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DNA Electrochemistry with Tethered Methylene Blue
Catrina G. Pheeney
and
Jacqueline K. Barton
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,
California 91125, United States
Abstract
Methylene blue (MB
), covalently attached to DNA through a flexible C
12
alkyl linker, provides a
sensitive redox reporter in DNA electrochemistry measurements. Tethered, intercalated MB
is
reduced through DNA-mediated charge transport; the incorporation of a single base mismatch at
position 3, 10, or 14 of a 17-mer causes an attenuation of the signal to 62 ± 3% of the well-
matched DNA, irrespective of position in the duplex. The redox signal intensity for MB
–DNA is
found to be least 3-fold larger than that of Nile blue (NB)–DNA, indicating that MB
is even more
strongly coupled to the
π
-stack. The signal attenuation due to an intervening mismatch does,
however, depend on DNA film density and the backfilling agent used to passivate the surface.
These results highlight two mechanisms for reduction of MB
on the DNA-modified electrode:
reduction mediated by the DNA base pair stack and direct surface reduction of MB
at the
electrode. These two mechanisms are distinguished by their rates of electron transfer that differ by
20-fold. The extent of direct reduction at the surface can be controlled by assembly and buffer
conditions.
INTRODUCTION
Since the discovery that DNA can efficiently serve to conduct electrical current, its
properties have been exploited across numerous platforms.
1–14
DNA-mediated charge
transport (CT) is exquisitely sensitive to perturbations in the intervening base stack,
including single base mismatches, lesions, or structural changes caused by proteins.
14–16
Electrochemistry experiments on DNA-modified electrodes have been particularly valuable
in probing ground state DNA CT and in the development of new DNA-based sensors.
11–19
To study and exploit the sensitivity of DNA CT, it is essential that the redox moiety makes
an electronic interaction with the base stack of the DNA.
17,20
Over the past decade, various reporters have been used for DNA electrochemistry; some
redox reporters have been well coupled and others not at all.
14–38
Early applications of
© 2012 American Chemical Society
Correspondence to: Jacqueline K. Barton.
The authors declare no competing financial interest.
NIH Public Access
Author Manuscript
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. Author manuscript; available in PMC 2013 May 01.
Published in final edited form as:
Langmuir
. 2012 May 1; 28(17): 7063–7070. doi:10.1021/la300566x.
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DNA-modified electrodes primarily depended on the use of noncovalent redox reporters.
Noncovalent methylene blue (MB) was widely used as a reporter for DNA CT as it
intercalates into the base stack.
11,13
Studies with noncovalent MB demonstrated the
exquisite sensitivity of the
π
-stack to subtle perturbations, and MB had the additional
advantage that it undergoes electrocatalytic signal amplification.
38
However, noncovalent reporters incurred a number of constraints, including the inability to
control probe placement and the stringent requirement for high quality DNA films. As such,
covalent tethering has been explored to address these issues. Covalent tethering of the
reporter results in its restricted mobility; therefore, it must be ensured that coupling between
the reporter and base stack is still feasible. The degree of coupling varies based on the
mechanism of interaction between the reporter and the base stack.
17
Coupling to the base
stack has been shown to be possible through various mechanisms including intercalation,
18
end-capping,
20
and direct conjugation to a nucleic acid base.
17
The degree to which the
reporter is coupled to the base stack determines the overall efficiency of DNA CT.
Daunomycin, when covalently tethered to the DNA through a short alkyl linkage, has been
shown to yield exceptionally strong electrochemical signals.
14,16,21
This evidence of strong
coupling agrees with the crystal structure that shows daunomycin is tightly intercalated into
the base stack.
39,40
Despite the exceptional coupling of daunomycin, the field of DNA-
mediated CT sensors has evolved to use other reporters as DNA modified with daunomycin
is difficult to prepare, is unstable, and has sequence constraints.
More recently, Nile blue (NB) has been used as an electrochemical reporter of DNA
CT.
12,17
The preparation of NB–DNA does not possess the synthetic limitations of
daunomycin, and it is well coupled to the base stack through direct conjugation of NB with a
modified uracil.
12
In the present study, we built upon this work by developing a system to
covalently tether MB, as it has previously been shown to strongly intercalate into the base
stack.
MB
–DNA was tethered through a flexible C
12
alkyl linkage to a modified uracil (Figure 1).
This linkage further improves the coupling of the reporter by providing the conformational
freedom for MB
to interact with the base stack through intercalation. However, with the
enhanced flexibility of the reporter, its ability to be reduced directly at the surface of the
electrode must be taken into account.
Various probes have been covalently tethered to the DNA through flexible alkyl linkages
and shown to be reduced directly at the electrode surface, including both MB and
ferrocene.
20,22–37
This has led to a new class of DNA-modified biosensors based upon
binding of either oligonucleotides
22–32
or DNA-binding proteins
33
that cause
conformational changes that attenuate the observed signal by physically diminishing the
surface accessibility of the reporter. These biosensors are based on the surface reduction of
the reporter, and thus the reporter does not couple with the base stack. Ferrocene has been
shown to be a common reporter for this platform given its poor coupling to the base stack
and yielding efficient reduction at the electrode surface.
37
More recently, MB-modified
DNA has been frequently used.
22–33
In this work, the mechanism of reduction for MB
covalently tethered to DNA through a
flexible C
12
alkyl linkage to a modified uracil is investigated (Figure 1). MB
–DNA,
covalently tethered through this flexible linkage, couples to the base stack through
intercalation. This is compared to the previously established reporter, NB–DNA, which is
covalently tethered through a short rigid linkage and couples to the base stack through direct
conjugation. MB
–DNA is shown not only to be reduced via DNA CT but is also capable of
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being reduced directly at the surface. We describe the conditions under which DNA CT is
the primary mechanism for MB
reduction.
EXPERIMENTAL SECTION
Synthesis of Modified NHS Ester Activated Methylene Blue
(Scheme 1)
All materials were used as purchased from Sigma-Aldrich.
2-Amino-5-(dimethylamino)phenylthiosulfonic Acid (3) Preparation—
3
was
prepared according to the procedure described by Wanger
41
by separately dissolving
aluminum sulfate octadecahydrate (43.6 mg, 65 mmol), sodium thiosulfate (22.0 g, 140
mmol), and zinc chloride (8.8 g, 63 mmol) in 100, 80, and 12 mL of water, respectively, and
added to
N,N
-dimethylphenylenediamine (
1
) (10 g, 73 mmol) in a 500 mL round-bottom
flask. The reaction mixture was then cooled to 0 °C while continuously stirring. Potassium
dichromate (5.0 g, 17 mmol) was dissolved in 30 mL of water and added dropwise to the
reaction mixture over 15 min. The reaction was kept at 0 °C for 2 h and then slowly allowed
to warm to room temperature. The precipitate was isolated by vacuum distillation and
washed with water, acetone, and ether. The purple solid (7.4 g, 30 mmol, 41% yield) was
confirmed as the desired product by
1
H NMR (DMSO-
d
6
) and was used in subsequent
reactions without further purification.
N-Methyl-N-(carboxypropyl)aniline (4) Preparation—
4
was prepared according to
the procedure described by Whitten.
42
N
-Methylaniline (
2
) (15.17 mL, 140 mmol) was
refluxed with ethyl-4-bromobutyrate (20.0 mL, 140 mmol) for 16 h in a 100 mL round-
bottom flask. The reaction mixture was then cooled to room temperature, and water (15 mL)
was added. The crude reaction mixture was then made basic by the dropwise addition of
saturated sodium hydroxide and extracted with ether (3 × 50 mL). The extract was washed
with water and dried over MgSO
4
, and the ether was removed under reduced pressure. Pure
N
-methyl-
N
-ethyl-4-butanoate aniline (16.3 g, 74 mmol, 53% yield) was isolated, as a clear
liquid, by vacuum filtration at 110 °C at 0.2 mmHg. The product was confirmed by
1
H
NMR and
13
C NMR (CDCl
3
) and an observed mass of 221.3 g/mol (calculated mass of
221.3 g/mol) in ESI-MS in acetonitrile: water:acetic acid (1:1:0.1%).
The ester product (9.4 g, 43 mmol) was subsequently hydrolyzed in 5% KOH (150 mL) by
refluxing for 2 h to form the desired carboxylic acid. The reaction was cooled to room
temperature and washed with ether (2 × 70 mL). Concentrated hydrochloric acid was added
dropwise to the aqueous layer to adjust the pH to 5.5, and then the product was extracted
with ether (3 × 100 mL) and dried over MgSO
4
, and the solvent was removed under reduced
pressure. The desired product,
4
(5.1 g, 26 mmol, 62% yield), was confirmed by
1
H NMR
and
13
C NMR (CDCl
3
) and an observed mass of 193.2 g/mol (calculated mass of 193.2 g/
mol) with ESI-MS in acetonitrile:water:-acetic acid (1:1:0.1%).
N-(Carboxypropyl)methlyene Blue (MB
) Preparation—
MB
was prepared by an
adapted procedure from Wagner.
41
3
(2.4 g, 9.7 mmol) and
4
(1.87 g, 9.7 mmol) were
combined and dissolved in a methanol:water mixture (200 mL:80 mL). The reaction was
heated to just below reflux (60 °C), and 50% w/w silver carbonate on Celite (10 g) was
slowly added. The reaction was then refluxed for 2 h. The reaction was left to cool to room
temperature and was vacuum filtered, and the solvent was removed under reduced pressure.
The desired product MB
(0.9 g, 2.5 mmol, 26% yield) was isolated by dry
chromatography
43
as conventional chromatography techniques were unsuccessful.
Impurities were eluted using 20 mL portions of chloroform:methanol:acetic acid
(100:15:1.5), and the blue band (MB
) was eluted using portions of
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chloroform:methanol:acetic acid (100:30:1.5). The final product was confirmed by an
observed mass of 356.3 g/mol (calculated mass of 357.13 g/mol) with ESI-MS in
acetonitrile:water:acetic acid (1:1:0.1%).
MB
–NHS Ester Preparation—
MB
(8 mg, 0.022 mmol) was dissolved in DMF (1 mL)
and combined with
N,N
-dicyclohexylcarbodiimide (9.3 mg, 0.045 mmol) and
N
-
hydroxysuccinimide (5.2 mg, 0.045 mmol). The reaction proceeded at room temperature for
24 h. The solvent was removed under reduced pressure, and the material was resuspended in
DMSO. Successful ester activation was confirmed by a mass of 454.5 g/mol (calculated
mass of 454.54 g/mol) in ESI-MS in acetonitrile:water:acetic acid (1:1:0.1%). Activated
ester was not found to be stable for extended periods of time; therefore, it was freshly
prepared directly before tethering to amino-modified DNA.
Synthesis of Modified Oligonucleotides
The synthesis and purification of NB and thiol-modified oligonucleotides were carried out
following the previously reported protocol.
17
Thiol-modified and NHS ester uracil analogue
phosphoramidites were purchased from Glen Research.
MB
-modified oligonucleotides were synthesized similarly to NB–DNA with the
substitution of an amino-C
6
-uracil analogue purchased from Glen Research. Amino-
modified DNA was purified using standard protocols and then coupled in solution to MB
NHS ester.
Amino-modified DNA was suspended in 200 μL of a 0.1 M NaHCO
3
solution in order to
buffer the reaction to a pH of 8.3–8.4. MB
–NHS ester was suspended in DMSO (100 μL)
and added to the DNA solution in roughly a 10-fold excess of MB
–NHS ester to amino-
modified DNA. The reaction was left to proceed for 12–24 h. A final round of purification
was performed by high-performance liquid chromatography (HPLC) using a 50 mM
ammonium acetate buffer/acetonitrile gradient with a PLRP-S Column (Agilent). The MB
DNA mass was confirmed by matrix-assisted laser desorption/ionization-time-of-flight mass
spectrometry. A mass of 5695 g/mol was found for well-matched MB
–DNA, agreeing with
with the calculated mass of 5695 g/mol (see Figure 2).
DNA stock solutions were prepared in low salt buffer (5.0 mM phosphate, 50 mM NaCl, pH
7) and quantified as previously reported. The extinction coefficient for single-stranded MB
-
DNA at 260 nm was corrected for the absorbance of MB
. This correction was performed by
adding the extinction coefficient of MB
at 260 nm (10 300 M
−1
cm
−1
) to the calculated
extinction coefficient for single-stranded DNA. All DNA solutions were thoroughly
deoxygenated with argon prior to annealing. Equimolar amounts of thiol-modified and
probe-modified oligonucleotides were combined and annealed by heating to 90 °C and
cooling to ambient temperature over 90 min to form duplexes.
Preparation of DNA Monolayers and Electrochemical Measurements
Multiplex chips, previously reported,
17
were employed for the electrochemical experiments.
Each chip contains 16 gold electrodes (2 mm
2
area) which were prepared with up to four
different kinds of DNA. Equimolar amounts of single-stranded thiol-modified and probe-
modified DNA were annealed prior to the electrode assembly. HPLC analysis of duplex
DNA stocks were performed prior to electrode assembly to ensure that there were no single-
stranded impurities (data not shown). The duplex DNA (25 μL of 25 μM) was then
assembled on the electrode surface overnight (20–24 h) in a humid environment to allow for
monolayer formation with or without 100 mM MgCl
2
. Once DNA films were assembled and
thoroughly washed with low salt buffer, the electrodes were backfilled with either 1 mM 6-
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mercaptohexanol (MCH) or 6-mercaptohexanoic acid (MHA) for 45 min in low salt buffer
with 5% glycerol. The electrodes were again washed to ensure removal of trace alkanethiols.
The electrodes were scanned in a common running buffer of either low salt buffer (5.0 mM
phosphate, 50 mM NaCl, and pH 7) or spermidine buffer (5.0 mM phosphate, 50 mM NaCl,
4 mM MgCl
2
, 4 mM spermidine, 50 μM EDTA, 10% glycerol, and pH 7). An analogous
buffer to spermidine buffer was prepared that lacked spermidine, and minimal changes were
observed in the electrochemistry (data not shown). Thus, electrochemical differences are
best attributed to the effect of adding spermidine. Electrochemical measurements were
performed with a CHI620D electrochemical analyzer and a 16-channel multiplexer from CH
Instruments. A three-electrode setup was used, with a common Ag/AgCl reference and a Pt
wire auxiliary electrodes placed in the central buffer solution.
Cyclic voltammetry (CV) data were collected at 100 mV/s, with the exception of the scan
rate dependence experiments where the scan rate is indicated. In order to minimize errors
associated with thiol oxidation and surface quality, all CVs overlaid and compared were
acquired on the same multiplexed chip with the thiol-modified strand being equivalent in all
duplexes.
The film density (
Γ
) of MB
–DNA and NB–DNA was calculated by the area under the
reductive peaks of CVs at 100 mV/s (eq 1).
(1)
In eq 1,
Q
is the area of the reductive signal,
n
is the number of electrons per redox event (
n
= 2 for both NB and MB
),
F
is Faraday’s constant, and
A
is the area of the gold electrode.
RESULTS
Electrochemistry of MB
–DNA with Intervening Mismatches
In this investigation MB
has been confined to the distal end of the duplex through a flexible
C
12
-linker appended off the terminally modified uracil (Figure 1). As seen in Figure 2, the
resulting CVs from scanning the electrodes in spermidine buffer exhibit strong reductive and
oxidative peaks with a midpoint potential of −290 mV versus Ag/AgCl. This is the same
reduction potential as freely diffusing MB indicating that covalently tethering MB
to the
DNA through a flexible alkyl chain does not alter its electronic properties.
44
The areas of the
reductive and oxidative signals were 7.8 ± 0.4 and 7.5 ± 0.3 nC, respectively. Surface-bound
species reduced by DNA CT have previously been shown to have cathodic/anodic signals
with ratios of nearly unity, which we have ascribed to the fact that the binding affinity of
MB for duplex DNA is lowered upon reduction.
19,38
In order to demonstrate that the reduction of MB
–DNA occurs via DNA CT, the signal
attenuation from the introduction of a single mismatched base pair (CA) intervening
between the surface and the probe was compared to that of well matched MB
–DNA. By
performing these experiments on multiplexed chips, any variation that can be observed from
backfilling the electrodes is removed. Under these conditions, any signal differential
obtained between well-matched and mismatched DNA on the same multiplexed chip can be
attributed to deficiencies in the CT properties of the mismatched DNA.
19
Introduction of a
signal base mismatch has been well documented to cause attenuations in DNA CT in both
photophysical studies and DNA electrochemistry studies.
1–16
Thiol-modified DNA was annealed to the complementary well-matched (WM) DNA
sequence and three different mismatched DNA sequences, where the position of the
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mismatch was varied. The mismatches varied from proximal to the electrode (MM3), in the
middle (MM10), and at the distal end (MM14) of the duplex (Figure 2). The four quadrants
of a multiplexed chip were used to simultaneously assemble each type of DNA overnight
without MgCl
2
(Figure 2). In this case the electrodes were passivated by backfilling with
MHA and scanned in spermidine buffer. The ratio of the mismatched to well-matched
reduction signal areas was quantified in all cases to determine the percent mismatch signal.
The signal area of well-matched MB
–DNA was found to be 7.8 ± 0.4 nC. For the various
mismatches, the percent signal remaining was 65%, 64%, and 59% for a mismatch
incorporated at position 3, 10, or 14, respectively. Under these assembly conditions, the
percent signal remaining, due to the incorporation of the CA mismatch, was essentially
equivalent regardless of the position in the duplex. The suppression of the MB
signal from
a single CA mismatch validated that MB
is well coupled to the
π
-stack and the electrons
that reduce MB
are traveling through the intervening DNA.
It should be noted that we observed signal attenuation from a CA mismatch at the 14th
position of the 17-mer, indicating that MB
must intercalate within 3 base pairs of where it
is covalently tethered; if MB
intercalates further away, there would be no observed signal
attenuation. This confinement of MB
is also consistent with what is predicted from model
building. Therefore, when MB
is tethered to the distal end of the duplex, any perturbation
to the base stack occurring below the top 7.2 Å will be reported.
Comparison with NB–DNA
MB
–DNA was then compared with the previously described DNA CT reporter, Nile blue
(NB). This comparison used electrodes assembled with 100 mM MgCl
2
, passivated with
MCH, and scanned in spermidine buffer. The area of the reductive signal for MB
–DNA is
significantly larger than the signal observed with NB–DNA, 12.5 ± 1.2nC compared to 4.3 ±
0.2 nC, respectively (Figure 3).
Additionally, when the same electrodes are scanned in low salt buffer, NB–DNA has an
average signal area of 0.2 ± 0.1 nC (Figure 3), while the reductive peak area for MB
–DNA
remains relatively unchanged at 11.8 ± 0.5 nC. Despite the area of MB
remaining
minimally affected, other peak characteristics were significantly altered. Switching to a low
salt buffer resulted in a broadening of the peak and an increase in the peak splitting. The
sharper peaks in spermidine buffer indicate that the film is more homogeneous. The peak
splitting between the oxidative and reductive peaks decreases from 140 mV in low salt
buffer to 30 mV in spermidine buffer. These peak changes are largely reversible; there is,
however, not a complete restoration of the peak observed in low salt buffer, indicating that
the spermidine is not fully removed.
Variation in Running Buffer for Optimized Mismatch Discrimination
The degree of signal attenuation upon introduction of a single mismatched base (MB
MM10 DNA) was compared in low salt buffer and spermidine buffer. The DNA was
assembled with 100 mM MgCl
2
and backfilled with MHA. Well-matched MB
–DNA
shows a reductive signal area of 11.1 ± 0.2 nC while mismatched MB
–DNA shows a signal
area of 4.0 ± 0.4 nC in spermidine buffer. The percent signal remaining after the
introduction of a CA mismatch is 36 ± 6% (Figure 4). When the same experiment is
performed with NB–DNA in spermidine buffer, a 50 ± 10% decrease is observed (data not
shown). This result agrees with the previously reported values with NB–DNA in that the
percent signal remaining due to a CA mismatch ranges from 30 to 60%.
12,19
When the same MB
–DNA electrodes were then examined in low salt buffer, the
mismatched reductive signal is significantly broadened and decreased in intensity, yielding
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an area of 0.5 ± 0.1 nC. This broadening decreases the percent signal remaining to only 7 ±
2% of the well-matched signal (9.1 ± 1.2 nC) (Figure 4). This same effect was observed in a
buffer equivalent to spermidine buffer but lacking spermidine (data not shown). Spermidine
binds in the grooves of duplex DNA, which rigidifies the duplex, resulting in sharper peaks.
However, spermidine should also decrease the binding affinity of MB
, which may account
for the decreased signal attenuation.
Variation in Assembly Conditions: Density of DNA Film
In addition to buffer conditions, the effect of varying the assembly conditions on mismatch
discrimination was investigated. The varying densities of DNA were obtained by assembling
with and without 100 mM MgCl
2
. In the absence of MgCl
2
, the negatively charged
phosphate backbone of DNA duplexes repel each other, yielding significantly lower film
densities.
13,20
MB
–DNA and NB–DNA films were therefore examined with and without
incubation with 100 mM MgCl
2
. With these assembly conditions, we find for MB
–DNA
and NB–DNA film densities of 1.3–3.2 and 0.8–1.6 pmol/cm
2
, respectively.
The percent of signal remaining after incorporation of a CA mismatch was then compared as
a function of film density in both low salt and spermidine buffer (Figure 5). When the
percent signal remaining is plotted against the film density, a roughly linear correlation is
observed. The mismatch signal attenuation improves with increasing density. This trend for
MB
–DNA is present in both low salt buffer and spermidine buffer.
The dependence of the mismatch signal attenuation on the film density suggests that there
might be a contribution of the signal that originates from direct reduction at the electrode
surface. DNA assembled under low density conditions (without MgCl
2
) has a higher surface
accessibility compared to DNA assembled under high density conditions (with MgCl
2
), thus
increasing the possibility of direct surface reduction.
Variation in Assembly Conditions: Backfilling Agent
In order to gain further insight, the backfilling agent utilized was varied. Backfilling is used
to decrease the background noise due to oxygen and to minimize nonspecific interactions
between the DNA and the gold surface. 6-Mercaptohexanol (MCH) is the most common
backfilling agent for DNA-modified electrodes. 6-Mercaptohexanoic acid (MHA) offers an
alternative backfilling agent with a more negative headgroup. The degree of signal
attenuation obtained, in spermidine buffer, was compared for MCH and MHA for electrodes
assembled with or without 100 mM MgCl
2
(Figure 6).
At either assembly concentration of MgCl
2
, switching from MCH to MHA improves the
signal attenuation observed due to mismatch incorporation. When electrodes were
assembled with 100 mM MgCl
2
and scanned in spermidine buffer, the percent mismatch
signal remaining improved from 52 ± 4% to 36 ± 6% by switching from MCH to MHA
(Figure 6). Increasing the negative charge of the surface, through backfilling with MHA,
increases the barrier between MB
–DNA and the electrode surface. The direct correlation
between decreased surface accessibility and increased mismatch signal attenuation provides
further evidence that, depending on assembly conditions, the signal observed from MB
DNA may be generated by two different mechanisms: DNA CT and direct surface
reduction.
Kinetics of MB
–DNA Reduction
The rates of electron transfer to the redox reporter for both MB
–DNA and NB–DNA were
estimated under various assembly conditions, passivated with MCH, by measuring the scan
rate dependence of the peak reduction potential and then applying the Laviron analysis.
45
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The Laviron analysis used to determine the rates of electron transfer has been routinely
employed for rate determinations in these systems.
12,21
Consistently, we find for DNA-
mediated electrochemistry that tunneling through the linker is rate-determining and the rate
is slower than with direct reduction. The same analysis was applied here to compare
reduction of the reporter by DNA CT to direct surface reduction. Electrodes assembled with
only single stranded (ss) MB
–DNA and NB–DNA were used to estimate the rate of
electron transfer by the direct surface reduction mechanism. In this case, the complementary
thiol-modified strand was omitted during assembly, ensuring that there could be no DNA-
mediated contribution to the observed signal; ssDNA does not efficiently conduct charge
and has a high affinity for the gold surface. For comparison, the rates of electron transfer for
the DNA-mediated reduction of MB
–DNA and NB–DNA were determined under
conditions where the signal attenuation due to an incorporated mismatch was maximal
(double-stranded (ds) DNA assembled with 100 mM MgCl
2
).
It has been previously established that the rate for ssNB–DNA is 10–30-fold faster than the
rate of electron transfer in dsNB–DNA, assembled with 100 mM MgCl
2
and acquired in
spermidine buffer.
12
The rate for dsDNA is limited by tunneling through the C
6
-alkane
linkage to the surface.
12
This previous result for NB–DNA was reproduced in this study
(Table 1). Furthermore, the rate of electron transfer for dsMB
–DNA, with 100 mM MgCl
2
,
is 20-fold slower than ssMB
–DNA in both spermidine and low salt buffers (Table 1). This
result confirms that dsMB
–DNA is reduced via DNA CT when assembled with 100 mM
MgCl
2
.
The rates of electron transfer were then examined under conditions where DNA CT is not
the sole mechanism for MB
–DNA reduction, evidenced by reduced signal attenuation from
a CA mismatch (dsDNA without MgCl
2
). At fast scan rates (5 V/s) for dsMB
–DNA, two
reductive peaks are resolved with a 15-fold rate differential in both spermidine and low salt
buffer (Figure 7 and Table 1). These data indicate that rates of electron transfer for these two
modes of reduction correspond with direct surface reduction and DNA-mediated reduction
of MB
–DNA. Well-matched and mismatched dsMB
–DNA were compared at 5 V/s, and
signal attenuation due to mismatch incorporation was observed only in the peak with a
slower rate of electron transfer, consistent with it being a DNA-mediated process (data not
shown). Alternatively, for dsNB–DNA only a single peak is observed in spermidine buffer
with a rate that is 20-fold slower than ssNB–DNA. This observation suggests that while
dsNB–DNA is only capable of being reduced via DNA CT, dsMB
–DNA may be reduced
by either a DNA-mediated pathway or direct surface reduction. The ability of MB
–DNA to
be reduced directly at the surface is likely due to the flexibility of the linkage through which
MB
is covalently tethered (Figure 7).
DISCUSSION
In this work, MB
is covalently tethered to DNA with a flexible C
12
alkyl linkage to a
modified uracil. MB
–DNA produces a reversible redox couple via DNA CT. Incorporation
of a single CA mismatch within a 17-mer can attenuate the mismatch signal to 7 ± 2% of the
well-matched signal. The degree to which the mismatch signal is attenuated was found to be
highly dependent on assembly conditions. Under conditions where DNA CT is the sole
available mechanism for MB
reduction, a 10-fold improvement in the mismatch sensitivity
is obtained, when compared to previously utilized nonamplified DNA-mediated
platforms.
12,19
MB
–DNA was found also to be capable of being reduced directly by the
surface of the electrode. The extent to which this mechanism contributes to the observed
signal was found to be directly influenced by assembly conditions.
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Therefore, MB
–DNA can both report on DNA CT through the intercalation of MB
into
the base stack and report on the surface accessibility of the reporter through direct surface
reduction of MB
. We can discern which mechanism of MB
reduction is dominating under
any given assembly condition by examining the signal sensitivity to
π
-stack perturbations or
by examining the rate of electron transfer.
The fact that assembly conditions can alter which mechanism is dominant for MB
reduction has relevance in the further development of DNA-based biosensors. Sensors using
direct surface reduction typically function through the formation of duplex DNA to attenuate
the observed signal by increasing the separation between MB
and the electrode.
22–32
Our
work shows that upon duplex formation a new pathway for dsMB
–DNA reduction has been
introduced, DNA CT, which can contribute to the residual observed signal.
Recent work performed to optimize these direct surface reduction-based platforms has
demonstrated that, upon duplex formation, no signal suppression is observed at slow scan
rates (100 mV/s), and the duplex signal yielded a rate of electron transfer slower than that of
the nonduplex DNA structure.
23
In order to optimize these devices, the scan rates were
increased to minimize the contributions from the dsMB
–DNA.
23
The trends observed in
this work are consistent with the work presented here and demonstrate that the DNA-
mediated reduction of duplex MB
–DNA can significantly contribute to the observed signal.
It is clear that both direct reduction and DNA-mediated reduction need to be considered as
important mechanisms in DNA-modified electrodes and that MB
–DNA is a viable reporter
for both systems. Furthering our understanding of the underlying mechanism for the
reduction of dsMB
–DNA is essential for exploiting both DNA CT and direct surface
reduction based biosensors to their full extent. Only when both mechanisms are considered
can the highest overall sensitivity be achieved.
Acknowledgments
This research was supported by the National Institute of Health (GM61077). The authors thank N. Muren for
discussions and her contributions in fabricating the multiplexed chips. This work was completed in part in the
Caltech Micro Nano Fabrication Laboratory.
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Figure 1.
Left: schematic illustration of MB
–DNA (top) and NB–DNA (bottom) monolayers bound
to an electrode. The reporter is appended to the distal base of the duplex. The intended path
for electrochemical reduction is indicated. Right: the chemical structure of both MB
and
NB covalently tethered to a modified uracil.
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Figure 2.
Electrochemistry of MB
–DNA with a single intervening CA mismatch. Left: cyclic
voltammetry was acquired at 100 mV/s with either a well-matched sequence (blue) or a
sequence containing a single mismatch (red). All four sequences (middle) of MB
–DNA
were assembled on the same multiplexed chip and assembled without MgCl
2
, passivated
with MHA, and scanned in spermidine buffer. Right: the area of the reductive signal was
used to quantify the signals observed, and the errors denoted are determined from the
variation across four different electrodes.
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Figure 3.
Comparison of the signal from NB–DNA (dotted) and MB
–DNA (solid). The electrodes
were assembled with 100 mM MgCl
2
and passivated with MCH. CVs were acquired at 100
mV/s, and all electrodes were scanned in both a low salt buffer (5.0 mM phosphate, 50 mM
NaCl, and pH 7) (gray) and a spermidine buffer (5.0 mM phosphate, 50 mM NaCl, 4 mM
MgCl
2
, 4 mM spermidine, 50 μM EDTA, 10% glycerol, and pH 7) (black). The CVs shows
the overall signal changes in response to the running buffer. The reductive peak areas were
quantified to show that the amount of MB
–DNA being reduced is relatively unchanged
regardless of the buffer. The errors denoted are determined from the variation across four
different electrodes.
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Figure 4.
Optimization of mismatch discrimination depending on the running buffer. Both WM MB
DNA (solid) and MM10 MB
–DNA (dotted) modified electrodes were assembled with 100
mM MgCl
2
, passivated with MHA, and scanned in both a low salt buffer (5.0 mM
phosphate, 50 mM NaCl, and pH 7) (right) and a spermidine buffer (5.0 mM phosphate, 50
mM NaCl, 4 mM MgCl
2
, 4 mM spermidine, 50 μM EDTA, 10% glycerol, and pH 7) (left).
The areas of the reductive peaks were used to quantify the reductive signal size (black) and
determine the percent signal remaining ([MM]/[WM] × 100) (gray) from the incorporation
of a single mismatch. The gray arrows denote the decrease in signal area with the optimal
percent signal attenuation being in low salt buffer. The errors denoted were determined by
the standard deviation from four electrodes averages, for each sequence of DNA, across
three chips.
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Figure 5.
Dependence of mismatch signal attenuation on film density for both MB
–DNA (black) and
NB–DNA (gray). Percent MB
–DNA signal remaining due to the incorporation of a CA
mismatch was determined in both low salt buffer (×) and spermidine buffer (squares).
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Figure 6.
Dependence on assembly conditions of the percent mismatch signal remaining for MB
DNA. Well-matched signal area (black) and the percent signal remaining (gray) were from
12 electrodes averaged across three chips. The backfilling agent (1 mM MCH or MHA) and
concentration of MgCl
2
are indicated. Scans were acquired in spermidine buffer.
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Figure 7.
Top: schematic illustration of reporter reduction mechanisms. Bottom: CV at 5 V/s (bottom)
for NB–DNA (left) and MB
–DNA (center) assembled without MgCl
2
and MB
–DNA
assembled with 100 mM MgCl
2
(right). Electrodes were backfilled with mercaptohexanol
and scanned in spermidine buffer. The red arrows indicate the peaks quantified in order to
determine the rate of the various processes for reporter reduction.
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