Hierarchical cystine flower based electrochemical genosensor for detection of Escherichia coli O157:H7

Article abstract of DOI:10.1039/c4ra04511d

This work reports on a facile and reproducible approach to synthesize novel organic flowers of cystine (CysFls) with high uniformity. These 3D flower-like structures have a purely hierarchical arrangement, wherein each petal is composed of several cystine molecules with an average size of 50 μM, as determined by transmission electron microscopy. The CysFls were self-assembled onto a gold electrode and were utilized as matrices for the covalent immobilization of an Escherichia coli O157:H7 (E. coli) specific probe oligonucleotide that was identified from the 16s rRNA coding region of the E. coli genome. This fabricated CysFl platform sought to provide improved fundamental characteristics to electrode interface in terms of electro-active surface area and diffusion coefficient. Electrochemical impedance spectroscopy revealed that this genosensor exhibits a linear response to complementary DNA in the concentration range of 10^-6 to 10^-15 M with a detection limit of 1 × 10^-15 M. Under optimal conditions, this genosensor was found to retain about 88% of its initial activity after being used for 6 times. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ra04511d

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Hierarchical cystine flower based electrochemical
genosensor for detection of Escherichia coli
O157:H7†
Cite this: RSC Adv., 2014, 4, 31047
b
a
Chandra Mouli Pandey,ab Ida Tiwari and Gajjala Sumana
*
This work reports on a facile and reproducible approach to synthesize novel organic flowers of cystine
(CysFls) with high uniformity. These 3D flower-like structures have a purely hierarchical arrangement,
wherein each petal is composed of several cystine molecules with an average size of 50 mM, as
determined by transmission electron microscopy. The CysFls were self-assembled onto a gold electrode
and were utilized as matrices for the covalent immobilization of an Escherichia coli O157:H7 (E. coli)
specific probe oligonucleotide that was identified from the 16s rRNA coding region of the E. coli
genome. This fabricated CysFl platform sought to provide improved fundamental characteristics to
electrode interface in terms of electro-active surface area and diffusion coefficient. Electrochemical
impedance spectroscopy revealed that this genosensor exhibits a linear response to complementary
DNA in the concentration range of 10ꢀ6 to 10ꢀ15 M with a detection limit of 1 ꢁ 10ꢀ15 M. Under optimal
conditions, this genosensor was found to retain about 88% of its initial activity after being used for 6 times.
Received 13th May 2014
Accepted 23rd June 2014
DOI: 10.1039/c4ra04511d
www.rsc.org/advances
properties of simplicity, rapidity, accuracy, low cost and porta-
bility.15,16 Electrochemical biosensors detect nucleic acid
1. Introduction
binding events on a solid-state transducer surface by moni-
toring electrochemical spectrum change.16–18
Micro-organisms, such as bacteria and viruses, are widely found
in food, soil, water and the intestinal tracts of humans and
animals.1 Some of these micro-organisms play a crucial role in
nature, but some can have intense negative effects on both
animals and humans.2 Among them entero-hemorrhagic
Escherichia coli (E. coli O157:H7) is a major food-borne and
waterborne pathogen that causes diarrhea, hemorrhagic colitis,
and hemolytic uremic syndrome.3,4 The traditional methods
reported for the detection of E. coli O157:H7 in food and water
include cultures using selective media,5 serotyping6,7 and PCR
amplication.8,9 Although these methods exhibit high accuracy,
but the operating procedure is complex, time consuming and
some techniques even have limited sensitivity and specicity.10
Hence, it is necessary to fabricate a sensitive, specic and rapid
detection technique for E. coli which would have implications in
environmental monitoring, food industry and clinical chem-
istry. Currently, surface plasmon resonance,11 quartz crystal
microbalance,12 and electrochemical13,14 transduction tech-
niques have been proposed as substitutes to these conventional
methods. Among them, electrochemical nucleic acid biosens-
ing has attracted considerable interest because of its unique
In the fabrication of high performance biosensors for clin-
ical diagnostics, the structure and morphology of the matrices
and the immobilization of bio-analytes for achieving rapid and
sensitive detection of bio-recognition is a major challenge.19 In
view of the motivating phenomena occurring at ordered
microstructures and nanoscale materials, signicant work has
recently been directed towards the synthesis of novel materials
and exploring their applications in a variety of important elds
including catalysis, point of care diagnostics for infectious
diseases and therapeutics.13,20,21 Presently, nanomaterials have
been widely used as signal amplication mediums to enhance
the limit of DNA detection.22,23 In this context, the synthesis of
polyaniline nanotubes has been reported that can detect
nucleic acids with a detection limit of 1.0 fM.24 The fabrication
of a nanoporous gold electrode using encoded gold nano-
particles achieved a detection limit of about 28 aM.25 A label-
free DNA sensor has also been prepared using gallium nitride
nanowires, revealing picomolar concentrations of target DNA.26
Wang et al. have fabricated a gold nanoower modied elec-
trochemical DNA biosensor capable of detecting target DNA
with a detection limit of 1 pM.27 There are reports on the
modication of electrodes by different nanomaterials to
improve DNA detection but the strategies adopted for these
modications are relatively complex. The modication of the
surface using surfactant or polymer matrix in the preparation
and assembly of nanomaterials oen causes some vague effects
aBiomedical Instrumentation Section, CSIR-National Physical Laboratory, New
Delhi-110012, India. E-mail: sumanagajjala@gmail.com; Tel: +91-11-45609152
bDepartment of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi-
221005, India
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra04511d
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in DNA detection.28 Thus, it is necessary to develop a simple
strategy for the construction of nanostructure modied elec-
trodes for improved and sensitive detection of DNA. In this
context, very few reports are available on the application of
hierarchical structures of organic and related compounds for
the fabrication of nucleic-acid biosensors. These hierarchically
structured materials not only possess structural organization at
various scales but they also lead to improved material proper-
ties and performance in terms of high surface area, synergistic
interactions, and multiple functionalities.29,30 Further, the
ability to design these hierarchical structures from materials
with self-assembling properties at the nanometer scale
opens up new opportunities for application in the eld of
biosensors.31,32
Previously, we reported the detection of nucleic acids using
micro-structured cystine dendrites, wherein the role of func-
tional groups and the morphology of dendritic cystine were
discussed.33 However, there is a lot of scope in tailoring the
order and morphology of the amino acid for enhancing
the sensitivity and stability of the biosensor. Therefore, in the
present work, detailed experiments have been carried out to
explore the complexation efficiencies of the binding of DNA
strands with probe molecules that are immobilized on cystine
owers (CysFls). These CysFls are being considered as model
systems because of their intriguing properties such as the ease
of preparation, less chemical handling, and they have a
common functionality at one end of the molecule, which is
coupled to a range of gradually differentiated functionalities at
the other. Further, this CysFl has been utilized to fabricate a
label free electrochemical genosensor by covalently immobiliz-
ing a specic probe oligonucleotide that is identied from the
16s rRNA coding region of the E. coli genome. This CysFl-based
platform was found to possess increased electro-active surface
area and high electron transport at the electrode–electrolyte
interface, enhancing the sensitivity of the genosensor.
2.2. Synthesis of cystine owers (CysFls)
For the synthesis of the CysFls, 100 mM L-cysteine aqueous
solution was prepared by maintaining the pH at 8.0 using
sodium bicarbonate. The solution was sonicated for 30 min at
ꢂ
25 C using an ultrasonic bath, and the crystal growth of the
CysFls was observed by maintaining the solution at 25 ꢂC for
12 h. For TEM analysis, the prepared CysFl was dropped onto a
carbon grid and dried at room temperature.
2.3. Fabrication of CysFl/Au electrode
The gold (Au) electrode (0.5 cm2 diameter) was washed in
boiling 2.0 M aqueous KOH for about 1 h, followed by ultra-
sonication in piranha solution (3 : 1H2SO4–H2O2) for 10 min.
The electrode was voltammetrically cycled and characterized in
0.2 M H2SO4 (ꢀ0.5 V to ꢀ1.4 V vs. Ag/AgCl) at a scan rate of
0.10 V sꢀ1 until a stable cyclic voltammogram was obtained. The
self-assembly of the CysFls was achieved by dipping a pre-
cleaned gold electrode into 100 mM CysFl solution overnight
(12 h) at 27 ꢂC. Aer that, the electrodes were removed and
washed repeatedly with de-ionized water.
2.4. Immobilization of probe DNA and hybridization with
target DNA
The fabricated CysFl/Au electrode was activated using EDC
(2 mM) and NHS (5 mM) and kept for 1 h in the dark. Subse-
quently, 40 ml of pDNA (10ꢀ6 M) was immobilized onto the
modied electrode at 100% humidity at room temperature
ꢂ
(ꢃ27 C) for about 6 h, followed by rinsing with Tris–HCl. The
hybridization studies were performed as a function of target
DNA concentration with complementary, non-complementary
and one-base mis-match sequences in a humid chamber for
about 20 min at 35 ꢂC. The proposed mechanism for the
synthesis of a CysFl and the fabrication of the nucleic acid
sensor for E. coli detection is shown in Scheme 1.
2.5. Extraction of DNA from bacterial clinical samples
2. Materials and methods
2.1. Chemicals and oligonucleotides
The extraction of DNA was conducted from a panel of strains
comprising E. coli, Salmonella typhimurium, Neisseria meningit-
ides, and Shigella dysenteriae. For this process, the suspensions
of the colonies were vortexed by pouring them into 100 ml of
sterile MilliQ water. The suspension was boiled for 10 min and
then centrifuged at 10 000 rpm for 5 min, followed by the
addition of an equal volume (100 ml) of 24 : 1 (v/v) chloroform–
iso-amyl alcohol. The solution was again centrifuged (12 000
rpm, 10 min), which resulted in the formation of an aqueous
layer containing DNA which was carefully pipetted out and
L-Cysteine (analytical grade, 98.5%), N-hydroxysuccinimide
(NHS),
N-ethyl-N-(3-dimethylaminopropyl
carbodiimide)
(EDC), and all other reagents and solvents were procured from
Sigma-Aldrich (India). Probe sequence specic to E. coli that
identied from the 16s rRNA coding region of the E. coli
genome, complementary, non-complementary and one-base
mismatch target sequences were procured from Sigma
Aldrich, Milwaukee, USA and are as follows:13 DNA probe
(pDNA): NH2-50-GGT CCG CTT GCT CTC GC-30 complementary
(cDNA): 50-GCG AGA GCA AGC GGA CC-30 non-complementary:
50-CTA GTC GTA TAG TAG GC-30 one-base mismatch: 50-GCG
AGA GAA AGC GGA CC-30.
maintained at ꢀ20 C prior to use.33
ꢂ
2.6. Pre-treatment of extracted DNA
All the bacterial clinical samples were prepared in Tris–EDTA
The bacterial culture samples of other water borne patho- buffer and were denatured by heating in a water bath (95 ꢂC) for
gens were provided by the All India Institute of Medical Sciences 5 min and were immediately chilled in ice to obtain the dena-
(AIIMS), New Delhi, India. The solutions of oligonucleotides tured single-stranded DNA. These aliquots of samples were
were prepared in Tris–EDTA buffer (1 M Tris–HCl, 0.5 M EDTA) subjected to sonication (15 min at 120 V) to break the long DNA
strands into smaller fragments.2
ꢂ
at pH 8.0 and stored at ꢀ20 C prior to use.
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Scheme 1 Schematic showing the synthesis and fabrication of CysFl and the pDNA/CysFl/Au bioelectrode.
phase, which is in good agreement with the standard value for
bulk hexagonal L-cystine (JCPDS 261776).33,34 FTIR spectroscopy
was used to characterize CysFl and the pDNA/CysFl/Au elec-
trode in the frequency region of 400–1800 cmꢀ1. The charac-
teristic bands of CysFl seen at 1490 cmꢀ1 are attributed to the
presence of amino groups. Intense absorption peaks at 1590
cmꢀ1, 1624 cmꢀ1 and 1420 cmꢀ1, 1490 cmꢀ1 may be assigned
2.7. Characterization
The structural and morphological investigations of CysFl were
carried out using X-ray diffraction (XRD, Cu Ka radiation,
Rigaku, miniax 2) and transmission electron microscopic
(TEM, Hitachi Model, H-800) studies. The scanning electron
microscopic (SEM) images were recorded using a JEOL-JSM-
6700F eld-emitting scanning electron microscope (FESEM,
15 kV). Contact angle (CA) measurements were obtained using a
contact angle meter (Data Physics OCA15EC). Fourier transform
infra-red (FT-IR) spectroscopy measurements were carried out
using a Perkin-Elmer spectrometer (Model Spectrum BX) at
+
to the asymmetric deformation of NH3 and the asymmetric
stretching of COOꢀ, respectively. Bands observed at 1420 cmꢀ1
,
1295 cmꢀ1 and 782 cmꢀ1 are attributed to CH2–CO deforma-
tion, CH2 wagging and rocking vibrations, respectively, con-
rming the presence of CH2 groups (Fig. 1B).32,33 Aer the
immobilization of pDNA on CysFl, the nitrogen base region
(1700–1500 cmꢀ1) in the DNA spectrum overlaps with the
amine signals of CysFl. The absorption bands seen at 1330 and
1133 cmꢀ1 may be attributed to the anti-symmetric and
symmetric stretching vibrations of the phosphate groups,
respectively (Fig. 1C). Further, the IR absorption peak at 1048
cmꢀ1 is because of the vibration of ribose (C–C sugar), and the
absorption at 903 cmꢀ1 is an indication of the immobilization
of DNA on the surface of CysFl.
ꢂ
25 C. Electrochemical analysis was carried out on an Autolab
potentiostat/galvanostat (Eco Chemie, Netherlands) using a
three-electrode cell with Au as the working electrode, platinum
as the auxiliary electrode and Ag/AgCl as the reference electrode
in phosphate buffer saline (PBS, 100 mM, pH 7.0, 0.9% NaCl)
containing 5 mM [Fe(CN)6]3ꢀ/4ꢀ
.
3. Results and discussion
3.1. Characterization of CysFl
The powder XRD pattern of CysFl is displayed in Fig. 1A. The
3.2. Microscopic analysis of the CysFl
main diffraction peaks of the CysFl structures appear at 18.8ꢂ,
28.4ꢂ, 33.0ꢂ and 34.3ꢂ corresponding to 100, 001, 112, and 116, TEM studies have been carried out to study the formation of
respectively. The diffraction peaks show a pure hexagonal CysFl. Fig. 2A shows that the CysFl consists of several hexagonal
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Fig. 1 (A) X-ray diffraction pattern of CysFl. FT-IR spectra of (B) CysFl/Au electrode, and (C) pDNA/CysFl/Au bioelectrode.
3.3. Morphological characterization of the CysFl/Au and
pDNA/CysFl/Au electrodes
The surface morphological studies of the CysFl/Au and DNA/
CysFl/Au bioelectrodes were investigated using SEM. Fig. 3A
shows the self-assembly of CysFl onto gold surface, where the
assemblies are formed in isolation and are not connected. The
high magnication image shows that each ower consists of
several hexagonal akes arranged in a patterned manner
(Fig. 3A, inset). Aer the immobilization of pDNA on the CysFl/
Au electrode, a shiny morphology is observed (Fig. 3B), showing
the complete coverage of CysFls by pDNA. This increase in the
chemically accessible area may be due to well-oriented func-
tional groups present in the CysFl which help in the covalent
binding of the amino terminated pDNA.
Fig. 2 TEM image showing (A) the formed CysFl at pH 8.0 and 100 mM
concentration and (B) the arrangement of hexagonal petals in CysFl.
structures arranged in an ornate manner, where the size of each
ower is 50 mM. High resolution TEM images show that the
thickness of each hexagonal ake is about 1 mM (Fig. 2B) and
they are arranged facial to each other. TEM studies reveal that
the growth of these CysFls start with the formation of cystine
hexagons, and with an increase in the reaction time these
primary hexagonal cystine crystals agglomerate and result in
the formation of ower-like structures.32 The formation of these
structures can be attributed to acoustic cavitations, and the
mechanism is described elsewhere.33 It appears that the inter-
molecular electrostatic interactions of hydrogen bonding are
responsible for the formation of CysFl, which depends on the
change in pH.35
Fig. 3 SEM image of (A) CysFl/Au electrode, and (B) pDNA/CysFl/Au
bioelectrode.
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Fig. 4 Contact angle measurements of (A) Au electrode, (B) CysFl/Au electrode, and (C) pDNA/CysFl/Au bioelectrode.
the electrolyte solution. Interestingly, when the CysFl/Au elec-
trode was activated with EDC/NHS, there was a decrease in the Rct
value, which may be attributed to the blocking of the negative
charges of the –COOꢀ groups of CysFl, and the available net
positive charges attract negative redox markers, resulting in the
decrease of Rct (16.7 U, curve (iv)).33 When the pDNA was
immobilized on the CysFl/Au electrode there was an increase in
the Rct value (47.54 U, curve (iii)) that could be attributed to the
repellence of the redox probe from the approaching electrode
surface by the negatively charged phosphate skeletons of DNA.
Further, when the pDNA is incubated with its cDNA sequence,
hybridization occurs and more negatively charged phosphate
backbones are introduced, resulting in an increase of Rct (149.3
U, curve (ii)).
3.5.2. Cyclic voltammetry studies. To investigate various
kinetic parameters, the cyclic voltammograms of the pDNA/
CysFl/Au electrode were recorded as a function of scan rate
(10–300 mV sꢀ1). It was observed that the magnitude of the
electrochemical response current [anodic (Ipa) and cathodic
(Ipc), Fig. S1A†] for the bioelectrodes is linearly dependent on
the scan rate and follows eqn (1) and (2). The separation of the
peaks suggests that the process is not perfectly reversible;
3.4. Contact angle (CA) studies for Au, CysFl/Au and pDNA/
CysFl/Au electrodes
To investigate the immobilization of pDNA onto CysFl/Au
electrode, CA measurements were carried out using the sessile
drop method. The change in contact angle reveals the
hydrophobic/hydrophilic character of the surface, which in turn
can be related to CysFl self-assembly and pDNA immobilization
on the CysFl/Au electrode. Table S1† shows the CA data of bare
Au, CysFl/Au and pDNA/CysFl/Au bioelectrodes. It can be seen
that the value of CA decreases aer the self-assembly of CysFl
(Fig. 4B) onto the Au electrode (Fig. 4A), indicating the presence
of numerous hydrophilic functional groups (–COOH, –NH2 and
–SH) in CysFl. CA measurements were also performed to
conrm the immobilization of pDNA onto the CysFl/Au elec-
trode, and it was found that with the progress of the immobi-
lization process, the surface becomes more hydrophilic because
of the presence of the phosphodiester backbone of pDNA. The
CA value subsequently decreases with time (4–18 h) and
becomes constant at 29.65ꢂ (Fig. 4C) aer about 6 h revealing
that the complete immobilization of the probe occurs in this
time frame.
3.5. Electrochemical characterization
3.5.1. Electrochemical impedance spectroscopy studies.
Electrochemical impedance spectroscopy (EIS) is a powerful
and sensitive tool for studying the charge transfer processes
occurring at the electrode-solution or modied electrode-
solution interfaces.36 In the Nyquist plot of impedance
spectra, the semicircle corresponds to the electron transfer
resistance process (Rct), which usually depends on the dielectric
and insulating features at the electrode–electrolyte interface.37,38
Fig. 5 shows the EIS studies of the CysFl/Au electrode, EDC/
NHS activated CysFl/Au electrode and pDNA/CysFl/Au bio-
electrode in phosphate buffer (100 mM, pH 7.0, 0.9% NaCl)
containing 5 mM [Fe(CN)6]3ꢀ/4ꢀ as a redox marker. The equiva-
lent circuit (Fig. 5, inset) and the calculated values for solution
resistance (Rs), constant phase element (CPE), and warburg
impedance (W) value, corresponding to different modications
are given in Table S2.† In the impedance spectra, the low
frequency semi-circle is the characteristic of an interfacial charge
transfer reaction. The surface modication of the Au electrode
with CysFl results in an increase of the Rct value (250.4 U, curve
(i)). This increase in the Rct value is because of the presence of
negative charges from the –COOꢀ groups of CysFl that interrupt
Fig. 5 EIS showing the Nyquist diagram (Zim versus Zre) for Faradic
impedance measured in 5 mM [Fe(CN)6]3ꢀ/4ꢀ PBS solution at pH 7.0 at
frequency ranging from 105 Hz to 0.1 Hz (i) CysFl/Au electrode (ii)
complementary DNA incubated on pDNA/CysFl/Au bioelectrode (iii)
pDNA/CysFl/Au bioelectrode (iv) EDC/NHS activated CysFl/Au
the interfacial electron-transfer rate between the electrode and electrode.
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however, stable redox peak current and position during
repeated scans at a particular scan rate suggests that CysFl-
based electrodes exhibit a quasi-reversible process.37 It was
also observed that the magnitude of both anodic (Ea) and
cathodic peak (Ec) potentials (Fig. S1B†) increases linearly as a
function of scan rate (eqn (3) and (4)), which reveals that elec-
tron transport from redox moieties to the electrode is very facile.
Ipa (A) [pDNA/CysFl/Au] ¼ 4.8957 ꢁ 10ꢀ5 (A) + 5.3112 ꢁ 10ꢀ5 A
(s mVꢀ1) [scan rate (mV sꢀ1)] R ¼ 0.9986, SD ¼ 1.208 ꢁ 10ꢀ5
(1)
Ipc (A) [pDNA/CysFl/Au] ¼ ꢀ1.1785 ꢁ 10ꢀ4 (A) ꢀ 3.542 ꢁ 10ꢀ5
A (s mVꢀ1) [scan rate (mV sꢀ1)] R ¼ 0.9920, SD ¼ 1.8612 ꢁ 10ꢀ5
(2)
Ea (V) [pDNA/CysFl/Au] ¼ 0.1302 (V) + 0.4606 (V)
ꢁ log[scan rate] R ¼ 0.9776, SD ¼ 0.0043 (3)
Ec (V) [pDNA/CysFl/Au] ¼ 0.1181 (V) ꢀ 0.0384 (V)
ꢁ log[scan rate] R ¼ 0.9885, SD ¼ 0.0025 (4)
On the basis of the linear slope of anodic peak currents on
the square root of potential sweep rates (Fig. S1B†), it is clear
that ion transport from the bulk to the electrode surface occurs
exclusively by diffusion.39 Further, the diffusion coefficient is
calculated by a concentration gradient of [Fe(CN)6]3ꢀ/4ꢀ ions
between the bulk and the interface, which can be calculated
using the Randles–Sevick equation:
Ip ¼ (2.99 ꢁ 105)a1/2n3/2ACD1/2 1/2
n
(5)
The diffusion coefficient for the pDNA/CysFl/Au electrode is
calculated to be 7.19 ꢁ 10ꢀ6 cm2 sꢀ1 and for the CysFl/Au
electrode it was found to be 1.02 ꢁ 10ꢀ5 cm2 sꢀ1. From the
abovementioned kinetics calculation it can be inferred that the
CysFl/Au assembly is expected to provide improved electron
transfer kinetics to the electrode interface.
Fig. 6 EIS response of (A) pDNA/CysFl/Au bioelectrode as a function
of cDNA concentration (10ꢀ6 to 10ꢀ15 M) in 5 mM [Fe(CN)6]3ꢀ/4ꢀ PBS
solution at pH 7.0. (B) Plot of the ratio of charge transfer resistance
after and before hybridization, Rct target/Rct pDNA of the pDNA/CysFl/
Au bioelectrode vs. the logarithm of target DNA concentrations.
3.6. Electrochemical response studies
3.6.1. Response studies of the pDNA/CysFl/Au bio-
electrode. The sensitivity of the pDNA/CysFl/Au bioelectrode
was investigated using EIS by varying the cDNA concentration
from 10ꢀ6 M to 10ꢀ15 M (Fig. 6A). It was found that with the
change in cDNA concentration, there was a change in the Rct
value. This signies that the hybridization reaction is occurring
at the bioelectrode because of which there is increased accu-
mulation of negatively charged phosphate backbones; hence,
the Rct value increases with the formation of double stranded
DNA. The difference (DRct ¼ Rct(cDNA) ꢀ Rct(pDNA)) between the
value of the pDNA immobilized electrode and that aer
hybridization with cDNA has been used as the measurement
signal. The analytical signal (DRct) shows a linear relationship
with the logarithmic value of complementary target DNA
concentration ranging from 10ꢀ15 to 10ꢀ6 M (Fig. 6B) and
follows eqn (6):
with a linear regression coefficient of 0.998. The detection limit
was calculated to be 1 ꢁ 10ꢀ15 M using the expression 3s, where
s was estimated as the standard deviation (n ¼ 10) of the
impedimetric signals obtained in the absence of bacteria. As the
CysFls contain ample functional groups for the immobilization
of biomolecules, a higher quantity of pDNA is immobilized onto
the CysFl/Au electrode, leading to an enhancement in hybrid-
ization efficiency. In this case, the pathway of electron transfer
to/from the electrode was very effectively blocked and the
changes in the Rct values were remarkably enhanced upon
hybridization, leading to a low detection limit. Further, the
association constant (Ka) between cDNA and the pDNA/CysNf/
Au bioelectrode was estimated from the slope of regression
equation and was found to be 0.243 Mꢀ1 (Fig. S2†).40,41 The
performance of the DNA hybridization detection based on the
pDNA/CysFl/Au platform were compared with other hierarchical
structure-based biosensors, as shown in Table 1. It was
DRct ¼ 161.47 + 9.07 log cDNA
(6)
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observed that the CysFl-based platform has a lower detection
limit and a wider detection range for nucleic acids.
3.7. Selectivity of the bioelectrode
The specicity of the pDNA/CysFl/Au bioelectrode towards
different target DNA sequences (complementary, non-
complementary and one base mismatch) and culture samples
of E. coli, S. typhimurium, N. meningitidis and S. dysenteriae was
studied using EIS (Fig. 7). Aer the incubation of bioelectrode
with E. coli spiked and E. coli culture DNA, there was a marked
increase in the semicircle diameter; the Rct value was nearly the
same as that of cDNA, conrming the process of hybridization.
When the pDNA/CysFl/Au bioelectrode was incubated with non-
complementary and culture samples of bacterial pathogens
DNA, there was a slight or negligible change in the Rct value with
respect to pDNA. Because the non-complementary DNA bases
do not match with the pDNA bases, it was expected that no
hybridization occurs, and the Rct should result in a semicircle
diameter similar to that of the pDNA/CysFl/Au bioelectrode
(Fig. 7, inset).33 A slight increase in Rct for S. typhimurium and N.
meningitidis DNA was observed, which may be because of
nonspecic DNA interactions with the pDNA/CysFl/Au bio-
electrode surface, leading to an increase in negative charges on
the electrode surface. When the pDNA/CysFl/Au bioelectrode
was exposed to the one base mis-match DNA sequence, there
was a slight increase in the semicircle diameter in comparison
to that of the pDNA. This may be because of the partial
hybridization of pDNA with the complementary bases of the one
base mis-match DNA, resulting in increased negative charge of
the electrode surface due to the higher blocking effect of the
surface or the repulsion of the marker ion.19 These results reveal
that the fabricated genosensor based on the CysFl/Au platform
is highly specic for the detection of E. coli O157:H7.
Fig. 7 Bar diagram showing the change in Rct values for (i) spiked E.
coli (ii) cDNA (iii) E. coli cultured (ii) one base mismatch (iii) S. typhi-
murium (iv) N. meningitidis (v) non complementary (iv) S. dysenteriae
immobilized onto (v) pDNA/CysFl/Au bioelectrode. Inset figure shows
the Nyquist plots for different cultured sample immobilized onto
pDNA/CysFl/Au bioelectrode in 5 mM [Fe(CN)6]3ꢀ/4ꢀ PBS solution at
pH 7.0.
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response of the bioelectrode observed was less than 10% when
ꢂ
it was stored at 4 C (Fig. 8B).
4. Conclusions
In summary, the present work emphasizes the synthesis of
cystine owers prepared using the acoustic cavitation method
and their application for E. coli detection. The structural and
morphological studies provided strong evidence for the
formation of CysFl and its self-assembly onto a gold electrode.
Label-free detection using an electrochemical impedance
spectroscopic technique reveals that the fabricated biosensor
with an appropriately optimized protocol can accurately detect
E. coli in the range of 10ꢀ6 to 10ꢀ15 M. This fabricated biosensor
was found to be selective for E. coli detection and retains
signicant amount of activity (88% of the initial activity) aer
using for 6 cycles. Our sensing strategy could be extended for
the detection of other micro-organisms and open up new
avenues for the designing of electrochemical nucleic acid
sensors.
Acknowledgements
We thank Prof. R.C. Budhani, Director, CSIR-NPL, New Delhi,
India for the facilities. C.M.P. is thankful to CSIR, India, for the
award of SRF. We thank Dr A.M. Biradar (NPL, New Delhi), Prof.
B.D. Malhotra (DTU, Delhi) and Dr A.K. Pandey (PGIMER,
Chandigarh) for interesting discussions. Thanks are due to Dr
Seema Sood (AIIMS, New Delhi) for providing the bacterial
culture samples. The nancial support received from DST,
(Grant no. DST/TSG/ME/2008/18) India is gratefully
acknowledged.
Fig. 8 (A) Percentage change in the Rct of the fabricated pDNA/CysFl/
Au bioelectrode towards E. coli detection was determined after each
regeneration cycle (6 cycles). (B) Bar diagram showing the stability of
the fabricated pDNA/CysFl/Au bioelectrode.
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