Electrochemical biosensor based on three-dimensional reduced graphene oxide and polyaniline nanocomposite for selective detection of mercury ions

Accepted Manuscript Title: Electrochemical biosensor based on three-dimensional reduced graphene oxide and polyaniline nanocomposite for selective detection of mercury ions Author: Yanqin Yang Mengmeng Kang Shaoming Fang Minghua Wang Linghao He Jihong Zhao Hongzhong Zhang Zhihong Zhang PII: DOI: Reference:

S0925-4005(15)00333-0 http://dx.doi.org/doi:10.1016/j.snb.2015.02.127 SNB 18202

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

27-12-2014 14-2-2015 27-2-2015

Please cite this article as: Y. Yang, M. Kang, S. Fang, M. Wang, L. He, J. Zhao, H. Zhang, Z. Zhang, Electrochemical biosensor based on three-dimensional reduced graphene oxide and polyaniline nanocomposite for selective detection of mercury ions, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.02.127 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Highlights: Preparation of Nanorod-like nanocomposite of three-dimensional reduced graphene oxide and polyaniline by an in situ chemical oxidative polymerization

ip t

method (3D-rGO@PANI).

cr

The presence of 3D-rGO within the nanocomposite further improves the specific surface area and electrochemical performance.

us

High sensitivity and selectivity toward Hg2+ within a concentration range from

Ac ce p

te

d

M

an

0.1 nM to 100 nM with low detection limit of 0.035 nM.

Page 1 of 30

an

us

cr

ip t

Graphical Abstract

M

Nanorod-like nanocomposite of three-dimensional reduced graphene oxide and polyaniline (3D-rGO@PANI) was synthesized via an in situ chemical oxidative

d

polymerization method and was then used as the sensitive layer of a DNA adsorbent

Ac ce p

te

for detecting Hg2+ in aqueous solution.

2

Page 2 of 30

Electrochemical biosensor based on three-dimensional reduced graphene oxide and polyaniline nanocomposite for

ip t

selective detection of mercury ions

cr

Yanqin Yang 1,2, Mengmeng Kang2, Shaoming Fang1, 2, Minghua Wang1, Linghao He2,

Henan Collaborative Innovation Center of Environmental Pollution Control and

an

1

us

Jihong Zhao1, 2, Hongzhong Zhang1, 2* , Zhihong Zhang 1,2*

Ecological Resoration,

Henan Provincial Key Laboratory of Surface and Interface Science,

M

2

Zhengzhou University of Light Industry, No. 166, Science Avenue, Zhengzhou 450001,

Ac ce p

te

d

China

Corresponding authors Tel.: +86-37186609676 Fax: +86-37186609676 ∗

E-mail addresses: [email protected] or [email protected]

3

Page 3 of 30

ABSTRACT: Nanorod-like nanocomposite of three-dimensional reduced graphene oxide and polyaniline (3D-rGO@PANI) was synthesized via an in situ chemical oxidative polymerization method and was then used as the sensitive layer of a DNA

ip t

adsorbent for detecting Hg2+ in aqueous solution. Amino-group-rich 3D-rGO@PANI

cr

exhibited high affinity toward the immobilization of T-rich DNA strands, which

us

preferred to bind with Hg2+ to form T-Hg2+-T coordination. Electrochemical impedance spectroscopy was applied to determine the difference in the

an

electrochemical performances during Hg2+ detection. The results demonstrated that the electrochemical biosensor based on 3D-rGO@PANI nanocomposite showed high

M

sensitivity and selectivity toward Hg2+ within a concentration range from 0.1 nM to 100 nM with low detection limit of 0.035 nM. The proposed nanosensor could be

te

d

applied for highly sensitive and selective determination of heavy metal ion in various environmental detections.

Ac ce p

Keywords: three-dimensional reduced graphene oxide, polyaniline, electrochemical

sensor, mercury ion detection

1. Introduction

Mercury is a highly toxic environmental pollutant with bioaccumulative properties,

high toxicity, and severe adverse effects on human health. To date, various analytical techniques, such as atomic absorption spectrometry [1], inductively coupled plasma mass spectrometry [2], capillary electrophoresis [3], and X-ray fluorescence spectrometry [4], have been performed for mercury quantification. However, these

4

Page 4 of 30

instrumentally intensive methods require expensive and sophisticated instrumentation. Thus, a simple and inexpensive method that not only detects but also quantifies heavy metal ions is desirable for the real-time monitoring of environmental, biological, and

ip t

industrial samples. The development of rapid, special, and cost-effective tools and

cr

tactics to detect Hg2+ ions has been attracting attention. Electrochemical sensors are

us

versatile tools for the determination of various analytes in different fields, such as food inspection, clinical diagnosis as well as environmental monitoring because they

an

are simple, rapid, portable and inexpensive [5-7]. However, the limited surface area of the working electrode is an important problem for electrochemical sensor since the

M

electrode is usually designed to be small to provide the portability for on-site monitoring and compatibility with trace amounts of sample. It always decreases the

te

d

sensitivity of electrochemical detection. Therefore, a few different methods for the electrochemical detection of heavy metal ions has been developed including

Ac ce p

electropolymerized ion imprinted polymer film modified glassy carbon electrode (GCE) [8, 9], polypyrrole-reduced graphene oxide nanocomposite modified GCE [10], graphene/polyaniline/polystyrene

(G/PANI/PS)

nanoporous

fiber

modified

screen-printed carbon electrode [11], and so on. Another strategy for improving electrode surface area is the use of nanostructured materials, including metallic nanoparicles [12] and carbon-based nanomaterials [13, 14]. As a two-dimensional single atom thick of carbon material, graphene has become a material of interest for electrode surface modification due to its outstanding electrical conductivity, high mechanical strength and large surface area. Comparing

5

Page 5 of 30

with the two-dimensional grapheme sheet, three-dimensional reduced graphene oxide (3D-rGO) has become an ideal material in electrochemical sensors because of its excellent physical and chemical properties, such as large specific surface area, conductivity,

and

good

mechanical

performances.

In

addition,

ip t

excellent

cr

graphene-based materials can be easily obtained via simple chemical processing of

us

graphite [15]. PANI, as a useful conducting polymer, has also been widely used in electrochemical applications because of its low cost, good environmental stability, electroactivity,

and

unusual

doping/dedoping

chemistry

[16].

an

interesting

Graphene@PANI composites have been recently highly applied in numerous

M

electronic, optical, and electrochemical devices as well as biosensors. The conventional methods for graphene@PANI composites preparation are in situ

te

d

electrochemical synthesis and in situ polymerization [17, 18]. When PANI is combined with graphene, the composites exhibit enhanced high performance, such as

Ac ce p

electrical conductivity and electrochemical stability, compared with pure PANI [19]. Particular metal ions can selectively bind to a few native or artificial bases in

DNA duplexes to form metal-mediated base pairs [20]. Thus, various assays for Hg2+ detection based on the unique T-Hg2+-T coordination chemistry have been developed

[21, 22]. Consequently, fabricating a functional layer that shows high affinity toward DNA immobilization is very important. DNA often anchors onto the PANI surface because of its high intensity of amino groups, which can be ionized as the positive charges to interact with the negative charges of DNA in aqueous solutions [23]. An electrochemical biosensor based on 3D-rGO@PANI nanocomposite for heavy ion has

6

Page 6 of 30

not been reported. In the present study, an amino group-rich nanocomposite was synthesized via an in situ chemical oxidative polymerization method and was used as the electrochemical biosensor to detect heavy metal ions (Scheme 1). The proposed

ip t

biosensors have two advantages over other routine methods: i) the presence of

cr

3D-rGO within the nanocomposite further improves the specific surface area and

us

electrochemical performance and ii) the high intensity of amino groups in PANI ensures sufficient DNA strands immobilized onto its surface, resulting in high

an

sensitivity for heavy metal ion detection.

M

2. Experimental section 2.1 Materials and Chemicals

te

d

Graphite powder (99.95%), aniline, ammonium per-sulfate, hydrazine hydrate (98%), and 1-octadecanethiol were purchased from Aladdin reagent (Shanghai,

Ac ce p

China). All commercial reagents were used directly without any further purification. Hg2+ stock solution was prepared by dissolving Hg(NO3)2 with 0.5% HNO3. All

solutions were prepared with Milli-Q water (≥18.2 MΩ·cm−1). DNA was obtained from SBS Genetech Co. Ltd. (Beijing, China). The sequence of oligonucleotide is listed as follows: 5'- CCC CCC CCC CCC TTC TTT CTT CCC CTT GTT TGT T-3'. 2.2 Preparation of phosphate buffer solution, electrolyte, and DNA solution Phosphate buffer solution (PBS) (0.1 mol·L-1) was used to prepare the DNA solutions. PBS contained 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 in 1 L of Milli-Q water. The electrolyte solution was prepared immediately

7

Page 7 of 30

before use by dissolving 1.65 g of K3[Fe(CN)6] and 2.11 g of K4[Fe(CN)6] in 1 L of PBS. All solutions were prepared immediately before the experiments and stored at 4 °C until use.

ip t

In order to investigate influence of the ionic strength of PBS, various

cr

concentration of NaCl (0, 140, 300, and 450 mM) were dissolved in PBS solution.

us

The pH values of the buffer solution were adjusted by addition of small amount of 0.1 M NaOH into the system and obtain the value of 3, 4.5, 7.4, 8.3 and 10.0,

an

respectively. 2.3 Preparation of 3D-rGO

M

Graphene oxide (GO) was prepared via a modified Hummers method [24]. First, GO was dispersed in 200 mL of Milli-Q water, followed by exfoliation with Cell

te

d

crusher at 900 W–1000 W for 40 min. An appropriate amount of H2O2 (30%) was added to the GO dispersion, which immediately became atrovirens. Afterward, the

Ac ce p

product was cleaned using 5% HCl and Milli-Q water until pH 7, and the product was freeze-dried in the freeze drier to obtain 3D-GO. About 100 mL of 3D-GO dispersion (1 mg/mL) and 3 mL of aqueous ammonia were then added to a 200 mL beaker with stirring for 2 h. Afterward, 2 mL of hydrazine hydrate was added in the beaker and stirred for 2 h. The mixture was then added in a 100 mL autoclave and kept at 100 °C for 24 h. Finally, 3D-rGO was obtained after 12 h of freeze drying. 2.4 Synthesis of 3D-rGO@PANI nanocomposite The 3D-rGO@PANI composites were prepared via situ polymerization of aniline in the presence ammonium persulfate [(NH4)2S2O8]. The mixture of the 3D-rGO (0.02

8

Page 8 of 30

g) and aniline monomers (0.2 g) was dispersed in 50 mL of Milli-Q water. About 1.35 g of persulfate was then added. The mixture in the reaction kettle was heated at 140 °C for 24 h. The final product was washed with Milli-Q water several times and dried

ip t

in a vacuum oven at 60 °C.

cr

2.5 Fabrication of the electrochemical biosensor based on the 3D-rGO@PANI

us

nanocomposite for detecting Hg2+

The gold electrode with the diameter of 3 mm was carefully cleaned before use.

an

The electrode was immersed in fresh ‘‘piranha’’ solution [a mixture of 7 mL of H2SO4 (98%) and 3 mL of H2O2 (30%)] for 3 min then washed with DI water and ethanol.

M

For Hg2+ detection, 10 μL of 3D-rGO@PANI composite (1.0 mg/mL) was dropped on the surface of the gold electrode. After drying at room temperature, the modified

te

d

electrode was immersed in 0.1 M PBS containing 100 nM DNA for 12 h. Then, the electrode modified with probe DNA was obtained.

Ac ce p

The composite electrode immobilized with DNA was incubated in 10 mM

phos-phate buffer solution (PBS) containing 0.5 M NaCl (pH 7.4) with different concentrations of Hg2+ at room temperature for 2 h, leading to the formation of the

double helix DNA structure through the specific T–Hg2+–T complexes. Then the

electrochemi-cal performance of the electrode was investigated by EIS in 4 mL of 10 mM PBS (pH 7.4) containing 5 mM K3[Fe(CN)6]@K4[Fe(CN)6] (1:1) mixture. Each

measurement was repeated at least 3 times, and the control experiments for various metal ions were performed under the same conditions. After the detection of Hg2+, the biosensor was regenerated by immersing the electrode into 4 mL of 10 mM PBS (pH

9

Page 9 of 30

7.4) containing 5 mM K3[Fe(CN)6]@K4[Fe(CN)6] (1:1) mixture and 1.0 mM EDTA for 2 h. After washed with 10 mM PBS (pH 7.4), the regenerated electrode could be

ip t

reused for another assay. 2.5 Characterization and measurements

cr

X-ray photoelectron spectroscopy (XPS) data were collected using an AXIS HIS

us

165 spectrometer (Kratos Analytical, Manchester, UK) with a monochromatized Al Kr X-ray source (1486.71eV photons). Field emission scanning electron microscopy

an

(FESEM) micrographs were taken using the JSM-6490LV scanning electron

3.6 Electrochemical measurements

M

microscope (Japan).

Electrochemical impedance spectroscopy (EIS) and cyclic voltammogram (CV)

te

d

were performed using a CHI660D electrochemical workstation (Shanghai CH Instrument Company, China). A conventional three-electrode cell was used, which

Ac ce p

included an Ag@AgCl (saturated KCl) electrode as reference electrode, platinum slides as counter electrode, and gold electrode as working electrode. A 5 mM K3[Fe(CN)6]@K4[Fe(CN)6] (1:1) mixture was used as a redox probe in PBS (pH 7.4,

containing 0.1 M KCl). The impedance spectra were measured in the frequency range from 10-2 Hz to 106 Hz in a potential of 0.22 V versus Ag/AgCl (saturated KCl), with a voltage amplitude of 5 mV. CV was measured in the following conditions: voltage range of -0.2 V– 0.8 V and pulse amplitude of 100 mV.

10

Page 10 of 30

3. Results and discussion 3.1 Sensor design The preparation processes of the DNA electrochemical biosensor based on

ip t

3D-rGO@PANI nanocomposites for detecting Hg2+ ions and the generation of the

cr

electrochemical signal was shown in Scheme 1. The composite AuNPs@PANI-GE

us

immobilized with the single strand DNA was used to determine Hg2+. DNA strand is composed of 2 complementary G–C base pairs and 14 mismatched C bases if folded. T–Hg2+–T base pairs will be formed and then cooperate to stabilize the duplex in

an

7

the presence of Hg2+, leading to the construction of a duplex-like DNA scaffold. The

M

concentration of Hg2+ ions added is directly relative to the variation of the EIS signal associated with the electrode.

te

d

3.2 Chemical components of 3D-rGO and 3D-rGO@PANI nanocomposite The chemical components of 3D-rGO and 3D-rGO@PANI nanocomposite were

Ac ce p

investigated and characterized via XPS. Fig. 1a shows the peak-fitted C 1s core-level

XPS spectrum of 3D-rGO. The main peak located at a binding energy of 284.6 eV is related to the C–C bonding. The composites are mainly composed of C, N, and O. The atomic percentages are as follows: C1s, 36.64%; N1s, 2.38%; and O1s, 60.52%. The C1s spectrum of the sample can be deconvoluted into four peaks (Fig. 1b). The main

peak located at a binding energy of 284.6 eV is related to the C–C bonding in defect-free graphite, and the peaks at 285.7, 286.6, and 288.8 eV are related to C-N, C-O, and C=O bonding, respectively [25]. The N 1s core level can be separated into three peaks (Fig. 1c). The binding energies centered at 399.4 eV are attributed to

11

Page 11 of 30

quinoid imine [=N–] and benzenoid amine [–NH–]. The peaks at 400.5 and 402.39 eV are attributed to cationic nitrogen atoms (=NH+) and protonated amine units (–NH+)

3D-rGO@PANI composite.

cr

3.3 Surface morphology of the 3D-rGO@PANI nanocomposite

ip t

in PANI [25]. These results demonstrate the successful synthesis of the

us

The morphology and structure of the 3D-rGO and 3D-rGO@PANI nanocomposite were characterized via FESEM, and the results are shown in Fig. 2. The

an

nanocomposite apparently has a different morphology from that of 3D-rGO and 3D-rGO@PANI. The 3D-rGO (Figs. 2a and b) prepared in this study has a typically

M

curved, layer-like, and three-dimensional structure with a size of tens of nanometers. Figs. 2c and d display distinct differences in morphology for 3D-rGO@PANI

te

d

nanocomposite. PANI particles seem to fully cover the 3D-rGO layer and form a flossy structure such that the layered structure of 3D-rGO can also be observed.

Ac ce p

Therefore, the nanocomposite of 3D-rGO@PANI was successfully obtained.

3.4 Electrochemical performance of the fabricated biosensor The impedance spectra were analyzed using Zview2 software. A modified

Randle’s equivalent circuit is shown in Fig. 3a. The circuit, which is often used to

model interfacial phenomena, includes the following four elements: (i) Rs, the ohmic

resistance of the electrolyte solution; (ii) Wo, the Warburg impedance caused by the diffusion of ions from the bulk electrolyte to the electrode interface; (iii) a constant phase element (CPE) between an electrode and a solution and is related to the surface condition of the electrode [26, 27]; and (iv) Rct, the electron transfer resistance, which

12

Page 12 of 30

exists if a redox probe is present in the electrolyte solution. After the optimization of experimental conditions during the procedure of the Hg2+ determination (See Supporting Information), the obtained parameters was used in the subsequent

ip t

electrochemical measurements. The bare gold electrode has a little semi-circular

cr

domain (Fig. 3a). The Rct value of the bare gold electrode is 0.18 kΩ, which implies a

us

very low charge-transfer resistance value. The diameter of the semicircle part increased after the 3D-rGO@PANI was modified onto the gold electrode surface, and

an

the Rct value is reduced to 0.65 kΩ. The DNA also increased the effect of the blocking layer on the electron transfer and resulted in the continuous decrease of the

M

electrochemical activity and increase in Rct (0.75 kΩ). When Hg2+ ions (10 nM) were added and the T-Hg2+-T bond was formed, the interfacial resistance remarkably

d

increased to 1.62 kΩ. After the addition of Hg2+, the free T-rich part in DNA

te

undergoes a conformational change to form an intramolecular T-Hg2+-T duplex and

Ac ce p

increase the film thickness, which in turn increased the Rct value [28]. The CV results

are in agreement with the results of the previous EIS study, which also suggests that the sensing interface was successfully fabricated according to Fig. 3b. 3.5 Sensitivity of the prepared electrochemical biosensor After DNA was immobilized on the developed sensor layer, different Hg2+

concentrations were subsequently incubated into the system (Fig. 4a). A similar trend during Hg2+ detection was observed. During Hg2+ detection, Rct increased gradually with increasing mercury ion concentration, which demonstrates that the method could have high sensitivity. Before the coordination of T-Hg2+-T was reached to a plateau,

13

Page 13 of 30

higher Hg2+ concentrations could lead to much more Hg2+ binding to the DNA strands, higher. It would increase the film thickness, resulting in the increment of the Rct value [28].

ip t

The quantitative behavior of this assay was assessed by monitoring the

cr

dependence of ΔRct on the amount of mercury ions. The differences in the Rct values

us

before and after the generation of a new layer adhesive (△Rct) could represent its relative amount [29]. ΔRct caused by the circulation of each solution was proportional

an

to the logarithmic concentration of Hg2+ ions from 0.01 nM to 100 nM with a regression equation of ΔRct = 574.7 + 391.9 logCHg2+ and a linear correlation

M

coefficient (R=0.9872) (Fig. 4b). The sensitivity of the developed biosensor was determined based on the values obtained for detection and quantification limits. The

te

d

limit of detection (LOD) is defined as the lowest concentration of analyte that can be detected with an acceptable accuracy. The LOD (S/N=3) was calculated to be 0.035

Ac ce p

nM [30]. Moreover, the developed DNA sensor for Hg2+ detection was compared with

other Hg2+ assay methods reported in literature (Table 1). We observed that the linear

range and LOD of the proposed DNA sensor were significantly improved, and a lower LOD was achieved.

3.6 Selectivity and repeatability of the developed biosensor In an additional set of experiments, we evaluated the selectivity of our system for metal ions. Solutions containing metal ions (Pb2+, Ni2+, Ca2+, Ag+, Co2+, Mn2+, Ca2+,

and Fe3+, each at 10 µM) were tested at conditions similar to those of Hg2+. Although the Hg2+ concentrations were 0.1 µM, which is even 10 times lower than other metal

14

Page 14 of 30

ions, a significant ΔRct was obtained, whereas almost negligible responses to other metal ions were observed (Fig. 5a). This phenomenon suggests that the sensor possessed excellent selective response to Hg2+ against other metal ions.

ip t

A simple chemical regeneration procedure of an electrode is very important for

cr

potential mass production and practical applications for the sensor. It should avoid the

us

complex mechanical cleaning of the electrode and time-consuming procedures for reproducing a new electrode. In the present work, the electrochemical biosensor based

an

on 3D-rGO@PANI could be renewed after EIS measurement by immersing the electrode in a stirred solution containing 1.0 M HNO3, 1.0 KCl and 1.0 mM EDTA for

M

1 min. The renewed 3D-rGO@PANI immobilized with DNA then showed high reproducibility in Hg2+ detection, and the relative standard deviation was 4.5% for 100

3.7 Water sample test

te

d

nM Hg2+ after 10 repetitive measurements (Fig. 5b). It indicated good repeatability.

Ac ce p

River water samples were tested to determine the recovery of the developed

biosensor for Hg2+ detection. First, the water sample collected from Xushui River (Zhengzhou, China) was filtered. Subsequently, standard Hg2+ solutions with different

concentrations (5, 20, and 50 nM) were added to the pretreated water sample. The spiked samples were analyzed separately using the designed biosensor. The results are shown in Table 2. Recovery values ranging from 101.2% to 103.4% were obtained, which indicated that the designed biosensor was applicable for Hg2+ analysis in real

water.

15

Page 15 of 30

4. Conclusions In summary, we successfully developed an electrochemical sensor for detecting Hg2+ by using 3D-rGO@PANI nanocomposite as a sensitive layer. The

ip t

3D-rGO@PANI nanocomposite was prepared via situ polymerization and was

cr

characterized via FTIR, XRD, XPS, and AEM. The electrochemical behavior of the

us

prepared sensor toward Hg2+ ions was evaluated via EIS. The results show excellent sensitivity, good selectivity, and repeatability of the proposed sensor. The LOD of

an

Hg2+ monitored by electrochemical measurements was 0.035 nM. Hence, the as-prepared DNA sensor based on the nanocomposite of 3D-rGO@PANI could be

M

regarded as an optional scheme for mercury ion determination.

d

Acknowledgements

te

This work was supported by Program for the National Natural Science Foundation of China (NSFC: Account No. 51173172 and 21104070) and Science and

Ac ce p

Technology Opening Cooperation Project of Henan Province (Account No. 132106000076). References

[1] Z. Fang, J. Růžička, E. Hansen, An efficient flow-injection system with on-line ion-exchange preconcentration for the determination of trace amounts of heavy metals by atomic absorption spectrometry, Analytica Chimica Acta, 164(1984) 23-29. [2] D. E. Nixon, K. R. Neubauer, S. J. Eckdahl, J. A. Butz, M. F. Burritt, Comparison of tunable bandpass reaction cell inductively coupled plasma mass spectrometry with conventionalinductively coupled plasma mass spectrometry for the determination 16

Page 16 of 30

of heavy metals in whole blood and urine, Spectrochimica Acta Part B: Atomic Spectroscopy, 59(2004) 1377-1387. [3] Y. Li, Y. Jiang, X. P. Yan, Probing Mercury Species-DNA Interactions by Capillary

ip t

Electrophoresis with On-Line Electrothermal Atomic Absorption Spectrometric

cr

Detection, Analytical Chemistry, 78(2006) 6115-6120.

us

[4] O. W. Lau, S. Y. Ho, Simultaneous determination of traces of iron, cobalt, nickel, copper, mercury and lead in water by energy-dispersive x-ray fluorescence

an

spectrometry after preconcentration as their piperazino-1,4-bis(dithiocarbamate) complexes, Analytica Chimica Acta, 280(1993) 269-277.

M

[5] X. Chen, Z. Ma, Multiplexed electrochemical immunoassay of biomarkers using chitosan nanocomposites, Biosensors and Bioelectronics, 55(2014) 343-349.

te

d

[6] Y. Li, J. Deng, L. Fang, K. Yu, H. Huang, L. Jiang, W. Liang, J. Zheng, A novel electrochemical DNA biosensor based on HRP-mimicking hemin/G-quadruplex

Ac ce p

wrapped GOx nanocomposites as tag for detection of Escherichia coli O157:H7,

Biosensors and Bioelectronics, 63(2015) 1-6. [7] G. Yan, Y. Wang, X. He, K. Wang, J. Su, Z. Chen, Zhihe. Qing, A highly sensitive electrochemical assay for silver ion detection based on un-labeled C-rich ssDNA probe and controlled assembly of MWCNTs, Talanta 94(2012) 178-183. [8] X. Fua, X. Chen, Z. Guo, C. Xie, L. Kong, J. Liua, X. Huang, Stripping voltammetric detection of mercury(II) based on a surface ion imprinting strategy in electropolymerized microporous poly(2-mercaptobenzothiazole) films modified glassy carbon electrode, Analytica Chimica Acta, 685(2011) 21-28.

17

Page 17 of 30

[9] X. Fu, Ju. Wu, L. Nie, C. Xie, J. Liu, X. Huang, Electropolymerized surface ion imprinting films on a gold nanoparticles/single-wall carbon nanotube nanohybrids modified glassy carbon electrode for electrochemical detection of trace mercury(II) in

ip t

water, Analytica Chimica Acta, 720(2012) 29-37.

ions

results

in

selective

response:

electrochemical

studies

on

us

metal

cr

[10] Z. Zhao, X. Chen, Q. Yang, J. Liu, X. Huang, Selective adsorption toward toxic

a polypyrrole/reduced graphene oxide nanocomposite, Chemical Communications,

an

48(2012) 2180-2182.

[11] N. Promphet, P. Rattanarat, R. Rangkupan, O. Chailapakul, N. Rodthongkum, An

M

electrochemical sensor based on graphene/polyaniline/polystyrene nanoporous fibers modified electrode for simultaneous determination of lead and cadmium, Sensors and

te

d

Actuators B: Chemical, 207(2015) 526-534. [12] Q. Fang, Y. Shen, B. Chen, Synthesis, decoration and properties

Ac ce p

of three-dimensional graphene-based macrostructures: A review, Chemical Engineering Journal, 264(2015) 753-771. [13] H. Zhu, Y. Xu, A. Liu, N. Kong, F. Shan, W. Yangd, C. J. Barrow, J. Liu, Graphene nanodots-encaged porous gold electrode fabricated via ion beam sputtering deposition for electrochemical analysis of heavy metal ions, Sensors and Actuators B: Chemical, 206(2015) 592-600. [14] H. Huang, T. Chen, X. Liu, H. Ma, Ultrasensitive and simultaneous detection of heavy metal ions based on three-dimensional graphene-carbon nanotubes hybrid electrode materials, Analytica Chimica Acta, 852(2014) 45-54.

18

Page 18 of 30

[15] X. Huang, Z. Yin, S. Wu, X. Qi, Q. He, Q. Zhang, Q. Yan, F. Boey, H. Zhang, Graphene-Based Materials: Synthesis, Characterization, Properties, and Applications, Small, 7(2011) 1876-1902.

ip t

[16] Q. Wu , Y. Xu , Z. Yao , A. Liu, G. Shi, Supercapacitors Based on Flexible

cr

Graphene/Polyaniline Nanofiber Composite Films, ACS Nano, 4(2010) 1963-1970.

us

[17] S. Ameen, M. S. Akhtar, H. S. Shin, Hydrazine chemical sensing by modified electrode based on in situ electrochemically synthesized polyaniline/graphene

an

composite thin film, Sensors and Actuators B: Chemical 173(2012) 177-183. [18] G. Wang, S. Zhuo, W. Xing, Graphene/polyaniline nanocomposite as counter

M

electrode of dye-sensitized solar cells, materials letters, 69(2012) 27-29. [19] X. Lu, Y. Ye, Y. Xie, Y. Song, S. Chen, P. Li, L. Chen, L. Wang, Copper

te

d

coralloid granule/polyaniline/reduced graphene oxide nanocomposites for nonenzymatic glucose detection, Anal. Methods 6(2014) 4643-4651.

Ac ce p

[20] G. Wang, H. Huang, X. Zhang, L. Wang, Electrically contacted enzyme based on dual hairpin DNA structure and its application for amplified detection of Hg2+,

Biosensors and Bioelectronics, 35(2012) 108-114. [21]

J.

Wan,

G.

Yin,

X.

Ma,

L.

Xing,

X.

Luo,

Highly

Sensitive

Electrochemiluminescence Detection of Mercury(II) Ions Based on DNA-Linked Luminol-Au NPs Superstructure, Electroanalysis, 26(2014) 823-830. [22] R. M. Kong, X. B. Zhang, L. L. Zhang, X. Y. Jin, S. Y. Huan, G. L. Shen, R. Q. Yu, An ultrasensitive electrochemical “turn-on” label-free biosensor for Hg2+ with AuNP-functionalized reporter DNA as a signal amplifier, Chemical Communications,

19

Page 19 of 30

(2009) 5633-5635. [23] H. Peng, L. Zhang, C. Soeller, J. Travas-Sejdic, Conducting polymers for electrochemical DNA sensing, Biomaterials, 30(2009) 2132-2148.

ip t

[24] W. S. Hummers Jr, R. E. Offeman, Preparation of Graphitic Oxide, Journal of the

cr

American Chemical Society, 80(1958) 1339-1339.

us

[25] W. L. Zhang, B. J. Park, H. J. Choi, Colloidal graphene oxide/polyaniline nanocomposite and its electrorheology, Chemical Communications, 46(2010)

an

5596-5598.

[26] T. J. Pajkossy, Impedance of rough capacitive electrodes, Journal of

M

Electroanalytical Chemistry, 364(1994) 111-125.

[27] R. De Levie, The influence of surface roughness of solid electrodes on

te

d

electrochemical measurements, Electrochimica Acta, 10(1965) 113-130. [28] He Gong and Xiaohong Li, Y-type, C-rich DNA probe for electrochemical

Ac ce p

detection of silver ion and cysteine, Analyst, 136(2011) 2242-2246. [29] A. Li, F. Yang, Y. Ma, X. R. Yang, Electrochemical impedance detection of DNA hybridization based on dendrimer modified electrode, Biosensors and Bioelectronics, 22(2007) 1716-1722.

[30] S. N. Topkaya, B. Kosova, M. Ozsoz, Detection of Janus Kinase 2 gene single point mutation in real samples with electrochemical DNA biosensor, Clinica Chimica Acta, 429(2014) 134-139. [31] S. Cai, K. Lao, C. Lau, J. Lu, “Turn-On” Chemiluminescence Sensor for the Highly Selective and Ultrasensitive Detection of Hg2+ Ions Based on Interstrand

20

Page 20 of 30

Cooperative Coordination and Catalytic Formation of Gold Nanoparticles, Analytical Chemistry, 83(2011) 9702-9708. [32] X. Wang, Y. Shen, A. Xie, S. Chen, One-step synthesis of Ag@PANI

ip t

nanocomposites and their application to detection of mercury, Materials Chemistry

cr

and Physics 140(2013) 487-492.

us

[33] Z. M. Dong, G. C. Zhao, Quartz Crystal Microbalance Aptasensor for Sensitive Detection of Mercury(II) Based on Signal Amplification with Gold Nanoparticles,

an

Sensors 12(2012) 7080-7094.

[34] X. Ding, L. Kong, J. Wang, F. Fang, D. Li, J. Liu, Highly Sensitive SERS

M

Detection of Hg2+ Ions in Aqueous Media Using Gold Nanoparticles/Graphene Heterojunctions, ACS Applied Materials & Interfaces, 5(2013) 7072-7078.

te

d

[35] X. Liu, Y. Tang, L. Wang, J. Zhang, S. Song, C. Fan, S. Wang, Optical Detection of Mercury(II) in Aqueous Solutions by Using Conjugated Polymers and Label-Free

Ac ce p

Oligonucleotides (pages 1471–1474), Advanced Materials, 19(2007) 1471-1474.

21

Page 21 of 30

ip t cr us

an

Scheme 1 Schematic diagram of the detection of Hg2+ ions using the developed

Ac ce p

te

d

M

electrochemical DNA biosensor based on 3D-rGO@PANI nanocomposite.

22

Page 22 of 30

(a) C 1s

(b) C1s

(c) N1s

C-C

=N-

C-C

C-N

280

284

288

292

280

285

290

295

392

Bingding energy/eV

cr

C=O

-NH-

ip t

=NH+ C-O

396

400

404

408

412

us

Fig. 1 (a) C 1s core-level XPS spectra of 3D-rGO and the (b) C 1s and (c) N 1s

Ac ce p

te

d

M

an

core-level XPS spectra of 3D-rGO@PANI nanocomposite

23

Page 23 of 30

(d)

ip t

(c)

cr

(b)

an

us

(a)

M

Fig. 2 (a and b) Typical SEM micrographs of 3D-rGO and (c-d) 3D-rGO@PANI

Ac ce p

te

d

nanocomposite

24

Page 24 of 30

(a)

(b) Au electrode 3D-rGO@PANI DNA 2+ 10 nM Hg

100 50 0

ip t

0.8

I/A 0.4

-50

Au electrode 3D-rGO@PANI DNA 2+ 10 nM Hg

0.0 1

2

3

-100

4

-0.3

0.0

Z'/kohm

cr

-Z''/kohm

1.2

0.3

0.6

0.9

Potential / V

us

Fig. 3 (a) EIS Nyquist plots and (b) CV curves of the different modified electrodes containing (i) bare gold electrode, (ii) gold electrode modified with 3D-rGO@PANI,

an

(iii) DNA immobilized onto gold electrode modified with 3D-rGO@PANI, and (iv)

M

Hg2+ detection based on the fabricated electrode in PBS containing 5 mM

Ac ce p

te

d

K3[Fe(CN)6]

25

Page 25 of 30

(b)

(a) 1.5 1.2

 Rct= 574.718+391.923logC( Hg

2+

1.0

)

R=0.9872

 Rct/kohm

0.9

100 nM

ip t

-Z''/kohm

1.5

0.6

0.5

0.3

1.5

3.0

4.5

-0.7

6.0

cr

0 nM

0.0

0.0

0.7

logC( Hg

Z'/kohm

1.4

2.1

2+

us

)

Fig. 4 (a) Nyquist plots of EIS for Hg2+ detection and (b) linear relationship between

Ac ce p

te

d

M

an

ΔRct and the logarithm of Hg2+ concentration

26

Page 26 of 30

(a)

7

(b)

3

1

5

4

3 0

2+

Ni

2+

2+

Cu

Ag

+

2+

Co Mn2+ Ca

2+

Fe

3+

Hg

0

2+

2

4

6

8

10

cr

Pb

ip t

2

Rct/ kohm

△Rct / kohm

6

Cycle number

us

Fig. 5 (a) Rct change in the presence of 10 µM of other metal ions and 0.1 µM Hg2+

an

ions and (b) reusability of EIS-based Hg2+ sensor challenged with 100 nM Hg2+ ions

Ac ce p

te

d

M

and washed with 50 mM cysteine

27

Page 27 of 30

Table 1 Comparison of sensitivities of different Hg2+ ion polymeric assay methods

Gold

Linear range

LOD

(nM)

(nM)

0.1 to 10

0.05

0.001 to 1000

0.001

Detection technique Chemiluminescence

Ref

Ag@polyaniline

Surface-enhanced

(PANI) core–shell

Raman scattering

nanoparticles QCM

us

Aptamer along

0.5 to 100

with Au-NPs Surface-enhanced

an

Au-NPs-rGO

0.1 to 6000

Raman scattering Fluorescence

M

poly(3-(3’-N,N,Ntriethylamino-1’-p ropyloxy)-4-meth

[33]

0.1

[34]

42

[35]

d

0.24

Ac ce p

hydrochloride)

0 to 667

[32]

te

yl-2,5-thiophene

[31]

cr

nanoparticles

ip t

Sensitive layer

28

Page 28 of 30

Table 2 Analysis of Hg2+ in water samples Found (nmol)

Recovery (%)

RSD (%)

5

5.06

101.2

1.96

20

20.68

103.4

3.29

50

51.05

102.01

2.06

Ac ce p

te

d

M

an

us

cr

River water

Add (nmol)

ip t

Sample

29

Page 29 of 30

Yanqing Yang is a lecturer in the department of polymer materials and engineering in Zhengzhou University of Light Industry. She obtained her master degree in Chemistry from the Zheng Zhou University, China (2008). Her research interests are in environmental engineering and nanocomposites.

cr

ip t

Mengmeng Kang is a graduate student at Zheng Zhou University of Light Industry. He obtained his undergraduate degree in Chemistry from the Zheng Zhou University of Light Industry, China (2011). He mainly engaged in the preparation of inorganic nanomaterials and the research of biosensors.

an

us

Shaoming Fang is a Professor in the Department of Materials and Chemical Engineering institute at Zheng Zhou University of Light Industry. His research interests are in the development of polymer and nanomaterials. He obtains his undergraduate degree at the Beihang University, China, and completed his doctoral work at the University of Hebei University of Technology, China. He is the dean of Materials and Chemical Engineering institute. He mainly engaged in polymer materials, functional materials and materials chemistry.

d

M

Minghua Wang is an associate professor in the department of polymer materials and engineering, Zhengzhou University of Light Industry. He obtained his master degree in Chemistry from the Zheng Zhou University of Light Industry, China (2008). Her research interests are in environmental engineering and nanocomposites.

te

Linghao He is a associate professor is the department of polymer materials and engineering in Zhengzhou University of Light Industry. Her research interests are in functional polymer materials and in nanocomposites. She obtained her Ph. D. from Zhengzhou University in 2011.

Ac ce p

Jihong Zhao is the professor of the department of Environmental Engineering, Zhengzhou University of Light Industry. She obtained her PhD from Research Center for Eco-Environmental Sxiences,Chinese Academy of Sciences, China (2003). Her research interests are in environmental engineering and microbiology.

Hongzhong Zhang is the professor of the department of Environmental Engineering, Zhengzhou University of Light Industry. He obtained his PhD from School of Civil and Environmental Engin eering, Beijing University of Science and Technology, China (2005). His research interests are in environmental engineering and nanocomposites. Zhihong Zhang is a Professor in the Department of Materials and Chemical Engineering institute at Zheng Zhou University of Light Industry. He obtains his undergraduate degree at the Zhengzhou University, China, and completed her doctoral work at Max-Plank Institute, German. After two years of postdoctoral work at the national university of Singapore she joined Zheng Zhou University of Light Industry in 2005, where she has since worked on plasma-polymerized polymer and biosensors.

30

Page 30 of 30