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Electrochemical DNA sensor Based on Polyaniline/graphene: HighSensitivity to DNA Sequences in a Wide Range
Received 00th January 20xx,
Accepted 00th January 20xx

a♀

a♀

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a

b

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Qing Zheng , Hao Wu , Zongxu Shen , Wenyu Gao , Yu Yu , Yuehui Ma , Weijun Guang ,cccaQuangui Guo , Rui Yan , Junzhong Wang *, Kejian Ding *DOI: 10.1039/x0xx00000x
www.rsc.org/

A free-label electrochemical DNA sensor was fabricated by deposition of polyaniline and pristine graphene nanosheet(P/Gratios) composites in different mass ratios, probe DNA and bovine serum albumin (BSA) layer by layer on the surface ofglassy carbon electrode (GCE). Electrochemical impedance spectroscopy (EIS) was employed to monitor every step offabrication of P/Gratios-based DNA sensors and to evaluate the detection results in terms of the hybridization ofcomplementary DNA, mutation DNA and non-complementary DNA. The results illustrate that the P/Gratios-based DNA
sensor could high efficiently detect complementary DNA from 0.01 pm to 1 µm and discriminate single-nucleotide
polymorphisms (SNPs). In the process of detection, double-stranded DNA (dsDNA), resulted from hybridization of probe
DNA, escaping from or remaining on the sensor surface, was monitored by changing the ratio of polyaniline (PANI) tographene, which was decided by the competition between electrostatic interaction and Brownian motion.
20,21

Introduction
Electrochemical DNA sensors have received intense
1-4
attention recently owing to several reasons. Firstly, simple
portable DNA detection tools are highly demanded in earlycancer diagnosis and the development of genetically modified
5,6
organisms.
Secondly, electrochemical signaling methods
involved in DNA sensors are simple, sensitive, specific and7-9
cost-effective. Thirdly, aptamers are short single-stranded
oligonucleotides being able to recognize and bind to specific10targets. They are analogous to antibodies but possess several
advantages including wider recognition range of targets,11
higher affinity, easier synthesis and modification. A lot ofworks have studied the immobilization of aptamers on the12
surfaces of various electrodes including Au,
carbon13
14
15
nanotube, nanodiamond, reduced graphene oxide and16
conjugated polymer. However, electrochemical DNA sensors
17
are still limited in sensitivity, stability, and cost of fabrication.
In the research of how to fabricate economic and functional
DNA sensors, graphene quickly came to the attention of many
18,19
scientists and researchers.
As a kind of two-dimensional
2material of sp bonded atomic carbon with long-range π cloud,graphene is of planar surface, high electrical and thermal
conductivity, strong mechanical strength and gooda.

College of Life sciences and Bioengineering, Beijing Jiaotong University, Beijing
100044, P R China.
b.
Key Laboratory of Carbon Materials, Institute of Coal Chemistry, Chinese Academyof Science, Taiyuan, 030001, P R China.
c.
Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing
100083, P R China.
† ♀ These authors have equal contribution to this work.
Electronic Supplementary Information (ESI) available: [more XRD,AFM,UVVis,SEM,Scheme images are Included]. See DOI: 10.1039/x0xx00000x

biocompatibility.
In addition, graphene is good at
22-24
adsorbing and sensing single-stranded DNA (ssDNA).
All
these make graphene a kind of promising material for
25,26
especially the fabrication of
applications in biosensors,
electrochemical DNA sensors because ssDNA would be stably
immobilized on the surface of graphene through π-π stacking
interaction between the conjugated π bonds of nucleotide
27
bases and the hexagonal units of graphene. In a DNA
biosensor, the immobilized-probe DNA selectively hybridizes
with target DNA and the whole process of hybridization ismonitored by electrochemical impedance spectroscopy (EIS) in28
the form of the change of charge transfer resistance (Rct).
Seemingly, the ending of hybridization would bring about thedecrease of Rct value in the result of double-stranded DNA(dsDNA, in the presence of target/probe DNA) escaping fromthe surface of biosensor, which is witnessed by many of
29,30
researchers and their experiments.
However, someresearchers oppositely suggested that the dsDNA still remainon the surface of biosensor leading to the raise of
31,32
impedemetric value.
This dispute on scientific facts
interests us so much that we set off to make efforts to comb
scientific reasons behind these conflicting facts and lookforward to contributing to the improvement ofelectrochemical DNA sensors.
It turns out to be not easy to realize this goal when the
researchers firstly met with the limitations of recent DNA
sensors which are built on the basis of reduced graphene oxidethat lacks of the native properties of graphene owing to many
33
defects. Fortunately, Wang et al succeeded in preparing
pristine few-layer graphene flakes through electrochemical
34
exfoliation of graphite at high yield. These graphene flakes
exhibit higher sensitivity to ssDNA than surface-enhanced laser

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desorption/ionization-mass spectrometry technique. In themeantime, the new technique also largely reduces the
fabrication cost of graphene electrodes so provides great
34,35
chance to produce such electrodes on a large scale.
Exceptthe invaluable improvement, however, we found thatgraphene-flakes are problematic because they are rarely able
to remain on electrodes in DNA water solution which would
lead to serious impact on the stability of the sensors and theaccuracy of their results. To solve this problem, we introduced
36
conductive polymer polyaniline,
an excellent organic
conductor with charges, to “stick” graphene flakes on glassy
carbon electrodes (GCE). Polyaniline involves outstanding
environmental stability and biocompatibility, which also
endowed polyaniline’s popularity in the exploitation of
37,38
39,40
biosensors,
especially the DNA sensors.
Thus, it ispossible for researchers to build a new biosensing platform for
impedance responses to target DNA oligomer by depositing
pristine graphene flakes and charged polyaniline on GCE insequence.
To avoid the non-specific adsorption of the target DNA, we
further modified electrodes with bovine serum albumin (BSA)
as an auxiliary medium, and probed into the interaction
between probe DNA and target DNA with help of EIS analyzing
the developed sensor’s sensing ability in terms of its
impedance response in the presence of K3/4[Fe(CN)6] solution.
When it comes to the conclusion, we found that the twoparties suggesting opposite results should not blame to eachother because both of them are partially right since theimpedance response of the DNA sensor is actually dynamic
depending on the mass proportion of polyaniline and29-32
To be clearer, we found out that the response isgraphene.
directly related to the mass proportion of polyaniline andgraphene. We proposed that the linear increasing response of
charge transfer resistance should result from dsDNA’s
remaining on the surface of the platform with muchpolyaniline while the linear decreasing response should be
assigned to dsDNA’s releasing off the sensor with dominativegraphene.

Experimental
Apparatus and chemicals
Electrochemical impedance spectroscopy measurements
were carried out in CHI660E electrochemical work station (CHI
instrument, Austin, TX, USA). A glassy carbon electrode (GCE)
was employed as the working electrode, a platinum wire as
the counter electrode and an Ag/AgCl electrode with asaturated KCl solution as the reference electrode. The work2
area of GCE was 7.065 mm . Scanning electron microscope(SEM) and transmission electron microscope (TEM) were
performed on a JSM-7001F FESEM (Field emission scanningelectron microscopy) and a JEM- 2100F microscope at anacceleration voltage of 200 kV, respectively. All of synthetic
oligonucleotides were purchased from Beijing Biomedical Co.,Ltd (Beijing, China).The sequences of oligonucleotides arelisted in Table 1.
Stock solutions of the oligonucleotides were diluted with TE
buffer (10 mM Tris-HCl, 1 mM EDTA, pH=8.0). Propylene
carbonate (PC, anhydrous, 99%), lithium perchlorate (LiClO4,
99.8%, powder), lithium chloride (LiCl, 99%), hydrochloric acid
(HCl, ~37%), ethanol (C2H6O, 99.8%), ammonia (28%),
Tetramethylammonium hydroxide（TMA, aqueous, 25wt%）
and N,N-dimethylformamide (DMF) were purchased from
Aladdin (Shanghai, China). The graphite powder (the average
particle size < 30 µm) was purchased from Xianfeng
Nanotechnology Co., Ltd (Nanjing, China). Polyaniline, in theform of emeraldine salt was purchased from Jianya New
Material technology co., Ltd (Shijiazhuang, China), and thenumber of aniline repeat units is ~480, which is measured bylight scattering. All chemicals were used with no further-1
purification. Water (18.2 MΩ cm ) used in the experiments
was generated by a Milli-Q water-purification system(Millipore, Bedford, MA, USA).
Synthesis of pristine graphene flakes
Graphene flakes were obtained by electrochemical
expansion of graphite lithium perchlorate in propylene
34
carbonate electrolyte. Graphite powder (5 g) in a porous
plastic tube (PPT) with aluminum wire was used as negativeelectrode and carbon rod as positive electrode. The working
solvent was 30 mg/mL of LiClO4 in propylene carbonate (PC)and high potential of 10 V was adopted in order to activate
Li/PC co-intercalation in graphite. After full electrochemicalcharging, the graphite powder was expanded and transferredfrom PPT to a glass Suslick cell (15 mL). The expanded graphite
was sonicated for 15 hours in concentrated 50 mg/mL of LiCl in
DMF (10 mL), PC (2 mL) and TMA (1 mL) solution. And then theabove intermixture was washed with HCl/DMF and Millipore
34
water several times. Finally, graphene powder was collected
after drying at 80°C.
Fabricationof
Polyaniline/Pristine
Graphene
Composites under various mass ratios (P/Gratios)

flake

The composites of polyaniline and graphene flakes wereTable 1. Sequences of all oligonucleotides
Single stranded DNA names
Probe DNA

Sequences
GCTCTAGAGCGGTTGGTGTGGTTGGTTTTTTTTTT (3’- 5’)Complementary DNA (Target DNA)CGAGATCTCGCCAACCACACCAACCAAAAAAAAAA (5’- 3’)

Mutant DNACGAGATCTCGCCAACGACACCAACCAAAAAAAAAA (5’- 3’)

non-complementary DNA

TAGTCCTGTGGTGGTTGAGTGGATACCCCCCCCCC (5’- 3’)

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synthesized by mixing graphene dispersion and polyaniline
dispersion in water with the assist of bath sonication. The
composites of polyaniline (P) and graphene (G) with different
mass ratios are signed as P/Gratios. For example, P/G1:100 is thecomposite of polyaniline and graphene with the mass ratio of
1/100. Typically, 1:1, 1:2, 1:10, 1:20 and 1:100 of P/Gcomposites were synthesized, respectively. After mixing
polyaniline and graphene flakes, the mixtures were sonicatedfor more than 8 hours with the help of an ultrasonic cleaner toimprove the uniformity and dispersion.
GCE-P/G electrode fabrication through GCE modification with
P/G composites
A glassy carbon electrode (GCE, 3.0 mm in diameter, Gauss
Union) was polished with alumina slurry to obtain a mirror-likesurface and then activated in 0.05 M H2SO4 by cycling thepotential from 0 to 2.0 V (vs. Ag/AgCl, sat. KCl) at a scanning
rate of 50 mV/s. The activated GCE was washed with a copious
amount of Millipore water and then dried with N2 gas. Then
2.5 μL of above prepared P/Gratio composites were dropped
and expanded into P/Gratio film on the surface of GCE. Finally,
the GCE-P/Gratio electrode was washed with Millipore water
several times and dried with N2 gas.
Immobilization of Probe DNA and BSA conjugation to the
GCE-P/Gelectrode (the sensor GCE-P/G-Probe)
The probe oligonucleotide was dissolved in TE buffer to-5obtain a 1e M solution. 2.5 μL of the Probe DNA solution was
deposited on the GCE-P/Gratios and dried with N2 gas for 4 h atroom temperature. Afterwards, the GCE-P/Gratios immobilizedwith probe DNA electrode (GCE-P/Gratios-Probe DNA) was
gently washed with Millipore water to remove excess and nonimmobilized Probe DNA. The electrode of GCE-P/Gratios
immobilized with probe DNA was immersed in bovine serum
albumin (BSA) solution for 2 hours to realize BSA conjugation
at room temperature. Then, the electrode was washed with
Millipore water. Finally, the DNA sensor (GCE-P/Gratios-Probe,that is, GCE-P/Gratios DNA sensor), modified by BSA, wascollected for next-step target DNA hybridization.
Hybridization of target DNA to the sensor GCE-P/G-probe
Beforeelectrochemical
impedance
spectroscopy
measurements, the GCE-P/Gratios-Probe were incubated in 300
μL of 2X SSC buffer containing target DNA with different
concentrations at 42 °C for 20 mins to allow target DNA
molecules completely to hybridize with the probe
30
oligonucleotides. After being washed with Millipore water
thoroughly, the electrode named GCE-P/G-probe-target wascollected for EIS analysis. The pUC19 DNA, a circular double
stranded DNA with 2686 base pairs, was extracted from thecompetent cells. The extracted DNA was cut into linear DNA
molecule by enzyme digestion. After that, the DNA samples
were denatured by heating at 95 °C for 10 mins and applied ice
shock for 1 min after heating. Finally, the samples werecollected for hybridization.
Electrochemical impedance spectroscopy measurements

All Electrochemical Impedance Spectroscopy (EIS)
measurements were made in an electrochemical cell
containing 3.5% NaCl, 10 mM K3[Fe(CN)6] and K4[Fe(CN)6].
Faradic impedance spectroscopy was conducted in thefrequency ranging from 100 kHz to 1 Hz with a sampling rate of
10 points per second. The amplitude of the applied sinusoidal
potential was 5.0 mV and the potential of + 0.25 V was limited
3−/4−
to the formal potential of the redox couple [Fe(CN)6]. The
reported result for every DNA sensor in this experiment was
the mean value of three parallel measurements. The
electrochemical cell was housed in a specially shielded cage toreduce stray electrical noise during the measurements and allof the measurements were carried out at room temperature.
An R(C(RW)) equivalent circuit (Nyquist plots) was used to fit
the obtained impedance spectra.

Results and discussion
Synthesis of P/Gratios composites
Graphene flakes with little defects were synthesized34
according to the already reported work. As shown in Fig. 1(a)and 1(b), highly conductive and single-crystalline few-layer
graphene flakes in size of several micrometers were clearlyvisible through SEM and TEM (ED pattern inset). XRD pattern(Fig. S1) of graphene confirmed its layered structures, in which
the peak seen at 26.2° corresponds to the interspacing
distance of the layers. No other phases were shown in thepattern, which means no impurity (i.e. lithium salt) was
remained in the graphene solution to affect the behavior of
the electrode and DNA adsorption. These graphene flakes canbe easily dispersed in solution and dropped onto GCE to makea graphene-based sensor, but a problem was met because thegraphene flakes tend to peel off the electrodes in DNA solution.
However, if the graphene flakes are mixed with polyaniline
which is a kind of conducting polymer, the problem can besolved. This should be attributed to stronger long-range π−πbonds and electrostatic interactions between graphene flakes
and charged polyaniline. Fig. 1(c and d) shows the typical SEM
and TEM images of PANI and graphene composite in the mass
ratio of 1:1 (named P/G1:1). It appears inter-stacking of flowerlike polyaniline and few-layer graphene flakes. Polyaniline canwork like “glue” sticking to graphene flakes. It can besuggested that the stability of graphene film would be
improved by mixing graphene with positively charged
polyaniline. The morphology of other composites of P/G withdifferent mass ratio and morphology of the ssDNA/BSA
modified electrode are shown in Fig. S2. As shown, the glue
sticking PANI gets less, and the size also reduces, along withthe decrease of the ratio of PANI to graphene. After theimmobilization of ssDNA and BSA, the PANI/graphene
composite (Fig. S2e) presents a rough surface appearance, due
to the existence of immobilized pony-size biomass. The
thickness of the film deposited was measured (Fig. S3.). As theresult, the concentration of PANI would not affect the film
thickness. The thicknesses of pure graphene and that in P/Gcomposites were both about 3.2 nm. The P/G composites were

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Figure 1. High quality of few-layer graphene nanosheets used for preparation of the hybrid of polyaniline and graphene platform.
(a, b) The images of few-layer graphene nanosheets: SEM (a), TEM and inset electron diffraction pattern (ED) (b). (c, d) The
hybrid of polyaniline and graphene.

Figure 2. Spectroscopy characterizations of the hybrids of polyaniline/graphene compared with the component of few-layer
graphene and polyaniline. (a) Raman (laser source of 488 nm), and (b) FTIR.

powerfully sonicated for more than 8 hours, and thencentrifuged under a speed of 5000 rpm. Therefore, theaverage thickness of the film was controlled.
We took advantage of spectroscopy techniques to analyze
the composites and compare the properties of each kind ofcomposites. Little defects of graphene flakes were confirmed
by Raman and FTIR spectra and these flakes are quite different
from reduced graphene oxide, as shown in Fig. 2. Fig. 2a shows-1that in the hybrid the peaks of D band (~1350 cm ) and G band
-1(~1570 cm ) of graphene were broadened and merged
together, and the defective signal of D band increased muchwhile the intensity of the 2D band decreased compared withthose of pristine graphene. This may result from the coupling

between graphene and polyaniline via strong long-range π−πand electrostatic interactions. FTIR spectra presented that the-1polyaniline features (1579 cm , quinonoid ring vibration; 1494
-1
-1
cm benzenoid ring vibration; 1301 cm C-N stretching-1
vibration; 1139 cm C-H in-plain vibration) were kept in the
hybrid. The absorbance of polyaniline in the hybrid is linearly
related to its concentration in the hybrid (Fig. S4a). The
increase of the mass ratio of polyaniline to graphene can
improve the colloidal dispersion of graphene in water (Fig.
S4b).
Construction of P/Gratios-based DNA sensor

As the scheme for the synthesis procedures of the sensor
presented (Fig. S5), the P/Gratios-based DNA sensor was

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Figure 3. (a) Nyquist plots of the hybrid of polyaniline/graphene (the mass ratio 1:1, named P/G1:1) based DNA sensor at various
stages: (1) glassy carbon electrode (GCE), (2) GCE modified with P/G1:1, and (3) GCE-P/G electrode immobilized by probe DNA. (b)Charge transfer resistance (Rct) reflects the dynamic adsorption curve of BSA on the surface of the P/G1:1–based sensor (Inset is
Nyquist plots).All the impedance data were recorded in the presence of 10 mM of K3/4Fe (CN)6] and 3.5% of NaCl.

Figure 4. (a) Nyquist plots for polyaniline/graphene (when the mass ratio is 1:1, it is noted as P/G1:1) based DNA sensor at various
stages: (1) GCE-P/G (black line), (2) GCE-P/G-Probe DNA (red line), (3) GCE-P/G-Probe DNA-BSA (green line), and (4) GCE-P/GProbe DNA-BSA-target DNA (blue line).The impedance was controlled by diffusion of the redox probe (of low frequencies) and byinterfacial electron transfer (high frequencies). (b) Dependence of Rct on the concentration of C-DNA for P/G1:1–based sensor. (c)Histograms representing the relative detection values obtained for P/G1:1–based sensor loaded with C-DNA, Nc-DNA, and M-DNA.

fabricated in three steps: firstly, bare GCE electrodes were
polished and cleaned; secondly, P/Gratios composites of a fixedmass ratio of polyaniline and graphene were deposited on thesurface of GCE electrodes; and thirdly, the GCE modified with
P/Gratios were immersed in the solution of probe-DNA to allow
the probe-DNA immobilize onto the electrode of GCE-P/Gratios.
It proves that high quality few-layer graphene flakes are ofoutstanding affinity to single stranded DNA owing to longrange π-π interaction, and the single stranded DNA probe-DNA
was thus immobilized onto the electrode GCE-P/Gratios. Here,
EIS, known as an effective method of probing into the featuresof surface-modified electrodes, was used to evaluate theelectrochemical properties of the P/Gratios-based DNA sensor ineach step and came up with Nyquist plots as shown in Fig. 3.
The curves of the Nyquist plots actually display two steps ofelectron movements: one is electron transfer, the semicircularportion of the curves, and the other is electron diffusion, thelinear portion of the curves. The typical R(C(RW)) equivalentcircuit, as shown in the inset of Fig. 3a, was used to fit EIS data

and determine the electrical parameters in each step of theprocess and the concentration of the target DNA. The
parameters of the circuit include Warburg element (Ws),electrolyte resistance (Re), charge transfer resistance (Rct) andconstant phase element (C).41 In this case, the non-specificadsorption of target DNA was taken into consideration due tothe non-uniformity of P/G. To overcome this drawback, thesensor was immersed in bull serum albumin (2.5 g/L) to allow
BSA self-assemble on the surface of P/Gratios-based sensor. As
shown in Fig. 3a, the semicircular domain of the curve of bare
GCE electrode is of short diameter so that the Rct of bare GCE
electrode should be low. After GCE was modified with P/G, thediameter of the semicircle increased and was more specific. Rctof modified electrode increases from 70 ohm to 220 ohm,
which is also in a very small region, indicating P/G composites
maintained the high electrical conductivity as pure graphene(Fig. S6). Due to the kinetics barrier between [Fe(CN)6]3−/4−
solution and the deposited probe, the Rct of the GCE-P/G
electrode increased dramatically from 220 ohm to 1,100 ohm

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when the probe was deposited. Finally, with the selfassembling of BSA, Rct continues to increase to a plateau
because the adsorption of BSA on the surface of GCE-P/G
electrode tends to saturate after 100 min (Fig. 3b).

important because it proves P/G1:1-based sensor’s capability of
discriminating single nucleotide polymorphisms (SNP) andmeanwhile inspires a new route to apply P/G composites.
The exploration of the new route is based on the influenceof the P/G mass ratios on the performance of the DNA sensors,
EIS analysis of the P/Gratios-based sensor
which means that the authorial research group attempts to
EIS allows analyzing interfacial changes originating from bio- build a dynamic DNA sensor with changeable mass ratios ofrecognition events on electrode surfaces through the P/G to widen its detection range and they decided that thiscapacitance and interfacial electron transfer resistance of the kind of DNA sensor should be named as P/Gratios-based DNA
conductive or semi-conductive electrodes. To test the sensors. The mass proportions of polyaniline and graphenesensitivity and selectivity of the resulting P/Gratios-based varied from 1:1, 1:2, 1:10, 1:20 to 1:100. Correspondently, theelectrochemical DNA sensor, complementary DNA sequences synthesized composites were marked as P/G1:1, P/G1:2, P/G1:10,(C-DNA), non-complementary DNA sequences (Nc-DNA) and P/G1:20 and P/G1:100. All of these P/Gratios composites weremutant DNA sequences (M-DNA) were used as targeted DNA employed to fabricate corresponding P/Gratios-based sensor
for the hybridization of the P/Gratios-based DNA sensor. Usually, through the same procedure. The performance of P/Gratiosdouble-stranded DNA (dsDNA) and single-stranded DNA based sensor was evaluated by the detection of C-DNA, as(ssDNA) possessed different linking affinities to the graphene- shown in Fig. 5. As one can see in Fig. 5a, the sensitivity ofbased substrate. Generally, the nitrogenous bases in ssDNA P/Gratios-based sensor is improved when the mass ratio of
readily formed π-π bond stacking with the hexagonal units of polyaniline to graphene decreases, and the sensor’s linear
graphene while dsDNA was less approachable to graphene due detection range of C-DNA concentration changes from [10-12,
to the shield of the nitrogenous bases in double-helix structure. 10-8] mol/L to [10-14, 10-10] mol/L. Meanwhile, the slope of the
It is generally accepted that the formed dsDNA escapes from curve of Rct changes from positive 194.4 to negative 48.8 when
the surface of DNA sensor and Rct decreases as the the mass ratio of P/G changes from 1/1 to 1/100 (Fig. 5b),concentration of C-DNA increasing when C-DNA is hybridized. which indicates that dsDNA, the result of hybridization of
However, this work illustrates that Rct exhibits a positive linear target ssDNA to probe ssDNA, prefers to remain on the sensor
relationship with the concentration of C-DNA in the range of surface when the mass ratio is higher than 1/10 while tends to
100 pM to 1 µm (Fig. 4a, b). That is, hybridized dsDNA release when the mass ratio is lower than 1/20. Then aremained on the surface of the sensor, which was quite conclusion is made that the ratio of 1/10 and 1/20 can be seen30
different from the reported works. Without BSA’s self- as the critical values when the slope angle of Rct is close to 0,
assembling on the surface of P/G1:1-based sensor, we obtained as readers can see in the Fig. 5a. That is, Rct didn’t change as
30
the similar results of the reported work as shown on the Fig. the concentration of C-DNA increased from 0.01 pM to 1 uM in
S7. The reverse mechanism of C-DNA detection involved to the the case of P/G1:20-based DNA sensor, which is apparently not
self-assembling of BSA is going to be discussed later. Fig. 4c is scientific, and so the concentration of C-DNA cannot be
the Rct of C-DNA, Nc-DNA and M-DNA (divided by the Rct of discriminated by P/G1:20-based DNA sensor.
P/Gratios-based sensor) detected by P/G1:1-based sensor. It’s not
Compared with P/G1:1-based sensor, P/G1:100-based sensor
hard to notice that Rct changed only subtly after DNA sensor was also employed to detect M-DNA, Nc-DNA and C-DNA andwas met with Nc-DNA, which means that dsDNA wasn’t the results seemed reversed to those of P/G1:1-based sensor if
effectively formed in this situation. As to M-DNA’s Fig. 4c is compared with Fig. 6c. In terms of the hybridization
hybridization, the Rct value is much lower than that of C-DNA of C-DNA and M-DNA, the Rct of P/G1:100-based sensor tended
and this variation seems apparent in Fig. 4c. This finding is

Figure 5. The plot of Rct against the logarithm of the concentrations (logC) of the detection range of the C-DNA forpolyaniline/Graphene hybrids with various mass ratios. (a) The linear relationship of Rct and logC at various ratio ofpolyaniline/graphene. (b) The tendency of R ct changed with mass ratio of polyaniline/graphene, which indicates selectivity.

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Figure 6. (a) Nyquist diagrams for polyaniline/graphene-based (the mass ratio 1:100, named P/G1:100) sensor loaded on GCE atvarious stages. P/G1:100 (1, black line), P/G1:100-Probe DNA (2, red line), P/G1:100-Probe DNA-BSA (3, green line), and P/G1:100-Probe
DNA-BSA-Target DNA(4, blue line). (b) Histograms representing the relative Rct values obtained with the different
oligonucleotides. (c) Histograms representing the relative detection values obtained for P/G1:100–based sensor loaded with CDNA, Nc-DNA, and M-DNA.
Table 1 Performance comparisons on analogical sensors of our work with reported ones recently

Main material

Detectingrange

LOD

MethodLiteratures

Graphene/Gold

1 pm ~ 1 µm

1 pm

DPV

Pandian et al.

Organic semiconductor

100 pm ~ 1 µm

100 pmTransistors

Bonfiglio et al.

Nano-gold

0.1 pm ~ 100 pm

0.1 pM

ECL

Zhang et al.
30

43

44

rGO/ Polyaniline

1 fm ~ 10 nm

0.25 fm

EIS

Jiao et al.

Graphene/ Aminopyrene

1 pm ~ 10 nm

0.45 pm

EIS

Wang et al.

P/G1:1- P/G1:100

0.1 pm ~ 1 µm

0.01 pm

EIS

this work

to decrease, which also means the results of the hybridizationof target DNA, dsDNA, were releasing off from the P/G1:100based sensor surface. The Rct of Nc-DNA remained high andalmost same as that of the DNA sensor, that is, on the one
hand the initial Rct of P/G1:100-based sensor was larger thanthat of M-DNA and C-DNA; and on the other hand, Nc-DNA
nearly did not hybridize with the probe-DNA. All these results
actually echoed the effect of P/G’s mass ratio illustrated in Fig.
4. However, it should be noted that the P/G1:100-based sensor
did not perform well on discriminating SNP. As shown in Fig. 6c,the Rct obtained from detecting M-DNA was just 2.6% higher
than that obtained from detecting C-DNA. It can be suggested
that the hybridization of probe DNA and single basemismatched DNA would also form dsDNA-like DNA polymers.
As a result, the ds-DNA would leave from the surface of sensor
and lead to the decrease of Rct.
It is worth noting that the newly proposed sensor breaks out
the traditional idea that one sensor should only be equippedwith a fixed ratio of polyaniline to graphene, but encourages
taking advantage of composites of P/G in different ratios tostrengthen the functions of sensors. For example, P/Gratiosbased sensor won’t be able to discriminate the concentrationof C-DNA in a wide range from 0.01 pM and 1 µM when theratio of polyaniline to graphene is settled as a specific number.
However, when the ratio of P/G is allowed to change, thewhole range from 0.01 pM and 1 µM for the concentration of

42

45

C-DNA became testable. Compared with the reported works,
the dynamic P/Gratios-based sensor shows great advantages
over the analogical DNA sensor developed since 2012, as listed
in Table 2. Our P/Gratios-based sensor succeeded to reduce LOD
to as low as 0.01 pM while widen the detecting range to befrom 0.01 pM to 1 µM.
Except being excited with the results in terms of theinfluence of varied mass ratio of P/G, we continued toexperiment on the impact of the conjugation of P/Gcomposites in different ratios and BSA on the performance of
P/Gratios-based sensor. To further explore and elucidate themechanism and anomalism in this field, pure polyaniline andpure graphene (prepared by the method of electrochemical
reduction of graphene oxide) were employed to modify GCE
and construct sensors. The electrochemical impedance
spectroscopy was also employed to monitor the process ineach construction step of pure polyaniline and graphenesensor, as shown in Fig. 7. When the polyaniline-modified GCEelectrode was immersed into 10 µM probe-DNA solution for 4
hours, there were no significant change of the value of Rct,which illustrates that few probe-DNA interacted withpolyaniline. However, when GCE electrode was modified withthe composites of polyaniline and graphene and thenimmersed into probe-DNA for 1 hour, the value of Rct changed
remarkably. Such response should be in the result of π-πstacking interaction between graphene and probe-ssDNA.

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Figure 7. Nyquist diagrams in same frequencies range after each step of different components immobilization on polyaniline (a)and graphene (b) deposited on GCE. (a) Nyquist diagrams of polyaniline (1, black line), polyaniline-probe ssDNA (2, red line).
polyaniline-probe ssDNA-BSA (3, blue line). (b) Nyquist diagrams of graphene (1, black line), graphene-probe ssDNA (2, red line),graphene-probe ssDNA-BSA (3, blue line).

Scheme 1. Schematic illustration of the experimental protocol. The platform can easily load the probe ssDNA and the detection
mechanism is affected by the ratio of polyaniline and graphene (P/G). (above) ds-DNA releases from the platform when the mass
ratio of P/G is less than 1/20, (below) ds-DNA remains on the platform when the ratio of P/G is larger than 1/10.

Thus, it proved that the probe-ssDNA and graphene wereunited by P/G-based sensor. The Rct enhanced when BSA
assembled on polyaniline electrode (Fig. 7a) while only slightly
changed when BSA was attached on graphene electrode (Fig.
7b), which suggested that BSA is easier to be adsorbed bypolyaniline than graphene. This is reasonable since polyaniline
has rich charged groups that can attract protein. To further
demonstrate this, the zeta potential of ss-DNA, BSA modified
P/G composites were measured, as shown in Fig. S8. The zeta
potentials decreased with the mass ratio of polyaniline tographene increase. This suggested more negatively charged
BSA was aggregated on the P/G composite particles. The
results fairly correspond with the EIS analysis. As a result,when polyaniline’s ratio rises in the composites of P/G, there
should be more BSA adsorbed to the surface of P/Gratios-based
sensor. This actually sheds light on the explanation for the
many results in this paper. Firstly, no matter whether the DNA
is probe-ssDNA or dsDNA in the result of hybridization, there
exists electrostatic interaction between DNA and polyaniline
or/and BSA. It is believable that the electrostatic interaction
plays an essential part in maintaining DNA on the surface ofthe sensor. Secondly, both probe ssDNA and dsDNA wouldinteract with graphene through π-π stacking attraction, but theπ-π stacking interaction between graphene and dsDNA is infact weakened due to the shield of the nitrogenous bases
which is of double-helix structure. And the strength of π-π

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Journal Namestacking interaction becomes so weak in the case of dsDNA
that this strength could be neglected in terms of its influenceon the position of dsDNA to the sensor’s surface. Different
with electrostatic interaction and π-π stacking interaction,
both of which maintain dsDNA on the surface of the sensor,
Brownian motion is the one that stands on their opposite sideand helps dsDNA escape from the sensor’s surface. Since π-πstacking interaction could barely influence the position of
dsDNA, whether it would remain or release from the surface of
P/Gratios-based sensor should be a result of the competition
between Brownian motion and the electrostatic interaction, as
shown in Scheme 1. The electrostatic force of the interaction
among dsDNA, polyaniline and BSA should be increasingly
attenuated with the decrease of the ratio of polyaniline tographene. The dsDNA releases from the sensor’s surface in theinfluence of Brownian motion when the ratio of P/G is as low
as 1:100; while dsDNA stays on the surface of sensor when theratio is high as 1:1. When the ratio reaches 1:20, there are
hardly any change of Rct detected, which means that there
should be a balance realized between Brownian motion andelectrostatic interaction. In this case, it becomes not authentic
to use the sensor to determine the concentration of target
ssDNA directly, let alone single nucleotide polymorphisms. In
addition, it is understandable that the sensitivity of sensor is in
direct proportion to the concentration of graphene.
Real sample analysis

As a biosensor, it is extremely important to verify its
application with extracted DNA from real samples. In Fig. 8, EIS
measurements show the signals obtained with the pUC19

DNA(target, i.e, real sample). It was observed that in thepresence of target the Rct value of P/G1:1 sensor (Fig. 8a) was
increased and the Rct value of P/G1:100 sensor (Fig. 8b) was
decreased, which confirmed the occurrence of hybridization. It
was also observed that the cyclic voltammetry curve peak of
P/G1:1 sensor (inset of Fig. 8a) decreased while the peak of
P/G1:100 sensor (inset of Fig. 8b) increased, indicating theformed dsDNA molecule remain or release from the surface. In
Fig. 8c and 8d, the Rct values of the sensors for synthetic
oligonucleotides were compared with that for extracted DNA
in the same concentration. As seen in Fig. 8c, the relative Rctvalue of P/G1:1 sensor for real sample was significantly higher
than that for synthetic oligonucleotides. This can be suggested
that longer target oligonucleotides would generate more
severe electron transfer effect. And as shown in Fig. 8d, with
the P/G1:100 sensor, the decrement in Rct value was slightly
lower for real sample detection than that for synthetic
oligonucleotides detection. Kalantari et al. reported that thetarget length (38, 107, 1788 and 2907 base pairs) can46
contribute to changes in the hybridization signal, which wasin good agreement with the P/G sensor.

Conclusions
In conclusion, we designed a dynamic P/Gratios-based DNA
sensor capable of detecting C-DNA in a range from 0.01 pm to
1 µm through changing ratios of polyaniline to graphene.
Moreover, SNPs are also detectable for such sensors. This
work also explores the reasons behind the dynamic
relationship between Rct and ratios of P/G, and concludes that

Figure 8. Nyquist diagrams of P/G1:1 (a) and P/G1:100 (b) sensors for the pUC19 DNA detection, inset was the corresponding cyclic
voltammetry curve. And the relative Rct value of the P/G1:1 (c) and P/G1:100 (d) sensors for synthetic/real sample DNA in the same
concentration.

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electrostatic interaction among dsDNA, BSA and polyaniline,
and Brownian motion should be the decisive forces for
dsDNA’s remaining on or releasing off the surface of thesensor. That is, one is able to control the movement tendency
of dsDNA by changing the proportion of polyaniline in thecomposites, which is helpful for the fabrication of novel
biosensors. And on this basis we proposed that the increasing
response of charge transfer resistance should result from
dsDNA’s remaining on the surface with much polyaniline while
the decreasing response should be assigned to dsDNA’s
releasing off the sensor with dominative graphene.

Acknowledgements
We gratefully acknowledge the support for this work from
Fundamental Research Funds for the Central Universities(S13JB00200, 2015JBM102), the National Natural Science
Foundation of China (Grants 11079010, 21373255), theHundred Talent Program of the Chinese Academy of Sciences(2013SCXQT01) and the Key Laboratory of Carbon Materials(KLCMKFJJ1401).

Notes and references‡*Corresponding authors.
Tel: 86-10-51688577. E-mail: dkjian@bjtu.edu.cn (K.J. Ding).
Tel: 86-351-4040407. E-mail: wangjz@sxicc.ac.cn (J.Z. Wang).
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