Survey of Redox-Active Moieties for Application in Multiplexed Electrochemical Biosensors

Article abstract of DOI:10.1021/acs.analchem.6b02376

Recent years have seen the development of a large number of electrochemical sandwich assays and reagentless biosensor architectures employing biomolecules modified via the attachment of a redox-active "reporter." Here we survey a large set of potential redox reporters in order to determine which exhibits the best long-duration stability in thiol-on-gold monolayer-based sensors and to identify reporter "sets" signaling at distinct, nonoverlapping redox potentials in support of multiplexing and error correcting ratiometric or differential measurement approaches. Specifically, we have characterized the performance of more than a dozen potential reporters that are, first, redox active within the potential window over which thiol-on-gold monolayers are reasonably stable and, second, are available commercially in forms that are readily conjugated to biomolecules or can be converted into such forms in one or two simple synthetic steps. To test each of these reporters we conjugated it to one terminus of a single-stranded DNA "probe" that was attached by its other terminus via a six-carbon thiol to a gold electrode to form an "E-DNA" sensor responsive to its complementary DNA target. We then measured the signaling properties of each sensor as well as its stability against repeated voltammetric scans and against deployment in and reuse from blood serum. Doing so we find that the performance of methylene blue-based, thiol-on-gold sensors is unmatched; the near-quantitative stability of such sensors against repeated scanning in even very complex sample matrices is unparalleled. While more modest, the stability of sensors employing a handful of other reporters, including anthraquinone, Nile blue, and ferrrocene, is reasonable. Our work thus serves as both to highlight the exceptional properties of methylene blue as a redox reporter in such applications and as a cautionary tale-we wish to help other researchers avoid fruitless efforts to employ the many, seemingly promising and yet ultimately inadequate reporters we have investigated. Finally, we hope that our work also serves as an illustration of the pressing need for the further development of useful redox reporters.

Full text of DOI:10.1021/acs.analchem.6b02376

Subscriber access provided by CORNELL UNIVERSITY LIBRARY
Article
A survey of redox-active moieties for application
in multiplexed electrochemical biosensors
Di Kang, Francesco Ricci, Ryan J. White, and Kevin W. Plaxco
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02376 • Publication Date (Web): 23 Sep 2016
Downloaded from http://pubs.acs.org on September 24, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth
Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 8
Analytical Chemistry
1
2
3
4
5
6
A survey of redox-active moieties for application in multiplexed elec-
trochemical biosensors
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
4
1, 2,
Di Kang , Francesco Ricci , Ryan J. White and Kevin W. Plaxco
*
1
2
Department of Chemistry and Biochemistry and Interdepartmental Program in Biomolecular Science and Engineering,
3
University of California Santa Barbara, Santa Barbara, California 93106,United States; Dipartimento di Scienze e Tecnolo-
gie Chimiche, University of Rome, Tor Vergata, Via della Ricerca Scientifica, 00133, Rome, Italy; Department of chemistry
4
and biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21250, United States
ABSTRACT: Recent years have seen the development of a large number of electrochemical sandwich assays and reagentless bio-
sensor architectures employing biomolecules modified via the attachment of a redox-active “reporter.” Here we survey a large set
of potential redox reporters in order to determine which exhibits the best long-duration stability in thiol-on-gold monolayer-based
sensors and to identify reporter “sets” signaling at distinct, non-overlapping redox potentials in support of multiplexing and error
correcting ratiometric or differential measurement approaches. Specifically, we have characterized the performance of more than a
dozen potential reporters that are, first, redox active within the potential window over which thiol-on-gold monolayers are reasona-
bly stable and, second, are available commercially in forms that are readily conjugated to biomolecules or can be converted into
such forms in one or two simple synthetic steps. To test each of these reporters we conjugated it to one terminus of a single-
stranded DNA “probe” that was attached by its other terminus via a six-carbon thiol to a gold electrode to form an “E-DNA” sensor
responsive to its complementary DNA target. We then measured the signaling properties of each sensor as well as its stability
against repeated voltammetric scans and against deployment in and reuse from blood serum. Doing so we find that the performance
of methylene blue-based, thiol-on-gold sensors is unmatched; the near-quantitative stability of such sensors against repeated scan-
ning in even very complex sample matrices is unparalleled. While more modest, the stability of sensors employing a handful of
other reporters, including anthraquinone, Nile blue, and ferrrocene, is reasonable. Our work thus serves as both to highlight the
exceptional properties of methylene blue as a redox reporter in such applications and as a cautionary tale –we wish to help other
researchers avoid fruitless efforts to employ the many, seemingly promising and yet ultimately inadequate reporters we have inves-
tigated. Finally, we hope that our work also serves as an illustration of the pressing need for the further development of useful redox
reporters.
15,16
Due to the ease, with which they are fabricated and em-
ployed, and their oft-impressive selectivity and detection lim-
its, electrochemical biosensors employing redox-reporter-
ferential measurement approaches [e.g., refs ]. To date,
however, the literature describing these sensors has almost
6
entirely utilized methylene blue , Nile blue, anthraquinone, or
1
2
6,17-21
modified oligonucleotides and polypeptides attached via
thiol self-assembled monolayer (SAM) formation onto gold
electrodes have seen significant recent attention. These include
ferrocene as reporters [e.g., refs
]. The expansion of this
relatively short list could improve the extent to which such
sensors can be multiplexed. Moreover, we have found that a
lack of redox reporters of similar stability hinders ratiometric
and differential error correction methods, which suffer if one
reporter degrades more rapidly than the other. Motivated by
these observations we have, over many years of work in this
area, characterized more than a dozen candidate redox report-
ers (Fig 1), all of which are either commercially available in
forms easily conjugated to biomolecules or are readily con-
verted into such forms via one or two simple synthetic steps
and all of which are active at potentials within the ~1 V win-
dow over which thiol-on-gold SAMs are reasonable stable.
Unfortunately, though, the overwhelming majority the result-
ing sensors were unstable thus we abandoned most of these
potential reporters without reporting their characterization in
the literature. Recently, however, we have become aware that
other researchers had similarly characterized and found un-
suitable many of the same potential reporters. In order to 1)
highlight the exceptional stability of the “best” redox reporter
in such applications, 2) identify other reporters that, while
non-optimal, still perform “well enough” for use in ratiometric
3
analytical approaches ranging from sandwich and intercala-
4
tion assays to reagentless sensors based on binding-induced
1
conformational changes [reviewed in ref , changes in collision
5
4,6
efficiency , or changes in through-DNA charge transfer . The
potential advantages of such sensors class are multifold. First,
many perform well even when deployed directly in complex
clinical samples, such as undiluted blood serum, saliva, or
7-9
crude cell lysates [e.g., refs ], and have even been used for
10
the continuous measurement of plasma drug levels . Second,
the approach is quite convenient, with sensor fabrication gen-
11
erally proving facile [e.g., ref ] and the electronics required to
1
2
interrogate them being small and inexpensive .
Beyond the above attributes an additional advantage of
SAM-on-gold-based electrochemical biosensors is the ability
to deploy multiple sensors on a single electrode by employing
reporters that signal at unique, non-overlapping reduction po-
13,14
tentials. This allows for improved multiplexing [e.g., refs
]
and for the introduction of error correcting, ratiometric or dif-
ACS Paragon Plus Environment

Analytical Chemistry
Page 2 of 8
or multiplexed sensors, 3) help future researchers avoid re- (AQ-NHS, empBiotech GmbH), anthraquinone-2-amidopentyl
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
peating yet again these same, largely failed stability studies
(none of which have previously been published, thus causing
unnecessary repetition of effort), and 4) motivate efforts to
identify new redox reporters suitable for use with thiol-on-
gold-based sensors, we describe here the results of our decade-
plus efforts.
carboxylic acid NHS ester (empBiotech GmbH), gallocyanine
carboxylic acid NHS ester (empBiotech GmbH), ferrocene-
amidopentyl carboxylic acid NHS ester (empBiotech GmbH),
phosphate buffer (Sigma-Aldrich), 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) (Sigma-Aldrich) and
fetal calf serum (Sigma-Aldrich) were all used as received.
For the majority of our studies we employed this sequence
as our E-DNA “probe.”
5’–HS-(CH ) –TGGATCGGCGTTTTATT-(CH ) –NH –3’
2
6
2 6
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
which we conjugated in-house to carboxyl-modified reporters
it to form an amide bond (see below). Five of the potential
reporters we characterized, however, were instead obtained as
DNA conjugates directly from a commercial synthesis house
(Biosearch, Inc., Novato, CA):
5
’–HS-(CH ) –TGGATCGGCGTTTTATT–(CH ) –(NH-
2 6 2 6
CO)-ROX–3’
5
’–HS-(CH ) –TGGATCGGCGTTTTATT–(CH ) –(NH-
2 6 2 6
CO)-dabcyl–3’
5
’–HS-(CH ) –TGGATCGGCGTTTTATT–(CH ) –(NH-
2
6
2 6
CO)-anthraquinone–3’
'–HS-(CH ) –ATTATTGATCGGCGTTTTAAAGAAG
5
2
6
–
(CH ) –(NH-CO)-methylene blue–3′
2 6
3’–HS-(CH ) –
2
6
AGACAAGGAAAATCCTTCAATGAAGTGGGTCG–
(CH ) –(NH-CO)-ferrocene-5’
2
6
As the target olignonucleotide for these sensors we em-
ployed the following unmodified DNA constructs as appropri-
ate:
5
5
5
’–AATAAAACGCCGATCCA–3’
’–TAAAACGCCGATC–3′
’–CGACCCACTTCATTGAAGGATTTTCCTTGTCT–3’
Synthesis of Succinimidyl Ester-modified reporters
Thionine. We mixed thionine (2.6 g) and 8-bromooctanoic
acid (3 g) in DMF (40 mL) to synthesis 3N-octanoic-acid-
modified thionine. The mixture was refluxed overnight (10 hr).
The crude product mixture was concentrated in vacuum, be-
fore being dissolved in methanol and filtered (Celite). We use
silica column chromatography (100:12:1.2 with chloro-
form/methanol/acetic acid) to purify the product (251 mg)
with poor yield (7.5%).
Figure 1. We characterized 13 potential redox reporters in total,
nine organic small molecules and two organometallic complexes.
Two of these, ferrocene and anthraquinone, were investigated
with and without an additional five-carbon linker and one, ferro-
cene, was investigated using both 3’ and 5’ linkages. All these
potential reporters are available in (or are easily synthesized as)
forms containing a carboxylic acid group for ready conjugation to
an amine-modified DNA.
To synthesize thionine succinimidyl ester, we mixed N,N'-
dicyclohexylcarbodiimide (DCC), N-hydrosuccinamide (NHS),
and 3N-octanoic-acid-modified thionine in dry dimethylfor-
mamide (DMF). The solution was stirred overnight under ar-
gon at room temperature. We concentrated the mixture in vac-
uum, and purified the product with silica column chromatog-
raphy (100:12:1.2 chloroform/methanol/acetic acid).
EXPERIMENTAL SECTION
Reagents
Anhydrous ferrous chloride (Sigma-Aldrich), sodium nitrite
(Sigma-Aldrich), Sodium cyclopentadienide (Sigma-Aldrich),
n-butyllithium (1.6 M in n-hexane) (Sigma-Aldrich), pentame-
thylcyclopentadiene (Sigma-Aldrich), 2-chlorobenzoyl chlo-
ride (Sigma-Aldrich), aluminum chloride (Sigma-Aldrich),
potassium t-butoxide (Sigma-Aldrich), methyl tertiary butyl
ether (Sigma-Aldrich), ferrocene-carboxylic acid, N-
Nile Blue. We first prepared 5-(dimethylamino)-2-
nitrosophenol
by
dissolving
826
mg
of
3-
(dimethylamino)phenol in concentrated hydrochloric acid (5
mL) at 0ºC, followed by the slow addition of 400 mg sodium
nitrite, stirring until the content solidified (about 40 min). The
precipitate was isolated via vacuum filtration and washed with
chilled 1 M hydrochloric acid. The crude product, a yellow-
brown solid, was dried in vacuum and used for the conjugation
reaction without further purification. In parallel we prepared
3-(naphthalen-1-ylamino) propanoic acid by dissolving 200
hydroxysuccinimide
(Sigma-Aldrich),
N-
hydroxysulfosuccinimide sodium salt (NHS), N-(3-dimethyl-
amino-propyl)-N’-ethylcarbodiimide (Fluka), 2,6-dichloro-p-
benzoquinone-4-chloroimine (Fisher), 2-hydroxyphenylacetic
acid (Fisher), Neutral red (Sigma-Aldrich), 3,7-bis(N-(3-
carboxypropyl)-N-methylamino)-phenothiazin-5-ium perchlo-
rate (MB -NHS, empBiotech GmbH), anthraquinone-NHS
ACS Paragon Plus Environment

Page 3 of 8
Analytical Chemistry
mg naphthylamine in DMF (15 mL) followed by the addition
Pentamethyferrocene. A suspension of FeCl in tetrahydro-
furan (THF) was vigorously stirred in the dark 1 hr to produce
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
of 5 mL of 6 M sodium hydroxide and equimolar amounts of
dissolved 3-bromo-propionic acid ethyl ester. We refluxed the
mixture for 12 hr. After the reaction finished, we dried the
crude reaction mixture in vacuum and purify the product using
silica column chromatography (20:1 hexane/methanol). To
obtain propionic acid modified Nile blue we first dissolved 34
mg of 5-(dimethylamino)-2-nitrosophenol and 44 mg of 3-
FeCl •THF. Separately, n-butyllithium was added drop-wise to
2
pentamethylcyclopentadiene in tetrahydrofuran in a dry ice
acetone bath and the mixture was then warmed and stirred at
room temperature for 2 hr. We then slowly transferred this
mixture into the FeCl •THF solution and stirred at room tem-
2
perature for 1 hr. Sodium cyclopentadienide was slowly added
to this via cannula before stirring over night (~16 hr). The
resultant pentamethylferrocene product was recrystallized
from pentane with a yield of 60%.
(naphthalen-1-ylamino)propanoic acid in DMF (10 mL), and
heated the reaction mixture to 90ºC for overnight. We dried
the crude reaction mixture in vacuum and purified it using
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
silica column chromatography (3:1 CHCl /methanol).
3
To obtain pentamethylferrocene carboxylic acid we first
2
,6-Dichlorophenal-indophenol. We dissolved 150 mg 2,6-
dichloro-p-benzoquinone-4-chloroimine in 2 mL methanol and
-hydroxyphenylacetic acid in 10 mL 100 mM K HPO . We
mixed 2-chlorobenzoyl chloride and AlCl in dichloromethane
3
for 1 hr at 0˚C. We cooled this to -40˚C and then slowly add
pentamethylferrocene in dichloromethane. We then warmed
the mixture to 15˚C over 60 min to generate 2-chlorobenzoyl-
pentamethylferrocene which we then poured onto crushed ice.
We next washed the organic phase with 1 M NaOH and dried
2
2
4
then mixed two solutions and stirred overnight at room tem-
perature. The resulting solution was dried under vacuum to
produce the crude purple blue product which we purified using
silica
column
chromatography
(100:10:1
chloro-
it against MgSO before filtering and evaporating the solvent.
4
form/methanol/acetic acid).
We purified the crude 2-chlorobenzoyl-pentamethylferrocene
using silica column chromatography (25:1 cyclohex-
ane/methyl tert-butyl ether). We then mixed the 2-
chlorobenzoyl-pentamethylferrocene with potassium tert-
butoxide and few drops of water in dimethylformamide (DMF)
and refluxed the mixture at 110˚C for 1 hr to produce the car-
boxylic acid. After cooling the mixture to 0˚C we then added 1
M HCl to obtain the red brown solid, which we washed with
water, filtered and dried.
20
Ferrocene. We use the previously described procedure to
convert ferrocene carboxylic acid to ferrocene succinimidyl
ester (Fc-NHS). In brief, we mixed a 5-fold excess of NHS
and EDC with ferrocene carboxylic acid in dichloromethane
(DCM). The solution was stirred overnight under argon (~12
hr) at room temperature. The resulting solution was then
washed with water and the organic phase was collected and
dried with MgSO , filtered, and evaporated. Finally, we used
4
silica column chromatography with diethyl ether to purify the
product (Fc-NHS).
Figure 2. Not all redox reporters are created equal. Dabcyl and ROX, for example, fail to produce clear oxidation and reduction peaks
when conjugated to DNA and interrogated using our standard square wave voltammetric parameters, and thionine exhibits two peaks in the
relevant potential window. We investigated three ferrocene-containing constructs: one in which the ferrocene is conjugated directly on to
an amine appended to the 5’ end of the DNA, a second in which the ferrocene is conjugated directly on to an amine appended to the 3’ end
of the DNA, and a third, ferrocene C5, in which there is an additional spacer between ferrocene and the amide linkage to the DNA. The
highly sloping baselines observed at potentials below -0.5 V and above 0.5 V (versus Ag/AgCl) are due to the reduction of oxygen and the
subsequent generation of reactive oxygen species (at low potentials) and the oxidation of gold (at high potentials). These same effects
cause significant degradation of the thiol-on-gold SAM; i.e., some redox reporters fail because they, themselves fail, and others fail be-
cause they report at potentials at which SAM stability is poor.
ACS Paragon Plus Environment

Analytical Chemistry
Page 4 of 8
To synthesize pentamethyferrocene succinimidyl ester we
added NHS and N-(3-dimethylamino-propyl)-N´-
ethylcarbodiimide hydrochloride (EDC) to a 25 mM solution
of pentamethylferrocene carboxylic acid in dichloromethane to
a final concentration of 60 mM each and stirred overnight
Our sensors were stored in buffer in sealed at room tempera-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ture. To test sensor robustness to repeated electrochemical
interrogations, sensors were subjected to multiple SWV scans
without target using a scan interval in both buffer and 20%
serum. To test sensor robustness to multiple testing cycles
were assessed by subjecting the sensors to a repeated cycle of
testing, testing in saturating target, and regeneration with a 30
s deionized water rinse. These cycles were repeated 5 times in
20% serum respectively.
(~12 hr) under argon at room temperature. The resulting solu-
tion was then washed with water and the organic phase was
collected and dried with MgSO , filtered, and evaporated. We
4
then purified the pentamethyferrocene succinimidyl ester
product using silica column chromatography (diethyl ether).
RESULTS AND DISCUSSION
Conjugating the reporters to the DNA probes
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The commercially available or easily synthetically accessi-
ble redox reporters we have characterized fall into either of
two classes: organic small molecules and organometallic com-
plexes. Among the former we have characterized methylene
blue, thionine, anthraquinone, anthraquinone with a five-
carbon linker (anthraquinone-C5), gallocyanine, Nile blue,
indophenol, neutral red, dabcyl, and carboxy-X-rhodamine
Conjugation of the redox reporters to the appropriate single-
stranded DNA was achieved via the coupling of the NHS-ester
redox reporter conjugate with the 5’-alkyl-amino modified
single stranded DNA. 10 μL of 200 μM 5’-alkyl-amino DNA
was added to 50 μL of a 0.5 M sodium bicarbonate solution
(
1
pH 8.5), and 1 μmole of the reporter-NHS was dissolved in
0 μL dimethyl sulfoxide (DMSO) . We mixed the DNA
2
2
(
ROX) (Fig. 1). Among the latter we have characterized ferro-
cene attached directly to either the 5’ or 3’ end of the DNA,
ferrocene linked to the DNA via a five-carbon linker (ferro-
cene-C5), and pentamethylferrocene. All told we have, over
more than a decade of working in the field of electrochemical
biosensors, characterized more than a dozen potential redox
reporters, each as with a carboxylic acid group supporting
ready conjugation to an amine-terminated DNA. Here we
summarize the results of our many years’ experience working
with these potentially promising reporters.
solution we prepared above and the reporter-NHS DMSO so-
lution, and incubated the mixture for 4 hours in dark at room
temperature. After conjugation the DNA was desalted using a
spin column (EMP-Biotech) and purified by RP-HPLC (C18
column). The stocked DNA solutions were stored at -20° C for
future use. The yield of the final conjugated product, estimated
using HPLC and gel electrophoresis (Fig. SI 1). Conveniently,
unmodified DNA is not electroactive at the potentials we em-
ployed and thus is silent in our experiments.
Electrode preparation and sensor fabrication
E-DNA sensors were prepared using established proce-
10
dures . In brief, prior to sensor fabrication, gold disk elec-
trodes (2 mm diameter, CH Instruments, Austin, TX) were
cleaned both mechanically (by polishing with diamond and
alumina oxide slurries successively) and electrochemically
(through successive scans in sulfuric acid solutions) as previ-
ously described. The linear probe DNA were reduced for 1 hr
at room temperature in 10 mM tris(2-carboxyethyl)phosphine
hydrochloride (Molecular Probes, Carlsbad, CA) and then
diluted to a final concentration of 1 μM in 50 mM phosphate,
100 mM NaCl buffer, pH 7.0, as was used in all the experi-
ments to follow unless otherwise noted). The gold electrodes
were incubated in this solution for 1 hr at room temperature,
rinsed with deionized water, and then incubated in 3 mM 6-
mercapto-1-hexanol in deionized water for 120 min. After
deposition of this molecule onto a gold electrode the electrode
surface is “backfilled” with 6-mercapto-1-hexanol to form a
continuous, mixed, self-assembled monolayer. Following this,
the electrodes were rinsed in deionized water and stored in
buffer for future use.
Figure 3. We challenged the reporter-conjugated probes that ex-
hibit clear reduction and oxidation peaks with saturating concen-
trations of their complementary target to test their signaling prop-
erties. All respond as expected to these targets, albeit with varying
signal gain. Of note signal gain in this class of sensors is a strong
function of both the intrinsic electron transfer rate of the reporter
and the square wave frequency and amplitude, thus likely ac-
counting for the variations in signal gain observed (all data were
collected under a single set of square wave parameters).
Sensor characterization
Fabricated sensors were interrogated using square wave
voltammetry (SWV) with a 50 mV amplitude signal at a fre-
quency of 60 Hz, in the absence and presence of fully com-
plementary target. For the latter measurements the electrodes
were incubated for 30 min with the target DNA at 1 μM in 50
mM phosphate, 100 mM NaCl buffer or 20% fetal calf serum
in the same buffer. Values with reported error bars represent
the average and standard deviations of measurements per-
formed on at least three independently fabricated electrodes.
Signal gain was computed by the relative change in SWV peak
currents with respect to background current (SWV peak cur-
rent in the absence of target).
To characterize the utility of these commercially or easily
synthetically available redox reporters we have, over a number
of years of research incorporated each into a simple E-DNA
electrochemical DNA sensor. Specifically, we employed E-
DNA sensors composed of linear DNA strands modified with
the relevant redox reporter on its 3’-terminus (except for fer-
rocene, for which we have explored two 3’ and one 5’ linkage)
and attached at its opposite terminus to a six-carbon alkane
ACS Paragon Plus Environment

Page 5 of 8
Analytical Chemistry
2
3
thiol monolayer to a gold electrode . Hybridization with a
target oligonucleotide reduces the efficiency with which the
attached redox reporter transfers electrons to the interrogating
electrode, leading to a significant decrease in faradaic current
when the system is interrogated using, for example square
wave voltammetry. For each of the resulting sensors we meas-
ured signaling (i.e., do we see a clear, single oxidation and
reduction peaks for the DNA-reporter conjugate), signal gain
ble against repeated voltammetric scans, here conducted in phos-
phate/NaCl buffer. (b) Unlike the other reporters we have charac-
terized, the signaling current from gallocyanine-based sensors
increases upon repeated scanning. The scans in these figures are
repeated every 20 s, and thus the x-axis are also proportional to
time. We find, however, that sensor decay (and, moreover, the
differences in the decay rate from one reporter to the next) are a
much stronger function of the number of scans than of time (data
not shown).
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
relative signal change upon target saturation), electrochemical
reversibility (i.e., its stability against multiple voltammetric
scans) and stability when exposed to a realistically complex
sample matrix (20% blood serum).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The properties of the potential reporters we have character-
ized vary widely. For example, three of the potential reporters,
thionine, dabcyl and ROX, failed to produce clear oxidation
and reduction peaks when conjugated to DNA (Fig. 2a). A
fourth potential reporter, indophenol, proved difficult for con-
jugation to DNA. Specifically, although free indophenol car-
boxylic acid is stable (and electrochemically active) under
basic conditions we found that it decomposed during our at-
tempts to conjugate it to an amine-terminated DNA. The redox
potential of a fifth candidate, neutral red, shifts to -0.7 V when
modified to support DNA conjugation, moving its potential
close to the redox potential of alkane thiols on gold and ren-
dering the resultant sensor unstable (data not shown). Finally,
a sensor employing a 3’ ferrocene reporter produced only a
very small peak current (Fig. 2), and thus we decide not to
carry it over to our next testing step. The remaining reporters,
in contrast, are all easily conjugated to amine-modified DNA
and produce clear redox peaks within the potential window
over which alkane-thiol-on-gold self-assembled monolayers
are stable.
Figure 5. (a) Sensors fabricated with methylene blue, ferrocene,
anthraquinone or Nile blue exhibit similar signal gain in response
to target binding whether deployed in simple buffer solutions or in
20% blood serum. (b) They all drift significantly, however, when
repeatedly scanned in 20% serum over the course of hours, with
methylene blue exhibiting the least drift. (c) Methylene blue-
Figure 4. With the notable exception of methylene blue, (a) the
redox reporters we have investigated are at least somewhat unsta-
ACS Paragon Plus Environment

Analytical Chemistry
Page 6 of 8
based sensors are likewise the most stable when the sensors are
The origin of the loss in single for most reporters is not clear,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
challenged with multiple cycles of hybridization (with saturating
target) and regeneration (via di-water wash) in 20% blood serum.
but investigation of their voltammograms and of prior litera-
ture descriptions of their chemistry provides some suggestions.
Some of the degradation is, no doubt, due to the poor stability
of some of the potential reporters. The ferrocenium ion, for
example, is known to be unstable in aqueous solution in the
All of the remaining redox-reporters in our set produce at
least reasonably high-gain E-DNA sensors under the standard
volammetric parameters we employ in our sensors (Fig. 3).
The stability of the resultant sensors to repeated oxidation-
reduction cycles, however, varies widely (Fig. 4). Sensors
fabricated with methylene blue, for example, are impressively
stable, exhibiting only 2% current loss after 100 square-wave
voltammetric scans in buffer. Sensors employing anthraqui-
none, Nile blue or 5’-linked ferrocene, in contrast, are only
modestly stable under these conditions, exhibiting ~50% sig-
nal loss after 100 scans. Sensors fabricated using 3’-linked
ferrocene, ferrocene-C5 and penta-methyl ferrocene linked are
still less stable, losing ~50% of their initial signal after only 50
scans. For most of the sensors we observe an exhibit steady,
monotonic decrease in current as the number of scans increas-
es. The signaling current of anthraquinone-based sensors,
however, drops off rapidly during the first few scans before
largely leveling off, and the signaling current from the gallo-
cyanine-based sensor increases significantly upon repeated
scanning. The origins of this increase are unknown to us.
[
24]
presence of oxygen
and the increasing current seen with
gallocyanine presumably occurs due to redox-driven modifica-
tion of the reporter. In addition, some of the degradation we
see is likely due to the instability of the SAM when repeatedly
scanned at high or low potentials. Specifically, a plot of stabil-
ity (as measured by the current remaining after 50 scans) ver-
sus the redox potential of the reporter suggests that the best
redox reporters all fall near the middle of the 1 V window over
which thiol-on-gold SAMs are stable (Fig. 6). Moreover, this
same plot illustrates an important point: there is a dearth of
potential reporters in what appears to be the “sweet spot” in
this plot (near 0 V versus Ag/AgCl), suggesting that efforts
aimed at filling this gap may prove fruitful.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CONCLUSIONS
Here we have shown that, although a number of redox ac-
tive moieties that are available in forms easily conjugated to
DNA also support high-gain E-DNA signaling, the stabilities
of the resulting sensors differ dramatically due either to the
inherent instability of the redox reporter or the instability of
thiol-on-gold SAMs at potentials below -0.6 V or above +0.4
V (versus Ag/AgCl). Sensors employing methylene blue, for
example, exhibit outstanding stability even in blood serum, a
complex, multi-component sample matrix. The next best re-
porters, in contrast, including ferrocene, anthraquinone, and
Nile blue, also produce high-gain sensors but exhibit rather
poorer stability against repeated voltammetric scanning at the
relevant redox potentials. In addition to highlighting the ad-
vantages of employing methylene blue in such sensors, our
results also suggest that more effort towards the development
of suitable redox reporters is needed if the multiplexing and
ratiometric error correction potential of electrochemical bio-
sensors are to be fully realized.
An advantage of E-DNA-type sensors is their excellent per-
1
formance in complex sample matrices, such as blood serum .
Thus motivated we have tested the performance of sensors
fabricated using the reporters methylene blue, 5’-ferrocene,
anthraquinone, and Nile blue when deployed in 20% blood
serum. The gain of each is effectively indistinguishable from
that seen in simple buffer solutions (Fig. 5a). And, once again,
we find that methylene blue is quite stable, exhibiting only
minor signal loss when scanned twice an hour for more than 8
hr under these conditions (Fig. 5b). Sensors employing 5’-
ferrocene, anthraquinone (see, also, Fig. SI 2), or Nile blue, in
contrast, loose 25-30% their original signal under these same
conditions. Finally, methylene blue-based sensors exhibit only
1
1% decay over four cycles of deployment in serum followed
by regeneration (Fig. 5c). Nile blue-based sensors, in contrast,
exhibit 20% loss and anthraquinone- and ferrocene-based sen-
sors exhibit about 50% loss under these same use-and-
regeneration conditions.
SUPPORTING INFORMATION
AUTHOR INFORMATION
Corresponding Author
*E-mail: kwp@chem.ucsb.edu
Author Contributions
The manuscript was written through contributions of all authors. /
All authors have given approval to the final version of the manu-
script.
ACKNOWLEDGMENT
This work was supported by the NIH (AI107936) and the Institute
for Collaborative Biotechnologies through grant W911NF-09-
0001 from the U.S. Army Research Office (KWP). The content of
the information does not necessarily reflect the position or the
policy of the Government, and no official endorsement should be
inferred.
Figure 6. A plot of sensor stability (here measured as relative peak
current remaining after 50 voltammetric scans) versus the redox
potential of the reporter suggests that reporters situated near the
middle of the ~1 V window over which thiol-on-gold SAMs are
stable exhibit the best performance.
ACS Paragon Plus Environment

Page 7 of 8
REFERENCES
Analytical Chemistry
(
13) Kang D.; Xia, F.; Zuo, X.; White, J.R.; Vallée-Bélisle, A.;
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Plaxco, K.W. NPG Asia Mat. 2012, 4, e1.
(
(
1) Lubin, A.A.; Plaxco, K.W. Acc. Chem. Res. 2010, 43, 496–505.
2) McQuistan, A.; Zaitouna, A.J.; Echeverria, E.; Lai, R.Y.Chem.
(
14) Xia, J; Daimin Song; Wang, Z.; Zhang, F.; Yang, M.; Gui, R.;
Xia, L.; Bi, S.; Xia, Y.; Li, Y.; Xia, L. Biosensors & Bioelectronics
015, 68, 55-61.
15) Du, Y.; Lim, B.J.; Li, B.; Jiang, Y.S.; Sessler, J.L.; Ellington
Commun. 2014, 50, 4690-4692.
(3) Zuo, X.; Xiao, Y.; Plaxco, K. W. J. Am. Chem. Soc. 2009, 131,
2
(
6
944-6945.
4) Boon, E.M.; Salas, J.W.; Barton, J. K. Nature Biotechnology
002, 20, 282-286.
5) Cash, K.J.; Ricci, F.; Plaxco, K.W. J. Am. Chem. Soc. 2009,
A.D. Anal. Chem. 2014, 86, 8010–8016.
(16) Ren, K.; Wu, J.; Yan, F.; Zhang, Y.; Ju, H. Biosensors & Bi-
oelectronics 2015, 66, 345-349.
(
2
(
(
17) Fan, C.; Plaxco, K.W.; Heeger, A.J. Proc. Natl. Acad. Sci.
U.S.A. 2003, 100, 9134-9137.
18) Gorodetsky, A.A.; Hammond, W.J.; Hill, M.G.; Slowinski, K.;
131, 6955-6957.
(6) Ikeda, R.; Kitagawa, S.; Chiba, J.; Inouye, M. Chem. Eur. J.
(
2
2
009, 15, 7048–7051.
7) Lubin, A.A.; Lai, R.Y.; Heeger, A.J.; Plaxco, K.W. Anal. Chem.
006, 78, 5671-5677.
8) Ferapontova, E.E.; Olsen, E.M.; Gothelf, K.V. J. Am. Chem.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Barton, J.K. Langmuir 2008, 24, 14282-14288.
(19) Pheeney, C. G; Barton, J.K. J. Am. Chem. Soc. 2013, 135,
(
1
4944–14947.
20) Balintová, J.; Pohl, R.; Horáková, P.; Vidláková, P.; Havran,
L.; Fojta, M.; Hocek, M. Chem. Eur. J. 2011, 17, 14063–14073.
21) Zuo, X.; Song, S.; Zhang, J.; Pan, D.; Wang, L.; Fan, C. J. Am.
(
(
Soc. 2008, 130, 4256–4258.
(9) Mahshid, S.S.; Camiré, S.; Ricci, F.; Vallée-Bélisle, A. J. Am.
Chem. Soc. 2015, 137, 15596–15599.
(
Chem. Soc. 2007, 129, 1042–1043.
(22) Kang, D.; Zuo, X.; Yang, R.; Xia, F.; Plaxco, K.W.; White,
R.J. Anal. Chem. 2009, 81, 9109-9113.
(
10) Ferguson, B.S.; Hoggarth, D.A.; Maliniak, D.; Ploense, K.;
White, R.J.; Woodward, N.; Hsieh, K.; Bonham, A.J.; Eisenstein, M.;
Kippin, T.; Plaxco, K.W.; Soh, H.T. Science Trans. Med. 2013, 5,
13ra165.
(11) Xiao, Y.; Lai, R.Y.; Plaxco, K.W. Nature Prot. 2007, 2, 2875–
880.
(
(
23) Ricci, F.; Lai, R.Y.; Heeger, A.J.; Plaxco, K.W.; Sumner, J.J.
Langmuir 2007, 23, 6827-6834.
24) Singh, A.; Chowdhury, D.R.; Paul, A. Analyst 2014,139 5747-
754.
2
(
2
5
12) Rowe, A.A.; Bonham, A.J.; White, R.J.; Zimmer, M.P.; Yad-
gar, R.J.; Hobza, T.M.; Honea, J.; Ben-Yaacov, I.; Plaxco, K.W. PLoS
One 2011, 6, e23783.
ACS Paragon Plus Environment

Analytical Chemistry
Page 8 of 8
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
For TOC only
8
ACS Paragon Plus Environment