV, indicating film degradation. Coulometric

measurements of the NB signals for monolayers labeled at either the top or bottom yield typical

DNA surface coverages of ~1–3 pmol/cm

(as compared to ~40 pmol/cm

or greater for densely

packed monolayers).

As expected for a surface-bound species, plots of peak current as a

function of scan rate are linear, and the ratios of the cathodic to anodic charge are approximately

unity (Supporting Information).

Overall, the electrochemical characteristics of the two types

of monolayers (functionalized at either the top or the bottom) are very similar, despite the ~45

Å difference in gold/NB separations. This observation is consistent with previous studies

involving site-specific labeling of DNA monolayers with redox-active intercalators.

13,39,40

On the basis of previous work involving electrocatalytic reduction of ferricyanide at DNA-

modified substrates via intercalated Methylene Blue (MB),

8,46

we explored the

electrochemistry of covalently bound NB in the presence of ferricyanide (Figure 2). The DNA

monolayers were backfilled with 11-mercaptoundecylphoshoric acid, a negatively charged

alkanethiol that completely passivates against ferricyanide (Figure 2 and Supporting

Information). Importantly, the presence of this underlayer makes it more likely that the DNA

is electrostatically repelled by the negatively charged surface,

ensuring an upright orientation

in a dilute film. For a DNA monolayer modified with NB at the top, only an asymmetric

cathodic peak is observed at the reduction potential of NB. However, for a DNA monolayer

modified with NB at the bottom, the catalytic peak is absent. These data strongly indicate that

only solution exposed NB is capable of reducing ferricyanide.

SECM of DNA Monolayers Modified with NB in Feedback Mode

To visualize the NB/ferricyanide catalytic cycle both locally and directly,

8,46

we carried out

SECM measurements at DNA monolayers covalently modified with NB (Figure 3). In these

experiments, Fe(CN)

−

is oxidized in solution at a platinum SECM tip, yielding a steady-

state, diffusion-controlled current. The SECM tip is then physically lowered toward the DNA-

modified gold substrate at a constant rate, and the current-distance feedback curve is recorded

at various substrate bias potentials. If reduction of tip-generated Fe(CN)

−

at the underlying

substrate is blocked by the monolayer, the steady-state tip current drops upon approach

(negative feedback) due to restricted diffusion of bulk ferrocyanide. If, on the other hand, the

rate of catalytic Fe(CN)

−

reduction via reduced NB at the modified substrate is rapid, redox

cycling between the tip and substrate occurs, yielding increased tip currents (positive

feedback).

As shown in Figure 3, films containing NB attached at the bottom of the DNA helices result

in complete negative feedback at substrate bias potentials of both 0 and

−

400 mV. At 0 mV,

bound NB remains in its oxidized state, and there is a relatively small (~200 mV) overpotential

for ferricyanide reduction. At

−

400 mV, however, the NB is fully reduced and the substrate

overpotential increases to ~600 mV. Despite this high driving force and the ability of reduced

NB to act as a redox catalyst, the SECM response mimics that expected for a purely insulating

substrate. This indicates sufficient electrostatic repulsion at the DNA/phosphate-terminated

surface to effectively block tip-generated ferricyanide not only from the underlying gold

electrode

26,28

but also from the reduced NB buried deep within the DNA film.

A markedly different response is observed for DNA monolayers modified with NB at the top

of the film. At substrate bias potentials of 0 mV, pure negative feedback is again observed.

However, when the substrate bias voltage is held at

−

400 mV (a value negative of the formal

reduction potential of NB), the response switches to positive feedback. Such a response signals

rapid CT between tip-generated Fe(CN)

−

and reduced NB, which is now bound at the solvent-

exposed periphery of the monolayer. Indeed, assuming that the tip current is controlled by NB-

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mediated CT through the DNA film, the approach curve can be modeled using well-known

analytical approximations for finite heterogeneous kinetics.

53–55

The fits in Figure 3 show

excellent agreement with the approximations both for the insulating monolayers (black solid

line for negative feedback) and for the finite heterogeneous kinetics of ferricyanide reduction

(red solid line for positive feedback). The one-parameter fit shown in Figure 3A for the DNA

film modified at the top corresponds to a surprisingly rapid apparent rate constant for

regeneration of ferricyanide of

app

= 6 ± 3 × 10

−

cm/s as measured at

−

400 mV versus SSCE

(over four independent trials).

Two pieces of experimental evidence further substantiate the proposed DNA-mediated

pathway for the SECM response. First, DNA quantification by ruthenium hexammine

assay

56–58

for DNA films modified with NB either at the top or at the bottom yields very

similar surface coverages (a minor contribution to the ruthenium hexammine signal is expected

from 11-mercaptoundecylphosphoric acid due to its single phosphate group). Although this

assay provides only a qualitative measure of surface coverage,

it indicates nevertheless that

both electrodes are covered with similar amounts of negative charge. Thus, for substrate

potentials of

−

400 mV, the large discrepancy in ET rate across DNA films modified with NB

at the top versus those modified at the bottom cannot be ascribed to relative differences in

electrostatic screening of ferricyanide.

Second, DNA films modified with NB at the bottom can support electrocatalytic reduction of

ferricyanide, but only upon addition of a redox mediator that has access to the film/solution

interface. Thus, as an additional control experiment, the approach curve in SECM feedback

mode was recorded for DNA monolayers modified with NB at the bottom before and after

addition of 1.5

M methylene blue (MB) to the solution (Supporting Information). While the

addition of MB does not change the observed negative feedback at a substrate potential of 0

mV, measurable positive feedback is found at substrate potentials negative of the MB redox

couple. Analogous behavior was recently observed by Zhou for DNA-modified electrodes in

the presence of MB in solution.

The positive feedback observed in our experiments with

MB is notably weaker versus that found for the monolayers covalently modified with NB at

the top and yields an apparent rate constant for ferricyanide reduction by noncovalent MB of

app

= 4.8 × 10

−

cm/s as measured at

−

300 mV versus SSCE, which is similar to that found

earlier.

Moreover, the MB controls yield essentially identical results with and without

backfilling (data not shown), revealing that MB cannot be simply incorporated within the 11-

mercaptoundecylphosphoric acid underlayer. This experiment indicates that the DNA film

modified with NB at the bottom supports long-range DNA-mediated CT, but measurable CT

occurs only as long as the Fe(CN)

−

in solution has access to the redox-active intercalator

within the film.

SECM Imaging of DNA Monolayer Morphology

Previously, undiluted DNA monolayers of the type utilized during the course of this study have

been characterized by atomic-force microscopy,

22–24

fluorescence spectroscopy,

59–61

surface-plasmon resonance,

and radio-active tagging.

12,63

These experiments (along with

electrochemical measurements) confirm that dense DNA films adopt an upright orientation

with respect to the surface. However, a distinct geometric orientation of a dilute submonolayer

film has been difficult to characterize conclusively with such techniques. To gain further insight

into the morphology of DNA monolayers modified with NB, SECM in imaging mode was

utilized to examine the DNA films. In general, the images indicate that the backfilled

monolayers investigated here are electrostatically smooth within the one micrometer resolution

of the SECM tip. The analysis of a large collection of images (hundreds collected over

numerous substrates) indicates that some electrodes are completely and uniformly covered by

the DNA film. On the other hand, some images feature domains on the electrode surface that

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are either DNA-free or contain thinner and less organized DNA assemblies. Indeed, such

partially covered or imperfect DNA monolayers were found to correlate well with incomplete

passivation of ferricyanide at the substrate, allowing for a detailed analysis of even these

imperfect surfaces with SECM.

Figure 4 shows images obtained for DNA substrates modified with NB at the top or bottom,

over an 85

m × 85

m scan area. The entire image is essentially flat and uniform with the

exception of the small area in the rear corner, which was generated by close approach of the

tip to the substrate upon initiation of the scan, thereby resulting in a partially uncovered surface.

Significantly, it is apparent that changing the substrate bias has only a small effect on the steady-

state current at the tip for films functionalized with NB at the bottom, yielding nearly identical

images at 0 and

−

400 mV. However, for the film functionalized with NB at the top, changing

the substrate bias modulates or “switches” the entire area under the scan with a resulting

significant enhancement of the steady-state current at

−

400 mV. These observations parallel

those obtained via the feedback mode of the SECM and illustrate the powerful imaging

capabilities of this technique.

Notably, the most efficient modulation occurs within regions of the DNA film that exhibit the

lowest currents at substrate potentials of 0 mV (Figure 4A,B). This can be best illustrated by

mapping the switching ratios, that is, the currents obtained at a substrate bias of

−

400 mV

versus a substrate bias of 0 mV for both types of films (Figure 4C,D). For example, the DNA

monolayer modified with NB at the top exhibits low currents at 0 mV over the entire scan area,

except for the partially uncovered corner (rear corner in Figure 4A,C); at a substrate bias of

−

400 mV, the smallest current amplification is observed in this uncovered spot. On the other

hand, the DNA monolayer modified with NB at the bottom exhibits low currents both at 0 and

−

400 mV over the well-covered areas of the scan. However, the poorly passivated region in

the corner of the film with NB at the bottom (rear corner in Figure 4B,D) exhibits partial

modulation when biased at

−

400 mV, hinting that the tip-generated Fe(CN)

−

can access either

some of the NB moieties or the bare gold surface. These observations demonstrate that the

SECM allows for investigation of imperfect DNA films because it affords the ability to

differentiate between catalytically active DNA spots and poorly passivated defects within the

film.

Detection of TATA Binding Protein by SECM

As confirmation of the DNA-mediated nature of this cycle, approach curves were recorded at

DNA monolayers featuring NB at the top before and after addition of TBP, a transcription

factor that bends the DNA by ~90°, thereby attenuating DNA CT upon binding.

12,13

Figure

5 shows a schematic illustration of the sequence-specific detection of TBP during an SECM

experiment. As previously described, the tip is polarized at a potential sufficient for direct

oxidation of Fe(CN)

−

while the potential of the substrate is varied. Prior to addition of TBP,

the ferricyanide/NB catalytic cycle is “turned on” at a substrate bias of

−

400 mV, resulting in

positive feedback (Figure 6A). Upon addition of TBP to a monolayer containing its binding

site (5

′

-TATA-3

′

), the ferricyanide/NB catalytic cycle is “turned off” at a substrate bias of

−

400

mV, resulting in negative feedback (Figure 6D). On the other hand, the feedback observed at

DNA monolayers lacking the binding site is unaffected by the addition of TBP (Figure 6B,C).

Therefore, the sequence-specific distortion of the DNA spacer by TBP inhibits the DNA-

mediated electrochemistry of Nile Blue.

To further explore the analytical capabilities of the SECM, TBP binding at DNA monolayers

was also investigated in imaging mode (Figure 7). Before addition of protein, a ~30% increase

in the steady-state current is observed over nearly the entire 85

m × 85

m scan area upon

modulation of the substrate potential from 0 to

−

400 mV (note that the partially uncovered area

in the rear corner exhibits the poorest current modulation in Figure 7A). Upon addition of 1

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M BSA to the solution, the steady-state tip current is slightly lowered (Figure 7B). However,

there is no effect on the switching behavior of the substrate with the degree of modulation

remaining at ~30%, and these observations are consistent with some nonspecific adsorption of

BSA at the tip and substrate.

Subsequently, the addition of 300 nM TBP further lowers the

steady-state tip current, presumably through nonspecific adsorption (Figure 7C), and,

importantly, this transcription factor eliminates “switching” over nearly the entire scan area,

indicating reliable detection of TBP binding in a localized area of the monolayer.

These results provide strong support for the DNA-mediated nature of the Nile Blue/ferricyanide

catalytic cycle. It is particularly striking that TBP binding eliminates positive feedback given

that bending the DNA brings NB (and presumably ferricyanide) closer to the surface. In fact,

upon binding, TBP should shield the negatively charged DNA and reduce the electrostatic

repulsion of ferricyanide from the negatively charged monolayer. If significant direct charge

transfer between the surface and tip-generated ferricyanide were occurring (as observed by

Bard and co-workers),

there would be an enhancement in the tip current at

−

400 mV upon

addition of TBP, yet the opposite effect is observed here.

Discussion

We have now utilized the SECM to demonstrate electrocatalytic signal amplification at DNA

monolayers modified with NB. Positive feedback was observed for DNA monolayers modified

with NB at the top and the substrate biased at potentials sufficiently negative to reduce NB.

However, only negative feedback was observed for DNA monolayers modified with NB at the

bottom, regardless of the substrate bias. These measurements have allowed us to extract an

apparent heterogeneous rate constant for ferricyanide reduction by DNA-bound NB of

app

6 ± 3 × 10

−

cm/s, indicating that such probe-modified DNA monolayers may behave somewhat

like conducting films.

Furthermore, we have shown the sequence-specific interruption of

the ferricyanide/NB electrocatalytic cycle upon addition of the TBP transcription factor;

positive feedback is completely eliminated upon addition of this protein. Taken together, these

data strongly indicate that the redox chemistry of NB is DNA-mediated.

Previous studies

22,24,59,60

have indicated that the individual duplexes within dense DNA

SAMs stand upright at potentials negative of the PZC, particularly in the presence of an

alkanethiol underlayer.

61,62

These findings were hinted by our observations for the

electrochemistry of MB at dense DNA monolayers, where only the MB constrained to the

periphery of the film was available for the MB/ferricyanide catalytic cycle.

8,46

The present

results for sparse films are fully consistent with an upright orientation of the DNA. By fixing

the location of the NB covalent probe, we have conclusively demonstrated that only those

monolayers that feature NB at the top of the film and close to the film/solution interface can

mediate catalytic ferrocyanide recycling at the probe tip.

The images obtained over micrometer areas of DNA monolayers modified with NB are

consistent with the approach curves in feedback mode. Notably, the imaging mode of the

SECM allows for the reliable investigation of imperfect DNA monolayers in multiple areas of

the substrate, making it an attractive complement to bulk voltammetric techniques. In addition,

the SECM can distinguish between areas with and without DNA by carefully monitoring the

ratio of the currents at various substrate biases. Therefore, unlike substrate voltammetry, the

SECM ensures that the observed electrochemical response is due solely to DNA-mediated

electrochemistry. This feature of SECM allows for the highly reliable detection of protein

binding, minimizing the possibility of false positives that can arise in experiments involving

only substrate voltammetry.

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As a surface characterization technique, the SECM also provides important information, which

is intermediate between centimeter scale electrochemical measurements and nanometer scale

AFM/STM measurements. For example, an SECM tip with a 10

m footprint will access

attomoles of DNA molecules (for uniform surface coverages of 1–3 pmol/cm

), thereby

simultaneously sampling a large number of molecules (relative to an AFM/STM) and avoiding

overwhelming contributions from defects in the monolayer (relative to substrate

electrochemistry). In addition, assuming a 1:1 DNA/protein stoichiometry, we are

consequently able to detect attomole quantities of the TBP transcription factor with the SECM,

underscoring the potential of this methodology for highly sensitive, sequence-specific

detection. Therefore, the scale and sensitivity of the instrument make the SECM inherently

compatible with multiplexed technologies such as DNA microarrays, indicating that it is a

potential alternative to fluorescence-based microarray technologies.

Overall, these results indicate that the investigation of DNA-mediated electrochemistry with

scanning electrochemical microscopy is a powerful and flexible methodology for the localized

investigation of DNA monolayers. Here, by backfilling with a negatively charged alkanethiol

and coupling NB with ferricyanide in a catalytic cycle, we have ensured that the DNA duplexes

adopt an upright orientation and that DNA CT dominates the electrochemical response

observed by SECM. In fact, if nonspecific protein adsorption at the tip can be minimized, the

micrometer size and reliability of the SECM could lead to sensitive monitoring of DNA/protein

interactions in a multiplexed format.

Acknowledgements

We are grateful to the NIH (GM61077 to J.K.B.) and the ACS-PRF (to K.S. and M.G.H.) for their financial support

of this research.

References

1. Boon EM, Barton JK. Curr Opin Struct Biol 2002;12:320. [PubMed: 12127450]

2. Delaney S, Barton JK. J Org Chem 2003;68:6475. [PubMed: 12919006]

3. O’Neill, MA.; Barton, JK. Charge Transfer in DNA: From Mechanism to Application. Wagenknecht,

H-A., editor. Wiley-VCH; Hoboken: 2005.

4. Giese B. Acc Chem Res 2000;33:631. [PubMed: 10995201]

5. Schuster GB. Acc Chem Res 2000;33:253. [PubMed: 10775318]

6. Drummond TG, Hill MG, Barton JK. Nat Biotechnol 2003;21:1192. [PubMed: 14520405]

7. Odenthal KJ, Gooding JJ. Analyst 2007;132:603. [PubMed: 17592577]

8. Kelley SO, Boon EM, Barton JK, Jackson NM, Hill MG. Nucleic Acids Res 1999;27:4830. [PubMed:

10572185]

9. Boon EM, Ceres DM, Drummond TG, Hill MG, Barton JK. Nat Biotechnol 2000;18:1096. [PubMed:

11017050]

10. Gorodetsky AA, Barton JK. Langmuir 2006;22:7917. [PubMed: 16922584]

11. Boal AK, Barton JK. Bioconjugate Chem 2005;16:312.

12. Boon EM, Salas JE, Barton JK. Nat Biotechnol 2002;20:282. [PubMed: 11875430]

13. Gorodetsky AA, Ebrahim A, Barton JK. J Am Chem Soc 2008;130:2924. [PubMed: 18271589]

14. Boon EM, Livingston AL, Chmiel NH, David SS, Barton JK. Proc Natl Acad Sci USA

2003;100:12543. [PubMed: 14559969]

15. Boal AK, Yavin E, Lukianova OA, O’Shea VL, David SS, Barton JK. Biochemistry 2005;44:8397.

[PubMed: 15938629]

16. Gorodetsky AA, Boal AK, Barton JK. J Am Chem Soc 2006;128:12082. [PubMed: 16967954]

17. DeRosa MC, Sancar A, Barton JK. Proc Natl Acad Sci USA 2005;102:10788. [PubMed: 16043698]

Gorodetsky et al.

Page 9

Langmuir

. Author manuscript; available in PMC 2009 December 16.

NIH-PA Author Manuscript

18. Gorodetsky AA, Dietrich LEP, Lee PE, Demple B, Newman DK, Barton JK. Proc Natl Acad Sci

USA 2008;105:3684. [PubMed: 18316718]

19. Ceres DM, Barton JK. J Am Chem Soc 2003;125:14964. [PubMed: 14653712]

20. Hihath J, Xu B, Zhang P, Tao N. Proc Natl Acad Sci USA 2005;102:16979. [PubMed: 16284253]

21. Wierzbinski E, Arndt J, Hammond W, Slowinski K. Langmuir 2006;22:2426. [PubMed: 16519433]

22. Kelley SO, Barton JK, Jackson NM, McPherson LD, Potter AB, Spain EM, Allen MJ, Hill MG.

Langmuir 1998;14:6781.

23. Zhou D, Sinniah K, Abell C, Rayment T. Langmuir 2002;18:8278.

24. Erts D, Polyakov B, Olin H, Tuite E. J Phys Chem B 2003;107:3591.

25. Turcu F, Schulte A, Hartwich G, Schuhmann W. Angew Chem, Int Ed 2004;43:3482.

26. Liu B, Li CZ, Kraatz HB, Bard AJ. J Phys Chem B 2005;109:5193. [PubMed: 16863184]

27. Wang K, Goyer C, Anne A, Demaille C. J Phys Chem B 2007;111:6051. [PubMed: 17487999]

28. Wain AJ, Zhou F. Langmuir 2008;24:5155. [PubMed: 18355100]

29. Stoica L, Neugebauer S, Schuhmann W. Adv Biochem Eng Biot 2008;109:455.

30. Bard, AJ.; Mirkin, MV. Scanning Electrochemical Microscopy. Marcel Dekker; New York: 2001.

31. Wang J, Zhou F. J Electroanal Chem 2002;537:95.

32. Komatsu M, Yamashita K, Uchida K, Kondo H, Takenaka S. Electrochim Acta 2006;51:2023.

33. Lie LH, Mirkin MV, Hakkarainen S, Houlten A, Horrocks BR. J Electroanal Chem 2007;603:67.

34. Palchetti I, Laschi S, Marrazza G, Mascini M. Anal Chem 2007;79:7206. [PubMed: 17696405]

35. Wong ELS, Gooding JJ. Anal Chem 2006;78:2138. [PubMed: 16579591]

36. Li X, Song H, Nakatani K, Kraatz HB. Anal Chem 2007;79:2552. [PubMed: 17279726]

37. Wong ELS, Gooding JJ. J Am Chem Soc 2007;129:8950. [PubMed: 17602630]

38. Di Giusto DA, Wlasoff WA, Giesebrecht S, Gooding JJ, King GC. J Am Chem Soc 2004;126:4120.

[PubMed: 15053597]

39. Kelley SO, Jackson NM, Hill MG, Barton JK. Angew Chem, Int Ed 1999;38:941.

40. Drummond TG, Hill MG, Barton JK. J Am Chem Soc 2004;126:15010. [PubMed: 15547981]

41. Liu T, Barton JK. J Am Chem Soc 2005;127:10160. [PubMed: 16028914]

42. Gorodetsky AA, Green O, Yavin E, Barton JK. Boconjugate Chem 2007;18:1434.

43. Okamoto A, Kamei T, Tanaka K, Saito I. J Am Chem Soc 2004;126:14732. [PubMed: 15535693]

44. Okamoto A, Kamei T, Saito I. J Am Chem Soc 2006;128:658. [PubMed: 16402854]

45. Inouye M, Ikeda R, Takase M, Tsuri T, Chiba J. Proc Natl Acad Sci USA 2005;102:11606. [PubMed:

16087881]

46. Boon EM, Barton JK, Bhagat V, Nersissian M, Wang W, Hill MG. Langmuir 2003;19:9255.

47. Zhang J, Kirkham J, Robinson C, Wallwork ML, Smith DA, Marsh A, Wong M. Anal Chem

2000;72:1973. [PubMed: 10815953]

48. Liu HH, Lu JL, Zhang M, Pang DW. Anal Sci 2002;18:1339. [PubMed: 12502086]

49. Ju H, Ye Y, Zhu Y. Electrochim Acta 2005;50:1361.

50. Schlereth DD, Schmidt HL. J Electroanal Chem 1995;380:117.

51. Sugawara K, Yamauchi Y, Hoshi S, Akatsuka K, Yamamoto F, Tanaka S, Nakamura H.

Bioelectrochem Bioenerg 1996;41:167.

52. Bard, AJ.; Faulkner, LR. Electrochemical Methods. Vol. 2. John Wiley & Sons; New York: 2001.

53. Mirkin MV, Arca M, Bard AJ. J Phys Chem 1993;97:10790.

54. Wei C, Bard AJ, Mirkin MV. J Phys Chem 1995;99:16033.

55. Lefrou C. J Electroanal Chem 2007;601:94.

56. Steel AB, Herne TM, Tarlov MJ. Anal Chem 1998;70:4670. [PubMed: 9844566]

57. Yu HZ, Luo CY, Sankar CG, Sen D. Anal Chem 2003;75:3902. [PubMed: 14572060]

58. Ceres DM, Udit AK, Hill HD, Hill MG, Barton JK. J Phys Chem B 2007;111:663. [PubMed:

17228925]

59. Rant U, Arinaga K, Fujita S, Yokoyama N, Abstreiter G, Tornow M. Nano Lett 2004;4:2441.

Gorodetsky et al.

Page 10

Langmuir

. Author manuscript; available in PMC 2009 December 16.

NIH-PA Author Manuscript

60. Rant U, Arinaga K, Fujita S, Yokoyama N, Abstreiter G, Tornow M. Langmuir 2004;20:10086.

[PubMed: 15518498]

61. Arinaga K, Rant U, Tornow M, Fujita S, Abstreiter G, Yokoyama N. Langmuir 2006;22:5560.

[PubMed: 16768474]

62. Yang X, Wang Q, Wang K, Tan W, Yao J, Li H. Langmuir 2006;22:5654. [PubMed: 16768490]

63. Gore MR, Szalai VA, Ropp PA, Yang IV, Silverman JS, Thorp HH. Anal Chem 2003;75:6586.

[PubMed: 14640732]

Gorodetsky et al.

Page 11

Langmuir

. Author manuscript; available in PMC 2009 December 16.

NIH-PA Author Manuscript

Figure 1.

Schematic illustration of SECM imaging of DNA monolayers modified with Nile Blue (NB)

at the top or the bottom; the negatively charged backfilling underlayer is also shown. The

sequence was 5

′

GC GTG CTT TAT ATC TC-3

′

(top NB) and 5

′

-TGC GTG CTT TAT ATC

C-3

′

(bottom NB), where the italicized U indicates the location of the NB moiety.

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Figure 2.

Cyclic voltammetry at 100 mV/s of DNA monolayers modified with NB at the top (blue) and

bottom (red) in 80 mM NaCl, 20 mM NaP

, pH = 7.2, 5 mM K

Fe(CN)

. The sequence is 5

′

GC GTG CTT TATATC TC-3

′

(top NB) and 5

′

-TGC GTG CTT TAT ATC

C-3

′

(bottom

NB), where the italicized U indicates the location of the NB moiety. Note that a catalytic peak

is only observed for a DNA monolayer modified with NB at the top.

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Figure 3.

SECM approach curves obtained for DNA monolayers modified with NB at the top and bottom

in 80 mM NaCl, 20 mM NaP

, pH = 7.2, 5 mM K

Fe(CN)

with a tip approach speed of 1

m/s. The tip approach current has been normalized by the tip steady-state current at an infinite

distance from the substrate. The normalized current is plotted against

, where

is the tip/

substrate separation and

represents the tip radius. For the DNA monolayer functionalized

with NB at the top, positive feedback is observed at

−

400 mV (A), and negative feedback is

observed at 0 mV (B). For the DNA monolayer modified with NB at the bottom, negative

feedback is observed both at 0 mV (C) and

−

400 mV (D). Theoretical curves are shown for

ideal negative feedback at an insulating substrate (black solid line) and for finite heterogeneous

kinetics with a rate constant of

app

= 4.6 × 10

−

cm/s for this specific substrate (red solid line).

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Figure 4.

SECM images obtained for DNA monolayers modified with NB at the top (A, C) and NB at

the bottom (B, D). The tip was polarized to 1 V, and the scans were performed in 80 mM NaCl,

20 mM NaP

, pH = 7.2, 5 mM K

Fe(CN)

at a speed of 10

m/s. The substrate bias was

−

400

mV (green in A, B) and 0 mV (blue in A, B). The switching ratios (the current obtained at

−

400

mV divided by the current obtained at 0 mV) are shown for monolayers with NB at the top (C)

and monolayers with NB at the bottom (D). The poorly covered areas of the DNA monolayer

are in the rear corners of A, B, C, and D.

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Figure 5.

Schematic illustration of the SECM-based, sequence-specific detection of TBP binding at DNA

monolayers modified with NB. The addition of TBP in the presence of its binding site (right)

attenuates the NB/ferricyanide catalytic cycle. Without the binding site for TBP, no protein

binding occurs and DNA-mediated CT proceeds.

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