Surface Characterization of New Azulene-Based CMEs for Sensing

Films of 2-(azulen-1-yldiazenyl)-5-phenyl-1,3,4-thiadiazole (T) were successfully deposited on glassy carbon surfaces to prepare chemically modified electrodes (CMEs). Their surface characterization was analyzed by electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM), and scanning electron microscopy (SEM). This complexing monomer has been deposited through direct electropolymerization in conditions established during the electrochemical characterization of T performed by cyclic voltammetry (CV), differential pulse voltammetry (DPV) and rotating disk electrode voltammetry (RDE). These methods put in evidence the high degree of asymmetry of oxidation and reduction curves, which is due to the irreversible processes occurring at opposite potentials. The film formation was confirmed by ferrocene redox assay probe. The properties of the electrodes modified with T (T-CMEs) were investigated for sensing heavy metal (HM) ions in water solutions, with promising results for Pb(II) among Cd(II), Cu(II), and Hg(II) ions.


Introduction
Knowledge on the relationships between structure, properties and performance is essential for chemically modified electrode (CME) applications in all areas of interest. They can be characterized by microscopic techniques that are used in various industrial applications, including topographic and dynamic surface studies [1][2][3][4][5] but also in medical diagnosis [6]. In the present study, the results are provided concerning surface characterization of new CMEs obtained by electropolymerization of the recently prepared [7] compound, 2-(azulen-1-yldiazenyl)-5-phenyl-1,3,4-thiadiazole (denoted T). This azulene derivative belongs to the diazene compounds, which can be used for technical purposes, such as preparation of nematic liquid crystals [8], compounds with special optical properties [9], photosensitive materials [10], and chromophores [11]. Such diazenes have been mainly characterized by UV-Vis spectroscopy. Some calculations for optical applications were also performed by DFT/TDDFT modeling [12].
Monitoring heavy metal (HM) ions in water and food is an extremely important task to avoid health problems associated with HM tendencies to accumulate in ecosystems. This can be monitored using methods with high sensitivity and accuracy. A modern alternative to these methods is based on electrochemical sensors that can be included in portable devices that allow on-site monitoring of HMs. Electrochemical sensors based on CMEs have a fast response and a high sensitivity compared to classical spectroscopic and optical techniques [13]. Given the advantages of CME-based sensors and our goal of determining HMs, it is important to find new molecule structures that lead to this type of sensor. This paper provides the data of the results obtained for CMEs based on the T ligand.
T (Figure 1) is part of the diazenes recently studied by our research group [7], which synthesizes various functionalized azulene derivatives for future applications [14]. Azulene is an interesting building block for the synthesis of new advanced materials [15], as it shows low ionization energy and high electron mobility among the classical electropolymerizable monomers (pyrrole, thiophene, etc.) due to its polarized structure. Our research team is concerned with the characterization of azulene ligands, and on building complexing modified electrodes based on azulene. The derivatives are tailored for a specific target that considers specific methods of synthesis. The main interest is to find several parameters that characterize the best ligand among many structures. The conjugated azulene structure allows the transfer of one electron from the seven-atom ring to the five-atom ring, giving a polar structure (dipole moment of 1.08 D) between the 6 π-electron cyclopentadienyl anion and a 6 π-electron tropylium cation. During anodic oxidation, azulene undergoes electrochemical polymerization at the 1 and 3 positions, leading to the formation of polymer films at the electrode surface [16]. Similar to other (azulen-1-yldiazenyl) heteroaromatic compounds, this ligand is a poor base, which is protonated at electron-rich azulene carbon atoms with the modification of the chromophore, and it could act as pH indicator [7]. In parallel, T revealed interesting properties as possible inhibitory activity against Escherichia coli or Mycobacterium tuberculosis as resulted from previous in silico studies conducted by molecular docking simulations. These promising preliminary results highlighted new opportunities of several structures derived from thiadiazoles coupled with azulene moieties as possible antimicrobial agents [17]. T is an azothiazole substituted azulene containing two parts: a polymerizing unit, which is the azulene, and a complexing unit, which is the thiazole. The azo group allows the conjugation between azulene and thiazole units, and the extra phenyl group connected to the thiazole extends this conjugation, leading to potentially favorable properties. It is known that derivatives containing the thiadiazole group have a complexing effect on heavy metals (HMs) [18,19], which is why 5-amino-1,3,4-thiadiazole-2-thiol has been used to modify the silica gel for HM ion removal [18]. Poly(2,5-dimercapto-1,3,4-thiadiazole) nanosheets have shown impressive properties in lead absorption, being promising materials in the fields of water treatment, sensors, and electrode materials [19]. The thiadiazoles are strongly absorbed on mercury, more than cobalt and copper, due to the same complexation effect [19]. The binding of thiadiazole to an azulene residue is an unexploited subject in the literature and creates the premise for recognition of HMs using CMEs based on T. The electropolymerization of similar azulenes [20] can occur by π-steaking [21]. Only few surface studies related to azulene-based CMEs are reported [22,23]. SEM investigation of polymerized polyazulene films can be found in the literature for polyazulene films used as supercapacitors [24], ion-to-electron transducer materials in solid-contact ion-selective electrodes [25], copolymers based on azulene and 3-thiophene acetic acid [26].
In this paper, the results are provided concerning the characterization by EIS, SEM and AFM of T-modified CMEs surfaces. T is characterized by voltametric techniques (CV, DPV, and EIS), and was used to prepare T-CMEs. They were examined by electrochemical methods and were finally tested in view of application for HM sensing. The study of the surface properties of T-CMEs revealed: (1) new aspects of film morphology obtained in different conditions and (2) the role of electrosynthesis potential in electrode nanostructuring, with direct consequences on sensing capacity.

Materials and Methods
The electrochemical experiments were performed using PGSTAT 12 AUTOLAB potentiostat (Utrecht, The Netherlands) connected to a three-electrode cell [19][20][21][22][23]. Bare or modified glassy carbon (GC) disks (3 mm diameter, Metrohm, Herisau, Switzerland) were used as working electrodes. Before each experiment, the working electrode was cleaned by polishing with diamond paste (2 µm), then rinsed with solvent, and dried with fine paper. The auxiliary electrode was a platinum wire, and the reference electrode was either Ag/10 mM AgNO 3 in 0.1 M TBAP/CH 3 CN (for electrochemical experiments performed in acetonitrile solutions), or Ag/AgCl, 3 M KCl (for electrochemical experiments performed in aqueous solutions).
The modified electrodes were prepared using controlled potential electrolysis (CPE) in millimolar solutions of T in acetonitrile (CH 3 CN) containing 0.1 M tetrabutylammonium perchlorate (TBAP), both from Fluka (Munich, Germany), as solvent and supporting electrolyte, respectively. The ligand T was synthesized according to the published procedure [7].
EIS curves were recorded with 10 mV AC voltage within 100 kHz-100 mHz frequency range on PARSTAT4000 potentiostat at open circuit potential. EIS spectra were processed using Z View 2.4 software from Scribner Association Inc. Derek Johnson.
Surface characterization of polyT modified electrodes carried out by AFM and SEM techniques was performed on GC disks (6 mm diameter) from OrigaLys (Les Verchères, France) as substrates.
Hitachi SU 8230 equipment (Tokyo, Japan) was used to perform SEM analysis at low acceleration voltage (5 kV) to avoid damaging the sample. The morphological properties of prepared CMEs were studied using a commercial multimodal AFM system (NTEGRA System, NT-MDT, Moscow, Russia). The tip of the n-type silicon cone mounted on a cantilever with a resonance frequency of 155 kHz and a stiffness of approximately 7 N/m was used for the topographic measurements, which were successfully performed at a size of 10 × 10 µm 2 in semi-contact mode. Images (512 × 512 lines) were collected at a rate of 0.3 Hz under ambient conditions. Using the processing software, the mean root square roughness (RMS) parameter of the acquired topographic images was calculated.
The electrochemical experiments were performed by CV, RDE, and DPV. CV investigations were usually run at a scan rate of 100 mV/s. DPV curves were recorded at 10 mV/s with a pulse height of 25 mV and a step time of 0.2 s, while RDE studies were performed at 10 mV/s for different rotation rates between 500 and 1500 rpm. All experiments for ligand characterization were performed at 25 • C under argon atmosphere in acetonitrile solutions. The potentials were finally referred to the potential of the ferrocene/ferricinium redox couple (Fc/Fc + ).

Immobilization of T Complexing Monomer on Electrode Surfaces
Electrochemical immobilization of T monomer on GC electrode was carried out in 1.2 mM solution of T in 0.1 M TBAP/CH 3 CN by CPE using different charge densities and potentials. All CPE potentials were located in the region of T anodic processes (as it is further detailed in Section 3.5). Several chronoamperograms are shown in Figure S1. By applying this technique, CMEs based on T (denoted T-CMEs) were prepared, ensuring the immobilization of T-ligand complexing units on the electrode surface as green films. After CPE, the modified electrodes were cleaned with acetonitrile, and transferred into 1 mM ferrocene solution in 0.1 M TBAP/CH 3 CN to check the film formation by ferrocene redox assay probe.
The comparative recorded CV curves on T-CMEs synthesized under different conditions and on the bare electrode are illustrated in Figure 2. The distortion of ferrocene signal reveals the coverage of GC electrode by films with different properties, depending on their synthesis conditions (potential value and charge density during CPE). From Figure 2a, it can be seen that the preparation potential significantly modifies the symmetrical ferrocene signal characteristics on the bare electrode, with an evident loss of reversibility on the modified electrode. For instance, on T-CME prepared at the potential of +1.5 V, the ferrocene anodic peak is diminished as compared to that on a bare electrode (dashed line), and its potential is shifted to +0.178 V (from +0.126 V). The ferrocene cathodic peak almost disappears. When the T-CME preparation was performed at +2.5 V, the anodic CV branch was more shifted than at +1.5 V toward positive potentials at +0.314 V, but the cathodic one is quite high, even if it is shifted to more negative potentials than at +1.5 V. This behavior suggests that the film prepared at +1.5 V is different (as structure and/or porosity) from that prepared at the potential of +2.5 V. It seems that higher potential during CPE leads to a more porous film, which ensures higher oxidation and reduction currents for ferrocene. For the application fields designed for these modified electrodes (see Section 3.5), the potential of +1.5 V was selected to prepare T-CMEs by CPE. This potential was assumed to ensure a good number of complexing units in view of further complexation and, consequently, a good detection of HMs (see Section 3.4) confirmed this fact. Figure 2b presents the CV curves of ferrocene on T-CMEs produced by CPE at a constant applied potential of +1.5 V, for different charges between 7 and 25 mC/cm 2 . They confirmed the coverage of the electrode with films of increased thicknesses in concordance with the charge used. As expected, the shape of the ferrocene signals changes as the thickness of films increases. Therefore, the voltammogram recorded on T-CME obtained at 7 mC/cm 2 is similar to that recorded on the bare electrode, indicating the deposition of a thin film. On the contrary, on T-CMEs prepared at the higher charge densities (21 and 25 mC/cm 2 ) the CV curves are close to the background. At 14 mC/cm 2 , the shape is intermediate. The charge of 21 mC/cm 2 was chosen for the preparation of T-CMEs for recognition experiments (see further) since it ensures a good coverage of the electrode (Figure 2b inset).

Studies by EIS
To obtain more information on the characteristics of polyT films T-CMEs, the films were prepared using either the same electropolymerization charge for various potentials (specimens P1, P2 and P3), or the same applied potential for different values of electropolymerization charge (specimens P4, P5, P6 and P7), as detailed in Table 1. The as-prepared modified electrodes were rinsed with acetonitrile and introduced into a transfer cell containing the supporting electrolyte (0.1 M TBAP solution in acetonitrile), and then the EIS curves were recorded at open circuit potential. +1. 5 25 Figures 3 and 4 present the resulted EIS spectra as Nyquist (a) and Bode (b) diagrams for the specimens from Table 1. As shown in these figures, the films behave as insulators. Each EIS curve has a semicircle in the relative high frequency range which continues in low frequencies. The diameters of the semicircles are associated with the polarization resistance (charge transfer resistance = Rct) of the film layer; therefore, the higher the diameter, the more insulating the film layer.   EIS data for each curve in Figures 3 and 4 were fitted using the equivalent circuit shown in Figure 5. This circuit has an ohmic resistance (Rsol) and a constant phase element (CPEdl), instead of true capacitance, and a charge-transfer resistor (Rct) in parallel. The resulting values for Rct and CPEdl corresponding to Figures 3 and 4, respectively, are presented in Tables 2 and 3. These values were obtained by fitting the impedance data with ZView software and the chi-squared (χ 2 ) values used to evaluate the fitting quality are shown, too. Table 2. Fitted parameters from the EIS curves of T-CMEs obtained by CPE at different potentials (for 14 mC/cm 2 ) using the equivalent circuit proposed in Figure 5.

T-CME Films Characteristics
Rsol(Ω) 10 Table 3. Fitted parameters from EIS spectra of T-CMEs obtained by CPE for different electropolymerization charge densities (at +1.5 V) using the equivalent circuit proposed in Figure 5. By examining the values in Tables 2 and 3, it can be seen that the films obtained at different potentials or charge densities are all insulating (P1-P7), as they have quite high values of Rct of 10 6 Ω order of magnitude. It suggests that the impedance response is dominated by the resistance of the polymeric phase deposited on the electrode surface. The specimen P3 has the lowest value of Rct (1.81·10 −6 Ω) from the specimens obtained at different potentials. In addition, its capacity is the lowest (1.84 µF).

T-CME Films Characteristics
From the specimens obtained at different charge densities (Table 3), a decrease of Rct value can be seen as the applied electropolymerization charge increased to 21 mC/cm 2 (P4 > P5 > P6). This unexpected behavior might be determined by a change in the morphology of the layer, during the growth of the film to a relatively high porosity, which could facilitate the charge transfer. For an applied charge of 28 mC/cm 2 (P7), a saturation occurs.
Taking into account the conclusions from Tables 2 and 3, T-CMEs electrochemically prepared at +1.5 V and 21 mC/cm 2 were selected for the investigation of the analytical response involved in the electrochemical detection of HMs, as detailed in Section 3.5.

Studies by SEM
Selected T-CMEs surfaces that were used for electrochemical detections of HMs were investigated by SEM to compare the morphology of polyT-films deposited on GC disks (6 mm diameter) using electropolimerization charge densities of 7, 14, 28 mC/cm 2 , respectively, corresponding to the charges used for T-CMEs characterization by electrochemistry in ferrocene solutions (performed on 3 mm diameter GC disks).
The polymer topography images acquired by SEM at different magnifications are seen in Figures 6 and 7. They show a relatively uniform arrangement of the polymer matrix surface. On each sample's surface, there are some clusters that deviate from planarity of the sample.   Figure 6 shows that cluster size increases with the polymerization charge at all magnifications, being well defined at the lowest charge density (7 mC/cm 2 ), and unclear at the highest charge (28 mC/cm 2 ).
The obtained images ( Figure 6) for the smallest charge density used (7 mC/cm 2 ) show that the surface has mechanical defects (scratches). These are due to the erosion of the electrodes during the preparation of surfaces with reproducible characteristics (by polishing the electrode on felt with diamond paste) before the preparation of the films. These defects are completely covered at electropolymerization charges of 14 and 28 mC/cm 2 . For the charge of 14 mC/cm 2 , the film is similar to an ordered matrix. Figure 7 shows that the morphology of the films depends on the potential at which the polymerization was performed. The polymer image for the film obtained at +1.5 V is the mostly nanostructured (chains situated at about 20 nm). At +0.9 V, the structure is also regular, but the distinction between the polymer chains is less visible, indicating that the formation process took place more slowly. At +2.5 V, the polymer has an amorphous aspect (all the irregularities are covered). This amorphous structure is certainly the result of an increased rate of polymerization at this potential. The polymerization times are of about 44, 71, and 60 s for +0.9, +1.5, and +2.5 V, respectively, from the chronoamperograms recorded during CPEs (Supplementary Figure S1). The shapes of these chronoamperograms are in agreement with the growing process of the polymer. At the potential step of +0.9 V, there is not much formation of the polymer at the beginning, and the current reaches the highest value when applying the step, and then it decreases to a plateau. When applying the potential level of +1.5 V, the current increases, but its initial value is much lower than at +0.9 V, which indicates that this potential favors the formation of a polymer that suddenly covers the electrode surface and the recorded current values decades to the value of the plateau current. When applying the +2.5 V step, a chronoamperogram with a maximum is obtained because the initiation of the polymerization takes place quickly and the current decreases more slowly, therefore the polymerization time is shorter than in the +1.5 V step.
The regular arrangement found at +1.5 V may be due to the π-steaking growth of the polymer [27] during the monomer electropolymerization, which can occur at this intermediate potential. This dependence of the polymer structuration on CPE potential was found also for other azulene derivatives [28].

Studies by AFM
T-CMEs surfaces were investigated by AFM to compare the topographic images of polyT-films deposited on GC disks (6 mm diameter) using different electropolymerization charge densities. These values correspond to the charge densities used for T-CMEs characterization by electrochemistry in ferrocene solutions performed on smaller GC disks (3 mm diameter). Figure 8 shows AFM images for polyT films deposited on GC electrodes by CPE using constant potential (+1.5 V) and various electropolymerization charge densities. As can be seen in Figure 8, the electropolymerization charge has influence on the electrode topography. RMS calculated from the acquired topographic images increases when the charge increases from 7 to 14 mC/cm 2 , but at higher charges (28 mC/cm 2 ), it decreases. This behavior agrees with SEM results from Figure 6 and corresponds to an ordered film for small charges and to an amorphous one for higher charges.

HMs Sensing
Electrochemical studies on T-CMEs have established the best conditions for obtaining complexing polyT films (see Section 3.6). To evaluate the ability of these modified elec-  Figure 9B gives the dependence of the stripping currents at the potentials on the ion concentrations in the tested solutions.
The best analytical signal from all the investigated cations was obtained for lead for which the detection in the accumulation solution is clearly possible over the concentration of 10 −6 M (Figure 9). Small DPV signals were obtained for cadmium, copper and mercury for a concentration over 5·10 −5 M. Figure 9B shows the DPV stripping peak currents for lead and cadmium are relevant signals in the investigated range of concentrations.  [28].
Several experiments using T-CMEs obtained at different electropolymerization potentials (+0.9, +1, +2.5 V) and prepared in similar conditions (using the same charge density of 14 mC/cm 2 ), were performed in tested water solutions containing only Pb(II) ions ( Figure 10). The higher peak obtained (of 12 µA) was for the CME prepared at +1.5 V. The plots in Figure 10 show that, for a preparation potential of +1.5 V, the response to Pb(II) complexation (12 µA) is six times better compared to about 2 µA at +0.9 V, and 1.5 µA at +2.5 V. This behavior indicates the highest number of complexing units accessible for Pb(II) complexation for this film. The result could be connected with the most nanostructured polymer noticed by AFM and SEM. T-CME signal can be further optimized by refining the preparation potential as noticed in other studies that have confirmed the crucial importance of electropolymerization potential in recognition experiments [28]. T-CMEs prepared at the same electropolymerization potential (+1.5 V) and using different charge densities (of about 7, 14 and 21 mC/cm 2 ) were conditioned as previously shown and tested as sensors for Pb(II) for synthetic water solutions containing only Pb(II) ions. Figure 11 shows the DPV curves obtained for T-CMEs prepared with different charge densities. The thicker film has the best response. This is the expected result, taking into account the higher number of complexing units in a thicker film. Figure 11. DPV stripping curves in acetate buffer at pH 5.5 measured T-CMEs obtained by CPE at +1.5 V using different polymerization charge densities; inset: DPV stripping current for Pb(II) dependence on the polymerization charge density.
From Figures 10 and 11, it can be concluded that the conditions that lead to the most sensitive T-CME for Pb(II) detection are the electropolimerization potential of +1.5 V and a charge density of 21 mC/cm 2 . Under these conditions, the detection limit for Pb(II) using T-based CMEs is 10 −7 M, which corresponds to about 20 µg L −1 . This limit is a modest limit compared to the performance of the latest modified electrodes in the literature [29] and is required to be improved in order to be able to generate commercial electrodes. This is a primary investigation, but further studies will allow the optimization of all parameters for obtaining good sensor for Pb based on this azulene derivative.

Electrochemical Characterization of T
Electrochemical characterization of T azulene derivative was performed by CV, DPV and RDE experiments on GC electrode. All these methods put in evidence the high degree of asymmetry of oxidation and reduction curves due to the irreversible processes occurring at opposite potentials.

CV and DPV
CV and DPV oxidation and reduction curves (with the starting points marked by → and ←, respectively, in Figure 12) were recorded at different concentrations of T (1-3 mM) in 0.1 M TBAP/CH 3 CN, starting from the stationary potential. DPV curves ( Figure 12A) show four oxidation (a1-a4) and four reduction (c1-c4) peaks. DPV currents for anodic and cathodic peaks increase with the concentration of T, except for a3 and a4. CV curves ( Figure 12B) show four anodic (a1-a4) and four cathodic (c1-c4) peaks, denoted according to DPV peaks. The CV peak currents increase with the concentration of T for a1-a3 and c1-c4, while it decreases for a4. According to this dependence on concentration it results that the peaks a1, a2, c1-c4 are related to electron transfers to T. a3 and a4 peaks are due to other processes such as formation or overoxidation of the polymer film generated during the scanning. The last ones are more intense at higher monomer concentrations, leading to more significant decreases of the currents at a given anodic potential in agreement with the chronoamperograms from Figure S1. The secondary processes are better seen in DPV, which is performed at lower scan rates (0.01 V/s) than CV (0.1 V/s). The DPV currents for a3 and a4 are smaller at T concentration of 2.4 mM than for 1.2 mM. The peak a4 appears after at about 1 V vs. a3, which shows that it may be due to either the formation of another structure of the film, or due to the overoxidation of the film formed at a3. The difference between the processes in a3 and a4 are confirmed by characterization of T-CMEs in ferrocene solution (see Section 3.1) and by SEM (see Section 3.3).  Figure 13 shows that c1, c2 and c3 peaks, noticed in the cathodic scans on the CV curves obtained on different potential domains, are reversible. These peaks have the response peaks c1 , c2 and c3 in the return sweeps (all situated at potentials of about 0.1 V in respect with the direct peaks). The cathodic peak c4 is irreversible, which is evident from Figures 12B and 13 (curve II). All processes in the anodic domain are irreversible. Table 4 presents the processes characteristics and the potential values for each peak, estimated from CV and DPV curves recorded for 1.2 mM solution of T.   Figure 14a presents the CV curves obtained at different scan rates (0.1-1 V/s) within the potential domains of the first anodic and cathodic peaks in 1.2 mM solution of T. The currents increase with the increase of the scan rate. Peak currents have a linear dependence on the square root of the scan rate (Figure 15b). The dependence on the scan rate confirms that c1 peak has reversible character. The response peak c1 vary more slowly with the scan rate than c1 (Figure 15b). The absolute slope of c1 peak (10.1·10 −5 ·A·V −1/2 ·s 1/2 ), estimated from the absolute values of the currents, is at about three times bigger than for c1 (2.79·10 −5 ·A·V −1/2 ·s 1/2 ). It is three-quarters from that of the anodic peak current a2 (14.4·10 −5 ·A·V −1/2 ·s 1/2 ). These slopes are in agreement with the number of electrons involved in the first oxidation and reduction processes, according to the mechanism for electrochemical oxidation and reduction of this class of derivatives [24][25][26][27][28].   Figure 15B). The nomination given for the peaks of the DPV curve (shown in Figure  15B) was kept for RDE processes. A high degree of asymmetry of anodic (0, +2.5 V) and cathodic (0, −3 V) processes can be noticed. There is no RDE wave for the processes in the anodic domain (corresponding to the DPV peaks a1, a2, a3). In the cathodic domain, there are two main waves for the DPV peaks c1 and c4. The peculiar shape of RDE curves in the anodic scan is certainly due to the formation of insulating polymer films that cover the electrode surface and lead to zero current on the whole anodic domain. The current values increase with the rotation rate for the two RDE cathodic waves w1 and w2. At potentials around c3 peak, the currents decrease with rotation rate similar to the case of film formation by anodic oxidation. It could be the case of cathodic electropolymerization of this azulene compound, but this aspect has not been yet investigated.

Global View of Results
Electrochemical immobilization of T monomer on GC electrode was carried out in millimolar solutions of T in 0.1 M TBAP/CH 3 CN by CPE using different values of electropolymerization potential (E) and charge density (Q) according to Tables 5 and 6, respectively. CMEs based on T were prepared and transferred into 1 mM ferrocene solution to check the film formation (Fc), or in 0.1 M TBAP/CH 3 CN for resistivity investigations (EIS). T-CMEs were also examined by SEM and AFM, and they were conditioned in acetate buffer 0.1M, pH = 5.5 and used for the analysis of HMs in aqueous synthetic solution (HMs sensing).  T-CMEs in ferrocene solution indicates the covering of the electrode with a thin film. The change of E significantly modifies the symmetrical ferrocene signal. The evident loss of reversibility of ferrocene on the modified electrode is dependent on E. The film formed at E = +1.5 V is different (as structure and/or porosity) from that prepared at the potential of +2.5 V. It seems that higher potential during CPE leads to a more porous film, which ensures higher oxidation and reduction currents for ferrocene. For the application fields, the potential of +1.5 V was selected to prepare T-CMEs by CPE. This potential was assumed to ensure a good number of complexing units in view of further complexation and, consequently, a good detection of HMs, confirmed by HMs sensing. For different Q values, the shape of the ferrocene signals changes as the thickness of films increases. The charge of 21 mC/cm 2 was chosen for the preparation of T-CMEs for recognition of experiments since it insures a good coverage of the electrode.
The study by EIS shows that the films behave as insulators. The fitting of EIS data allowed the calculation of the constant phase element (CPEdl) impedance, and chargetransfer resistance (Rct) of the layers. For instance, for the films obtained at the potential of +1.5 V, the lowest value of Rct (1.81·10 −6 Ω) and the lowest capacity (1.84 µF) were obtained. Rct values decreased with electropolymerization charge. This unexpected behavior might be determined by a change in the morphology of the layer, during the growth of the film to a relatively high porosity, which could facilitate the charge transfer. T-CMEs electrochemically prepared at +1.5 V and 21 mC/cm 2 were selected for the investigation of the analytical response involved in the electrochemical detection of HMs.
SEM investigations of T-CMEs surfaces showed a relatively uniform arrangement of the polymer matrix surfaces and the presence of some clusters. The film is similar to an ordered matrix for the charge of 14 mC/cm 2 . The morphology of the films depends on E. SEM analysis allowed to evidence the most nanostructured surface. The shapes of these chronoamperograms agree with the growing process of the polymer and are connected to the polymerization times. A regular arrangement was found at +1.5 V, which may be due to the π-steaking growth during the monomer electropolymerization. This dependence of the polymer structuration from SEM results for films prepared at various CPE potentials agree with that for other azulene derivatives.
AFM investigation of polyT films indicates a major influence of Q on the electrode topography which has a maximum at 14mC/cm 2 . This behavior agrees with SEM results and indicates an ordered film for smaller charges and an amorphous one for higher charges.
HMs sensing investigation of T-CMEs indicates the major influence of E and Q on DPV peak current for Pb(II) ions analysis. Small DPV signals were obtained for cadmium, copper, and mercury. The analytical signal for Pb(II) was examined. The best analytical signal from all the investigated cations was obtained for lead (linear dependence of the Pb(II) peak current (iPb(II)) on concentration was found between 10 −6 and 10 −5 M). The highest signal was obtained for +1.5V and 21 mC/cm 2 indicating the highest number of complexing units accessible for Pb(II) complexation for this film. This last result relates to the most nanostructured polymer noticed by AFM and SEM. T-CME signal can be further optimized by refining the preparation potential as noticed in other studies that have confirmed the crucial importance of electropolymerization potential in recognition experiments.
All techniques used for the evaluation of T ligand capacity to build new complexing structures confirm that T-CMEs films are good for the analysis of Pb(II). Further experiments to optimize the preparation conditions will lead to the decrease of the detection limit for Pb. Previous experiments with other azulene-based CMEs have shown good reproducibility of the CME preparation and detection processes of these electrodes, which are useful in large-scale practical applications [27].

Conclusions
2-(azulen-1-yldiazenyl)-5-phenyl-1,3,4-thiadiazole (T) was studied by electrochemistry and the best potentials to which this ligand can be electropolymerized. T-modified electrodes for HM ions analysis were obtained. All CV, DPV, and RDE studies put in evidence the importance of electropolymerization potential on the properties of the modified electrodes. Surface studies by EIS, SEM and AFM confirmed regular arrangement of the complexing polymer at a certain potential. The corresponding T-CMEs gave promising results in the analysis of HM ions. The attained detection limit for Pb(II) using T-based CMEs is of about 20 µg L −1 , and it is required to be improved to generate commercial electrodes.
The study of the surface properties of T-CMEs revealed (1) new aspects of film morphology obtained in different conditions, and (2) the role of electrosynthesis potential in electrode nanostructuring with direct consequences on sensing capacity.