Journal of The Electrochemical Society, 2020 167 037529

Review—Approaches for the Electrochemical Interrogation of

DNA-Based Sensors: A Critical Review
z
Miguel Aller Pellitero, Alexander Shaver, and Netzahualcóyotl Arroyo-Currás
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore,
Maryland 21202, USA

The desire to improve and decentralize diagnostic platforms to facilitate highly precise and personalized medicine has motivated the
development of a large number of electrochemical sensing technologies. Such a development has been facilitated by electrochemistry’s
unparalleled ability to achieve highly specific molecular measurements in complex biological fluids, without the need for expensive
instrumentation. However, for decades, progress in the field had been constrained to systems that depended on the chemical reactivity
of the analyte, obstructing the generalizability of such platforms beyond redox- or enzymatically active clinical targets. Thus, the
pursuit of alternative, more general strategies, coupled to the timely technological advances in DNA sequencing, led to the development
of DNA-based electrochemical sensors. The analytical value of these arises from the structural customizability of DNA and its ability
to bind analytes ranging from ions and small molecules to whole proteins and cells. This versatility extends to interrogation methods,
as DNA-based sensors work through a variety of detection schemes that can be probed via many electroanalytical techniques. As a
reference for those experienced in the field, and to guide the unexperienced scientist, here we review the specific advantages of the
electroanalytical methods most commonly used for the interrogation of DNA-based sensors.
© The Author(s) 2019. Published by ECS. This is an open access article distributed under the terms of the Creative Commons
Attribution 4.0 License (CC BY, http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse of the work in any
medium, provided the original work is properly cited. [DOI: 10.1149/2.0292003JES]

Manuscript submitted September 17, 2019; revised manuscript received November 12, 2019. Published December 19, 2019. This
paper is a Critical Review in Electrochemical and Solid State Science and Technology (CRES3 T). This article was reviewed by
Ganesh Venkatachalam (ganelectro@gmail.com). This paper is part of the JES Focus Issue on Sensor Reviews.

Electrochemical biosensors are powerful analytical tools that com-
bine the specificity of biomolecular recognition with the low detection
limits achievable via electroanalytical methods.1 These devices, which
convert a biorecognition event into an electric signal,2 can support
both discrete and continuous measurements in even the most com-
plex and clinically relevant biological environments such as, for ex-
ample, whole human blood.3–5 Moreover, electrochemical biosensors
can be readily microfabricated to be portable,6 low-cost,7 convenient,8
and easy to integrate with the existing infrastructure of cloud-based
mobile electronic devices.9 This fabrication versatility is evidenced
by the many electrochemical biosensors successfully implemented in
food control,10 environmental safety,11 energy harvesting,12 and hu-
man health monitoring.13 Thus, electrochemical biosensors truly offer
the opportunity to achieve versatile molecular measurements at the
point-of-need.14
The large diversity of biophysical interfaces and detection ap-
proaches employed in electrochemical biosensors allows for their clas-
sification in many ways. One functional approach, based on the nature
of the biorecognition event, divides them into two distinct groups:15
biocatalytic sensors (Fig. 1A), which electrochemically monitor the re-
activity of the analyte with a biorecognition element (e.g., enzymes16,17
or bacteria18,19 ); and non-reactive, affinity-based sensors, which rely
on the specific binding of the analyte with a biorecognition receptor
(e.g., antibodies (Fig. 1B)20,21 or nucleic acids (Figs. 1C, 1D)22,23 ).
While the development of enzyme-based sensors precedes all oth-
ers and successfully created a commercial market for electrochemical
biosensors,14 affinity-based sensors emerged as a complementary tech-
nology offering the possibility to sense targets for which enzymatic or
direct electrochemical detection were not possible.
Nucleic acids offer unique features that make them ideal biorecog-
nition elements for affinity-based sensors. Specifically, unlike anti-
bodies, nucleic acids are easy to produce via solid phase synthesis24
and are less sensitive to temperature fluctuations.25,26 They can bind to
a large variety of molecular targets, ranging from ions27,28 and small
molecules29,30 to whole proteins31,32 and cells,33,34 and offer the pos-
sibility to do so in a reversible manner. Moreover, they can be easily
engineered to adopt specific folded conformations,35,36 which can be
exploited to maximize sensor signaling. These features have naturally
directed the focus of biosensor research efforts to DNA-based plat-
forms, to almost the same level of scientific production as enzymatic
z
E-mail: netzarroyo@jhmi.edu
sensors (Fig. 1E). We note that, while the number of reports on DNA
sensors in general has increased at a pace similar to that of enzyme-
based sensors (Fig. 1E), works on electrochemical DNA-based sensors
have not achieved the same publication rate (Fig. 1F). This discrep-
ancy presumably arises because of the diversity and complexity of
detection schemes employed in such sensors, the many electrochem-
ical methods used to interrogate them, and a lack of understanding of
the measurement advantages that each interrogation technique offers.
Thus, there is a need to review specific advantages that each family
of electrochemical methods offers to the interrogation of DNA-based
sensors.
Architectural considerations regarding sensor design and fabrica-
tion have been discussed at great length for the various existing DNA-
based approaches by prior publications,22,37–41 and are not the focus
of this review. Moreover, comprehensive analytical descriptions of
equations pertaining to electrochemical methods, including method
theory, and sensor-relevant parameters have also been published.42 In-
stead, we identified a need to critically discuss the value that different
electrochemical methods bring to the interrogation of DNA-based sen-
sors. Specifically, we discuss here methods that measure the current
output of the sensor upon application of an electrical voltage; namely
amperometry, linear and pulsed voltammetry, alternating current (AC)
methods and electrochemical impedance in the context of convenience
of signaling output, time resolution, and relevant system parameters
measured. This review is not exhaustive and primarily focuses on re-
cent developments in interrogation approaches. Our goal is to provide
a practical reference to aid in the selection of methods and technique
parameters that give optimal sensor readouts.
The Anatomy of DNA-Based Sensors
The anatomy of DNA-based biosensors varies depending on the
mechanism of detection but generally contains three common ele-
ments: 1) an electrode-blocking self-assembled monolayer (SAM)
used to prevent the occurrence of undesired electrochemical reac-
tions and to confer biocompatibility to the bioelectronic interface; 2)
a SAM functionalized with biomolecular receptors; and 3) a redox re-
porter sensitive to analyte-binding events (Fig. 2). The redox reporter
can be added to the bulk solution after target binding22,38,43 (Fig. 1C)
or covalently-attached to the receptors37,44 (Figs. 1D, 2). While the
former approach is only useful for ex-situ, single-point detection, the
latter supports reagentless, reversible, and continuous electrochemical
 Journal of The Electrochemical Society, 2020 167 037529

Figure 1. Electrochemical biosensors rely on biomolecular receptors to achieve highly specific detection of targets in complex media. They can be classified as
biocatalytic sensors (A), where solvated redox reporters shuttle electrons from the active site of an enzyme to the electrode surface; or as affinity-based sensors
(B-D). Affinity-based sensors can rely on the use of antibodies (B) or DNA molecules (C,D) as the biorecognition element. For the latter, when the reporter
is covalently attached to the biomolecular receptor, the target is detected via binding-induced changes in the electron transfer rate between the reporter and the
electrode surface. The signal of such sensors, however, is limited by the finite number of molecular receptors that can occupy the electrode surface. To overcome this
limitation and achieve lower detection limits, some affinity-based sensors are modified with an amplifying element (B,C); e.g., an enzyme or an inorganic catalyst
such as Ru(NH3 )6 3− , which is electrochemically regenerated by a solvated reporter to amplify currents. (E) The number of publications related to DNA sensors in
general has increased to the same numbers per year as works focused on biocatalytic sensors. Here, for example, we show data extracted from SCOPUS using the
search terms “DNA/RNA/Nucleic acid sensor”, “Enzym∗ sensor”, and “Immunosensor/antibody sensor”. (F) The same trend is not quite seen for electrochemical
DNA-based sensors. We argue that this discrepancy arises due to a combination of factors including the complexity and great diversity of detection schemes and
interrogation methods, and a lack of understanding of the measurement advantages that each interrogation method offers. The data shown here were extracted from
SCOPUS using the terms “DNA/RNA/Nucleic acid electrochemical sensor”, “Enzym∗ electrochemical sensor”, and “Immunosensor/antibody electrochemical
sensor”.

sensing. However, the addition of redox reporters or mediators to the
bulk solution enables electrocatalytic amplification of the signal,43
which can be exploited to decrease detection limits and improve mea-
surement signal-to-noise ratios. Thus, the architecture of these DNA-
based sensors is tightly connected to their detection mechanism, the
analyte to be measured, the matrix where the analyte is present, and
the time and spatial resolutions required by the sensing application.
DNA-based electrochemical sensors produce a similar signal out-
put for a variety of detection schemes. For example, nucleic acids
(e.g., miRNA or circulating-tumor DNA) are usually detected by mea-
suring changes in electron transfer rate occurring upon hybridization
with redox reporter-modified receptor strands23,38 (Fig. 1D). Similarly,
small molecule targets are detected by measuring changes in electron
transfer rate due to binding-induced structure switching of receptor
strands.37 Both detection schemes have gained increased scientific at-
tention in the last two decades, in part because of the ease with which
nucleic acid receptors can be selected and synthesized.45–48 Also, un-
like biocatalytic sensors, DNA-based detection does not depend on the
 Journal of The Electrochemical Society, 2020 167 037529
Figure 2. The general anatomy of affinity-based sensors. Although sensor ar-
chitecture varies depending on detection scheme, the majority of affinity-based
electrochemical sensors share three common elements: a mixed self-assembled
monolayer (SAM) containing electrode-passivating chemistry (here illustrated
with 6-mercaptohexanol molecules) and a tether for the biomolecular recep-
tor, and a redox reporter, either attached to the receptor (here illustrated with
a derivative of methylene blue) or solvated. The example architecture shown
here specifically corresponds to an electrochemical, aptamer-based sensor.
specific chemical reactivity of the analyte and, thus, offers a unique
approach to achieving the general sensing of a great variety of impor-
tant clinically relevant molecules that cannot be otherwise detected
biocatalytically.
Shape of the Calibration Curve and Limits of Detection
Because the main objective of electrochemical DNA-based sen-
sors is to detect and quantify specific targets in relevant samples, any
newly developed sensor must be calibrated, preferably at the point of
manufacture,49 by constructing dose-response curves. The shape of
those curves should be independent of the interrogation method used
but, in contrast, is highly dependent on sensor architecture. Thus, we
thought it important to briefly discuss the physics of target-receptor
binding involved in DNA-based sensing.
The interface of DNA-based sensors can be approximately de-
scribed as consisting of two states: one with free and one with target-
bound receptors.50–52 By making the assumption that only one binding
site is available at each receptor, the electrochemical response of these
sensors must necessarily behave following the physics of single-site
Figure 3. The adsorption isotherms relevant to DNA-based electrochemical sensing. The binding of target to biomolecular receptors may obey the physics of
single-site binding, modeled by the Langmuir isotherm (A). If target affinity for the receptor is high (few nM or below), binding can be modeled by a linear isotherm
(B). Finally, for the case of receptors with multiple binding sites, target binding is better modeled by the Hill-Langmuir isotherm (C).
binding.53,54 That is, the relative fraction of target-bound receptors is
modeled by the Langmuir adsorption isotherm:55
[T ]
θ= [1]
KD + [T ]
where θ represents the target-bound fraction, T is the concentration
of the target, and KD the dissociation constant of the receptor. This
adsorption model (Fig. 3A) assumes that (1) all probes on the surface
have the same binding affinity for the target; (2) the DNA probes
do not interact with each other; and (3) the extent of target binding
is limited to a single monolayer. Many DNA-based sensors produce
signals that can be modeled using Eq. 1. However, in cases of extremely
high target affinity, DNA-based sensors can produce linear responses
(Fig. 3B) that become limited by the number of receptors present on
the surface of the sensor.56 Furthermore, in cases where the receptor
offers multiple target-binding sites, and such binding sites interact
with one another, the system is better represented by a modification
of Eq. 1 called the Hill-Langmuir isotherm57,58 (Fig. 3C):
[T ]n
θ= [2]
KD + [T ]n
where n is the Hill coefficient, which establishes the extent of interac-
tions between such binding sites.
The above-described physics set the theoretical bar that must be
addressed when reporting new DNA-based sensors. Specifically, it is
crucial to demonstrate the receptor-target binding stoichiometry and
affinity (i.e., their KD ) based on the sensor architecture employed. Be-
cause the Langmuir isotherm has a useful dynamic range (here defined
as the range of target concentrations that change receptor occupancy
from 10% to 90%) of only 81-fold centered at the KD ,54 DNA-based
sensors producing linear calibration curves that exceed such dynamic
range indicate more complex and, presumably, non-specific interac-
tions. In such cases, proper description of the sensing mechanism (and
how it affects the signaling of the interrogation method employed) is
necessary to justify the shape of the calibration curve.
The ultimate goal of DNA-based sensors is to measure environmen-
tally relevant levels of target in the exact medium where the target is
found. For disease- or health-diagnostic platforms, this means achiev-
ing target measurements in the clinically-relevant range for viruses,
cells, and biomolecules; i.e., aM – nM,59 and for small-molecule
drugs; i.e., nM – μM.60 To achieve these low levels of detection, the
scientific community is investing significant efforts on the develop-
ment of highly sensitive sensor interfaces and signal-amplification
strategies. These include, for example, modifying probe density,61 the
nature of the blocking monolayer,62 the redox reporter63 and the mi-
croscopic architecture of the electrode,64,65 the electrode surface area
to increase the moles of DNA receptors,66–69 coupling a catalytic pro-
cess to electron transfer events to amplify binding-induced signals43,70
or achieving binding-induced receptor polymerization to also am-
plify signaling.71 Computational methods have also been reported that
achieve lower detection limits via noise processing strategies.72 Al-
though the ultimate limit of detection is inherently set by the receptor
KD and signal amplification mechanism, it can also be improved via
 Journal of The Electrochemical Society, 2020 167 037529
Table I. Time constants relevant to the interrogation of DNA-based
electrochemical sensors.
Physicochemical Phenomena Time scale Reference
Double-layer charge/discharge 10s to 100s of μs 53
Field-induced actuation of DNA motion 1 to 10s of ms 76
Mass transport of redox reporter 1 to 100s of ms 77
Standard electron transfer rate of reportera 1 to 100s of ms 78
Rates of association/dissociation 10s of ms 79
a The vast majority of DNA-based sensors use the redox couple methy-
lene blue/leucomethylene blue, which has a k0 significantly slower than
that for ferrocenes or other commonly used redox reporters.80
careful selection of the electrochemical interrogation method and its
parameters.72,73
The Time Constants Involved in DNA-Based Sensing
The application of a voltage bias to the bioelectronic interface
of DNA-based sensors generates electrokinetic, faradaic, mass and
charge transport phenomena that affect the output of electrochemi-
cal interrogation methods (Table I). Whether the voltage perturba-
tion is a discrete (e.g., chronoamperometry) or a continuous function
of time (e.g., voltammetry), the electrolyte responds to the perturba-
tion by dynamically aligning to the electric field.74 This effect gen-
erates double-layer charging/discharging currents in electrochemical
measurements.75 Moreover, the voltage perturbation may cause field-
induced movement actuation of the negatively-charged DNA back-
bone – that is, DNA will move toward and away from the electrode
surface depending on the voltage applied.76 This effect will likely
perturb the frequency of electron transfer which, in turn, affects the
signaling currents measured. Beyond field-induced modulation of the
electrolyte and DNA strands, mass transport of the reporter to the
electrode also affects electron transfer. Furthermore, DNA secondary
structures and SAM length affect the currents measured, as do the
standard electron transfer rate (k0 ) of the redox reporter and the rates
of receptor-target ligand association/dissociation. All of these factors
make the signaling of DNA-based interfaces strongly interrogation-
frequency dependent.
The interrogation-frequency dependence of DNA-based sensors
limits their ability to address important biological questions. For ex-
ample, there is significant interest in achieving chemically-specific
measurements of neurotransmitters in live brains to expand our un-
derstanding of health and disease-induced neuromodulation.81 Ideally,
such measurements should be performed with faster interrogation fre-
quencies than the rates of neuronal firing in the brain, which naturally
occur at time scales of 10s of ms.82,83 However, this is impossible to
achieve with, for example, state-of-the-art electrochemical, aptamer-
based (E-AB) sensors,84 because they rely on methylene blue as their
redox reporter,85–88 which undergoes electron transfer at rates of 100s
of ms,89 slower than fast neuronal firing. A better approach to this end
could be the use of label-free sensors like aptamer-based field effect
transistors,90 which can theoretically achieve faster response times
than E-AB sensors as they are not limited by the intrinsic electron
transfer rate of a reporter. However, unlike E-AB sensors, deploying
field effect transistors in vivo has proven an important challenge be-
cause of their inherent sensitivity to changes in electrolyte composition
and status over time. In any case, the approximate time constants pro-
vided in Table I should serve as a guide and reminder that choosing the
right interrogation technique and parameters is crucial to achieving the
time resolution needed for any particular application of DNA-based
sensors.
The best electrochemical interrogation method for a given appli-
cation is thus determined by the time-resolution, specificity, conve-
nience of signaling output, and sensitivity needed. Chronoamperom-
etry, for example, offers sub-second time resolution91 but requires
considerable training and experience for the correct interpretation of
chronoamperometric currents. Voltammetry, in contrast, is much eas-
ier to interpret, but its time resolution can be worse than amperometry,
as it requires the sweeping of voltage ranges (except in the case of
fast-scan voltammetry,91 which has other technical challenges related
to signal calibration). Below, we provide a discussion on the specific
value that each of the most common electrochemical techniques offers
to the interrogation of DNA-based sensors.
Interrogating Sensors with Sub-Second Time Resolution via
Amperometry
Amperometry employs the simplest voltage program used to in-
terrogate electrochemical biosensors. Here we consolidate all am-
perometric methods into techniques that measure current versus
time following the application of discreet voltage steps (Fig. 4A).
We note, however, that there are important technical differences
in the duration and number of voltage pulses (and in analyti-
cal value) between, for example, constant-voltage amperometry at
ultramicroelectrodes,92 stepped-voltage chronoamperometry52,93 and
intermittent-pulse amperometry.94,95
Because of the non-transient nature of their voltage program, am-
perometric measurements offer simple signaling outputs (current de-
cays, Fig. 4B) that can be used to determine kinetic parameters of
target-binding events. Moreover, the simplicity of the voltage program
allows the interrogation of bioelectronic interfaces at millisecond52,94
and even sub-millisecond92 frequencies, making them ideal for mon-
itoring fast, transient and dynamic processes.96
The signaling output of amperometrically-interrogated DNA-
based sensors varies depending on whether the redox reporter is
present in solution or tethered to the electrode surface (Fig. 4B).
Moreover, it also varies depending on whether the detection mech-
anism is based on catalysis or not. For DNA-based sensors that rely
on, for example, binding-induced electrode blockage,97 the current vs
time response observed upon application of a voltage step will de-
cay under diffusion control and is accurately modeled by the Cottrell
equation98 (Fig. 4C). For sensor platforms that depend on homoge-
neous catalysis,31,99 the current may decay or increase depending on
the catalytic rate of the homogeneous reaction (Fig. 4D). Finally, for
DNA-based sensors with surface-bound reporters, the current will ex-
ponentially decay to zero within hundreds of milliseconds or faster
due to complete oxidation or reduction of all the reporters (Fig. 4E).
Thus, amperometric methods not only offer fast sensor interrogation
frequencies but their signals also directly report on the kinetics of
electron transfer.
The kinetic sensitivity of amperometric interrogation can be ex-
ploited to determine the electron transfer rate of a given sensing in-
terface. For example, Dauphin-Ducharme and colleagues89 have used
chronoamperometry to study the dependence of the electron transfer
rate from methylene blue reporters bound to the terminal (3’) end
of single-stranded DNA chains (Figs. 5A–5C). Their results demon-
strate that DNA chain dynamics affect the apparent electron transfer
rate at chain-lengths longer than 10 nucleotides (Fig. 5D). This result
is expected because longer DNA strands are more affected by the dif-
fusional transport described in Table I. Moreover, the same group has
discussed how converting chronoamperograms to chronocoulograms
(by simply multiplying current by time) can be exploited to deter-
mine the relative populations of DNA receptors that undergo electron
transfer at different rates (for example, single stranded vs hybridized
DNA)100 (Fig. 5E). These reports highlight the analytical value of
chronoamperometry for the interrogation of DNA-based sensors.
The kinetic sensitivity of chronoamperometry can also be ex-
ploited to overcome signal drift in DNA-based electrochemical mea-
surements. Drift is observed as a continuous change in the relative
signal of the sensors that is independent of target concentration. For
example, the signal of DNA-based sensors deployed in whole blood
tends to decrease (i.e., drift) over time, presumably due to progres-
sive degradation of the sensor interface.101 To overcome this problem,
 Journal of The Electrochemical Society, 2020 167 037529

Figure 4. Chronoamperometric interrogation of DNA-based sensors. (A) A voltage step is applied to the electrode, driving the electrochemical conversion of
the redox reporter. (B) The current output varies depending on the architecture of the sensing layer. A progressive current decay is observed in systems that use
reporters in solution, which is modeled by the Cottrell equation (dotted line). A fast decay to zero is observed when the reporter is anchored to the electrode surface
due to its complete depletion (solid line). Last, a stationary current is observed in systems where the reporter in solution reacts with a catalyst attached to the target
DNA strand (dashed line). (C) For electrode blocking-based detection, target binding hinders electron transfer leading to a decrease in the magnitude of the current
decay. (D) The target molecule can be labeled with a catalyst that reacts with reporters in solution, generating a stationary current proportional to the amount of
target. (E) In systems where the DNA probe has been modified at one of its ends with the reporter, target binding increases the reporter’s electron transfer rate,
which translates into shorter decay times in the chronoamperograms.

Arroyo-Currás and colleagues have demonstrated that chronoampero-
metric lifetime measurements (Figs. 6A, 6B) are inherently resistant to
such drift.52 This resistance arises because the apparent rate of electron
transfer of DNA-based sensors can be, to some extent, independent of
the state of the interface. Thus, while the total amplitude of chronoam-
perometric currents drifts significantly (due to, for example, loss in to-
tal number of redox reporters), the lifetime of their exponential decay
is independent of this amplitude and thus largely drift-free (Fig. 6C).
Because of this property, chronoamperometry can support continuous,
drift-free detection of analytes in untreated biological fluids.52
Serial and fast chronoamperometric interrogation can be applied to
extract the rates of receptor-target binding. An example of this was re-
ported by Santos-Cancel and colleagues, who used intermittent-pulse
amperometry to measure the association rates of small molecule tar-
gets (the antibiotic tobramycin and adenosine triphosphate) to DNA
receptors94 (Fig. 6D). This work claims interrogation frequencies as
fast as one chronoamperogram every 2 ms (Fig. 6E). Such fast mea-
surement frequencies enable the acquisition of hundreds of datapoints
and allow the determination of binding kinetics with 95% precision
or higher (Fig. 6F). We foresee that the amperometric interrogation
of DNA-based sensors will also be exploited in the future to investi-
gate the kinetics of fast physiological processes in the body, such as
inflammatory response or neurotransmitter release and modulation.
Employing Cyclic Voltammetry for Sensor Characterization and
Sensing
Cyclic voltammetry uses a voltage program that allows the easy
characterization of the bioelectronic interface of DNA-based sensors.
Specifically, in cyclic voltammetry, the electrode voltage is linearly
swept at a constant scanning rate between an initial and a final value,
then swept back to the initial voltage while continuously sampling the
current (Fig. 7A). The resulting voltammogram (Fig. 7B) reports on
the combination of ionic and faradaic charging/discharging processes
occurring throughout the voltage sweep (Table I), which is seen as a
series of peaks or waves. Because such processes are a function of the
organization and composition of the interface, a cyclic voltammogram
is a powerful tool for the electrochemical characterization of DNA-
based sensors.53,77,99,102 For example, cyclic voltammetry can be used
to determine the surface concentration of double and single stranded
DNA.103 It can also be employed to determine the extent of passivating
monolayer surface coverage and its homogeneity (i.e., to identify the
presence of defects such as collapsed sites or pinholes).42,104 Addition-
ally, by interrogating the sensor interface at different scanning rates,
cyclic voltammetry can be employed to extract important electrochem-
ical parameters of the redox reporter such as its diffusion coefficient (if
present in solution phase)105 or its standard electron transfer rate.106
 Journal of The Electrochemical Society, 2020 167 037529

Figure 5. Sensor architecture has an impact on electron transfer between a tethered redox reporter and the electrode. (A) Here, for example, we show a redox
reporter (methylene blue), anchored to the 3’ end of an immobilized DNA strand with length equal to a N number of nucleotides. To transfer electrons, the reporter
must first diffuse to the electrode surface. (B) The chronoamperometric interrogation of this interface first charges the double layer, then drives the reduction of
methylene blue. The latter is seen as a sigmoidal decay in a log-log plot. (C-D) The rate of electron transfer is limited by the length of the DNA strand, with a clear
transition to diffusion-limited rates when N > 10 nucleotides. (E) The apparent electron transfer rate, expressed in Hz, varies between single and double-stranded
DNA. Single stranded DNA is more flexible and reaches the electrode surface more frequently, thus presenting a faster electron transfer rate (∼200 Hz) relative to
double stranded DNA (3 Hz). This effect is easily observed in the chronocoulogram presented here. Figures adapted with permission from Refs. 89,100.

Beyond its analytical applications, cyclic voltammetry can also be
used to directly interrogate DNA-based sensors.
The signaling output of sensors interrogated via cyclic voltamme-
try strongly depends on architecture and detection mechanism. For
example, when the detection scheme relies on solution-phase redox
reporters – e.g., systems based on hexacyanoferrate or hexaammine
ruthenium salts – the output cyclic voltammogram has a characteris-
tic duck shape that changes in magnitude following a binding event
(Figs. 7C, 7D). Many examples of this sensing approach have been
reported, which use cyclic voltammetry to study the blocking effect of
a non-redox active protein that binds to anchor DNA receptors, caus-
ing a decrease in the voltammetric currents (Fig. 7C) (e.g., see Refs.
107,108); or measure the signal amplification (current increase) de-
rived from the catalytic reaction between a DNA-bound reporter and
a second reporter in solution (Fig. 7D) (e.g., see Refs. 28,109,110).
Because the currents measured in these systems depend on the rates
of reporter diffusion to the sensor surface, protein-DNA binding or
catalytic reaction, the scanning rate must be adjusted as a function of
the time constants of these processes (Table I).
When the sensor architecture involves surface-bound reporters -
such as in the case of E-AB32 or DNA-scaffold sensors111 - the voltam-
metric currents are limited by the finite number of reporters attached
to the sensor surface. In this situation, the currents raise to a maximum
and fall back to background in a gaussian looking peak (Figs. 7B, 7E
and 8B, 8C), with their height or area relating to the target concentra-
tion, and their width depending on specific factors such as the nature of
the redox reporter, the thickness of the passivating layer, or the length
of the labeled DNA probe.104 This sensor design, typically based on
DNA-bound methylene blue or ferrocene reporters, is attractive for
applications that require reagentless, continuous sensing29,30 because
both the molecular recognition (the DNA strand) and the signaling
(the reporter) elements are covalently attached to the electrode sur-
face. Therefore, the addition of exogenous reagents in not needed for
molecular detection. When these sensors are interrogated via cyclic
voltammetry, the signaling output depends strongly on the scanning
rate and the time constants of the different processes occurring at the
sensor interface (Table I).112 For example, voltammograms scanned at
rates slower than the apparent electron transfer rate of the system show
complete conversion of the surface-bound redox reporter with mini-
mal peak-to-peak separation between the forward and reverse scans
(Figs. 7E and 8B).112 Moreover, in these measurements, the voltammo-
gram’s peak current linearly correlates with scanning rate (Figs. 8C,
8D). In contrast, when the sensors are interrogated at scanning rates
that exceed the time constant of electron transfer, the electrochemical
response becomes limited by the frequency with which the reporter can
transfer electrons to the sensor (Fig. 8B). In such a case, the voltam-
mograms present peak-to-peak separations that linearly increase with
the square root of the scanning rate112 (Figs. 8C, 8E). The exact limit
at which the response of the measurement changes depends on the
composition of the assembled layer.106,113 Thus, the structure of the
bioelectronic interface is also important to consider when choosing an
electrochemical interrogation method.
One important caveat to using cyclic voltammetry for the in-
terrogation of sensors employing surface-bound reporters is the
 Journal of The Electrochemical Society, 2020 167 037529

Figure 6. Chronoamperometry achieves drift-free and sub-second-resolved DNA-based sensing. (A) The difference in electron transfer rate between bound and
unbound DNA receptors (Fig. 4E) can be measured as differences in current decay lifetimes. (B) Such lifetimes can be related to the concentration of the target
in the sample. (C) Because chronoamperometric lifetimes are a function of the fractional population of bound vs unbound receptors, they are less sensitive to
progressive changes of the sensor interface relative to total current amplitude (the current plateau in, for example, Fig. 5C), which depends on the total number of
receptors. Thus, the chronoamperometric interrogation of DNA-based interfaces supports drift-free sensing. (D) A different interrogation mode, called Intermittent
Pulse Amperometry, can be used to achieve few-millisecond-resolved measurements of target binding kinetics. (E) The output of each periodic pulse produces one
forward and one reverse chronoamperograms, which can be subtracted to produce a differential current that is directly proportional to the concentration of target.
(F) Monitoring such a differential current in real time allows the highly precise measurement of target-receptor association kinetics. Figures edited with permission
from Refs. 52,94.

inherent low signal-to-noise ratio of this technique. This problem
arises from the fact that linearly polarizing the electrode surface causes
progressive charging of the electrical double layer, which appears in
a voltammogram as charging current (for example, see the thickness
of the CVs in Fig. 8B). Depending on the density and organization
of the blocking monolayer used in these sensors, the magnitude of
the charging current could be comparable or greater than the faradaic
current produced by the electrochemical conversion of the reporter. To
overcome this limitation, we recommend the use of a different set of
voltammetric techniques based on pulsed-voltage programs, which en-
able the discrimination between charging and faradaic processes when
needed.
Achieving Charging Current-Free Sensing with Pulsed-Voltage
Methods
Most clinically relevant targets exist at concentrations in the range
of aM to nM54 and are likely to produce only small changes in the
signaling current output of DNA-based sensors, making their detec-
tion strongly affected by charging currents. Thus, techniques that
minimize the contribution of double-layer charging to the currents
measured are particularly valuable for clinical applications. Specifi-
cally, pulsed voltammetric techniques overcome this issue by apply-
ing periodic voltage perturbations – such as voltage steps in differ-
ential pulse voltammetry (DPV) (Fig. 9A) and square-wave voltam-
metry (SWV) (Fig. 9B) or sinusoidal functions in alternating-current
voltammetry (ACV) (Fig. 9C) – instead of the conventional linear
perturbations applied in chronoamperometric and cyclic voltammet-
ric techniques. Because these methods employ periodic perturbations,
parameters like pulse width, signal frequency, and amplitude affect
the currents (and interfacial processes) measured. Such parameters
can be exploited to eliminate charging currents and simultaneously
enhance the current generated by reversible redox systems such as the
oxidation/reduction of a redox reporter, leading to mostly faradaic,
high signal-to-noise measurements (Fig. 9D) and, consequently, lower
detection limits.114 These features have made pulsed techniques
the preferred interrogation approach for DNA-based electrochemical
sensors.72
Pulsed techniques have been extensively used to interrogate DNA-
based sensors that exploit solvated redox reporters for target detection.
A few example approaches include the interrogation of sensors where
target-binding physically blocks electrode access to the reporter, de-
creasing the peak current,115–117 and sensors with a sandwich-type
configuration where the signal generated by target-binding is enzy-
matically or chemically amplified,118–120 enabling remarkably low de-
tection limits.121 However, because of their unique ability to discrim-
inate between charging and faradaic currents, pulsed methods are of
most value for the interrogation of sensors relying on surface-attached
reporters.27,122–125
Pulsed methods are not only attractive for their low detection limits,
but also because their current output is convenient and easy to inter-
pret. For example, all three methods discussed in Fig. 9 produce sharp,
gaussian-like peaks that increase or decrease in magnitude upon target
binding, depending on the technique parameters employed (Fig. 9D).
Because the shape and magnitude of the currents measured by these
methods are so strongly dependent on parameters like waveform fre-
quency and amplitude, the construction of multi-parameter maps is a
great resource to optimize their signaling (Fig. 9E). One approach to
 Journal of The Electrochemical Society, 2020 167 037529

Figure 7. Interrogation of DNA-based sensors via cyclic voltammetry. (A)
The voltage program is a triangular waveform starting from a value where no
charge transfer is observed (Ei ), to a value where charge transfer is limited by
the rate of electrochemical conversion of redox reporters (Eswitch ), and then
back to Ei . (B) The current output is a voltammogram characterized by having
a duck-shaped current profile when the reporters are in solution (solid line) or a
symmetric, gaussian-shaped profile for surface-bound reporters (dashed line).
(C-D) Voltammograms representing unbound (solid line) and bound (dotted
line) states for electrode-blocking-based detection (C, see Fig. 4C) and catalytic
detection (D, see Fig. 4D). (E) Voltammograms obtained at low (solid line) and
high (dotted line) scan rates for a surface-bound reporter.
building such maps is to graph the peak current in the z-axis of a three-
dimensional cartesian coordinate, where the y and x axes correspond
to frequency and amplitude, respectively. Such maps reveal parameter
combinations that lead to signal ON or OFF sensor responses upon
target binding (Fig. 9F), which can be used to, for example, achieve
calibration-free sensing.126
A second important attribute of pulsed methods is their ability to
support drift free sensing. As described above, the waveform frequency
and amplitude can be tuned to specifically probe the effect of target
binding on the fractional populations of bound vs. free receptors80,127
(Fig. 10A). Moreover, previous reports have shown that waveform
frequency pairs can be found which produce peak currents that drift
in concert when the sensors are interrogated serially. Thus, by sim-
ply subtracting the signals recorded at such frequency pairs, pulsed
methods enable continuous, drift-free measurements of targets in com-
plex biological fluids.27,128 This approach, called Kinetic Differential
Measurements (KDM), was first demonstrated for DNA-based sen-
sors confined to microfluidic channels and challenged with target in
a continuous stream of blood30 (Fig. 10B). However, by fabricating
DNA-based sensors with a geometry factor small enough to fit inside
the veins of rats, more recent reports have demonstrated the applica-
bility of KDM to the continuous measurement of specific targets in
vivo29,52,100 (Figs. 10C, 10D).
Up to this point, the described amperometric and voltammetric
methods are all limited in measurement frequency by the rate at which
the redox reporter can transfer electrons. One alternative to overcome
this limitation is, of course, to fabricate sensors with reporters that
transfer electrons faster than the commonly used methylene blue.
However, doing so is not straightforward, as changing the chemistry of
the reporter always affects the properties of the sensor’s biomolecular
interface; for example, by making it less stable under continuous elec-
trochemical interrogation.44 A second, and less explored alternative,
is to use reporter-free interrogation methods such as electrochemical
impedance spectroscopy, which we discuss next.

Figure 8. Voltammetric behavior of DNA-based sensors with surface-bound reporters. (A) Here, for example, we illustrate the behavior of a sensor based on DNA
hybridization. In the unbound, folded state, the proximity of the reporter to the surface allows electron transfer. In contrast, the bound, hybridized state hinders
electron transfer. (B) This is reflected in the shape of the voltammograms obtained for each case, which shows a decrease in peak currents and broader peak-to-peak
separation for the bound, hybridized state. (C) The cyclic voltammetry scan can be tuned to probe the different time scales and processes involved in signaling.
For example, here we show that scan rates slower than the diffusional approach of the reporter to the electrode produce symmetric, sharp voltammograms. At rates
that exceed the diffusional motion, however, the voltammograms become broader and the peak-to-peak separation increases. The scan rate threshold depends of
course on the architecture of the interface. (D) For low scan rates, a linear relationship is observed between the scan rate and the peak current, indicative of electron
transfer limited by surface confinement. (E) For higher scan rates, the electron transfer is limited by the motion of the reporter toward the electrode, and diffusion
control is observed. Figures adapted with permission from Refs. 106,112.
 Journal of The Electrochemical Society, 2020 167 037529

Figure 9. Interrogation of DNA-based sensors via pulsed-voltage methods. (A) In DPV, a voltage pulse of a certain width and amplitude is applied to the electrode.
The current is measured immediately before and at the end of the pulse, and the difference between the two currents is used to create a voltammogram. (B) In SWV,
a square waveform is superimposed on a voltage staircase. The current is measured at the end of each square pulse, producing forward (If ) and reverse currents
(Ir ). The absolute values of both currents are subtracted to obtain a differential voltammogram. (C) In ACV, a sinusoidal waveform is superimposed on a linear
ramp and the current is measured at a regular frequency. The reference parameter values shown in panels A-C were estimated from reported DNA-based sensors.
(D) Depending on, for example, technique frequency, the output voltammogram for all three techniques can give a signal ON or OFF output upon target binding.
(E) Current maps can be constructed to identify parameter combinations that produce the largest signal ON or lowest signal OFF outputs. (F) Those parameter
combinations can then be employed to build dose-response curves. Here, for example, we show that the combination of 25 mV amplitude and 20 Hz frequency,
and 25 mV – 750 Hz from the map in panel E, produce the largest signal OFF and ON responses, respectively, for the sensor interrogated. Figures adapted with
permission from Ref. 73.

Performing Reporter-Free Sensing via Impedimetric Detection
Electrochemical impedance spectroscopy (EIS) offers the unparal-
leled ability to interrogate DNA-based sensors with or without the use
of redox reporters. This ability exists because the currents measured in
EIS are sensitive to surface processes that affect the electrical proper-
ties of such sensors: specifically, their interfacial capacitance (Cdl ) and
electron transfer resistance (Ret ).129 The technique is based on over-
lapping a sinusoidal function (AC voltage) of low amplitude (typically
≤ 10 mV) to a constant DC voltage on the sensor surface (Fig. 11A)
while measuring in real time the AC current output as a function of
angular frequency. This frequency is swept from values ranging from
roughly 10−2 Hz to 106 Hz, thus obtaining impedance spectra that can
be represented in Nyquist (Fig. 11B) and Bode plots (Figs. 11C, 11D).
Because the waveform frequency can be finely tuned, EIS enables
the study of interfacial phenomena at different time scales (Table I),
being particularly sensitive to double-layer-related processes.130 For
in-depth descriptions of EIS, and its application to the interrogation of
biosensors in general, we refer the reader to previously published, com-
prehensive reviews.131,132 Here, instead, we discuss a few key concepts
that are relevant to the specific interrogation of DNA-based sensors.
Arguably the major motivation to use EIS for the interrogation
of DNA-based sensors is its ability to perform reporter-free detec-
tion. EIS achieves this by monitoring binding-induced changes to
the double-layer capacitance driven by the mere interaction between
immobilized DNA receptors and a target.133–135 Thus, by monitor-
ing binding- induced changes to the double-layer capacitance at, for
example, constant angular frequency, EIS allows the continuous mea-
surement of fluctuating concentrations of target.136,137 This reporter-
free approach is called non-faradaic impedance spectroscopy.138,139
Mechanistically, it is thought that target-binding changes the surface
density of water molecules and ions at the sensor interface, thereby
changing the double-layer thickness and, consequently, the interfa-
cial capacitance, which can be related with the amount of target in
the sample (Figures 12A–12C).140,141 However, the exact detection
mechanism is often hard to define for a given sensor architecture and
depends strongly on medium conditions like the concentration, pH,
and composition of the electrolyte employed.141 Moreover, other chal-
lenges with this detection approach include the difficulty of developing
signal-amplification strategies, and the non-specificity of capacitance-
based detection—i.e., any entities interacting with the sensor interface
can potentially change its capacitance—hindering its use in complex
samples, such as blood, which are prone to blocking the electrode
surface.
An approach to achieving higher specificity and signal amplifica-
tion in EIS is the use of reporter-based detection. Two modalities are
often described in the literature: 1) the use of “labeled” receptors,
which consist of reporters covalently attached to DNA strands an-
chored to the electrode surface, and 2) the use of “label-free” strategies,
which typically consist of DNA sensors that rely on electron transfer
from solvated reporters externally added to the sample141 or, in cer-
tain cases, on the intrinsic redox chemistry of the target molecule.142
 Journal of The Electrochemical Society, 2020 167 037529

Figure 10. Pulsed methods are kinetically sensitive to differences in electron transfer between receptor populations. (A) By tuning the frequency of the pulsed
waveform, it is possible to electrochemically discriminate between two populations of DNA receptors: one free and one bound to target. (B) Conveniently, for
some frequency pairs the signal drifts in concert, allowing to differentially correct sensor drift in real time, an approach called Kinetic Differential Measurements
(KDM). Here, for example, we show real-time drift correction of a concentration step for an E-AB sensor confined to a microfluidic channel. (C-D) This approach
to drift correction works even when the sensors are deployed in situ in the veins of live rats. Here we illustrate this by showing the E-AB measurement of two
bolus injections of a drug, here tobramycin, into a rat. The top graph shows concerted drift for the different frequency measurements which can be differentially
corrected at the bottom to achieve drift-corrected, real-time sensing in vivo. Figures adapted with permission from Refs. 29,30,147.

Irrespective of modality, these approaches are known as faradaic
impedance spectroscopy. The use of labeled receptors offers the pos-
sibility of achieving continuous, real-time sensing since, in theory, the
addition of exogenous reagents is not needed for detection. However,
receptor labeling itself requires extra synthetic steps, and a deep knowl-
edge of the molecule is required so that its binding capacity remains
unaltered after modification.143 In contrast, label-free detection may
be less prone to reporter-induced fouling of the receptor. For exam-
ple, electron transfer from the redox couple [Fe(CN)6 ]4− /[Fe(CN)6 ]3−
is affected by binding-induced electrostatic changes in the backbone
of DNA.132,144,145 The binding of the target molecule to the immobi-
lized DNA strand alters the conformation and charge distribution of
the interface, further modifying this electron transfer resistance and,
consequently, allowing the relation of this parameter to the amount of
target in the sample (Figures 12D–12F).
Conclusions
DNA-based electrochemical sensors emerged as an alternative to
enzyme-based and antibody-based measurements for analytes that
cannot be detected by such approaches (i.e. when the analyte is not the
substrate of a known enzyme or if there is no antibody that can recog-
nize it). The analytical value of DNA for electrochemical biosensing

Table II. Merits and Demerits of Electrochemical Methods Most Commonly Used for the Interrogation of E-AB Sensors.

Acquisition Reporter-Free Eliminates

Technique Time (s) Drift-Free Measurements? Measurements? Charging Current?

Cyclic Voltammetry 10−1 – 1 Not demonstrated No No

Chronoamperometry 10−3 – 1 Yes, via current decay lifetime measurements No No
Differential Pulse Voltammetry 1 – 30 Not demonstrated No Yes
Alternating Current Voltammetry 10−1 – 1 Not demonstrated No Yes
Square Wave Voltammetry 1 – 30 Yes, via differential frequency measurements No Yes
faradaic Impedance Spectroscopy 10 – 100 Not demonstrated No Yes
Non-faradaic Impedance Spectroscopy 10 – 100 Not demonstrated Yes No
 Journal of The Electrochemical Society, 2020 167 037529

ploit the analytical potential of DNA-based sensors, it is essential

Figure 11. Interrogation of DNA-based sensors via electrochemical
impedance spectroscopy. (A) A sinusoidal voltage is applied (solid line)
at a fixed frequency, generating the corresponding current (dashed line). This
is repeated for a wide range of frequencies. (B) The imaginary part of the
impedance is plotted against the real part for each sampled frequency to
obtain the Nyquist plot. The value of the impedance and its phase angle at a
certain frequency can be obtained from the module and the angle of the vector,
respectively. (C-D) Bode plots provide another way of representing the data
obtained in an EIS measurement. In this case, the impedance and the phase
angle are plotted against the applied frequency, offering a straightforward
manner to view the impedance at a certain frequency value. (B-D) Spectra
obtained for a non-faradaic (dashed line) and a faradaic (solid line) system.
derives from the relative facility with which DNA can be organized
in specific conformations146 and on its binding versatility— DNA can
bind with high specificity to ions,27,28 small molecules,29,30 proteins
or nucleic acids31,32 and even whole cells.33,34 However, to fully ex-
to have a clear, deep understanding of the features and advantages
that different electrochemical methods offer for their interrogation.
The selection of the most suitable technique for a given application
naturally depends on biosensor architecture, detection mechanism,
time-resolution needed, and required sensitivity. Here, we discussed
the advantages of different interrogation techniques considering sen-
sor configuration (i.e., if a redox reporter is added to the system and
whether that reporter is tethered to the sensing layer or free in solu-
tion) and the time scales of the physicochemical phenomena occurring
at the interface during sensing (Table I). Beyond simply providing a
technical description of methods, which has been done in many pre-
vious reviews,42 here we point to specific measurement advantages
that are somewhat unique to each technique (Table II). For example,
we recommend chronoamperometry as the method of choice if fast,
sub-second measurements are needed. Cyclic voltammetry is a great
tool for analytical characterizations of sensor interface. However, the
value of the latter for sensor interrogation can be limited by its inabil-
ity to discriminate between charging and faradaic currents. In contrast,
pulsed-voltage methods offer charging-free sensing and high detection
limits. Finally, if the reporter itself sets the limit in temporal resolution
for sensing, electrochemical impedance supports reporter-free sens-
ing. We also discuss applications of the above-mentioned techniques
to achieve calibration-free and drift-corrected sensing.
The ideal interrogation method for future sensor developments will
be dictated by the analytical application. As future perspectives we
speculate that voltage-pulsed methods will be the choice for applica-
tions related to decentralized diagnostics (including point-of-care de-
tection). We say this because the convenient output of these methods,
a current peak, can be easily interpreted by technicians with minimal
training or automated systems. Moreover, pulsed methods are the least
affected by charging currents, which can foul the outcome of electro-
chemical measurements. We also speculate that cyclic voltammetry
will continue to be the method of choice for the electrochemical char-
acterization of bio interfaces; although, it will likely play a key role
for in-vivo measurements in the brain, alongside chronoamperometry.

Figure 12. Impedimetric behavior of DNA-based sensors. (A) An illustrative example of a non-faradaic DNA-based sensor in which the immobilized aptamer
binds the target molecule, modifying the morphology of the double-layer, (B-C) which leads to a change in its capacitance that can be related with the target
concentration. (D) faradaic sensors, on the other hand, rely on the use of a redox compound to report the binding of the DNA probe with its target. (E-F) Upon
binding, the electron transfer of the reporter with the electrode changes, and the electron transfer resistance can be related with the amount of target in the sample.
Figures adapted with permission from Refs. 139,148.
 Journal of The Electrochemical Society, 2020 167 037529

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