Supplementary data
S1. DNA isolation from culture
DNA has been isolated from a panel of strains comprising of control strains of Neisseria
gonorrhoeae (WHO C), Klebsella pneumoniae, Staphylococcus aureus, Escherichia coli and a
clinical strain of Neisseria sicca for specificity studies. The isolation has been done by
suspending colonies in 100 µl sterile MilliQ water and vortexing the mixture followed by boiling
for about 10min and subsequently centrifuging at 8000 rpm for about 5 min. To this, equal
volume (100 µl) of 24:1 (v/v) chloroform: iso-amylalcohol is added followed by centrifuging at
12000 rpm for about 10 min. The aqueous layer containing the DNA is carefully pipetted out
into another eppendorf. This extracted DNA is kept at -293 K prior to being used.
S2. DNA extraction from urethral and endocervical swab patient samples
Urethral and endocervical swab patient samples have been supplied by the STD Clinics,
Dermatology OPD of the All India Institute of Medical Sciences (AIIMS), New Delhi, India.
The swabs have been collected in triplicate for microscopy, culture and DNA extraction,
respectively. Endocervical/urethral swabs have been plated on two selective media, (one with
and one without inhibitors). The culture plates are incubated in 5 % CO2 atmosphere for 24 h to
48 h at 309 K and are inspected for presence of N. gonorrhoeae colonies. Suspected colonies are
subjected to Gram stain, oxidase, superoxol test and rapid carbohydrate utilization test (RCUT)
for confirmation. DNA has been extracted by using QIAamp DNA mini kit from N. gonorrhoeae
culture, pus sample spiked with N. gonorrhoeae culture and patient sample.
S3. Urethral and endocervical swab patient samples preparation for direct detection of
complementary target DNA
N. gonorrhoeae culture, pus sample spiked with N. gonorrhoeae culture and patient
sample solution in TE buffer is denatured by heating in a water bath (368 K) for 5 min to obtain
denatured single-stranded DNA followed by sonication (15 min at 120 V, 2 A and 24 KHz
frequency using ultrasonic cleaner, Vibronics Pvt. Ltd., Mumbai, India) to break the long DNA
strands into smaller fragments of the DNA. N. gonorrhoeae culture, pus sample spiked with N.
gonorrhoeae culture and patient samples are utilized for the hybridization studies using aDNAPANI–Fe3O4/ITO nanobioelectrode.
S4. EIS measurements
The electrochemical impedance spectroscopy measurements have been performed using
an Autolab Potentiostat/Galvanostat (Eco Chemie, Netherlands) with a Frequency Response
Analyzer (FRA) Module in the frequency range 105 Hz to 10 mHz. A conventional three
electrodes cell has been used with aDNA-PANI-Fe3O4/ITO as the working electrode, Ag/AgCl
as reference electrode and platinum as the counter electrode in PBS of pH 7.0 containing 5 mM
[Fe(CN)6]3-/4-. All the solutions used for the electrochemical measurements are autoclaved and
the electrodes are always rinsed with de-ionized water prior to measurements. However, the
DNA immobilized bioelectrodes are rinsed with PBS at pH 7.0.
S5. EIS instrumentation
Electrochemical impedance spectroscopy is known to be a promising technique for label
free detection of desired bioanalytes. EIS can be used to measure total impedance of the desired
surface. For this purpose a small potential perturbation (a DC potential on which a sinusoidal
shaped potential signal with small amplitude is superimposed) is applied to the system and a
corresponding response in the form of current is measured as a function of frequency. The ratio
of potential at a particular frequency and response current gives the value of impedance of the
system and the data can be plotted using different measurement models, like equivalent circuits
or mathematical models. The application of equivalent circuit is based on the similarity between
an electrochemical cell with an electrical circuit comprising resistors, capacitors and inductors.
In the equivalent circuit, electrical double layer formed between the working electrode and the
electrolyte solution represents a parallel plate capacitor (Cdl) and the resistance to the charge
transfer in the electrode-solution interface to a resistance can be represented by a resistor (RCT) in
parallel to the capacitor. The resistive effect on the migration of ions in solution is represented by
the solution resistance (Rs).
The impedance of an electrochemical system can be represented by a phase diagram in
which the impedance shows a real (resistive) and an imaginary (capacitive) component.
For the equivalent circuit the real (Z′) and imaginary (Z′′) system impedance components can be
written as:
Z '  Rs 
RCT
2
1   2Cdl2 RCT
(1)
2
Cdl2 RCT
Z''
2
1   2Cdl2 RCT
(2)
where, Rs is the solution resistance, RCT is the charge transfer resistance, ω is the angular
frequency and Cdl is the electrical double layer capacitance.
Supplementary eqs 1 and 2 lead to:
2
R 

R 
2
 Z ' Rs  CT   Z ' '   CT 
2 

 2 
2
(3)
Eq 3 represents a circle with a diameter of RCT. The plot of real vs imaginary part of the
impedance at different frequencies gives an impedance spectrum known as Nyquist or Cole-Cole
plot, in which the diameter represents the charge transfer resistance (RCT) of the electrochemical
system, the linear portion represent the Warburg resistance.
S6. X-ray diffraction studies
Supplementary Fig. 1 shows the X-ray diffraction pattern of PANI-Fe3O4 nanocomposite
(JCPDS–International center diffraction data, PDF cards 3-864 and 22-1086). For the PANIFe3O4 nanocomposite, the peak appearing at 22.3o representing the (020) Bragg reflection is
stronger, implying that the molecular chains of the PANI are in increased ordered state in the
presence of Fe3O4 and the crystallinity of the PANI in the composite is higher. Further, five
strong bands appear with maximum intensity at 30.1o, 35.4o, 42.9o, 57.5o and 62.7o representing
Bragg reflections from (220), (311), (400), (511) and (440) planes of Fe3O4 showing the growth
of Fe3O4 in all five planes. This result indicates the presence of Fe3O4 nanoparticles in the PANI–
Fe3O4 nanocomposite.
1.4k
Intensity (a. u.)
1.2k
1.0k
800.0
600.0
400.0
200.0
20
30
40
50
60
70
80
(2-Theta)
Supplementary Fig. 1. XRD of PANI-Fe3O4 nanocomposite
S7. Electrochemical studies using CV
The electrodes have been characterized using CV measurement in 0.05 M Phosphate
buffer saline (PBS) solution (pH 7.0, 0.9 % NaCl). Supplementary Fig. 2 shows the CV obtained
for PANI-Fe3O4/ITO nanoelectrode (i), Glu-PANI-Fe3O4/ITO nanobioelectrode (ii) and for the
aDNA-PANI-Fe3O4/ITO nanobioelectrode (iii). The redox peak is found to decrease after Glu
modification of PANI-Fe3O4/ITO, due to the insulating nature of the Glu layer. The further
decrease in the redox peak height after aDNA immobilization indicates successful
Current (A)
immobilization of aDNA on to the Glu-PANI-Fe3O4/ITO nanobioelectrode.
300µ
(i) PANI-Fe3O4/ITO
150µ
(ii) Glu-PANIFe3O4/ITO
0
(iii) aDNA-PANI-Fe3O4/ITO
-150µ
-300µ
-0.4
0.0
0.4
Potential (V vs Ag/AgCl)
0.8
Supplementary Fig. 2. CV of (i) PANI-Fe3O4/ITO nanoelectrode, (ii) Glu-PANI-Fe3O4/ITO
nanobioelectrode and (iii) aDNA-PANI-Fe3O4/ITO nanobioelectrode in 0.05 M PBS (pH 7.0, 0.9
% NaCl).
S8. Electrochemical studies using DPV
Supplementary Fig. 3 (a) describes results of response studies of the bioelectrode using
DPV for detection of c-DNA, nDNA) and oDNA synthetic oligomers using aDNA-PANIFe3O4/ITO nanobioelectrode by monitoring the redox activity of methylene blue (MB). MB as
redox hybridization indicator can be used to distinguish single stranded and double-stranded
form of DNA. MB is known to associate with the unpaired nitrogenous bases of single-stranded
DNA as compared to the double-stranded DNA. It can be seen that MB peak is almost negligible
after hybridization with c-DNA suggesting presence of duplex DNA at the bioelectrode surface.
The presence of the MB peak of almost same height as that for aDNA-PANI-Fe3O4/ITO
nanobioelectrode has been observed after incubation with nDNA and oDNA synthetic oligomers
indicating absence of duplex at the surface. Supplementary Fig. 3(b) describes response of the
aDNA-PANI-Fe3O4/ITO bioelectrode after hybridization with c-DNA concentration ranging
from 1×10-18 M to 1×10-6 M. It can be seen that MB peak height increase with decrease in the cDNA concentration indicating enhanced number of double-stranded DNA molecules at the
surface (Supplementary Fig. 3(b)). It has been observed that increase in the MB peak with
respect to c-DNA concentration (The variation of the MB peak current has been found to obey
supplementary eq. (4) with value of regression co-efficient as 0.98733:
I(Target) = 1.9995 [log (Target concentration)] + 8.6285
(4)
The presence of the MB peak of the lower (closer) height as that for aDNA-PANI-Fe3O4/ITO
nanobioelectrode has been observed for c-DNA concentration ≤1×10-18 M indicating absence of
duplex at the surface. Therefore, it can be concluded that 1×10-17 M is the detection limit.
25µ
(iii) Non-comp
target DNA
(ii) Comp
target DNA
20µ
Current (A)
30µ
(i) aDNAPANI-Fe3O4/ITO
(iv) One base
mismatch targetDNA
(a)
15µ
10µ
25µ
(xiv)
20µ
15µ
c-DNA
conc (M)
10µ
5µ
5µ
0
-0.40
(i) aDNAPANI-Fe3O4/ITO
(A)
Current (A)
30µ
(ii)
0
-0.35
-0.30
-0.25
-0.20
Potential (V vs Ag/AgCl)
-0.15
-0.4
-0.3
-0.2
Potential (V vs Ag/AgCl)
Supplementary Fig. 3. (a) DPVs of aDNA-PANI-Fe3O4/ITO nanobioelectrode after treatment
with c-DNA, nDNA, oDNA, (b) DPV of aDNA-PANI-Fe3O4/ITO nanobioelectrode (i), after
hybridization with compDNA concentration 1×10-6 M (ii), 1×10-7 M (iii), 1×10-8 M (iv), 1×10-9
M (v), 1×10-10 M (vi), 1×10-11 M (vii), 1×10-12 M (viii), 1×10-13 M (ix), 1×10-14 M (x), 1×10-15 M
(xi), 1×10-16 M (xii), 1×10-17 M (xiii), 1×10-18 M (xiv), at step potential of 3 mV and modulation
amplitude of 50 mV, in 20 µM MB, pre-treatment at +0.1 V for 10 s, 0.05 M PBS (pH 7.0, 0.9%
NaCl)
-0.1