Multi-walled Carbon Nanotubes/Manganese Dioxide Nano- flowers-like/Polyaniline Nanowires Nanocomposite Modified Electrode: A New Platform for a Highly Sensitive Electrochemical Impedance DNA Sensor


 We describe in this report a development of label-free electrochemical DNA sensor based on a novel nanostructured electrode of multi-walled carbon nanotubes (MWCNTs)/ nano-flowers-like manganese dioxide (MnO2)/polyaniline nanowires (PANi NWs) nanocomposite. The nanocomposite was synthesized in-situ onto an interdigitated platinum microelectrode (Pt) using a combination of chemical and electrochemical synthesis methods: chemical preparation of MWCNTs/MnO2 and electropolymerization of PANi NWs. The fabricated MWCNTs/MnO2/PANi NWs was then used to develop a label-free electrochemical DNA sensor for a specific gene of Escherichia coli (E.coli) O157:H7 detection. The MWCNTs/MnO2/PANi NWs modified Pt electrode’s surface can facilitate for probe DNA strands immobilization and, therefore the electrochemical signal of the DNA sensors has been improved. The electrochemical impedance spectroscopy (EIS) measurements were conducted to investigate the output signals generated by the specific binding of probe and target DNA sequences. Obtained results indicated that the developed electrochemical biosensor can detect the target DNA in the linear range of 5 pM to 500 nM with a low limit of detection (LOD) at 4.42 × 10 –13 M. The research results demonstrated that the MWCNTs/MnO2/PANi NWs nanocomposite-based electrochemical DNA sensor has a great potential application to the development of highly sensitive and selective electrochemical DNA sensors to detect pathogenic agents.


Introduction
In recent decades, conducting polymers have been attracted the much attention of scientists worldwide for biosensing applications thanks to their unique properties [1][2][3][4]. Pure conducting polymers are formed in various structures such as nanowires, nanotubes and nano-thin films to obtain a large surface area and a high efficiency [5,6]. The combination of conducting polymers with dopants can provide additional advantages, such as high conductivity, large surface areas, environment-friendly features, stability and applicability in biosensors [7][8][9].
Nanocomposite materials created from conducting polymers such as polyaniline (PANi), polypyrrole (PPy), poly(3,4-ethylenedioxythiophene) (PEDOT), and inorganic nanomaterials have excellent characteristics that are not obtained by individual components, including high conductivity, high stability, and high electroactive surface area, leading to new promising applications [10][11][12][13]. These nanocomposites show advantageous properties of inorganic nanomaterials distributed in continuous polymer networks [14,15] and improvement of specifications of conducting polymers, including changes in electron structure of polymer chains, enhancement of charge transfer, and changes in conductivity of polymer chains [16,17]. Actually, several inorganic nanomaterials have been used to dope into the polymer networks, such as PANi/Ni [18], PANi/Au [19], PANi/WO3 [20], PANi/Mn2O3 [21], PANi/MnO2 [22] and PANi/MWCNTs [8] nanocomposites. In general, these nanocomposites can be synthesized by different techniques including chemical, electrochemical, photochemical and mechano-chemical methods and these developed materials have been applied for many applications such as energy storage, electrochemical biosensors, electrochemical sensors, FET-based biosensors, and coating and metal protecting [1]. To apply of the inorganic nanomaterial/conducting polymer nanocomposites for development of electrochemical biosensors, these fabricated nanomaterials have to be coated onto the electrodes using a drop-casting and/or an electrochemical method insitu on the working electrode [2,23]. In which, the electrochemical techniques are often used due to well controlling and high reproducibility. For examples, several PANi nanocomposites-based sensitive electrochemical DNA sensors have been developed such as PANi-graphene nanocomposite [24], silver nanoparticles decorated PANi nanowires [2], carbon dot/ZnO nanorod/PANi composite [25], PANi/gold nanoparticles [26], Sm2O3 NPs-rGO/PANi composite [27]. These developed PANi-based electrochemical DNA sensors have shown sensitivity at nM to fM level for DNA detection, however, the fabrication steps are still laborious. Therefore, in this study, the MWCNTs/MnO2/PANi NWs nanocomposite was synthesized in-situ on Pt microelectrodes by a novel combined chemical-electrochemical technique, and was used for the first time to develop a label-free electrochemical DNA sensor for rapid detection of pathogenic bacteria.

Interdigitated Pt electrodes
The interdigitated Pt microelectrodes were fabricated using a standard photolithography technique with a finger width of 10 μm and a gap size of 20 μm. The fabrication process was conducted by sputtering 10 nm Ti and 200 nm Pt on a 100 nm thick silicon dioxide (SiO2) layer thermally grown on top of a silicon wafer. The configuration and fabrication process of these electrodes were discussed in our previous work [28].

Instrumentations
Scanning electron microscopy (SEM) images and energy dispersive X-ray spectroscopy (EDX) spectra of the synthesized materials were investigated using a Nova NanoSEM 450 microscope.
The structure of the fabricated samples was examined using Fourier transform infrared spectroscopy (FT-IR) spectra measured with a Shimadzu IRAffinity-1S FTIR spectrometer.
Electrochemical measurements were performed using the PGSTAT302N AutoLab electrochemical workstation (Netherlands). A three-electrode configuration consists of the interdigitated Pt electrode as a working electrode (WE), a Pt plate as a counter electrode (CE), and a Ag/AgCl electrode (SCE) in 3M KCl solution (Metrohm) as a reference electrode (RE).

Fabrication of MWCNTs/MnO2/PANi NWs nanocomposite-based Pt microelectrodes
The multi-walled carbon nanotubes were firstly oxidized by a mixture of H2SO4/HNO3 by following typical protocol: a 21.8 mg of MWCNTs was refluxed with 2 mL of mixture  water, and was dried at room temperature.

DNA hybridization detection
To prepare of electrochemical DNA sensor, a 10 μL of 10 μM DNA probe in PBS solution was    (Fig. 3A), PANi NWs (Fig. 3B), MWCNTs/PANi NWs (Fig. 3C), MWCNTs/MnO2 (Fig. 3D) and MWCNTs/MnO2/PANi NWs (Fig. 3E and Fig.   3F) modified on the surface of the Pt microelectrodes. It can be seen that the MWCNTs are highly uniform with diameters ranging from 100 to 200 nm (Fig. 3A). Fig. 3B shows the PANi NWs with 150-160 nm in diameters which were formed directly on the Pt electrodes by the CA method. The size of the PANi NWs is uniform, and the nanowires are distributed throughout the surface of the WE. Moreover, it can be seen in Fig. 3C that, the obtained PANi NWs are relatively uniform and distributed on the MWCNTs layer. On the other hand, Fig. 3D shows that the MWCNTs are surrounded by the MnO2 material with the unique flower-like structure. Finally, it can be seen in the Pt microelectrode surfaces (Fig. 4A, from curve a to curve e, respectively). As can be seen in MWCNTs/PANi NWs materials (Fig. 4B, curve a and curve b, respectively). This result can be explained that the addition of dopant components (MWCNTs/MnO2) causes the transfer of part of quinoid rings to benzenoid rings, leading to an interesting result that the conductivity of the obtained nanocomposite will increase. Besides, the bands associated with the C-N stretching and bending modes are observed at 1300 and 1243 cm -1 [33,34]. The band observed at 3216 cm -1 is assigned to the N-H stretching mode [33]. And, the band associated with the S-O vibration mode is also found at 701 cm -1 [35]. These results indicate that in these samples (Fig. 4B, from curve a to curve c), the PANi material was formed, and H2SO4 was doped into PANi NWs. So, the above

Direct immobilization of DNA probe on Pt/MWCNTs/MnO2/PANi NWs electrodes
The electrochemical impedance spectroscopy (EIS) measurements were used to investigate the efficiency of the DNA probe immobilization on the Pt/MWCNTs/MnO2/PANi NWs electrode due to the electrical impedance changes of the electrode surface caused by the immobilization process of DNA capture probe strands. Figure 5A shows the Nyquist plots of the Pt/MWCNTs/MnO2/PANi NWs electrodes before (curve a) and after (curve b) DNA capture probe immobilization. In comparison with the case that there is only the MWCNTs/MnO2/PANi NWs nanocomposite deposited on the working electrode surface (Fig. 5A, curve a), the impedance of the sensor after DNA probe immobilization increases significantly (Fig. 5A, curve b). It can be seen in  [26,37]. In addition, the MWCNTs/MnO2/PANi NWs nanocomposite with high conductivity also plays an important role in enhancing of signal transmission from DNA hybridization to the transducer, thus, the sensibility of the electrochemical DNA sensor is expected to be improved significantly.

DNA sensors
The selectivity of the DNA sensor based on the Pt/MWCNTs/MnO2/PANi NWs electrode has been evaluated. Fig. 5B shows the Nyquist plots of the DNA sensor before (curve a) and after (curve

Conclusions
The

Conflict of interest
There are no conflicts of interest to declare.