Electrochemically Effective Surface Area of a Polyaniline Nanowire-Based Platinum Microelectrode and Development of an Electrochemical DNA Sensor

Electrochemical DNA sensors based on nanocomposite materials of polyaniline nanowires (PANi NWs) have been published in the literature. However, it is interesting that there are very few research studies related to the development of electrochemical DNA sensors based on PANi NWs individually. In this study, PANi NWs were synthesized site-specifically on a Pt microelectrode with only 0.785mm 2 area using an electropolymerization procedure. The electrosynthesis allows direct deposition of PANi NWs onto the Pt microelectrode in a rapid and cost-effective way. The good properties of PANi NWs including uniform size, uniform distribution throughout the Pt working electrode, and H 2 SO 4 doping which improved the conductivity of the PANi material were obtained. Especially, the electrochemically effective surface area of the PANi NW-based Pt microelectrode determined in this work is nearly 19 times larger than that of the Pt working electrode. The PANi NW layer with large electrochemically effective surface area and high biocompatibility is consistent with the application in electrochemical DNA sensors. The fabricated DNA sensors show advantages such as simple fabrication, direct detection, high sensitivity (with the detection limit of 2.48 × 10 − 14 M), good specificity, and low sample volume requirement. This study also contributes to confirm the role of PANi NWs in DNA probe immobilization as well as in electrochemical signal transmission in the development of electrochemical DNA sensors.


Introduction
DNA sensors have demonstrated a wide range of applications in detection of pathogenic viruses and bacteria, disease diagnosis, and food safety monitoring [1][2][3][4][5]. Particularly, electrochemical DNA sensors have shown many advantages such as high sensitivity, good selectivity, rapid detection time, reasonable cost, and high compatibility with lab-on-achip systems [6][7][8]. In the development of electrochemical DNA sensors, many studies have been carried out to improve the sensitivity of sensors, to simplify the sensor fabrication process, and to miniaturize the analytical system [9][10][11]. In these strategies, the microelectrode modi cation using nanostructured materials is an important solution that contributes to promoting the research and application of electrochemical DNA sensors [12][13][14].
Nanostructured conducting polymers with excellent properties, such as good electrical conductivity, environmental durability, ease of preparation, and ability to integrate with electronic components, are ideal materials for various applications including nanoelectronic devices, catalysts, electron eld emitters, actuators, membranes, biomedical devices, rechargeable batteries, supercapacitors, sensors, and especially, electrochemical DNA sensors [15][16][17][18][19][20]. In the eld of electrochemical DNA sensors, one-dimensional nanostructures of polyaniline (PANi) including PANi nanowires (PANi NWs), PANi nanotubes, PANi nano bers, and PANi nanorods have attracted research interest due to their large surface area, tunable electrochemical properties, and high biocompatibility [21]. An electrochemical DNA sensor operates based on the principle that interactions between the probe and target DNA sequences will cause changes in the sensor's electrochemical signals. In the fabrication of electrochemical DNA sensors, the PANi nanomaterials can play important roles as intermediate material layers between DNA probe sequences and metal microelectrodes [22]. ese PANi nanomaterials can act as linking agents for the immobilization of DNA probe sequences on the microelectrode surface and can enhance the signal transmission from biointeractions to transducers [22]. erefore, the one-dimensional PANi nanostructures can be used to modify the microelectrode surface in order to fabricate electrochemical DNA sensors with high sensitivity [12,23]. e one-dimensional PANi nanostructures can be fabricated using chemical and/or electrochemical routes [24][25][26]. In the development of electrochemical DNA sensors based on onedimensional nanostructures, electrosynthesis methods have shown many advantages compared to chemical methods, such as rapidity, ease of mass production, uniformity, and low cost [27]. Besides, electrosynthesis methods allow one-dimensional nanostructures to grow directly on electrode surfaces, thereby overcoming the disadvantage of chemical methods, which is the difficulty in binding the synthesized nanomaterials with electrode surfaces [22]. Moreover, when using electrosynthesis methods, the size of one-dimensional nanostructures and the thickness of nanomaterial layers formed on electrode surfaces can be easily controlled by changing synthesis conditions [25,28]. erefore, the application of nanocomposite materials containing electrosynthesized PANi NWs in the fabrication of electrochemical DNA sensors has been published in the literature [12,23,29]. However, there are very few research studies related to the development of electrochemical DNA sensors based on PANi NWs individually.
In this work, PANi NWs were synthesized directly on a Pt microelectrode with only 0.785 mm 2 area using an electrochemical procedure. Electrochemically effective surface area of the Pt/PANi NW microelectrode, which is an important parameter affecting the sensitivity of an electrochemical sensor, was determined using data from electrochemical measurements. en, a simple and effective electrochemical DNA sensor was developed using the optimized Pt/PANi NW microelectrode.
e Pt/PANi NW microelectrode with unique structural and electrochemical properties was expected to simplify the DNA probe immobilization, to improve the electrochemical signals of the fabricated DNA sensor, and to reduce the volumes of analytical samples. is work will also contribute to confirm the role of PANi NWs in the development of electrochemical DNA sensors.

Integrated Pt Microelectrodes.
e integrated Pt microelectrode consisting of a 0.785 mm 2 working electrode (WE) and a 5.0 mm 2 counterelectrode (CE) was deposited on a SiO 2 /Si substrate using the cathode sputtering technique. Its configuration and fabrication process were discussed in our previous study [27]. e fabrication process is briefly described as follows: first, a silicon wafer was thermally oxidized to create an insulation oxide layer, and a photoresist layer was then coated on the surface of SiO 2 ; second, a photomask was placed onto a mask aligner and exposed to an UV light; third, the wafer was immersed in a development solution to remove undesired parts of photoresist, and then desired parts on the wafer were hardened by thermal annealing in the air. After that, the sputtering technique was applied to deposit a platinum membrane.

Instrumentations.
A PGSTAT302N Autolab electrochemical workstation connected with an Ag/AgCl electrode in 3 M KCl solution (as a reference electrode, RE) and the fabricated Pt microelectrode (consisting of the WE and the CE) was used to perform electropolymerization of PANi NWs and to conduct electrochemical measurements. A Nova NanoSEM 450 microscope was used to investigate the surface morphology and chemical composition of PANi NWs through scanning electron microscopy (SEM) images and energy dispersive X-ray spectroscopy (EDX) spectra. A LabRAM HR 800 Raman was used to study the structural properties of PANi NWs.

Electropolymerization of PANi NWs on Pt Working
Electrodes (WEs). An electrolyte solution containing 0.05 M aniline and 0.5 M H 2 SO 4 was blown with N 2 gas for 15 minutes to remove the dissolved oxygen. e chronoamperometry (CA) method with an applied voltage of 0.9 V vs. Ag/AgCl RE was used to electropolymerize PANi NWs on the Pt WEs. After that, the Pt/PANi NW WEs were gently washed with deionized water and were dried at room temperature.

Immobilization of DNA Probe on Pt/PANi NW WEs and
Detection of DNA Target Using PANi NW-Based Electrochemical DNA Sensors. Each of the Pt/PANi NW WEs was coated with 5 μL of a DNA probe solution (10 μM in PBS, pH 7.4). e immobilization of DNA probe was kept for 2 hours at room temperature. e Pt/PANi NW/DNA probe microelectrodes were then washed with deionized water to remove DNA probe sequences with weak binding to PANi NWs and were dried at room temperature.
After that, each of the fabricated DNA sensors was dropped with 5 μL of complementary DNA target solution with different concentrations (from 1.0 × 10 −13 M to 1.0 × 10 −8 M) in PBS (pH 7.4). e DNA hybridization was conducted for 2 hours at room temperature. en, electrochemical impedance spectroscopy (EIS) spectra of the DNA sensors were recorded in a solution consisting of K 3 Fe(CN) 6 /K 4 Fe(CN) 6 (0.005 M) and 0.1 M·KCl in PBS (pH 7.4). e frequency range was from 10 5 Hz to 0.1 Hz, the AC potential was 5 mV, and the DC potential was 160 mV. In the EIS spectra, the increase in the electron transfer resistance ΔR ct (ΔR ct � R ct,i− R ct,0 ), where R ct,i and R ct,0 are the electron transfer resistances in the presence and absence of the DNA target, respectively, is used as the DNA hybridization signal.

Structural and Electrochemical Characterization of PANi
NWs Electrosynthesized on Pt Microelectrodes. PANi NWs were electropolymerized directly on Pt WEs using the CA method.
e CA curves recorded during these electropolymerization processes with different sets of time are shown in Figure 1(a). As can be seen in Figure 1(a), curve a-d, the current increases when the time for electropolymerization increases from 300 to 600 seconds. e maximum current value is obtained when the electropolymerization time is 600 seconds (Figure 1(a), curve d).
e formation of conductive polymer layers with good electrical conductivity on Pt WEs led to the increase in the current density. e high current density would be favorable for signal transmission from the biological interaction to the transducer in the development of electrochemical DNA sensors. However, when the electropolymerization time continued to increase (higher than 600 seconds), it appeared that the formed PANi layer was too thick to adhere to the Pt WE's surface and was peeled off during the washing of the Pt/PANi NW electrode with deionized water. erefore, the selected polymerization time for the subsequent electrosynthesis processes was 600 seconds. e EIS spectra in Nyquist form of the Pt/PANi NW WEs recorded in K 3 Fe(CN) 6 /K 4 Fe(CN) 6 (0.005 M) and 0.1 M KCl solution corresponding to the different electropolymerization times are also shown in Figure 1(b). In Figure 1(b), curve a-d, the charge transfer resistance (R ct ) value decreases from 1647 Ω (Figure 1(b), curve a) to 75 Ω (Figure 1(b), curve d) when the electropolymerization time increases from 300 to 600 seconds. e R ct value is the smallest (75 Ω) when the electropolymerization time is 600 seconds (Figure 1(b), curve d).
e EIS spectra in Figure 1(b) are completely consistent with the CA results in e Pt/PANi NW WE (Figure 1(c), curve b) has a higher peak current than the Pt WE ( Figure 1(c), curve a). PANi NWs with high conductivity grew directly on the Pt WE; as a result, the peak current of the Pt/PANi NW WE increased. e use of highly electroactive PANi NWs to modify the Pt WE was expected to improve the electrochemical active area for DNA probe immobilization and to enhance the electrochemical signal of DNA sensors. e SEM image of PANi NWs electrosynthesized on the Pt WE is shown in Figure 2(a). e average diameter of PANi NWs is 102 nm (Figure 2(b)). e size of PANi NWs is relatively uniform, and PANi NWs are distributed throughout the WE surface. e uniform distribution of PANi NWs on the WE surface will play an important role in enhancing the repeatability of electrochemical DNA sensors. e synthesized PANi material with nanowire structure will be more favorable for the DNA probe immobilization than the PANi film form [22,30]. Figure 2(c) shows the Raman spectrum of PANi NWs formed on the Pt WE surface. e characteristic peaks for the PANi material are consistent with the descriptions in the literature. e band related to the C-H bending vibrations corresponding to the quinoid and benzenoid rings is at 1163 cm −1 [31]. e bands at 1257, 1352, and 1477 cm −1 are assigned to the C-N stretching mode of polaronic units, the C-N + vibration of polaronic structures, and the C�N stretching mode of the quinoid rings [12,31,32], respectively. e band associated with the C-C stretching mode of the benzenoid rings is at 1591 cm −1 [31]. Besides, the bands at 748, 783, 807, and 837 cm −1 are associated with the out-of-plane C-H motions which depend on the torsion angle between two aniline rings of the emeraldine form of PANi [32][33][34].
e Raman spectrum demonstrates that the PANi material was successfully electrosynthesized on the Pt WE and existed in the emeraldine form, which is the most conductive form of PANi. e EDX spectrum of PANi NWs on the Pt WE is shown in Figure 2 NWs. e doping of PANi NWs with H 2 SO 4 will play an important role in improving the conductivity of PANi NWs [35,36]; thus, it will help to enhance the electrochemical signals of DNA sensors.
Electrochemically effective surface area (A) of WEs is an important factor that improves the electrochemical response and the sensitivity of electrochemical sensors [37]. In this study, the electrochemically effective surface area of the PANi NW-based Pt WE was determined by its CV data recorded at different potential scan rates from 30 to 80 mV·s −1 in K 3 Fe(CN) 6 (Figure 3(a)). From these CV data, the dependencies of the anodic and cathodic peak potentials on the natural logarithm of the potential scan rate were investigated, and their corresponding linear t lines for each set of experimental data are shown in Figure 3 and E pc vs. ln], It can be seen in Figure 3(b) that the peak potential di erence ΔE (ΔE E pa − E pc ) increases as the potential scan rate ] increases. is result indicates that the electrochemical processes happening on the Pt/PANi NW electrode surface are quasireversible [38]. On the other hand, the dependencies of E pa and E pc on ln] in which v (V s −1 ) is the potential scan rate are linear, and they are expressed by the following equations [38,39]: where E 0 (V) is the formal standard potential, α is the charge transfer coe cient, n is the number of transported electrons, k 0 (cm·s −1 ) is the electron transfer rate constant, D (cm 2 ·s −1 ) is the di usion coe cient, R is the gas constant (R 8.314 J·mol −1 ·K −1 ), T is the working temperature (T 298 K), and F is Faraday's constant (F 96480 C·mol −1 ). e values of α and n can be calculated from the slopes of E pa vs. ln] and E pc vs. ln], and the results are that the value of α is 0.460 and the value of n is 0.901. e value of n veri es that the oxidation of Fe(CN) 6 4− and the reduction of Fe(CN) 6 3− occurring on the Pt/PANi NW electrode surface are through monoelectronic steps. e di usion coe cients (D O and D R ) of ferri-and ferrocyanide ions in 0.1 M KCl medium at 25°C are 7.20 × 10 −6 cm 2 ·s −1 and 6.66 × 10 −6 cm 2 ·s −1 , respectively [40]. From the intercepts of E pa vs. ln] and E pc vs. ln] and E 0 (E pa + E pc )/2 0.17 V [38], the values of k 0 were calculated as 1.436 × 10 −3 cm·s −1 and 1.360 × 10 −3 cm·s −1 for the anodic and the cathodic branches, respectively, and the average value was 1.398 × 10 −3 cm·s −1 .
ese results further con rmed that, in the considered scan rate range, the oxidation of Fe(CN) 6 4− and the reduction of Fe(CN) 6 3− which occurred on the Pt/PANi NW electrode surface are quasireversible.
Besides, from the CV data expressed in Figure 3(a), the relationship between the cathodic peak current and the square root of the potential scan rate is plotted in Figure 3  Journal of Nanotechnology It can be seen in Figure 3(c) that the cathodic peak current depends linearly on the square root of the potential scan rate. On the other hand, the electrochemically e ective surface area, A (cm 2 ), of the Pt/PANi NW electrode can be determined by using the Randles-Sevcik equation for quasireversible reactions as follows [41]: where ] (V·s −1 ), α, n, and D (cm 2 ·s −1 ) are mentioned in equations (3)-(6) above, C 0 (mol·cm −3 ) is the concentration of redox species, and I pc (A) is the cathodic peak current. Based on equation (8) and the slope of the linear dependence of the cathodic peak current on the square root of the potential scan rate (Figure 3(c)), the value of A of the Pt/ PANi NW electrode was calculated to be 0.147 cm 2 . us, the electrochemically e ective surface area of the Pt/PANi NW WE is nearly 19 times larger than that of the Pt WE (0.785 × 10 −2 cm 2 ). e above CV, SEM, Raman, and EDX results show that in this work, PANi NWs were formed exactly on a Pt WE with very small area (0.785 × 10 −2 cm 2 ) using the simple and e ective CA method. e obtained PANi NWs with onedimensional nanowire structure and high electrochemically e ective surface area will be able to signi cantly enhance the electrochemical response of the WE and to improve the electrochemical DNA sensor's sensitivity compared with conventional membrane materials [35].

Direct Immobilization of DNA Probe on Pt/PANi NW Microelectrodes and DNA Hybridization Detection Using
PANi NW-Based Electrochemical DNA Sensors. It can be seen in Figure 4 that the R ct value of the Pt/PANi NW/DNA probe electrode (5767 Ω; Figure 4 (curve b)) increases signi cantly compared to that of the Pt/PANi NW electrode (75 Ω; Figure 4 (curve a)).
ese results reveal that the immobilization of probe DNA sequences onto the Pt/PANi NW WEs was e ectively and simply performed, due to the ability to create links between the amino groups of PANi NWs and the phosphate groups of the probe DNA sequences [42] and due to the PANi nanowire structure with porous surface characteristics and high electrochemically e ective surface area.
It can be seen in Figure 5(a) that the impedance values of the DNA sensors after hybridization with di erent concentrations of complementary target DNA (from 1.0 × 10 −13 M to 1.0 × 10 −8 M, from curve b to curve g, respectively) increase signi cantly in comparison with the impedance value of the DNA sensor before hybridization with DNA target (curve a). DNA strands contain negatively charged phosphate groups, while the synthesized PANi NW material is a conducting polymer with p-type charge carriers (holes). us, when the DNA hybridization occurred on the sensor surface, the density of the charge carriers of PANi NWs was reduced, leading to the decrease in the conductivity and the increase in the impedance of the sensor.
e R ct value exhibited by a semicircle region at high frequency range on an EIS spectrum depends on changes of the sensor surface where DNA hybridization happens; thus, it is selected as the output signal for DNA target detection.  [43]. Moreover, as shown in Figure 5(b) (curve b), the response signal at the highest studied concentration (1.0 × 10 −8 M) of the noncomplementary target DNA is negligible. e above results indicate that the fabricated DNA sensors using PANi NWs express high sensitivity and good selectivity. Table 2 exhibits the comparison of the fabricated DNA sensor with some others in the previous studies. e electrochemical DNA sensor based on PANi NWs in this work shows many advantages such as direct detection, high sensitivity, low detection limit (2.48 × 10 −14 M), good selectivity, and small volumes of both the DNA probe and DNA target samples (only 5 μL) for the DNA probe immobilization and DNA target detection, respectively. Moreover, the sensor fabrication process proposed in this study is simple and e ective with the one-step electrochemical method for the direct synthesis of PANi NWs on the Pt microelectrode's surface, as well as the direct immobilization of DNA probe on the PANi NW material. erefore, the electrochemical DNA sensor proposed in this study has many potential applications such as detection of pathogenic viruses and bacteria, disease diagnosis, and food safety monitoring. However, similar to the polymerization chain reaction (PCR) techniques, the DNA sensor also works with pretreated biological samples, in which the target DNA sequences need to be extracted before being detected by the Pt/PANi NW microelectrode.

Conclusion
In this paper, PANi NWs were synthesized directly onto the Pt WEs (with only 0.785 mm 2 area) using the simple onestep electrochemical approach. In particular, the electrochemically e ective surface area of the Pt/PANi NW WE is nearly 19 times larger than that of the Pt WE. e Pt/PANi NW microelectrodes with high electrochemically e ective surface area, high biocompatibility, and ease of miniaturization facilitated the probe DNA immobilization and enhanced the DNA sensor's electrochemical signals. e detection limit of the sensors was 2.48 × 10 −14 M. e DNA sensors exhibited advantages including simple fabrication, direct detection, high sensitivity, good speci city, and easy miniaturization of analytical systems.
Data Availability e data used to support the ndings of this study are included within the article.