Direct Electrochemical Bioconjugation on Metal Surfaces

Article abstract of DOI:10.1021/jacs.7b06385

DNA has unique capabilities for molecular recognition and self-assembly, which have fostered its widespread incorporation into devices that are useful in science and medicine. Many of these platforms rely on thiol groups to tether DNA to gold surfaces, bu

Full text of DOI:10.1021/jacs.7b06385

Article
pubs.acs.org/JACS
Direct Electrochemical Bioconjugation on Metal Surfaces
Ariel L. Furst,† Matthew J. Smith,† and Matthew B. Francis*,†,‡
†Department of Chemistry, University of California, Berkeley, Berkeley, California 94720-1460, United States
‡Materials Sciences Division, Lawrence Berkeley National Laboratories, Berkeley, California 94720-1460, United States
S
* Supporting Information
ABSTRACT: DNA has unique capabilities for molecular recognition and self-assembly, which have fostered its widespread
incorporation into devices that are useful in science and medicine. Many of these platforms rely on thiol groups to tether DNA to
gold surfaces, but this method is hindered by a lack of control over monolayer density and by secondary interactions between the
nucleotide bases and the metal. In this work, we report an electrochemically activated bioconjugation reaction as a mild, reagent-
free strategy to attach oligonucleotides to gold surfaces. Aniline-modified DNA was coupled to catechol-coated electrodes that
were oxidized to o-quinones using an applied potential. High levels of coupling could be achieved in minutes. By changing the
reaction time and the underlying catechol content, the final DNA surface coverage could be specified. The advantages of this
method were demonstrated through the electrochemical detection of the endocrine disruptor bisphenol A, as well as the capture
of living nonadherent cells on electrode surfaces by DNA hybridization. This method not only improves the attachment of DNA
to metal surfaces but also represents a new direction for the site-specific attachment of biomolecules to device platforms.
azide groups to alkynes, widely known as “Click Chemistry”,
provides the most common method for coupling the DNA
strands to these surfaces.17,21−24 Unfortunately, these methods
can be problematic because they require redox-active catalysts
that damage DNA and complicate electrochemical sensing
applications or long coupling times of over 24 h. Elegant
electrochemically activated couplings using p-quinones as
Diels−Alder participants have been reported for small
molecules and short peptides,25−27 but these approaches have
not been applied to the coupling of larger proteins or
oligonucleotides.
Described herein is a new direct electrochemical bioconju-
gation reaction to couple nucleic acids to electrode surfaces
with high chemoselectivity and efficiency. The reaction
generates DNA monolayers in a matter of minutes without
the use of additional reagents and, importantly, enables tuning
of the amount of DNA coverage. The advantages of this
method for preparing diagnostic platforms are highlighted
through the electrochemical sensing of the endocrine disruptor
bisphenol A and the capture of living, nonadherent cells on
INTRODUCTION
■
Nucleic acids are a privileged class of biomolecules with
unmatched versatility for molecular recognition and self-
assembly. Beyond their central role in living systems, synthetic
oligonucleotides are essential for applications in gene
detection,1−3 specific analyte binding,4 catalysis,5,6 the assembly
of three-dimensional structures,7−9 and the capture of living
cells bearing sequence complements.10,11 Many of these
applications depend on the coupling of DNA oligomers with
inorganic surfaces, such as nanoparticles12,13 and gold electro-
des,14 typically through thiol groups introduced at the strand
termini.15,16 Though ubiquitous, this assembly method is
hindered by secondary interactions between the nucleobases
and metal surfaces, which complicate the formation of well-
defined, homogeneous monolayers with consistent coverage.
Additionally, adequate spacing between biomolecules is critical
for effective biosensing17,18 and nanoparticle assembly
applications.19,20 Thus, a growing number of alternative
synthetic approaches have involved the preassembly of mixed
monolayers containing chemically active head groups,21,22
which passivates the metal surface to prevent interactions
with the DNA bases and enables control over the number of
coupling sites available. Generally, a 1,3-dipolar cycloaddition of
Received: June 19, 2017
© XXXX American Chemical Society
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Figure 1. Overall strategy for direct electrochemical oxidative coupling on gold surfaces. (a) The structures of the coupling partners used in this
study are shown. (b) Mixed monolayers containing either 1 or 2 were first formed on gold electrodes (X = NO2 or OH; Y = O or NH). An applied
potential oxidizes the coupling groups, which in turn react with anilines introduced on the DNA strands (3).
electrode surfaces by DNA hybridization. Both applications
were found to be dependent on the surface coverage of DNA.
In addition to improving the attachment of oligonucleotides to
metal surfaces, this new technique represents a promising
direction for the site-specific attachment of biomolecules to
device platforms.
chemical version of the reaction involving azidophenols has
been developed for surface patterning applications.35
The reliance of these reactions on the generation of an
oxidized intermediate suggested that a reagent-free electro-
chemical variant could be developed by attaching o-
iminoquinone or o-quinone precursors to electrode surfaces.
To test this hypothesis, thiols 1 and 2 (Figure 1a) were
prepared and combined in varying ratios with 6-mercaptohex-
anol. It was envisioned that the nitro group of 1 could be
reduced immediately before the coupling reaction to access the
o-iminoquinone. Monolayers were assembled on the surfaces of
gold rod electrodes by exposure to ethanolic solutions of the
thiol mixtures at RT for 12 h (Figure 1b). The DNA-coupling
partners (3) were prepared through the reaction of 5′-amine
terminated strands with the NHS ester of 4-azidodihydrocin-
namic acid, followed by azide reduction with TCEP, as
previously reported.34
Electrochemical Behavior of Catechol-Containing
Monolayers. Surfaces coated with o-nitrophenols were first
evaluated for the coupling reaction. Preliminary studies
indicated that the nitro groups could be reduced smoothly to
anilines, but the resulting o-aminophenol groups exhibited poor
electrochemical reversibility upon subsequent redox cycling.
Reductions were tested both electrochemically and using
sodium dithionite. This is likely due to self-coupling of the o-
iminoquinones with o-aminophenols that had not yet been
reduced, a result that is consistent with the previous
observation that the oxidation step is rate-limiting in these
coupling reactions.36 Previous experiments in our group have
similarly observed poor reaction performance when o-
iminoquinones are generated on polyvalent surfaces, such
viral capsids. As a result, the aniline groups are typically
incorporated in such locations. Although this issue could likely
be solved through sufficient dilution of the o-nitrophenols on
the gold surface, irreversible signals were observed even with
10% o-nitrophenol was present in the monolayer. The o-
nitrophenol monolayers were not evaluated further.
EXPERIMENTAL SECTION
■
General Procedure for DNA Modification of Electrodes.
Catechol functionalized gold surfaces were modified with ssDNA. A 20
μL drop of 50 μM aniline-modified DNA in PBS (pH 7.2) was placed
on the center of the electrode. For the rod electrodes, the gel-tip
reference electrode and a platinum counter electrode were inserted
into the DNA-containing liquid drop. Constant potential amperometry
at a potential of 0.3 V for 240 s was generally used to attach aniline-
modified DNA to the surface. For the AUTR disposable electrodes,
the reference and auxiliary electrodes incorporated on the surface were
used. These electrodes were activated at 0.35 V for 240 s to induce
DNA coupling. Following the application of a potential, electrode
surfaces were rinsed with PBS and Nanopure water. Further detail for
experimental procedures is available in the Supporting Information.
Quantification of DNA on Electrode Surfaces.17,23,40,41 DNA-
modified electrodes were subjected to electrochemical measurement
with 20 μM ruthenium hexammine in 0.1 M Tris buffer (pH 7.6). At
this concentration of ruthenium hexammine, no signal is observed on
monolayers containing no DNA and is at a concentration conven-
tionally used for low-density DNA monolayers.14,17,23 Cyclic
voltammetry scans were obtained at a scan rate of 100 mV/s.
RESULTS AND DISCUSSION
■
Selection of the Coupling Partners. Previous inves-
tigations in our laboratory have explored the use of oxidative
bioconjugation reactions for the rapid coupling of diverse
compounds to biomolecules.28 These strategies are dependent
on the addition of anilines to o-iminoquinones and o-quinones
generated in situ with a chemical oxidant, such as potassium
ferricyanide or sodium periodate.29−31 Protein N-termini have
also been found to participate as nucleophiles in this reaction.32
This class of reactions exhibits very high chemoselectivity and is
compatible with especially low biomolecule concentrations in
aqueous media. In previous work, these reactions have
successfully been applied to the attachment of biomolecules
to aniline-coated gold nanoparticles33 and glass surfaces34 in the
presence of potassium ferricyanide. In addition, a photo-
In contrast, the electrochemical characterization of o-
catechol-containing monolayers revealed reversible signals
over multiple electrochemical cycles (Figure 2a). The
consistent reversibility demonstrates that minimal self-coupling
occurs between the o-quinones on the surface. The redox signal
that does not disappear over multiple rounds of cyclic
B
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encumbered thiol, allowing a maximum number of catechol
groups to be incorporated while maintaining the lateral van der
Waals interactions.39 The maximum number of catechol groups
was 105 2 pmol/cm2.
DNA Coupling to Monolayers and Quantification.
Constant potential amperometry (CPA) was used to activate
the catechol-modified surfaces for aniline-DNA attachment
(Figure S2). The potential for activation was chosen based on
the anodic peak current (310 mV versus AgCl/Ag and 350 mV
versus a Ag pseudoreference). Two of the major advantages of
this reaction over other chemical methods to attach DNA to
preformed mixed monolayers are that this reaction is one-pot
(the DNA to be coupled is added prior to oxidation of the
catechol) and reagentless (no additional catalyst or oxidant is
required for the reaction except for electrochemical activation).
After the coupling reaction, the amount of coupled DNA was
detected using ruthenium hexammine, which electrostatically
interacts with the phosphate backbone of each strand.17,40,41
Quantifying the ruthenium hexammine enabled the determi-
nation of the DNA surface coverage, which can be difficult to
measure accurately using nonelectrochemical methods, such as
fluorescence or radioactivity. Using this readout, the reaction
was optimized for both DNA concentration and the duration of
applied potential (Figure S3). Low concentrations (50 μM) of
DNA yielded efficient coupling, and increasing the concen-
tration beyond 50 μM did not improve the surface yield. The
surface coverage of aniline-modified DNA was found to be
proportional to the underlying catechol (Figure 3a, red trace).
Maximum coupling could be achieved in 4 min, with that
length of time being used for the experiments described below.
As experimental controls, no coupling was observed for
unmodified DNA strands or aniline-terminated strands that had
been acylated when exposed to the electrochemically activated
(EC) catechol surfaces, Figure 3b. As a positive control, aniline-
terminated DNA strands were also coupled to catechol surfaces
without applied potential but in the presence of K3Fe(CN)6
(KFC) for 30 min, albeit it with lower overall yield. While
anilines react especially rapidly with catechols, both aliphatic
amines and thiols are also known to participate in the reaction
at pH > 7.31,32 The coupling efficiencies of commercially
available thiol-terminated and amine-terminated DNA strands
were evaluated similarly, and the DNA surface coverage was
also found to be proportional to the underlying catechol
coverage. In both cases, however, lower overall coverages were
obtained than for aniline DNA. For thiolated duplexed DNA
self-assembled as a dense monolayer, reported DNA surface
coverages range from 30 to 50 pmol/cm2. The maximum DNA
coverage achieved in these experiments was 20 pmol/cm2,
which compares well with yields using strain-promoted Click
coupling chemistry (but after 24 h).17 The addition of Mg2+
salts to the coupling buffer was not found to influence the total
amount of DNA on the surface, in contrast to surface assembly
of thiolated duplexed DNA. Additionally, equal amounts of
DNA were found to couple at a pH range between 7.0 and 7.5.
Formation of Whole Cell Thin Films through DNA
Hybridization. The study of single cells and small groups of
cells has garnered major interest in recent years because of the
potential to model human diseases, such as cancer.43 A major
challenge to the study of many types of cells is their lack of
innate adhesion to surfaces. Even adherent cells, which are
often bound to “RGD”-coated surfaces42 through integrin
binding, require significant amounts of time or culturing to
obtain confluent layers. In previous reports, we have shown that
Figure 2. Electrochemical behavior of catechol monolayers. (a)
Reversible oxidation occurs at a potential of 0.29 V (40% catechol).
The asterisk denotes an artifact from workup and is not an
independent redox peak. (b) The surface coverage of catechol can
be determined using cyclic voltammetry. The number of catechol
groups reflects the starting thiol ratio used for monolayer formation.
Error bars represent SD for n = 3 replicates.
voltammetry indicates that the self-coupling occurring with the
o-iminoquinones is likely not occurring to a large extent on
these surfaces. Importantly, the final amount of catechol that
assembles in a given monolayer can be controlled by varying
the initial ratio of mercaptohexanol to 2, which was quantifiable
from the overall electrochemical signal from the catechol
(Figure 2b and SI Figure S1). Based on literature precedent, it
is hypothesized that the resultant mixed monolayer is
homogeneous.37,38 Interestingly, more catechols were incorpo-
rated using 10% 6-mercaptohexanol in the self-assembly step
than when the catechol thiol was applied alone. This is likely
due to improved monolayer packing by the less sterically
C
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Figure 4. Attaching living cells to DNA-functionalized gold electrodes.
(a) Synthetic DNA strands were attached to the surfaces of yeast and
nonadherent mammalian cells. The cells attach to surfaces bearing
complementary sequences. (b) SEM micrographs of Ramos cells on
transparent gold electrodes. (c) Cell adhesion was dependent on the
presence of the proper complement. (d) The optimal amount of
surface DNA varied by cell type. Error bars represent SD for n = 3
replicates.
technology was originally developed on gold surfaces,10 but
low levels of cell coverage were observed. The use of the
electrochemical oxidative coupling method to introduce the
capture strands is advantageous for this application because it
allows both the control and the quantitation of the DNA
strands on the surface. This allows the effects of capture strand
density on cell binding efficiency to be determined for the first
time.
The surfaces of three different cell types were modified with
a particular sequence of DNA, and catechol-substituted,
optically transparent gold electrodes were modified with
varying amounts of the complementary strand as described
above. Nonadherent mammalian cells (Jurkat and Ramos) were
tested, as were Saccharomyces cerevisiae.45 The cells were treated
with fluorescein diacetate prior to imaging to allow quantitation
using fluorescence imaging. To observe the cell morphology
after binding, the mammalian cells were subsequently fixed on
electrode surfaces and imaged by scanning electron microscopy
(SEM, Figure 4b).
Figure 3. Electrochemical coupling of DNA strands to catechol
monolayers on gold electrodes. (a) The total amount of DNA coupled
to the surface was found to depend on the percentage of catechol in
the monolayer. (b) DNA strands with different 5′-functional groups
(none = OH, AN = aniline, NH2 = aliphatic amine, SH = thiol) were
exposed to gold surfaces coated with catechols from a 50% starting
thiol mixture. Some strands were capped (p-iminoquinone for AN,
acetyl for NH2, and maleimide for SH) to block the terminal
functional groups. To couple the strands, a potential of 0.31 V was
applied for 240 s (EC), or 2 mM K3Fe(CN)6 (KFC) was added for 30
min. The surface DNA was quantified using ruthenium(III)
hexammine. Error bars represent SD for n = 3 replicates.
live cells can be modified to display synthetic DNA strands on
their surfaces, allowing their efficient capture by surfaces
bearing the strand complements, Figure 4a.10,11,44 This
In each case, the cells were observed to bind to the electrode
surfaces when the correct strands were present. Surfaces
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bearing no capture strands or noncomplementary sequences
(identical sequences, sequence C2, on both the cells and the
electrode surface) did not bind to the cells (Figure 4b,c). As has
been previously reported, the cells maintained their morphol-
ogy upon binding to electrodes via DNA hybridization.
Interestingly, the optimal DNA coverage for cell capture was
found to vary by cell type, Figure 4c. Both Jurkat and Ramos
cells were found to bind optimally to electrodes prepared with
an underlying catechol concentration of ∼40%. In contrast, S.
cerevisiae were found to bind optimally to DNA surfaces with
underlying catechol concentrations of ∼80%. The ability of the
mammalian cells to bind to surfaces with a lower DNA
coverage is likely due to the greater overall number of DNA
contacts that can be formed over their larger contact area,
relative to yeast. This effect was seen for both Ramos and Jurkat
cells. All three cell types exhibited reduced binding efficiency at
the highest levels of DNA coverage, possibly due to increased
levels of strand repulsion with the excess unbound strands. The
larger degree of negative charge on surfaces with high DNA
coverage could also serve to repel the negatively charged
mammalian cell surfaces more strongly than the yeast. While
these effects are under continued investigation, these experi-
ments clearly show the benefit of being able to tune and
determine the DNA density for optimal cell binding.
Specific Detection of BPA Using an Electrode-Bound
Aptamer. Bisphenol A (BPA) is a component of plastics and
epoxies, many of which are used for food or beverage storage.
BPA is an agonist for the human estrogen receptor α (ERα)
and binds to this receptor with a low-micromolar affinity,
making concentrations of ppm potentially problematic. Studies
have implicated this compound in a variety of disorders and
diseases, including obesity, infertility, early puberty, and
cancer.46,47 Because of its ubiquity and potential for detrimental
effects on human health, its rapid and facile detection is of the
utmost importance. To date, most techniques for the detection
of BPA either rely on the detection of total estrogenic
activity48,49 of a sample or on chromatographic-based
separations.50,51 Aptamers offer sensitivity and selectivity
without some of the difficulties associated with the application
of antibodies for detection.
In previous work, a DNA aptamer has been reported for the
selective binding of BPA detected by electrochemical sensing
on carbon nanotube−gold nanoparticle composites.52 Con-
formational changes in the DNA strand occurring upon BPA
binding increase surface blocking, therefore producing a
detectable electrochemical signal. The reported detection
limit with these composite nanorods was sub-nanomolar, yet
this method of detection was not evaluated on a 2-dimensional
electrode. It has previously been reported that 3-dimensional
features on electrode surfaces alter both coverage and detection
limits.53 To test the compatibility of this detection system both
with 2-dimensional, commercial electrodes and with the direct
electrochemical coupling technique, the BPA aptamer sequence
was appended with an aniline moiety and coupled to the
catechol monolayers at 310 mV for 4 min. Quantification using
ruthenium hexammine indicated a coverage of 18 1.2 pmol/
cm2. Following electrode preparation, the electrochemical
Figure 5. Specific detection of bisphenol a (BPA) using DNA
aptamers oxidatively coupled to gold electrodes. (a) The current
varied with the concentration of BPA. The inset shows the square
wave voltammetry data, and the larger graph reports the maximum
peak currents observed. (b) The response was specific for BPA and
depended on the aptamer sequence. Error bars represent SD for n = 3
replicates.
in the aptamer sequence upon BPA binding is reported to block
more of the surface from the especially high concentration of
ferricyanide. As more BPA binds, more of the surface is blocked
due to rigidification of the aptamer.52 The concentration of
3−/4−
Fe(CN)6
in solution was optimized such that the surface
was not passivated against it prior to BPA addition, but upon
BPA addition, passivation increased, decreasing the observed
signal. The maximum peak height (current) from SWV was
found to depend on BPA concentration (Figure 5a).
Concentrations as low as 50 nM could be detected clearly (1
pmol of BPA in a 20 μL sample). Based on the data obtained,
the detection limit of our system was 10 nM, with a dynamic
range of 10 nM to 5 μM, well within the biologically relevant
range of BPA contamination. The linear range of the sensor is
from 10 nM to 1 μM with concentration on a logarithmic scale.
response to BPA was monitored by square wave voltammetry
3−/4−
(SWV) with Fe(CN)6
in solution, which was used to
minimize the capacitance in the signal and maximize the signal-
to-noise ratio. As BPA was added, the signal decreased (Figure
5a, inset). The original reference for this aptamer provides a
hypothesis for its function. Briefly, the conformational change
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The aptamer-based BPA detection was found to be sequence-
specific; a scrambled aptamer sequence was tested, and no
signal decrease was observed upon BPA addition (Figure 5b).
Furthermore, exposure of the electrodes to bisphenol B, C, AF,
and G (BPB, BPC, BPAF, and BPG, each at 1 μM) confirmed
that the binding specificity of the aptamer sequence was
maintained upon surface attachment. As with cell adhesion, an
optimal surface coverage of DNA was found for BPA detection;
if DNA coverage was not at the optimal level, signal decrease
was significantly attenuated in the presence of BPA (starting
with a 75% catechol monolayer, Figure S5).
This electrochemically activated coupling method is ideal for
surface modification due to its ease of use, biocompatibility, and
reagentless surface activation. It also has a unique ability to
establish and quantify the level of DNA coverage, which was
found to be advantageous for multiple applications. The
efficiencies of both cell adhesion through DNA hybridization
and BPA detection by DNA aptamers were found to depend
greatly on the surface coverage of DNA, with individual
optimization necessary for each situation. We have optimized
surfaces for the binding of three nonadherent cell types: Jurkat
cells, Ramos cells, and S. cerevisiae. Additionally, especially low
concentrations of BPA were detected with our platform using
optimized DNA aptamer-modified electrodes. It is likely that
this convenient method will find use for many different
applications that require well-defined DNA monolayers on
conductive surfaces.
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.7b06385.
Methods and materials, cyclic voltammetry and electro-
chemical activation of catechol-modified electrodes,
optimization of aniline−DNA attachment to catechol
electrodes, images of cell-modified electrodes, optimiza-
tion of BPA detection, and current density for detection
of BPA using DNA aptamers (PDF)
AUTHOR INFORMATION
Corresponding Author
*mbfrancis@berkeley.edu
■
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ORCID
Matthew B. Francis: 0000-0003-2837-2538
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by Agilent and the NSF (Grant CHE-
1413666). A.L.F. was supported by the A. O. Beckman
Postdoctoral Fellowship. M.J.S. was supported by NSF SAGE
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