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ae

Current Medicinal Chemistry, 2018, 25, 4102-4118

REVIEW ARTICLE
eISSN: 1875-533X ISSN: 0929-8673

Current
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Medicinal
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Electrochemical Nucleic Acid-Based Biosensing of Drugs of Abuse and
Pharmaceuticals
The
International
Journal for
Timely In-depth
Reviews
in Medicinal
Chemistry

BENTHAM
SCIENCE

Susana Campuzano, María Pedrero and José M. Pingarrón*

Departamento de Química Analítica, Facultad de CC. Químicas, Universidad Complutense de Madrid, E-

28040 Madrid, Spain

Abstract: Background: Studies on the interactions of DNA with small molecular drugs
are currently performed both to explore their mechanism of action and to develop new
drugs. Electrochemical biosensors offer a very promising alternative to more complex
conventional techniques for drug determination due to rapidness, low cost, simplicity,

tio ly
high sensitivity and compatibility with use in different settings. In this review, selected
electrochemical nucleic acid-based biosensing methods described so far for the determi-

bu n
nation of pharmaceuticals and illicit drugs are briefly overviewed, discussing their basics

n
tri O
and main features. A section pointing out general conclusions and future directions in this
field is also provided.
is se
Results: The 42 selected contributions described electrochemical platforms to determine
ARTICLE HISTORY drugs of interest by monitoring their specific interactions with nucleic acids (DNA and
aptamers), DNA damage and specific DNA-protein interactions. The highlighted ap-
rD U

Received: May 23, 2017

Current Medicinal Chemistry

Revised: September 06, 2017 proaches reported the use of electrodes unmodified or modified with nanomaterials and/or
Accepted: September 17, 2017
polymers in which DNA-drug interaction was followed by electrochemical detection of
fo al

DOI:
10.2174/0929867324666171121103156
DNA puric bases, active drug or diffusion-free markers, and monitoring changes in the
surface layer morphology/permeability and charge transfer resistance using different elec-
ot on

trochemical techniques.
Conclusion: Although electrochemical nucleic acid biosensing approaches constitute an
N rs

interesting option for drugs determination in terms of cost, simplicity and miniaturized in-
strumentation, validating exhaustively their performance in real samples against conventional
Pe

methodologies and implementing them into portable and automatic high throughput devices,
together with exploring novel electrode modifications with nanomaterials and polymers and
studying in more detail their multiplexing ability for analysis of a large number of analytes, is
still needed.
Keywords: Electrochemical sensors, nucleic acid, drugs, DNA damage, nanomaterials, polymers.

1. INTRODUCTION [1]. Once metabolized in the organism, these drugs

are excreted and may end as pollutants in the envi-
Drug analysis has played classically an important
ronment from where they can even reach the food
role in Analytical Chemistry due to significance in drug
chain. Moreover, their intake can sometimes lead to
quality control and wide impact on public health.
dependence and addiction and even to physical and
Drugs of abuse and pharmaceuticals are designed either
mental damage, their abuse meaning a significant
to be highly active and interact with receptors in hu-
public health problem [2]. Therefore, the development
mans or to be toxic for many pathogen organisms
of sensitive, simple, rapid, and reliable methods for
the determination of active ingredients in illegal or
*Address correspondence to this author at the Departamento de
Química Analítica, Facultad de CC. Químicas, Universidad Com- legal drugs in pharmaceutical industries is valuable
plutense de Madrid, E-28040 Madrid, Spain; E-mail: pinga- and necessary [3].
rro@quim.ucm.es

; /18 $58.00+.00 © 2018 Bentham Science Publishers

Nucleic Acid Bionsensing of Drugs
Although many analytical techniques including
fluorescence spectroscopy, high performance liquid
chromatography (HPLC), capillary electrophoresis,
enzyme-linked immunosorbent assay (ELISA) and sur-
face plasmon resonance (SPR), are currently available
for measuring drugs of abuse and pharmaceuticals,
their application has some well-known drawbacks be-
ing time consuming, high cost, requirement of sample
pretreatment, expensive apparatus and highly qualified
personnel [4]. In this sense, electrochemical biosensors
offer an interesting alternative in the field of pharma-
ceutical analysis due to their remarkable features, such
as high sensitivity and selectivity, simplicity, reasona-
bly price, reasonable accuracy and precision, relatively
short analysis times, small sample volume requirement,
compatibility with turbid samples, easily miniaturized
and inexpensive instrumentation and, usually, no need
tio ly
for complex sample pretreatment such as derivatization
bu n
or time-consuming extraction steps, because of the
lower sensitivity of electroanalytical methods to matrix
n
tri O
effects [3-13]. Among electrochemical biosensors, in
recent years, considerable interest has been devoted to
is se
DNA sensors to develop new methods for the determi-
nation of illicit drugs, antibiotics and vitamins. Electro-
rD U
chemical DNA biosensors comprise a nucleic acid rec-
ognition layer immobilized onto an electrochemical
transducer that detects the changes occurring in the
fo al
DNA structure during interaction with other comple-
ot on
mentary oligonucleotides or with small molecules [8,
12-17].
N rs
In order to obtain the biochemical mechanisms of
prevention and treatment of human diseases, great ef-
Pe
forts have been made to investigate the interaction be-
tween DNA and drug molecules. Moreover, nucleic
acids interaction with drugs is a fundamental issue in
life phenomena [15]. The function of DNAs can be ar-
tificially modulated by small molecule ligands that re-
versibly bind to them inhibiting their activity. There-
fore, the studies on the binding nature of these small
molecules with DNA are interesting not only in under-
standing the interaction mechanisms and dynamics, but
also for speeding up the rational design of new phar-
maceutically active compounds drugs [6, 18, 19]. By
observing the electrochemical signal related to DNA-
drug interactions, it is possible to propose the mecha-
nism of interaction, adduct conformation, nature of the
complex formed, and to evaluate the binding constant
[20, 21].
The interactions of DNA with drug molecules may
be arranged either in solution or on an electrode surface
upon immobilization of either DNA or the drug mole-
cule. The DNA-trapped compounds can be detected
Current Medicinal Chemistry, 2018, Vol. 25, No. 33 4103
directly, if they are electroactive molecules, or through
changes in an electrochemical signal related to DNA
[22]. In a modified electrode, changes of current inten-
sity and/or peak potential in the electrochemical re-
sponse of DNA nitrogenous base residues can reflect
alterations in the double-stranded DNA (dsDNA) struc-
ture. In the solution phase, the interaction of com-
pounds with dsDNA can be monitored by alterations in
the electrochemical response of the target compound
[23]. In both cases the concentration of these drugs can
also be determined using the drug-DNA interaction
[12].
Binding of small molecules to DNA occurs cova-
lently and non-covalently and results in their pre-
concentration on the DNA sensors surfaces, leading to
an enhanced sensitivity for the drug and a better peak
definition [24]. The non-covalent bonding may involve
intercalation between the stacked base pairs of native
dsDNA, non-specific electrostatic interactions with the
negative-charged sugar-phosphate out of the DNA
structure and binding interactions with both minor and
major grooves of the DNA double helix [22, 23, 25].
These reactions cause changes in the structure of DNA
and the base sequence, leading to perturbation of DNA
replication [26]. Intercalation and groove binding are
the two most common direct binding modes of small
molecules to DNA. Intercalation, which is an enthalpi-
cally driven process, encompasses inserting planar or
nearly planar aromatic ring systems between DNA base
pairs with concomitant unwinding and lengthening of
the DNA helix [26]. The intercalation of compounds
into DNA as an initial step leading to the formation of
covalent adducts is an especially undesirable feature,
since the intercalated compounds can lead to mutations
and carcinogenesis [23]. In contrast, groove binding,
which is predominantly entropically driven, involves
direct interaction of the compound with the edges of
the base pairs in the major or minor grooves of dsDNA,
extending to fit over many base pairs, and with very
high sequence specificity. This binding mode does not
perturb the duplex structure in a large extent [22, 26,
27]. Minor groove binding makes intimate contacts
with the walls of the groove and, as a result of this in-
teraction, numerous hydrogen binding and electrostatic
interactions occur between a drug and DNA bases and
its phosphate backbone [25, 28].
The interaction between the drug and DNA can be
irreversible, causing subsequent destabilization, break-
ing of the hydrogen bonds, and opening of the double
helix. This would lead to exposure of the DNA purine
bases guanine and adenine, which can be oxidized at
around +0.8 V and +1.1 V, respectively, on carbon
4104 Current Medicinal Chemistry, 2018, Vol. 25, No. 33
electrodes [7]. Therefore, direct monitoring of changes
in the DNA's electrochemical properties, such as de-
crease in the oxidation of guanine and adenine signals,
which are explained by the damage or shielding of the
oxidizable group of these electroactive bases due to the
adsorption of the drug [6, 21, 29], can be used to evalu-
ate the interaction of small molecules with DNA on the
electrode surface or to monitor the DNA damage
through a simple and label-free approach [30, 31]. Car-
bon electrodes have been extensively used for this pur-
pose, in particular pencil graphite electrodes (PGEs),
carbon paste electrodes (CPEs) and glassy carbon elec-
trodes (GCEs) [21]. Other electrodes have also been
used in the investigation of the interaction between
drugs and DNA, including the hanging mercury drop
electrode (HMDE), gold electrode (AuE), and screen-
printed carbon electrodes (SPCEs) [28]. It should be
tio ly
mentioned in this point that there is an increasing ten-
bu n
dency to develop electrochemical DNA biosensors us-
n
ing disposable electrochemical substrates such as PGEs
tri O
and SPCEs because of their mass production, low cost,
potential for miniaturization, facility of automation,
is se
and easy implementation in simple and portable
equipment [11, 16]. The electrochemical techniques
rD U
more often used include voltammetry, amperometry,
chronopotentiometry and others based on measuring
fo al
the changes in redox potential or current before and
after the DNA-interaction [9, 32, 33]. Although these
ot on
techniques have been widely used, they show some
limitations due to the interference of redox potential or
N rs
current of both DNA and drug molecules overlapping
with each other. Moreover, as electrochemical reac-
Pe
tions of either DNA or drug molecules may result in
damage to their bioactivity and properties, other elec-
trochemical techniques not involving the electrochemi-
cal reaction of DNA or the target compound, such as
electrochemical impedance spectroscopy (EIS) or the
voltammetric measurement of an external redox probe
such as [Fe(CN)6]4-/3- or methylene blue (MB), have
been used to study the interaction between DNA and
drug molecules [32].
To meet the requirements of trace determination and
since DNA immobilization on the electrode surface
plays an important role in the performance of DNA
biosensors, different nanomaterials (multiwalled car-
bon nanotubes (MWCNTs), bamboo-like MWCNTs
(bMWCNTs), SiO2/Al2O3/Nb2O5, TiO2 nanoparticles
(NPs), Fe3 O4 NPs, gold-coated Fe2 Ni@Au magnetic
nanoparticles and SiO2 NPs, among others) and poly-
mers (chitosan (CHIT), poly-(5-amino-2-mercapto-
1,3,4-thiadiazole) (PAMT), polyaniline (PANI)), with
well-known electronic and high redox reversibility
Campuzano et al.
properties have been used [3, 34]. Moreover, the sensi-
tivity of DNA-sensors can be further improved by cou-
pling the DNA-drug interaction with enzymatic oxida-
tion by using external active redox compounds able to
work also as efficient mediators of electron transfer on
enzymes [33].
Although less explored than DNA-based electro-
chemical sensors based on the detection of DNA-drug
interaction, other electrochemical sensors relying on
monitoring DNA damage and on the use of aptamers or
specific DNA-protein interactions have been developed
for the determination of drugs of abuse and pharmaceu-
ticals. This article reviews methods reported in the last
5 years for the determination of pharmaceuticals and
illicit drugs using electrochemical DNA sensors. Al-
though most common strategies involve the preparation
of integrated DNA sensors, a few examples of method-
ologies involving DNA-drug interaction in solution are
also considered. Moreover, a critical analysis of the
state-of-the-art, the existing challenges and future di-
rections in this topic are also discussed.
2. ELECTROCHEMICAL SENSORS BASED ON
DRUG-DNA INTERACTION
2.1. DNA Electrochemical Sensors at Non-modified
Electrodes
Due to the great advantages offered by electro-
chemical techniques, electrochemical DNA sensors
have been widely applied in the determination of drugs
such as antidepressants [17, 20], antibiotics [23, 24,
35], vitamins [7, 36], or anticancer drugs [6, 10, 11, 13,
16, 21, 29, 32, 37, 38], based on their interaction with
DNA. The developed methods are mainly based on the
differences observed in the electrochemical behavior of
the drugs in the absence and presence of dsDNA as
follows: (1) the dramatic decrease/increase in the oxi-
dation/reduction peak current of the redox active drug
which selectively binds with dsDNA; (2) the de-
crease/increase in the oxidation/reduction peak current
of the electroactive DNA bases such as guanine or ade-
nine; (3) the shifts in the formal potentials of the redox
couple caused by the intercalation of nucleic acid-
binding molecules into dsDNA [31]. Table 1 summa-
rizes the main features of the selected DNA electro-
chemical sensors reported for drug determination based
on drug-DNA interaction.
Guo et al. [32] developed an electrochemical
method for determining the anti-tumor drug tamoxifen
(TAM) using zero current potentiometry and immobi-
lizing calf thymus dsDNA on a CP surface. The inter-
action of dsDNA with TAM was measured by the
Nucleic Acid Bionsensing of Drugs Current Medicinal Chemistry, 2018, Vol. 25, No. 33 4105

Table 1. DNA-based electrochemical sensors reported for drug and pharmaceuticals determination based on drug-
DNA interactions.

Electrochemical
Binding Drug/Type of
Electrode Strategy Sample Technique (signa- L.R. LOD Ref.
mode drug
ling compound)

DNA electrochemical sensors at non-modified electrodes

CP connected
in series
between the
clips of work- 2.0×10−7
Interaction of TAM
ing and TAM / antitu- Zero current poten- - 1.1×10−7
with calf thymus −6 [32]
counter elec- mor tiometry 8.0×10 M
dsDNA/CP
trodes of a M
potentiostat
and a refer-
ence electrode
Interaction of vita-

tio ly
min B6 with Spiked real 0.10- 0.040
GCE Vitamin B6 CV (vitamin B6) [36]
salmon ssDNA- tablets 6.00 mM mM

bu n
GCE

n
tri O
Interaction of BZP
BZP /
GCE with calf thymus Intercalation DPV (BZP) [20]
antidepressant
dsDNA-GCE
is se
Interaction of TET Spiked phar-
0.30-
with UV- calf Electrostatic TET / antibio- maceutical
GCE DPV (TET) 90.00 0.27 μM [35]
thymus dsDNA- interaction tic formulations
μM
rD U

GCE and milk

Intercalation
Interaction of ETO Spiked
fo al

and electros- ETO / antitu- 5.0×10−9

SPCE with salmon sperm human serum DPV (ETO) [16]
tatic interac- mor M
dsDNA-SPCE samples
ot on

tion
Interaction of DTIC Spiked
DTIC / antitu- 2.0×10-8
SPCE with salmon sperm Intercalation human serum DPV (DTIC) [11]
N rs

mor M
dsDNA-SPCE samples
Intercalation
Pe

Interaction of NTZ Spiked

and groove NTZ / antipara- 0.1-25.0 0.025
SPCE with salmon sperm human serum DPV (NTZ) [39]
binding sitic μM μM
dsDNA-SPCE samples
modes
Interaction of FLV Pharmaceuti- 1.00-
FLV / antican- 0.41 µg
PGE at fish sperm Intercalation cal dosage DPV (guanine) 20.00 µg [29]
cer mL-1
dsDNA- PGE forms mL-1
Interaction of LPR Pharmaceuti- 0.005-
LPR / antican- 0.0014
PGE at fish sperm cal dosage AdSDPV (guanine) 0.20 [6]
cer ppm
dsDNA- PGE form ppm
Interaction of LPT SWV (LPT+ct-
LPT / antican-
PGE at calf thymus Intercalation dsDNA anodic peaks [25]
cer
dsDNA-PGE combination)
Interaction of Su-
Spiked chili
dan II with calf Sudan II / DPV (guani- 0.5-6 μg 0.43 μg
PGE Intercalation and ketchup [26]
thymus dsDNA- carcinogen ne+adenine) mL−1 mL−1
sauce
PGE
Interaction of folic
Groove bin- Folic acid Wheat flour 0.1-10.0 1.06×10−
PGE acid with salmon DPV (adenine) 8 [7]
ding /vitamin and spinach μM M
sperm dsDNA-PGE
(Table 1) contd….
4106 Current Medicinal Chemistry, 2018, Vol. 25, No. 33 Campuzano et al.

Electrochemical
Binding Drug/Type of
Electrode Strategy Sample Technique (signa- L.R. LOD Ref.
mode drug
ling compound)

Interaction of
9.0×10-7-
mangiferin with Mangiferin / Urine sam- DPV (guani- 3.9×10-7
PGE 4.5×10-5 [10]
salmon sperm antitumor ples ne+adenine) M
M
dsDNA-PGE
Spiked phar-
Interaction of taxol maceutical
2.0×10-7-
with salmon sperm Taxol / anti- preparations, DPV (guani- 8.0×10-8
PGE Intercalation 1.0×10-5 [13]
dsDNA-modified cancer human blood ne+adenine) M
M
PGE serum and
urine samples
Intercalative
Interaction of PRX
and electros- PRX / antican-
PGE with fish sperm DPV (guanine) [21]
tatic interac- cer
dsDNA-PGE
tion
Interaction of CIP

tio ly
Electrostatic 1.0-10.0
GCE with calf thymus CIP / antibiotic AdSDPV (CIP) 0.117 μM [24]
interaction μM
dsDNA-GCE

bu n
4.50×10-

n
tri O
Interaction of ARP 7
ARP / antipsy- DPV (guani- -
GCE with calf thymus [17]
chotic ne+adenine) 1.12×10-
dsDNA-GCE 6
is se M
Interaction of mor-
phine with dsDNA Intercalative Spiked urine
rD U

immobilized onto and electros- Morphine / and blood 0.05-500 0.01

AuE DPV (morphine) [27]
mercapto- tatic interac- analgesic plasma sam- μmol L-1 μmol L-1
fo al

benzaldehyde- tion ples

modified AuE
ot on

Interaction with Spiked synt-

0.500 - 0.440
BDD calf thymus dsDNA Intercalation CIP / antibiotic hetic urine DPV (CIP) [23]
60.0 μM μM
in solution samples
N rs

Interaction of keta- Spiked

Groove bin- Ketamine /
CPE mine with dsDNA- human serum DPV (guanine) 1.98 nM [40]
Pe

ding analgesic
CPE samples
Interaction of ETZ
with calf thymus
ETZ / antican-
CMCPE dsDNA in solution SWV (ETZ) + EIS [37]
cer
and detection at
CMCPE
Interaction of
6-TG tablets
salmon sperm 16.0 -
6-TG / antican- and spiked AdSV + SWV (6-
HMDE dsDNA with 6-TG Intercalation 360.0 1.1 μM [38]
cer blood serum TG)
at the surface of μM
samples
HMDE
0.7-40
Electrostatic
μM 0.07 μM
Interaction of mor- mode (code-
Morphine and (morp- (morp-
phine and codeine ine); Interca- Spiked urine DPV (morphine and
SPCE codeine / anal- hine); hine); [22]
with calf thymus lative interac- samples codeine)
gesic 2.3-40 0.23 μM
dsDNA-SPCE tion mode
μM (codeine)
(morphine)
(codeine)
(Table 1) contd….
Nucleic Acid Bionsensing of Drugs Current Medicinal Chemistry, 2018, Vol. 25, No. 33 4107

Electrochemical
Binding Drug/Type of
Electrode Strategy Sample Technique (signa- L.R. LOD Ref.
mode drug
ling compound)

DNA electrochemical sensors using polymer-modified electrodes

Interaction of MC 4.47 μg
with calf thymus MC / antican- DPV (MC and gua- mL-1
PGE [28]
dsDNA- CHIT-IL- cer nine) (13.37
PGEs μM)
Pharmaceuti-
Interaction of NFT cal dosage
Intercalation
with NFT / antibio- forms and 2-25 mg 0.65 mg
GCE and electros- DPV (guanine) [8]
dsDNA/PAMT/GC tic spiked human L−1 L−1
tatic binding
E serum sam-
ples
1×10−10-
1×10−8
and
1×10−8-
0.01 nM
2×10−4
(doxoru-

tio ly
M
bicin),
Interaction of an- Spiked com- (doxoru-
Anthracycline 0.1 nM

bu n
thracycline drugs mercial EIS+CV bicin);
GCE Intercalation drugs / antitu- (dauno- [42]
with PANI/DNA- preparation of ([Fe(CN)6]3-/4-) 5×10−101

n
tri O
mor rubicin);
modified GCE doxorubicin ×10−5 M
0.2 nM
(dauno-
(idarubi-
rubicin);
is se cin)
1×10−9-
1×10−4
M (ida-
rD U

rubicin)
Interaction of 5-FU
1.0-8.5
fo al

with dsDNA im-

5-FU / antibio- Pharmaceuti- and 8.5- 0.31 mg
GCE mobilized at Intercalation DPV (guanine) [12]
tic cal sample 50 mg L- L-1
ot on

P(BCP)-modified 1
GCE
Spiked phar-
N rs

Interaction of GEM
maceutical
with fish sperm
Groove bin- GEM / antican- dosage forms 1-30 mg 0.276 mg
GCE dsDNA immobi- DPV (guanine) [30]
L
1 L
1
Pe

ding cer and human

lized at P(PDCA)-
serum sam-
modified GCE
ples
Interaction of
BNN-17 with fish
Spiked 0.213-
sperm dsDNA Groove bin- BNN-17 / 0.063
GCE human serum DPV (guanine) 32.03 [34]
immobilized at ding anticancer μmol L−1
samples μmol L−1
P(AT)-modified
GCE
DNA electrochemical sensors using electrodes modified with nanomaterials
Interaction of PMZ
20-
within DNA- PMZ / antipsy- Pharmaceuti- 5.9×10−6
MWCNTPE Intercalation SWV (PMZ) 100×10−6 [9]
SiAlNb chotic cal samples M
M
MWCNTPE
Carbon paste
composed of Interaction of AMT
AMT / analge- Pharmaceuti- 1,080
graphite and with Intercalation DPV (AMT) 0.12 μM [46]
sic cal samples μM
SiO2/Al2O3/N GRT/SiAlNb/DNA
b 2 O5
(Table 1) contd….
4108 Current Medicinal Chemistry, 2018, Vol. 25, No. 33 Campuzano et al.

Electrochemical
Binding Drug/Type of
Electrode Strategy Sample Technique (signa- L.R. LOD Ref.
mode drug
ling compound)

Spiked se-
Calf thymus
rum, plasma DPV (guani- 1.0-80 0.44 μg
MWCNTPE dsDNA- Intercalation Vitamin B1 [14]
and urine ne+adenine) μg mL−1 mL−1
MWCNTPE
samples
Interaction of PMZ
PMZ / antihis- Pharmaceuti- 0.10-6.0
GCE with dsDNA- Intercalation AdSDPV (PMZ) 23 nM [44]
taminic cal product nM
bMWCNT/GCE
Interaction of atro- 0.6-30.0
pine sulfate at Atropine sulfa- Spiked blood μM and
DPV (guani-
PGE dsDNA-PDDA- Intercalation te / antimusca- serum and 30.0- 30.0 nM [3]
ne+adenine)
TiO2NPs- rinic urine samples 600.0
MWCNTs/PGE μM
Interaction of GCV
Spiked serum 80-
with
GCE Intercalation GCV / antiviral and urine SWV (GCV) 53,000 20 nM [19]
DNA/Fe3O4/MWC

tio ly
samples nM
NTs/GCE

bu n
Interaction of DOX
with DOX / che- 0.003-5 8 μg mL-

n
tri O
AuDE Intercalation DPV (Fe2+/3+ redox) [47]
dsDNA/Fe2Ni@Au motherapeutic mg mL-1 1

/rGO/CYS/AuDE
is se
Interaction of TAM
i-motif DNA struc-
TAM / antitu- 0.01-1
CPE ture immobilized Intercalation SWV (TAM) 0.06 μM [45]
rD U

mor μM
onto a
SiO2NPs/CPE
fo al

Interaction of 6-MP
with salmon sperm 6-MP / anti- DPV (guani- 0.2−100
ot on

PGE Tablets 0.08 μM [48]

dsDNA/PP/MWCN cancer ne+adenine) μM
Ts/PGE
N rs

AdSDPV: adsorptive stripping differential pulse voltammetry; AdSV: adsorptive stripping voltammetry; AMT: amitriptyline; ARP: aripiprazole; AuDE: gold-
disk electrode; AuE, gold electrode; bCNT: bamboo-like multi-walled carbon nanotubes; bMWCNTs: bamboo-like multiwalled carbon nanotubes; BDD: bo-
Pe

ron-doped diamond electrode; BNN-17: 2-(2-phenyl ethyl)-5-methylbenzimidazole; BZP: buzepide methiodide; CHIT: chitosan; CIP: ciprofloxacin; CMCPE:
CPE modified with sepiolite clay; CP: carbon paste; CPE: carbon paste electrode; CV: cyclic voltammetry; CYS: cysteamine; DOX: doxorubicin; DPV: diffe-
rential pulse voltammetry; DTIC: dacarbazine; EIS: electrochemical impedance spectroscopy; ETO: etoposide; ETZ: etoposide; FLV: fulvestrant; 5-FU: 5-
fluorouracil; GCE: glassy carbon electrode; GCV: ganciclovir; GEM: gemcitabine; GRT: graphite; HMDE: hanging mercury drop electrode; IL: ionic liquid;
KANA: kanamycin; LPT: lapatinib; LPR: leuprolide; L.R.: linear range; LOD:, limit of detection; MB: methylene blue; MC: mitomycin C; 6-MP: 6-
mercaptopurine; MWCNTPE: multiwalled carbon nanotube paste electrode; NFT: nitrofurantoin; PAMT: poly-(5-amino-2-mercapto-1,3,4-thiadiazole); PANI:
polyaniline; P(AT): poly-3-amino-1,2,4-triazole-5-thiol; PDDA: poly-dialyldimethylammonium; PGE: pencil graphite electrode; PMZ: promethazine; PP:
polypyrrole; P(PDCA): poly(2,6-pyridinedicarboxylicacid); PRX: NSAID piroxicam; NTZ: nitazoxanide; rGO: reduced graphene oxide; SD: sulfadiazine;
SPCE: screen-printed carbon electrode; SWV: square wave voltammetry; TAM: tamoxifen; TET: tetracycline; 6-TG: 6-thioguanine.

change in interfacial potential at the dsDNA/CP/ solu-
tion interface in aqueous solutions containing TAM.
When linear sweep potential was applied to the
dsDNA/CP and the corresponding i-E curve was re-
corded, interfacial potential offset applied potential
partially, making the i-E curve displaced along the po-
tential axis. The displacement of the i-E curve was
checked by measuring the potential where the circuit
current was equal to zero in the i-E curve. The so called
zero current potential values were linear with log
[TAM] in the concentration range from 2.0×10−7 to
8.0×10−6 M with a limit of detection (LOD) of 1.1×10−7
M. This method is especially attractive when electro-
chemical oxidation peaks of both DNA and the target
drug overlap preventing the determination using con-
ventional detection protocols.
A voltammetric sensor for the determination of vi-
tamin B6 using a salmon single-stranded (ssDNA)-
modified GCE was proposed by Lai et al. [36]. Meas-
uring the oxidation signal of vitamin B6 by cyclic volt-
ammetry, a linear range from 0.10 to 6.00 mM, a LOD
of 0.040 mM and good recoveries in spiked real tablets
were provided by this approach. A similar method us-
ing a modified calf thymus dsDNA-GCE was proposed
Nucleic Acid Bionsensing of Drugs
for the differential pulse voltammetric determination of
the antidepressant buzepide methiodide (BZP) [20].
The electrochemical oxidation of tetracycline (TET)
measured by DPV at calf thymus dsDNA immobilized
on a GCE by UV-irradiation was proposed by Gholi-
vand et al. [35] for the determination of this antibiotic.
This UV-irradiated DNA film was water-insoluble,
resistant to nuclease hydrolysis and maintained the B-
form structure of DNA in water, so that the base pairs
were vertical for the axis of double helix. The opti-
mized electrode showed a linear dynamic range of
0.30-90.00 μM and a LOD of 0.27 μM and was used
for the analysis of spiked pharmaceutical formulations
and milk.
Radi et al. developed voltammetric methods for the
determination of three drugs which interact through
tio ly
different binding modes with dsDNA: the antitumor
drugs etoposide (ETO) [16] and dacarbazine (DTIC)
bu n
[11] and the antiparasitic drug nitazoxanide (NTZ)
n
tri O
[39]. The developed strategies involved salmon sperm
dsDNA immobilized on an anodically activated SPCE.
is se
The electrochemical detection implied the measure-
ment of the DPV oxidation signal of the drug bound to
the salmon sperm dsDNAs. The DNA sensors provided
rD U
LODs of 5.0×10−9 M (ETO), 2.0×10-8 M (DTIC) and
0.025 μM (NTZ) and demonstrated applicability for the
fo al
analysis of spiked human serum samples.
ot on
Ozkan’s research group studied the interaction of
different anticancer drugs: fulvestrant (FLV) [29], le-
uprolide (LPR) [6] and lapatinib (LPT) [25] at dsDNA
N rs
modified PGEs. FLV determination was performed by
measuring the DPV current decrease of the guanine
Pe
oxidation which resulted in a linear dependence within
the 1.00-20.00 µg mL-1 range and a LOD of 0.41 µg
mL-1. The proposed method was applied to the deter-
mination of this drug in injectable solutions. In the case
of LPR, apart from monitoring the decrease in the gua-
nine DPV signal, a more sensitive approach was devel-
oped using differential pulse adsorptive stripping volt-
ammetry which showed a linear dependence with LPR
concentration over the 0.005-0.20 ppm range and with
a LOD of 0.0014 ppm. The applicability of these fish
sperm dsDNA-PGEs for the analysis of FLV and LPR
was demonstrated by analyzing pharmaceutical dosage
forms. LPT determination involved the measurement
by square wave voltammetry of the three anodic peaks
resulting from the combination of the 3 originally from
LPT and the 2 corresponding to the DNA after the in-
teraction of the drug with the calf thymus dsDNA.
Salmon sperm dsDNA-modified PGEs were also
used by Ensafi et al. for the determination of the car-
cinogen dye Sudan II [26]. The peak current measured
Current Medicinal Chemistry, 2018, Vol. 25, No. 33 4109
by DPV for adenine and guanine was linearly depend-
ent on Sudan II concentration over the 0.5-6 g mL−1
range with a LOD of 0.43 g mL−1, and demonstrated
applicability to the analysis of spiked chili and ketchup
sauces.
A highly selective electrochemical biosensor for the
determination of folic acid (one of the most important
vitamins for normal human metabolic functions) using
a salmon sperm dsDNA modified-PGE was developed
by Mirmoghtadaie et al. [7]. By monitoring the adenine
oxidation signal by DPV, folic acid could be deter-
mined in the 0.1-10.0 μM concentration range with a
LOD of 1.06×10−8 M. This salmon sperm dsDNA-PGE
was successfully applied to the determination of folic
acid in wheat flour and spinach real samples.
Label-free electrochemical biosensors using salmon
sperm dsDNA modified PGEs were developed by Tajik
et al. for the determination of the flavonoid mangiferin
[10] and the anticancer drug taxol [13]. Both methods
monitored the decreases in the guanine and adenine
oxidation peaks measured by DPV after interaction
with the drugs. The biosensors provided linear dynamic
ranges of 9.0×10-7-4.5×10-5 M and 2.0×10-7-1.0×10-5 M
and LODs of 3.9×10-7 M and 8.0×10-8 M for mangif-
erin and taxol, respectively, and were applied to their
determination in spiked pharmaceutical preparations
(paclitaxel injection), human blood serum and urine
samples. A similar approach was used by Çeşme et al.
[21] to evaluate the interaction of the nonsteroidal anti-
inflammatory drug piroxicam with a fish sperm
dsDNA-modified PGE.
The determination of the antibiotic ciprofloxacin by
differential-pulse anodic stripping voltammetry at a
calf thymus dsDNA-GCE was proposed by Diab et al.
[24]. A linear relationship between the peak current
and the antibiotic concentration was found over the
1.0-10.0 μM with a LOD of 0.117 μM.
Kurbanoglu et al. [17] proposed the use of a GCE
modified with calf thymus dsDNA for the electro-
chemical determination of the antipsychotic agent
aripiprazole (ARP). Measuring the peak currents of
deoxyguanosine and deoxyadenosine moieties by DPV,
a linear decrease with increasing the ARP concentra-
tion was observed between 4.50×10-7 and 1.12×10-6 M.
A sensitive biosensor for morphine was developed
by Talemi et al. [27] based on the intercalative and
electrostatic interaction of morphine with NH2 -
terminated ds-DNA covalently immobilized onto the
residual aldehyde group of a mercapto-benzaldehyde-
modified AuE. By monitoring the oxidation signal of
morphine by DPV a linear dependence from 0.05 to
4110 Current Medicinal Chemistry, 2018, Vol. 25, No. 33
500 μmol L-1 and a LOD of 0.01 μmol L-1 morphine
were obtained. Results demonstrated also that this sen-
sor could be applied to the determination of morphine
in spiked urine and blood plasma samples.
Garbellini et al. [23] reported the voltammetric de-
termination of ciprofloxacin through its interaction
with calf thymus dsDNA in solution on a cathodically
pretreated boron-doped diamond electrode (BDD). By
monitoring the electrooxidation of this fluoroquinolone
using DPV, a linear response was found over the 0.500
to 60.0 μM. A LOD of 0.440 μM and successful de-
termination in spiked synthetic urine samples were re-
ported. However, the use of the cathodically pretreated
BDD electrode did not improve significantly the ana-
lytical characteristics reported previously by Diab et al.
[24] for a calf thymus dsDNA-GCE.
tio ly
A dsDNA (a short sequence of p53 gene)-CPE was
used to determine the analgesia drug ketamine by
bu n
monitoring the oxidation signal of guanine using DPV
n
tri O
[40]. This biosensor provided a LOD of 1.98 nM and
successful applicability to the analysis of spiked human
serum samples.
is se
A sepiolite clay modified CPE was employed to
rD U
study the etoposide (ETZ) interaction with calf thymus
dsDNA in solution by SWV (ETZ oxidation signal)
fo al
and at the dsDNA-modified CPE by EIS [37]. A de-
crease in the ETZ SWV peak current was observed as a
ot on
result of the interaction of this drug with the base pairs
of dsDNA in homogeneous solution. This interaction
was also confirmed by EIS after the incubation of the
N rs
dsDNA-CPE with ETZ.
Pe
An electrochemical DNA sensor for the determina-
tion of the anticancer drug 6-thioguanine (6-TG) at a
HMDE was proposed by Mirmomtaz et al. [38]. The
method was based on the interaction of salmon sperm
dsDNA with 6-TG at the surface of the HMDE in neu-
tral buffer. An adsorptive stripping voltammetry proto-
col was applied for accumulation of ds-DNA at the
HMDE, and subsequent SWV was used for measuring
the reduction signals of 6-TG. This electrochemical
method provided a low LOD (1.1 μM), a dynamic
range of 16.0 to 360.0 μM and successful performance
for the determination in 6-TG tablets and spiked blood
serum samples.
Feizbakhsh et al. [22] reported an electrochemical
sensor for the simultaneous determination of morphine
and codeine analgesia drugs constructed using SPCEs
modified with calf thymus dsDNA coupled with an
electro-membrane microextraction (EME) step. By
measuring the oxidation signals of both drugs using
DPV, linear ranges of 0.7-40 μM and 2.3-40 μM and
Campuzano et al.
LODs of 0.07 μM and 0.23 μM were obtained for mor-
phine and codeine, respectively. This method demon-
strated to be effective for the simultaneous determina-
tion of both drugs in spiked urine samples.
A DNA-based electrochemical device was used to
determine β-lapachone (β-lap) following the DNA
damaging activity induced by the Fenton reaction in the
presence of this anticancer agent and subsequent base
excision repair (BER) activity by formamido
pyrimidine glycosylase (FPG) [41]. This electrochemi-
cal strategy tracked the β-lap-induced, NAD(P)H: qui-
none oxidoreductase 1 (NQO1)-dependent DNA dam-
age and its subsequent repair activity by simulating
features of the cellular environment in a controlled
format (Fig. 1). For this purpose, authors prepared 16-
electrode devices with fully well-matched, undamaged
DNA monolayers bearing redox probes (Nile Blue,
NB) on gold electrodes. These devices utilized a clamp
and gasket assembly that supported two wells over the
chip, dividing them in two halves of 8 electrodes: one-
half of this chip was the drug side, with all cofactors
necessary for generation of β-lap-induced H2 O2, DNA
damage, and damage repair and the other half corre-
sponded to the control side where all cofactors except
the drug were present. In this arrangement β-lap, in the
presence of biologically-high levels of NQO1 and
NADH produced superoxide that converted to H2 O2 ,
which, in the presence of iron in the vicinity of DNA
caused oxidative DNA damage, such as 8-oxoguanine.
This damage was repaired by FPG, an 8-oxoguanine
repair enzyme, which removed the damage base thus
lowering the SWV signal of the NB. This chip-based
platform enabled the determination of ß-lap at thera-
peutic levels and the detection of sub-lethal levels of
this drug, providing a unique synthetic platform for
tracking of drug-induced damage repair processes nor-
mally confined inside cells.
2.2. DNA Electrochemical Sensors Using Polymer-
modified Electrodes
In recent years, conducting polymers have attracted
much attention because of their electrical, thermal, envi-
ronmental, optical, chemical, and biological properties.
Some conducting polymers such as CHIT, PAMT or
PANI, have been widely used as immobilizing substrates
for nucleic acids in the development of electrochemical
DNA-based sensors for drugs determination [8].
An electrochemical DNA biosensor, prepared by
immobilization of calf thymus dsDNA on a CHIT/ionic
liquid modified-PGE, was developed for the determina-
tion of the anticancer drug mitomycin C (MC). This
Nucleic Acid Bionsensing of Drugs
Fig. (1). DNA-based electrochemical device developed to follow the DNA damage induced by the Fenton reaction in the pres-
ence of β-lap. Reprinted from [41] with permission.
biosensor, which measured for the first time the oxida-
tion signals of MC and guanine in the same DPV scan,
allowed a LOD of 4.47 μg mL-1 (13.37 μM) [28].
Another electrochemical DNA biosensor was pre-
tio ly
pared by adsorption of dsDNA on a PAMT modified
bu n
GCE for the determination of the antibiotic nitrofuran-
n
tri O
toin (NFT) [8]. Using this approach, the decrease in the
guanine oxidation peak current measured by DPV was
linearly proportional to the concentration of NFT in the
is se
range of 2-25 mg L−1 and the LOD was found to be
0.65 mg L−1. The practical utility of this biosensor was
rD U
demonstrated by the analysis of pharmaceutical dosage
forms and spiked human serum samples.
fo al
Shamagsumova et al. [42] developed an electro-
ot on
chemical biosensing strategy for anthracycline antitu-
mor drugs based on the use of GCEs covered with
PANI obtained by electropolymerization in the pres-
N rs
ence of native DNA and oxalic acid as doping agents.
The decrease in the electron transfer resistance and
Pe
suppression of redox probe current ([Fe(CN)6]3-/4-)
measured by EIS and CV after incubation of the sensor
with the drugs were used for the determination of the
drugs. While the calibration curve for doxorubicin
showed two linear sections 1×10−10-1×10−8 and 1×10−8 -
2×10−4 M, only one linear concentration range was ob-
served for daunorubicin (5×10−10-1×10−5 M) and idaru-
bicin (1×10−9-1×10−4 M). The method allowed LODs
of 0.01 nM (doxorubicin), 0.1 nM (daunorubicin) and
0.2 nM (idarubicin). The usefulness of the biosensor
was demonstrated for the determination of daunorubi-
cin in a spiked commercial preparation of doxorubicin.
Zeybek et al. [12] developed a sensitive electro-
chemical DNA biosensor for the antineoplastic drug 5-
fluorouracil (5-FU) based using a GCE modified with
poly (bromocresolpurple) (P(BCP)) and fish sperm
dsDNA. The P(BCP) film was electrosynthesized by
CV and the dsDNA was electrochemically immobilized
on the surface of the P(BCP)-modified GCE. The de-
Current Medicinal Chemistry, 2018, Vol. 25, No. 33 4111
crease in the guanine oxidation peak current measured
by DPV observed after the interaction of dsDNA and
5-FU (based mainly on intercalation and partially on
electrostatic effect in the experimental conditions se-
lected) was used to determine the antibiotic concentra-
tion. Two linear regions were observed for 5-FU de-
termination (1.0-8.5 and 8.5-50 mg L-1) and the calcu-
lated LOD was 0.31 mg L-1. This biosensor was applied
for the determination of 5-FU in a commercial injection
solution form. Same authors developed similar meth-
ods for the determination of the anticancer drugs gem-
citabine (GEM) [30] and 2-(2-phenylethyl)-5-
methylbenzimidazole (BNN-17) [34] by using
poly(2,6-pyridinedicarboxylicacid) (P(PDCA)) and
poly-3-amino-1,2,4-triazole-5-thiol (P(AT)) modified
GCEs, respectively. In these strategies the guanine oxi-
dation peak current was linearly proportional to the
concentrations of GEM in the 1-30 mg L-1 range and of
BNN-17 in the 0.213-32.03 μmol L−1 range. The esti-
mated LOD values were 0.276 mg L-1 and 0.063 μmol
L−1 for GEM and BNN-17, respectively. The applica-
bility of these biosensors for the analysis of spiked
commercial injection formulations [30] and human se-
rum samples [30, 34] was successfully demonstrated.
2.3. DNA Electrochemical Sensors Using Electrodes
Modified with Nanomaterials
Through the development of nanotechnology and
nanoscience, different nanomaterials (MWCNTs,
bMWCNTs, SiO2/Al2 O3/Nb2 O5, TiO2 NPs, Fe3 O4NPs,
gold-coated Fe2 Ni@Au magnetic nanoparticles and
SiO2 NPs) have been used as electrode modifiers to im-
prove the analytical performance of DNA-based elec-
trochemical sensors for drugs determination [43].
MWCNT paste electrodes (MWCNTPEs) and
MWCNTs or bMWCNTs-modified electrodes have
demonstrated to possess high conductivity and adsorp-
tion capacity as well as excellent electrocatalytic prop-
erties when used to implement nucleic acid-based elec-
4112 Current Medicinal Chemistry, 2018, Vol. 25, No. 33
trochemical sensors [9, 15, 18, 44]. MWCNTs and
Fe3 O4NPs have been combined in a practical strategy
to improve the electrode performance, this combination
resulting in synergistic effects (unique structure, high
chemical stability, high surface area, relatively good
conductivity and desirable biocompatibility) toward
target analysis [19]. Although inorganic silica nanoma-
terials, such as SiO2/Al2 O3/Nb2 O5 and SiO2, are incor-
porated in the composites generally with the aim of
increasing the stability of the immobilized electroactive
species, additional properties of this nanomaterials
such as their good biocompatibility, easy preparation,
enhanced adsorption capacity, acid-base chemistry, and
thermal stability, can also be advantageously exploited,
for example, for the accumulation of electroactive spe-
cies prior to electrochemical detection. Furthermore,
the interesting surface chemistry of silica nanomaterials
tio ly
allowed them to be grafted with a variety of functional
bu n
groups leading to a considerable enhancement of their
n
surface properties [9, 45].
tri O
Marco et al. [9] reported a simple and rapid SWV
is se
method for determination of the antipsychotic drug
promethazine (PMZ) in raw material and a pharmaceu-
tical formulation using a DNA-modified MWCNTPE.
rD U
The composite biosensor relied not only on the
MWCNTPE but also on an inorganic material, SiO2/
fo al
Al2 O3/Nb2O5 (SiAlNb), where the herring sperm DNA
was adsorbed. The method allowed the determination
ot on
of PHZ in a 20-100×10−6 M linear range with a LOD of
5.9×10−6 M, and was successfully applied for the PMZ
N rs
determination in bulk pharmaceutical and pharmaceuti-
cal formulations containing this drug. Same group de-
Pe
veloped a DPV method with a biosensor using a com-
posite of carbon paste composed of graphite (GRT) and
silica modified with niobium oxide, alumina and ad-
sorbed DNA (GRT/SiAlNb/DNA) for the analgesic
drug amitriptyline determination [46]. A linear range
from 10 to 80 μM, a LOD of 0.12 μM, and successful
applicability to the analysis of a bulk pharmaceutical
and pharmaceutical formulations containing amitrip-
tyline was reported.
An electrochemical sensor for the determination of
vitamin B1 was developed using a MWCNTPE modi-
fied with calf thymus dsDNA [14]. The oxidation sig-
nals of guanine and adenine were measured by DPV and
showed a linear range of 1.0-80 μg mL−1 and a LOD of
0.44 μg mL−1. This biosensor was successfully applied
to the analysis of spiked serum, plasma and urine.
The determination of PMZ, has also been accom-
plished with a GCE modified with bMWCNTs dis-
persed in calf thymus dsDNA (dsDNA-
Campuzano et al.
bMWCNT/GCE) using AdsDPV with medium ex-
change [44]. The dsDNA-bMWCNT/GCE allowed the
quantification of PMZ in a linear range between 0.10-
6.0 nM with a LOD of 23 nM and was successfully
used for the analysis of a pharmaceutical product con-
taining PMZ.
Ensafi et al. [3] developed an electrochemical sen-
sor for the determination of atropine sulfate, prescribed
for systemic anesthesia and used also as an anti-
arrhythmic drug, based on the immobilization of
salmon sperm dsDNA onto a PGE modified with
MWCNTs, titanium dioxide nanoparticles and poly-
dialyldimethylammonium chloride. By measuring by
DPV the guanine and adenine oxidation responses, the
biosensor allowed the determination of atropine sulfate
in the linear ranges of 0.6 to 30.0 μM and 30.0 to 600.0
μM with a LOD of 30.0 nM and demonstrated success-
ful applicability to the analysis of spiked blood serum
and urine samples.
Paimard et al. [19] described the determination of
the antiviral drug ganciclovir (GCV) using a biosensor
prepared by adsorbing DNA onto a
Fe3 O4NPs/carboxylated MWCNTs-modified GCE. By
monitoring the electrochemical oxidation of GCV by
SWV, a linear relationship between peak current and
the concentration of GCV was found over the 80-
53,000 nM range with a LOD of 20 nM. Satisfactory
quantification of GCV in spiked serum and urine sam-
ples was reported.
Nanostructured surface-enhanced Raman scattering
spectroscopic (SERS)-electrochemical biosensors
modified with a dsDNA sequence of the breast cancer
gene BRCA1 were explored for drug determination
[47]. A gold-disk electrode (AuDE) modified with a
cysteamine (CYS) self-assembled monolayer was fur-
ther coated with reduced graphene oxide (rGO) and
decorated with plasmonic gold-coated Fe2 Ni@Au
magnetic nanoparticles functionalized with dsDNA
(dsDNA/Fe2 Ni@Au/rGO/CYS/AuDE) (Fig. 2) was
used for the determination of the model chemothera-
peutic drug doxorubicin (DOX). Measuring the oxida-
tion signal of the Fe2+/3+ redox probe using DPV, the
nanobiosensors provided a semilog linearity with DOX
concentration over 3 orders of magnitude (0.003 to 5
mg mL-1) and a LOD of 8 μg mL-1.
Heydari et al. [45] reported the use of CPE sensors
modified with SiO2 nanoparticles and cysteine as im-
mobilization scaffolds of i-motif DNA structure (a
highly ordered structure formed by the oligonucleotides
containing tracts cytosines) of human telomeric DNA
Nucleic Acid Bionsensing of Drugs
Fig. (2). SERS/electrochemical nanobiosensor developed by using an AuDE substrate modified with a CYS SAM, rGO and
Fe2Ni@AuNPs functionalized with dsDNA for the determination of DOX. Reprinted and adapted from [47] with permission.
sequences to perform the electrochemical determina-
tion of TAM (the most important hormonal agent for
treatment of breast cancer). By measuring the electro-
chemical oxidation of TAM attached to the i-motif
DNA using SWV, the biosensor allowed a linear dy-
namic range between 0.01-1 μM and a LOD of 0.06
μM which is lower than that obtained previously (see
tio ly
section 2.1) by Guo et al. using a potentiometric calf
thymus dsDNA/CP sensor (LOD 1.1×10−7 M) [32].
bu n
n
tri O
A DNA biosensor using a PGE modified with
polypyrrole (PP)/functionalized MWCNTs (MWCNTs/
COOH) has been reported for the determination of 6-
is se
mercaptopurine (6MP) anticancer drug [48]. The bio-
sensor involved the immobilization of salmon sperm
rD U
dsDNA on the PP/MWCNTs/PGE at a constant poten-
tial and the measurement of the oxidation responses of
fo al
guanine and adenine by DPV. The decrease in the gua-
nine peak current was linear with 6-MP concentration
ot on
over the 0.2−100 μM range with a LOD of 0.08 μM.
The sensor was employed to determine the analyte in
N rs
6-MP tablets.
Pe
2.4. DNA Electrochemical Sensors Involving the Use
of Enzymes
Other reported strategies for the determination of
drugs with DNA-based electrochemical sensors imply
the use of electroactive substances, able to work as ef-
ficient mediators of electron transfer on enzymes, in-
teracting with DNA covalently and non-covalently,
such as phenothiazine dyes like MB [5]. For example,
Evtugyn et al. [33] developed an electrochemical sen-
sor for the determination of sulfonamides and anthra-
cyclines based on the use of a GCE modified with
dsDNA and horseradish peroxidase (HRP) and the
phenothiazine dyes MB and Methylene Green as elec-
trochemical markers. Authors demonstrated that while
sulfonamides diminished the surface concentration of
MB accessible for HRP reaction, anthracyclines re-
leased the intercalated marker and increased the volt-
ammetric signal obtained by enzymatic reduction of
H2 O2. The DNA-HRP sensor made possible the detec-
tion of 0.002 nM sulfamethoxazole, 0.1 nM sulfadiaz-
Current Medicinal Chemistry, 2018, Vol. 25, No. 33 4113
ine, 0.01 nM sulfamethazine, 0.1 nM sulfaguanine,
0.05 µM rubomycin and 0.08 µM doxorubicin. Another
amperometric biosensor based on the use of HRP, the
H2 O2/MB system and calf thymus dsDNA was applied
for sulfonamides determination [5]. In the absence of
sulfonamides, the presence of calf thymus ssDNA led
to a decrease in the MB cathodic amperometic signal
upon the addition of H2O2/MB, as a consequence of
binding of MB with free guanine bases in the ssDNA.
Conversely, in the presence of sulfonamides a lower
decrease in the signal was observed because of their
competition with MB for the DNA binding sites. Using
this approach a LOD of 0.019 μM (4.9 μg L−1) was
reported for sulfamethoxazole.
3. APTAMER-BASED ELECTROCHEMICAL
SENSORS
Aptamers are artificial ss- RNA or DNA oligonu-
cleotides screened from synthetic DNA/RNA libraries
[43]. Comparing to immunological and chemical rec-
ognition molecules, aptamers have better target versa-
tility, which include both small molecules, such as
drugs, and large molecules, such as proteins and cells.
In addition, aptamers are more stable to denaturation
and degradation than antibodies and are easy to pro-
duce and modify through chemical synthesis [49]. Ap-
tamers have been used in connection with electro-
chemical sensors to determine antibiotics such as ka-
namycin (KANA), tetracycline (TET), phenylethano-
lamine (PHL), clenbuterol (CLB), ractopamine (RAC),
salbutamol (SAL) and procaterol (PRO).
Wang et al. [4] reported the first approach combin-
ing a signal-on electrochemical DNA sensor with a
multiple recycling amplification strategy for the deter-
mination of antibiotics. The method implied an ingen-
ious design of a hairpin aptameric probe (HP1) which
was selectively opened in the presence of the target
antibiotic (KANA) and triggered the polymerase-
assisted target recycling amplification which resulted in
autonomous generation of secondary target. The multi-
ple recycling amplification strategy used involved po-
lymerase-catalyzed target recycling amplification
4114 Current Medicinal Chemistry, 2018, Vol. 25, No. 33 Campuzano et al.

a* c*
a*
antibiotics a b c
a b c
a a* c*
b c b c a b c
a* a* a
c*
a b c b c
bc
5’ 3’
antibiotics 5’

HP1 5’ 3’
3’ 5’
primer
a*
phi29 Signal-on Signal-off
Nt. Alw I 5’ 3’ 5’ 3’

ExoIII

tio ly
a* e e
HP2

bu n
Helper 5’ 5’

n
tri O
3’ Au 3’ Au
Fig. (3). Schematic illustration of the signal-on electrochemical sensor based on target-aptamer binding triggered multiple recy-
is se
cling amplification for antibiotics determination. Reprinted from [4] with permission.
rD U

(highlighted in blue in Fig. 3) and ExoIII-assisted sec- anti-TET/OA/CPE and anti-TET/OA/Fe3O4 NPs/
ondary target recycling amplification (highlighted in MBCPE aptasensors, respectively, using DPV detec-
fo al

orange in Fig. 3). It is worth noting that the produced tion. The applicability of these aptasensors was evalu-
secondary target, apart from hybridizing with other ated in the analysis of a tablet drug, spiked milk, honey
ot on

HP1, also displaced the Helper from the electrode and blood serum samples.
which helped MB labeled HP2 form a "close" probe Chen et al. [49] developed a label-free aptasensor
structure, increasing the MB signal (measured by
N rs

prepared by self-assembling of a 22 nucleotides ssDNA

DPV), and trigger a new recycle with the aid of Exo on a gold electrode for 5 β-agonists determination:
Pe

III. This method allowed a wide linear range (5 fM to
100 pM) and a low LOD (1.3 fM) for KANA determi-
nation and was successfully applied to the analysis of
spiked milk samples.
Two different aptasensors, were developed and
compared for the determination of tetracycline (TET)
(a part of a drug family of antibiotics that are antibacte-
rial, antiprotozoal, anticancerous, and antimalarial)
[43]. The performance of these two aptasensors, using
the aptamer (anti-TET) immobilization onto modified
carbon paste/oleic acid (anti-TET/OA/CPE) and mag-
netic bar carbon paste/Fe3O4 @oleic acid nanoparticle
(anti-TET/OA/Fe3O4 NPs/MBCPE) electrodes, was
evaluated using EIS and DPV. By using EIS, linear
ranges of 1.0×10-12-1.0×10-7 and 1.0×10-14-1.0×10-6 M
and LODs of 3.0×10-13 and 3.8×10-15 M were provided
by anti-TET/OA/CPE and anti-TET/OA/Fe3O4 NPs/
MBCPE aptasensors, respectively. LODs of 2.9×10-11
and 3.1×10-13 M and linear ranges of 1.0×10-10-1.0×10-7
M and 1.0×10-12-1.0×10-6 M were obtained with the
phenylethanolamine (PHL), clenbuterol (CLB), racto-
pamine (RAC), salbutamol (SAL) and procaterol
(PRO). In a 15 min detection time and using DPV in
the presence of K3[Fe(CN)6], this aptasensor provided
LODs of 0.04 ng mL-1 (RAC), 0.35 pg mL-1 (CLB), 1.0
pg mL-1 (PHL), 0.53 pg mL-1 (SAL) and 1.73 pg mL-1
(PRO), linear ranges of: 0.1-10.0 ng mL-1 (RAC), 0.1-
5.0×102 pg mL-1 (CBL), 5.0-1.0×103 pg mL-1 (PHL),
0.1-10.0 pg mL-1 (SAL) and 10.0-1.0×103 pg mL-1
(PRO), and successful applicability to the analysis of
spiked pork samples.
4. ELECTROCHEMICAL SENSOR BASED ON
THE USE OF SPECIFIC DNA-PROTEIN
INTERACTION
Based on the mechanism of operator DNA-repressor
protein interaction by antibiotics, very recently a mul-
tianalyte electrochemical microfluidic platform has
been implemented for the determination of tetracycline
and streptogramin [50]. The main feature of this
Nucleic Acid Bionsensing of Drugs Current Medicinal Chemistry, 2018, Vol. 25, No. 33 4115

a) Glucose H2O2 b) Glucose

Syringe
pump c)
Amperometric
Avidin-Glucose reservoir
oxidase
GOx
GOx detection at +450 mV
Biotinylated antibiotic Pt working electrode
GOx
repressor protein
2e-
GOx
Fluorescein-labeled
Operator DNA
Anti-fluoresce in
Antibody H2O2 2H+ + O2
Blocking BSA BSA
Chip capillary Channel surface Electrochemical cell

d) e)
120
Analyte concentration
Ipeak Stop-flow signals
Current density in mA cm -2

I0
100 1.56 ng ml-1
6.25 ng ml-1
25 ng ml-1
80 100 ng ml-1
400 ng ml-1
1600 ng ml -1
60 NSB

40

tio ly
20
Stop-phase

bu n
n
tri O
0
150 155 160 165 170 175 180 185 1 10 100 1000
Time in s Analyte concentration in ng ml-1
is se
Fig. (4). Measurement principle used in an electrochemical antibiotics sensing platform based on DNA-proteins specific inter-
action. (a) Scheme of the protocol used to perform the determination based on the interaction of repressor protein with DNA
rD U

reporters. (b) CAD drawing showing the sealing of the individual inlets with a PMMA piece and double-sided tape enabling
connection to a glucose reservoir and a syringe pump via vacuum cups. (c) Reaction mechanism of the H2O2 oxidation at the Pt
working electrode resulting in a concentration dependent current. (d) Stop-flow peaks from a simultaneous amperometric read-
fo al

out of eight immobilization sections with different analyte concentrations. Additionally, the signals for no antibiotic (I0) and
ot on

the nonspecific binding (NSB) are gauged. (e) Resulting on-chip calibration curve. Reprinted from [50] with permission.

method is a repressor protein (TetR or PIP) that bound The microfluidic platform demonstrated IC50 of 48.78
N rs

to a specific operator DNA (tetO or Pir). In the pres- and 29.02 ng mL−1 and LODs of 6.33 and 9.22 ng mL−1
ence of the target antibiotic, the proteins suffered a for tetracycline and pristinamycin, respectively, and
Pe

conformational change and could not bind to their des- applicability for the simultaneous determination of both
ignated operator DNAs. Antifluorescein antibodies ad- antibiotics in spiked human plasma in less than 15 min.
sorbed on the surface of the microfluidic platform
channels were used as spacer and capture biomolecules GENERAL CONCLUSIONS AND OUTLOOK
of the respective operator DNAs, labeled with fluo- Nowadays, it is unquestionable the interest in drugs
rescein at their 5´-end. The antibiotic sensitive re- determination both for understanding their mechanism
pressor proteins were biotinylated to facilitate a subse- of action and in the development of more efficient
quent binding to an avidin-glucose oxidase (GOx) con- ones. In this field, nucleic-acid-based electrochemical
jugate (Fig. 4a). The biotinylated antibiotic-sensitive sensors have demonstrated to offer excellent features in
repressor proteins and avidin-GOx were added to the terms of simplicity, sensitivity, rapidity, requirement of
sample and introduced in the microfluidic network
portable and cost-effective instrumentation and possi-
(Fig. 4b). Subsequently, unbound proteins were re-
bility to perform multiplexed determination, which
moved in a washing step. The electrochemical signal
make them much closer than state of the art method-
readout was achieved by supplying the network with
ologies to implement user-friendly devices to perform
glucose solution and detecting amperometrically the
decentralized and routine drug analysis. This paper has
H2 O2 generated at a Pt working electrode (Fig. 4c).
reviewed the most recent literature on electrochemical
Real peak signals generated from the H2 O2 oxidation
sensors based on the use of nucleic acids developed for
and resulting calibration curve obtained at this sensing
drugs determination through selected examples high-
platform are displayed in Figs. (4d and e), respectively.
lighting the most used and promising approaches.
4116 Current Medicinal Chemistry, 2018, Vol. 25, No. 33
The selected contributions describe the development
of electrochemical platforms designed to determine
different drugs of interest by monitoring the specific
interactions of the target drug with nucleic acids (DNA
and aptamers), DNA damage and specific DNA-protein
interactions. The discussed methods demonstrated that
drugs interact with DNA mainly by intercalation be-
tween the stacked base pairs of native DNA and elec-
trostatic interactions with the negative-charged nucleic
sugar-phosphate structure. Among the different elec-
trode substrates (GCE, CPE, PGE, SPCE, AuE, BDD
and HMDE) used, PGE and CPE have been the most
popular due to the possibility to regenerate their sur-
faces without memory effects, their ease of preparation
and cost effectiveness. These electrodes have been used
unmodified or modified with nanomaterials and/or
polymers to improve their immobilization capabilities
tio ly
and electrocatalytic properties. Although many differ-
bu n
ent electrochemical techniques (EIS, DPV, SWV, CV,
n
DPASV and zero current potentiometry) have been
tri O
reported, DPV and SWV monitoring of the purine
DNA bases or the target drug are by far the most used.
is se
Moreover, it is important to mention that EIS and zero
current potentiometry, although less explored, consti-
rD U
tute interesting alternatives to avoid the electrochemi-
cal interference when oxidation signals of guanine and
fo al
drugs overlap or when the electrochemically redox re-
actions of either DNA or drug molecule would result in
ot on
the damage to their bioactivity and properties.
However, it is important to remark that although the
N rs
advantages offered by electrochemical biosensors make
up the weaknesses of conventional methodologies in
Pe
very important aspects such as cost and simplification
of instruments or procedures, at the moment they can-
not replace them to be conventionally used due to the
lack of exhaustive validation. Therefore, efforts should
be focused on validating the reproducibility and reli-
ability of electrochemical biosensors against conven-
tional methodologies and on implementing the biosen-
sors into portable and automatic devices which allow
the analysis of a large number of samples with minimal
manipulation by the operator, essential requirements in
the practical routine. The development and application
of new nanomaterials and polymers will be required
also to enhance biosensing performance in terms of
sensitivity and selectivity. Another important challenge
is more testing in real samples. Although very low
LODs have been claimed in most reports, they have
demonstrated applicability mostly in spiked real sam-
ples; therefore, more effort should be also made on ap-
plying the biosensors to the analysis of samples with
endogenous content of the target drug. Moreover, al-
Campuzano et al.
though some approaches for dual detection have been
reported, the simultaneous determination of multiple
drugs still remains unsolved and novel methods should
be designed and explored for multiplexed analysis of a
large number of analytes.
Despite all the issues pointed out to be addressed,
taking into account the large number of different ap-
proaches and the advantages offered to develop sensi-
tive, simple and fast responding drug determination
systems without requiring time-consuming extraction
or pretreatment steps, it is expected that electrochemi-
cal biosensors continue to play a key role in the charac-
terization, determination and development of new
drugs in the future.
CONSENT FOR PUBLICATION
Not applicable.
CONFLICT OF INTEREST
Financial support of the CTQ2015-64402-C2-1-R
(Spanish Ministerio de Economía y Competitividad
Research Project) and S2013/MT3029 (NANOAVAN-
SENS Program from the Comunidad de Madrid) are
gratefully acknowledged.
ACKNOWLEDGEMENTS
All authors have contributed substantially in the
preparation of this review. M. Pedrero was in charge of
conducting an exhaustive literature search and classifi-
cation, and S. Campuzano wrote the first draft which
was thoroughly revised by J.M. Pingarrón. The three
authors have equally contributed on the final article
formatting and revision.
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