Screen-Printed Electrodes Based on Conductive Inks of Polyaniline/Graphene Hybrids and Their Application to Progesterone Detection

Screen-printed electrodes (SPEs) were produced using conductive inks based on hybrids of polyaniline (PAni) and reduced graphene oxide (rGO). Cyclic voltammetry (CV) showed peaks characteristic of the PAni redox behavior in acidic media for all the modified SPEs. Electrochemical impedance spectroscopy (EIS) showed a significant decrease in the charge-transfer resistances, from 930 Ω for SPE/G:PAni to 544 Ω for SPE/G:PAni-rGO1 and to 303 Ω for SPE/G:PAni-rGO2 with just 0.06% and 0.12% in mass of rGO, respectively, in the final mass composition of the conductive inks. The SPEs were evaluated in the determination of progesterone (P4) hormone in neutral medium (phosphate buffer solution, pH 7.0). The CV results showed higher current signals at SPE/G:PAni-rGO1 compared with SPE/G and SPE/G:PAni, indicating a synergistic effect of PAni-rGO1 in the determination of P4. EIS also showed significant changes in the electrochemical double-layer capacitances in the presence of P4. The limits of detection (LOD) and quantification (LOQ) were, respectively, 211 nmol l−1 and 703 nmol l−1. This method is a simple, scalable and low-cost alternative for the fabrication of electrodes based on PAni-rGO hybrids, with synergic properties, aiming for future applications in sensors.

Screen-printing technology has attracted attention in the development of flexible and disposable electrochemical devices due to characteristics such as easy production, relatively low cost and being suitable for large-scale production. [1][2][3][4] Methods for the manufacturing of screen-printed electrodes (SPEs) include aerosol jet printing, 5 inkjet printing, 6 3D printing, 7 hand painting, 8 and serigraphy. 3,4 These techniques are also versatile since they allow the development of SPE that incorporate numerous substances in the ink composition. 4 Reports have described the use of flexible substrates such as paper, 8,9 PET (polyethylene terephthalate), 3,4,10 scotch tape, 11 and Kapton ® polyimide films. 12 Carbon conductive materials such as graphite, 3,4 graphene 13,14 and carbon nanotubes 15,16 have been used in the production of new inks. Graphene has received attention due to its many special properties, such as high electrical conductivity, large specific surface area, flexibility as well as high mechanical strength, and chemical stability, 17 thus being an interesting material for the development and application of flexible SPE.
Polyaniline (PAni) is one of the main conducting polymers (CPs) used in electronic, optical, and electrochemical applications due to its low cost, good environmental stability, good electroactivity, and reversible control of electrical properties by both charge-transfer doping and protonation. [18][19][20] Some reports concern the application of PAni as a non-specific sensor, but showing different interactions with different analytes. 2,[21][22][23] . Therefore, these graphene-based PAni nanohybrids have attracted much interest. The modification of SPEs with PAni and graphene has been reported employing methods such as drop casting, 24,25 electropolymerization, 21,26 inkjet printing technology, [26][27][28][29] and serigraphy, 17 aiming towards applications in sensors, 24,29 biosensors, 25,26 or supercapacitors. 17,30,31 Hybrids based on graphene and CPs exhibit superior thermal, electrical, mechanical, optical and electrochemical properties compared with the neat polymer or graphene. 19 However, the fabrication of flexible SPEs by serigraphy, using conductive inks based on hybrids of PAni and graphene, still almost unexplored.
Xu et al., 17 developed a SPE employing the serigraphy method by utilizing a series of nanographene platelets (NGP) and PAni inks with different mass ratios and polytetrafluoroethylene (PTFE) as resin matrix, for application as a supercapacitor device. The NGP/ PAni inks were prepared with 90%, 60%, 45%, 36% or 30% of PAni, and 10% of PTFE, the rest being NGP produced by ball milling. The mixture was deposited by serigraphy onto plastic substrates, such as PET, and on carbon fabric substrates. The electrochemical properties of the films were investigated by cyclic voltammetry (CV), galvanostatic charge-discharge, and electrochemical impedance spectroscopy (EIS). The CV results showed that all NGP/PAni electrodes have a larger capacitive response than the pure NGP modified electrode and that the peak potentials changed with the mass ratios. EIS of NGP/PAni electrodes showed a pure capacitive behavior at lower frequencies. It was also shown that the combination of high NGP conductivity with PAni reversible redox properties is necessary to achieve supercapacitors with a long life. The electrode NGP/PAni labeled 1:1.5 (36% NGP, 54% PAni and 10% PTFE), showed the best specific capacitance of 269 F g −1 , a power density of 454 kW kg −1 and an energy density of 9.3 Wh kg −1 , operating in 1.0 mol l −1 H 2 SO 4 electrolytes. The authors concluded that SPE NGP/PAni based electrodes are promising for use in supercapacitor devices.
Hormone imbalance in the body can cause infertility problems and malformation of the reproductive system. Progesterone (P4), a steroid hormone, is an important bioactive substance and plays an important role in stabilizing and maintaining pregnancy in mammals. [32][33][34] Thus, the pharmaceutical industry produces P4 as the active ingredient in many drugs. 35,36 However, when high doses of P4 are consumed by humans, generally the body absorbs only small amounts and the rest is excreted as waste into sewage water, being an environmentally emerging contaminant. 37,38 The electroanalytical determination of P4, is generally performed at very negative potentials in alkaline solution within the pH range from 8 to 12. 36,[39][40][41] In order to improve the electroanalytical z E-mail: fabio.simoes@unifesp.br performance, especially sensitivity, LOD and selectivity, nanomaterials have been used for modification of the electrodes. Among these are carbon-based nanomaterials 34,35,42 and CP 43 as well as their nanocomposites. 44 In the literature, to the best of our knowledge, there are no papers that report modified SPE based on PAni and graphene hybrids fabricated by the serigraphy technique with application to the determination of hormones such as P4.
Thus, this work was focused on producing and characterizing new, simple, low-cost combined, and flexible SPEs, using conductive inks based on PAni-rGO hybrids deposited onto polyester sheets by the serigraphy technique, and exploring the versatility of these modified SPEs based on PAni and graphene, illustrated by P4 determination.
Instrumentation.-The morphologies of rGO, PAni and PAni-X %rGO were examined by scanning electron microscopy (SEM), using a model 6610LV (JEOL) with acceleration voltages of 10 kV and 15 kV. A potentiostat-galvanost at model PGSTAT 128N, from AUTOLAB, with NOVA (version 1.11) software was used for CV and EIS experiments.
Preparation of rGO, PAni and hybrids.-rGO, PAni, and their PAni-rGO hybrids were prepared as described in our previous work. 45 GO was obtained by the modified Hummers method and then reduced using L-AA. PAni was chemically polymerized and the PAni-rGO hybrids were obtained using the same procedure except that before the chemical polymerization, amounts of 2.5% and 5.0% of rGO (mass ratio relative to the aniline monomer) were dispersed for 30 min using an analog cell disruptor (Branson Sonifer ® model 250), producing the respective hybrids (PAni-rGO1 and PAni-rGO2, respectively).
Fabrication of the flexible SPEs.-The conductive inks were prepared by mixing 50% of alkyd resins and 50% of graphite (G) for the auxiliary and the counter-electrodes and mixing 50% of alkyd resins, 4 45% of graphite and 5.0% of PAni or its hybrids (PAni-rGO) for the working electrode. The reference electrode was painted with silver ink (Electron Microscopy Sciences) to obtain the final sensor. The choice of 5.0% in mass of PAni or PAni-rGO in the conductive ink was based on the optimized procedure previously described by Consolin-Filho, et al. 23 and Simões, et al., 22 in the preparation of carbon paste electrodes. It was found in, 23 that carbon paste electrodes with PAni concentrations greater than 5.0% lead to a slight shift in the cathodic peak potential, indicating saturation of the polymer on the electrode surface. The compositions of the conductive inks are shown in Table I.
The conductive inks were diluted in toluene (1.15 ml g −1 ) to reduce the viscosity and to improve the spreading over the substrate. The SPE masks (Fig. 1a) were designed using AutoCAD ® software, delimiting a geometric area of 0.214 cm 2 for the working electrode. The masks of the SPE circuits were printed onto glossy photo paper and deposited onto a polyester sheet substrate, previously abraded using 1200 abrasive paper and washed with 70% ethanol. The conductive inks were spread manually over the substrates using a silicon spatula. After 5 min of drying in air, the masks were removed. The resulting SPE/G, SPE/G:PAni, SPE/G:PAni-rGO1 and SPE/G:PAni-rGO2 were cut individually (Fig. 1b).     46 where s d is the standard deviation of the curve and b is the slope of the analytical curve.

Results and Discussion
Characterization of the flexible SPEs by Scanning Electron Microscopy.- Figure 2 shows the SEM images of the SPEs fabricated with the conductive inks based on PAni and its hybrids with rGO. In Fig. 2a, the graphite structure formed by several layers of bonded graphene sheets can be seen, and the surface is totally covered by conductive ink of the SPE/G working electrode. Figure 2b shows a uniform distribution of the graphite within the PAni chain. 47 Besides this, the fibrous structure and agglomerate of the PAni 18,30 can be seen mixed with the graphite structure formed by graphene sheets. The images of SPE/PAni-rGO1 (Fig. 2c) and Fig. 2d (SPE/PAni-rGO2) evidence the rGO sheets, forming a flatter part (circled) that are graphite layers, as well as exhibiting thinner sheets, suggesting the presence of thin graphene sheets (circled), besides the graphite structure. These images also show structures that can be associated with PAni growing on the surface of the rGO sheets. 30,48 Electrochemical characterization of the flexible SPEs.- Figure 3 shows (a) CVs and (b) complex plane impedance spectra for SPE/G, SPE/G:PAni, SPE/G:PAni-rGO1 and SPE/G:PAni-rGO2 in acidic medium (0.5 mol l −1 H 2 SO 4 ). The CVs in Fig. 3a show that SPE/G:PAni and both hybrid modified SPE (SPE/G:PAni-rGO1 and SPE/G:PAni-rGO2) present a redox behavior characteristic of protonated PAni in its conducting form. A well-defined anodic peak, near to 0.25 V, is attributed to the oxidation of leucoemeraldine to emeraldine, and a less pronounced peak at 0.51 V, to the oxidation of emeraldine to pernigraniline in the protonated state. 12,45 It was also observed that, even with low proportions of PAni or PAni-rGO hybrids in the ink (5.0% in mass), the voltammetric behavior was similar to electropolymerized PAni films. 21,45,49 The CV for SPE/G:PAni-rGO2 was slightly different to those of SPE/G: PAni and SPE/G:PAni-rGO1, indicating that an increase of the rGO mass ratio above 5.0%, can negatively influence the PAni redox behavior, as already reported in our previous work. 45 Figure 3b shows complex plane spectra from EIS experiments for SPE/G, SPE/G:PAni SPE/G:PAni-rGO1 and SPE/G:PAni-rGO2 at OCP (0.268 V, 0.087 V, 0.096 V and 0.126 V vs Ag/AgCl, respectively). The SPE/G spectrum has an inclined straight line at low frequency that is attributed to a capacitive behavior. The SPE/G: PAni shows a semicircle in the region of high and medium frequencies (about 828 Hz to 2 Hz) that can be attributed to an electron transfer resistance in parallel with a capacitance, followed by a linear segment at lower frequencies (about 2 Hz to 100 mHz), attributed to ionic diffusion processes in the modifier layer that will exhibit non-homogeneities at the electrode/electrolyte interface, and some porosity owing to PAni. 50 The SPEs based on PAni-rGO hybrids (SPE/G:PAni-rGO1 and SPE/G:PAni-rGO2) show a similar EIS behavior with semicircles in the region of high and medium frequencies (3.4 kHz to 5 Hz). However, the smaller radius compared to the spectrum for SPE/G:PAni (without rGO) indicates smaller charge-transfer resistances. The linear segments at lower frequencies (from 5 Hz to 100 mHz) are attributed to ionic diffusion as for SPE/G:PAni; the hybrid based modified SPE also have porous surfaces as seen in the SEM images in Figs. 2c and 2d. 50 Figure S1 shows the Bode diagrams for the SPEs. The middle part of the Bode diagram for SPE/G:PAni shows a higher phase angle than SPE/ PAni-rGO1 and SPE/PAni-rGO2, indicating a higher electrontransfer resistance. In its turn, the SPEs with the hybrids showed the decrease of the phase angles with increase of rGO loading, resulting in faster charge-transfer processes. 45 The impedance spectra were fitted using an equivalent circuit (Fig. 3b inset). The circuit elements were the cell resistance (R Ω ), a charge transfer resistance (R ct ) in parallel with a constant phase element (CPE 1 ), followed by a CPE 2 . CPE 1 is a non-ideal capacitance for the double layer CPE 1 = -[(Ciω) α ] −1 where α can vary between 0.5 and 1.0. CPE 2 can represent ionic diffusion at low frequencies in the modifier layer of modified electrodes with porosity that leads to charge separation. 50 In Table II, the calculated values of R ct for the SPE based on the hybrids (SPE/G:PAni-rGO1 and SPE/G:PAni-rGO2) were smaller (544 Ω and 303 Ω, respectively) compared with the SPE/G:PAni without rGO (R ct = 930 Ω).This significant decrease of R ct , even using rGO mass ratios as low as 0.0625% for the SPE/G:PAni-rGO1 and 0.125% for the SPE/G:PAni-rGO2, were up to three times lower than for SPE/G:PAni. Thus, these combined and flexible SPEs based on the PAni-rGO hybrids, using a lower mass ratio of PAni or its PAni-rGO hybrids in the ink composition (about 5.0%) and with just 0.0625% of rGO (SPE/G:PAni-rGO1) showed a significant reduction of the charge transfer resistances, without any significant changes in the PAni redox behavior.
Evaluation of the electrochemical detection of progesterone (P4).-The SPE/G, SPE/G:PAni and SPE/G:PAni rGO1 were evaluated for the detection and quantification of P4 in neutral medium (phosphate buffer solution, pH = 7.0) using CV and EIS. Figure 4 shows a comparison between the CV results for the SPEs in the phosphate buffer solution, and with addition of 1.59 × 10 −4 mol l −1 of P4. As expected, since PAni is in its insulating form (pH = 7.0), no redox pair was observed for the modified SPEs. With the addition of P4, a large increase of the capacitive currents was observed and was significantly greater for the PAni-rGO hybrids.
Different conducting polymers can selectively interact with different analytes in sensor applications. For example, carbon paste electrodes modified with PAni or Polypyrrole (PPy) were evaluated for the determination of the pesticides 2,4-D, methylparathion (MP), paraquat (PQ), bentazon (BZ) and glyphosate (GLY) also in neutral medium (pH = 7.0) and using CV as electrochemical technique. 22 As well as P4, the electroanalytical determination of 2,4-D, GLY or BZ is difficult, since no redox behavior is observed at unmodified electrodes. However, the PAni modified electrode was capable of detecting BZ, GLY and 2,4-D while PPy detected GLY and PQ. Sorption studies using UV-vis spectrophotometry showed that the pesticides with acidic characteristics (2,4-D, GLY and BZ) were readily sorbed on the dedoped PAni and this interaction was attributed to a possible secondary doping mechanism. 51,52 Thus, the increase of the capacitive currents observed is probably due to a strong interaction by a sorption mechanism between P4 and PAni. Moreover, the highest capacitive currents observed for SPE/G:PAni-  Table II. See text for further details. Table II. Values of the calculated elements for the equivalent circuit used to fit the impedance spectra of Fig. 3b for the electrodes: SPE/G, SPE/G: PAni, SPE/G:PAni-rGO1 and SPE/G:PAni-rGO2. See text for explanation of symbols.

SPE
R rGO1 shows the improvement of sensitivity for this hybrid even using a low mass proportion of rGO (0.06%) in the ink composition. This synergism was attributed to the better charge transfer as demonstrated by EIS.
With the objective of studying the influence on the interfacial region of the modified SPEs impedance spectra were obtained without and with the presence of P4 (1.59 × 10 −4 mol l −1 ). Figure  S2 shows complex plane plots for SPE/G, SPE/G:PAni and SPE/G: PAni-rGO1 in phosphate buffer pH = 7.0 at OCP (Fig. S2-a Figure S2-a shows that the spectrum for SPE/G:PAni as well as the hybrid SPE/G:PAni-rGO1 only include inclined linear segments, without any semicircle, since PAni is in its non-conducting form (dedoped). With the addition of P4, (Fig. S2-b) the spectra exhibit semi-circles in the region of higher and medium frequencies (100 kHz to 1 kHz). Table S1 shows the calculated values of the elements fitted by the equivalent circuit. It is seen that SPE/G:PAni-rGO1 has a higher value of R ct (148 Ω) than SPE/G:PAni (90 Ω) and SPE/G (31 Ω). In addition, the Bode diagram for the SPE/G:PAni-rGO1 ( Fig. S3-a) showed a small increase in phase angle at higher frequencies (100 kHz to 10 kHz) compared to the other SPE/G and SPE/G:PAni in the presence of P4. In Fig. S3-b, there can also be seen an increase in the impedance magnitude |Z| at medium (10 kHz to 1 kHz) and high frequencies (100 kHz to 10 kHz) for SPE/G:PAni-rGO1. These results indicate that the interaction of PAni with P4 is favored by a synergic effect of the hybrid PAni-rGO1 compared with its unmodified counterparts.
Due to the improved sensitivity, SPE/G:PAni-rGO1 was chosen to perform quantitative analysis with additions of P4 in the concentration range from 31.8 to 318 μmol l −1 (10 mg l −1 to 100 mg l −1 ). The determination of P4 at SPE/PAni-rGO1 was performed in phosphate buffer (pH = 7.0). The CVs (Fig. 5a) show an increase in the current with increase of P4 concentration. The CVs show a large capacitive current attributed to the P4 absorption in the surface of the working electrode that also increases with concentration of the hormone. The potential chosen for plotting the analytical curve was 0.15 V vs Ag/AgCl/KCl sat because besides coinciding with the PAni oxidation peak it is also the potential region that presents the largest capacitive current. The analytical curve (Fig. 5b) shows a linear dependence on concentration at SPE: G/PAni-rGO1, I (μA) = 0.017 C + (−0.025), where C is the concentration of P4 in the solution and R 2 = 0.98). The relative standard deviation (RSD) was 0.001 μA and the values of LOD and LOQ were 211 nmol l −1 and 703 nmol l −1 .
As already mentioned, the electroanalysis of P4 is difficult and generally performed at very negative potentials and in alkaline solutions. 34,53 However, the electroanalytical performance for the determination of P4 can be improved using nanomaterials such as carbon nanotubes, 35 or metallic nanoparticles 35,53 that are usually combined with active molecules such as amino-acids, poly-L-serine 35 and poly(sulfosalicylic acid) as well as biosensors (immunosensors and aptasensors). 44,54 Table III shows a comparison between some non-enzymatic modified carbon-based electrodes and this work. The main difference is that the literature reports are based on GCE modified by casting and or electropolymerized films including nanoparticles. However, this work is based on a cheap and easily scalable modified SPE (SPE:G/PAni-rGO1) that is also flexible, disposable and combined in a 3-electrode device. The LOD is comparable to the    electrode that used imidazole as active molecule 34 also not using an aminoacid or metallic nanoparticles however using non-pulsed technique such as CV in neutral medium (pH = 7.0) at a low potential (0.15 V). Thus, we believe that this work can be the basis for future modifications aiming at the electroanalytical determination of P4 using printable and scalable electrodes.

Conclusions
A new, easily scalable, disposable, cheap, flexible and combined three-electrode SPE device was produced using conductive inks based on hybrids of PAni grafted with rGO (PAni-rGO). All the PAni based SPEs showed similar redox behavior but with a significant improvement of charge-transfer for the PAni-rGO hybrids even with low mass proportions of rGO in the conductive inks, 0.06% for SPE/PAni-rGO1 and 0.12% for SPE/ PAni-rGO2. The applicability of the SPEs was evaluated in the electroanalytical determination of P4 in neutral medium (pH = 7.0, phosphate buffer) by cyclic voltammetry. The CVs showed a strong interaction of P4 with PAni, manifested by an increase of the capacitive currents that were attributed to sorption followed by a secondary doping mechanism of PAni. The higher capacitive currents using SPE:G/ PAni-rGO1 were attributed to better charge-transfer efficiency of the rGO hybrid. The LOD and LOQ were 211 nmol l −1 and 703 nmol l −1 , being comparable to non-enzymatic electrodes but without the use of metallic nanoparticles. The experiments are performed in neutral medium (pH = 7.0), at lower potential than other reports (0.15 V) and use a non-pulsed technique. This developed SPE based on a conductive ink with just 5% of PAni hybrid and 0.06% in mass of rGO shows a high synergistic effect that augurs well for future applications of such PAni based sensors.