In our experiments, 1 mL of a Dimethyl sulfoxide (DMSO)-Hematin solution (100 mg/mL) and 200 mg of EDOT(3,4-ethylenedioxythiophene) were added to 20 mL of 1:40 PSS/TSA solution pH 2 (70,000 g/mol PSS obtained by RAFT). The emulsion was stabilized for 2 hours under magnetic stirring, then pyrrole (Py) (0.1 molar fraction, to adjust the molar fraction of monomers as EDOT 0.9 - Py 0.1) as added and constant stirring was maintained at 1000 rpm for 2 hours at room temperature. Polymerization begins by microdosing 200 µL of 30% H₂O₂ for 1 minute, keeping the system at 0˚C. The temperature and stirring conditions (0˚C, 250 rpm) were sustained for 18 hours. Each copolymer was frozen at −25˚C and lyophilized for 24 hours, then resuspended in cold acetone, filtered on a 0.25 µm PTFE membrane, and multiple acetone washes were running over the product until a colorless filtered liquid was obtained. Finally, the product was dried at 70˚C for 12 hours. The synthesis of the homopolymers and copolymers without PSS, doped only with p-Toluenesulfonic acid (TSA), was carried out keeping the same synthesis conditions described above.

The biomimetically synthesized conjugated copolymer (CCBS) chosen for electrophoretic deposition was EDOT-PY in a molar fraction 0.9 - 0.1, as well as their respective homopolymers, all doped with PSS according to the synthesis methodology described above. Colloidal dispersions were prepared at a concentration of 10 mg/mL using a 500 mM PBS (phosphate buffer solution pH 7) aqueous solution as a medium dispersant and sonicated for 15 minutes. The deposit was carried out using a glass container at a capacity of 90% (25 mL of CCBS dispersion), with a surgical grade stainless-steel plate work electrode (0.1 mm thick, 0.75 cm² area) separated by 1 cm from a Pt wire coil as a counter electrode (4 cm). Before each experiment, the electrodes were washed by sonication in isopropanol for 30 seconds, then dried with compressed air. The electrophoretic deposit (EPD) was performed at 12 V (constant voltage source PowerPac Biorad) at room temperature with magnetic stirring (100 rpm). Different deposit times (5 - 120 min) were evaluated. After each deposit, each electrode was dried with compressed air and stored in hermetic packaging. The amount of mass deposited on the stainless-steel electrode was recorded for the different deposit times of the EPD method described above. At different intervals, the electrode was weighed using an analytical balance (SE2ultra-microbalance Sartorius with a resolution of 0.1 mg). The electrical resistance presented by the deposit at each interval was measured using a Caitlyn electrometer.

The cyclic voltametric (CV) measurements were carried out using a potentiostat (Rodeostat, IO Rodeo Smart Lab Technology) and a three-electrode cell where the working electrode (WE) corresponds to an electrode manufactured by any of the mentioned methods or a tablet of 7 mm diameter bulk polymer. The counter electrode (CE) consists of a platinum wire and Ag/AgCl was used as a reference electrode (RE). The electrolytes used were 0.5 M NaCl and 0.5 M PBS. Once the electrodes were submerged in the electrolyte, N₂ was bubbled in for 20 minutes. The operating conditions consisted of a potential window of −0.7 V to 0.7 V, and sweep speeds of 100 mV/s for 10 cycles.

The specific capacitance was determined by cyclovoltammetry with the configuration cell and the electrolyte mentioned above. A potential window was set at −0.7 V to 0.7 V and a sweep speed of 100 mV/s, which was calculated according to the equation

C s = S / 2 Δ U V (1)

where Cs is the specific capacitance, S is the integrated area of the voltametric curve (expressed as mA·V/cm²), ∆U is the potential window used, and V is the sweep speed (expressed as V/s).

Scanning electron microscopy (SEM) images and elemental analysis (EDX) mapping were obtained using a LEO (Zeiss) 1540XB FIB scanning electron microscope with a gun emission field of 3 to 15 kV and a 5 mm working distance from the sample holder to the optical system. The EDX microanalysis was obtained using an Oxford Inca Energy 350X-MAX 50, with linear resolution of 127 eV in Mn Kα from 1 to 100,000 cps., The detection and quantification of elements are from atomic number 4 (Beryllium).

The transmission electron microscopy (TEM) images were taken on a Titan Fei Thermofischer instrument with an acceleration voltage of 200 kV. The samples were prepared by immersing lacey-carbon grids in an isopropanol suspension, allowing them to dry in a vacuum oven for 2 hours at 60˚C.

Fourier transform infrared (FTIR) spectra were acquired using a Thermo Fischer Scientific FTIR Spectrophotometer in the attenuated total reflectance (ATR) mode using a diamond crystal. The sample does not require preparation. The powder is placed on the surface of the glass and the measurement is carried out. The spectra were acquired taking an average of 32 scans with a resolution of 4 cm^-1within a range between 400 cm⁻¹ to 4000 cm⁻¹.

The powder x-ray diffraction (PXRD) patterns were acquired using a Brucker D8 Advance Diffractometer with a Cu Kα radiation source (λ = 1.5418 Å). The powdered samples were placed in a standard sample holder. Measurements were made at intervals of 0.02˚ at a scanning speed of 10˚/min from 2θ = 2˚ to 82˚.

The ECG was recorded with a MP36R Biopac unit and an RHD2000 electrode adapter plate. Electrophysiological ECG recordings were performed using an EDOT-PY 0.9 - 0.1:PSS/PO₄ EPD electrode, and a Biopac electrophysiological commercial-grade Ag/AgCl electrode was used as a control. The tests were carried out with a 3-electrode configuration: the reference electrode (RE) was placed on the right ankle, the working electrode (WE) and counter electrode (CE) were placed on the left and right arm respectively, 5 cm from the wrists. Each test used a trio of equal electrodes (flat stainless steel 1.5 × 0.5 cm electrodes). An electrophysiological gel drop was added to each electrode before starting the test. The test subject (female, average weight) remained seated and at rest while the signal was allowed to stabilize for 15 minutes, and any type of movements was avoided to not add breathing or shaking artifacts.

The value of the signal-to-noise ratio (SNR) was extracted from the electrophysiological ECG recordings. A MATLAB function is applied to the data to obtain the root mean square (RMS) value of the signal of interest divided by RMS value of electronic noise according to the following formula:

SNR = 10 log ( RMS : signal ) 2 ( RMS : noise ) 2 (2)

The thermal noise voltage was calculated with the equation:

Vn = 4 kTBR (3)

where k is the Boltzmann’s constant in J/K, T is the temperature in degrees K, B is the noise bandwidth value, and R is the impedance in Ohms.

TableS1 (supporting information) presents the main bands identified in the different polymers analyzed. When analyzing PEDOT-Hematin, the appearance of a band related to the asymmetric stretching C = C of the ring [23] is observed at 1539 cm⁻¹. Likewise, some signals associated with the symmetric stretching of the ring were also not observed, which could mean a decrease in conjugation in the chain related to defects such as branching and crosslinking, which in general terms affects the conductivity of the polymer. In general, when comparing the spectra of PEDOT or Polypyrrole (PPY) (Figure 1), most of the bands have remarkable similarity regardless of the chemical skeletons of PPY and PEDOT, which indicate that the hematin does not exert a significant effect on the chemical structure of the homopolymers of PEDOT and PPY just as a catalyst would. Figure 1 shows the Fourier transform infrared (FTIR) spectra of the EDOT-PY copolymers: 0.9 - 0.1, 0.7 - 0.3, 0.5 - 0.5 and 0.3 - 0.7 biomimetically synthesized in the presence of Hematin. In the case of the PEDOT PSS spectrum, the existing bands corroborate the chemical structure of the polymer reported in the literature. Contributions from PSS are also observed, specifically from the −SOx group, which indicates that the doping process with PSS was successful. The spectrum of PPY:PSS shows the characteristic bands observed for PPY doped with TSA as well as how groups of the polymer were observed. When analyzing the bands of the EDOT-PY:PSS copolymer (TableS1), the formation of the copolymer is corroborated, with respect to the molar composition. Similarities can be observed in practically identical positions, which confirm the chemical structure of the synthesized copolymer and reproduces what was observed in the case of doping with TSA. This can be attributed to the increase in doping ions. According to Sandoval et al., the change in the structure of the doping causes small variations in intensity and position of some bands, especially at frequencies in the region between 1500 - 1400 cm⁻¹. This coincides with our experimental observations, and some may even disappear, specifically those related to neutral segments of the chain. The variations registered in the FTIR spectrum may indirectly be an indication of the increase of doping in the synthesized structure as well as in the presence of polyanions and an organic acid just as it is with TSA. One interesting characteristic of the spectra is that regardless of the dopant type,

the region between 1200 and 1000 cm⁻¹ is equivalent to the different products. This is not a strange result since it would be expected that a structure with a certain chemical character is not dependent on the dopant to retain similar chemical functionalities. Thus, it confirms that the EDOT-PY copolymers showed their own FTIR spectral fingerprint under the conditions of the synthesis investigated.

It is worth mentioning that depending on the molar relationship, there are positional variations in the region between 1500 and 1400 cm⁻¹. This could mean changes in the doping of the chain and in the conjugation of the skeleton, since at a lower content of PEDOT, the bands related to C = C the stretching of the ring change, and in the case of the copolymer 0.5 - 0.5, they decrease even more remarkably, so much that they cannot be assigned with precision. On the other hand, in the region that comprises 1300 - 1000 cm⁻¹, there is a high correlation of the S vibrational modes of the present functionalities that indicate equivalence at the structural level.

In the case of simultaneous doping with TSA and PSS, it is determined that they present comparable spectral patterns. This result illustrates the direct effect that co-doping has. The explanation is likely that a considerable increase in p-type doping of the structure has the consequence of homogenizing the oxidation state of the chain and gives rise to practically identical vibrational modes, because they result from equivalent molecular segments. However, a slight decrease in the intensities of the bands between 1500 - 1400 cm can be observed in the 0.5 - 0.5 copolymer. This is not as drastic as the case of single doping with PSS, and such an understanding could be relevant for demonstrating the change in the oxidation state of the polymer through the previously mentioned variation in the bands, and which has been suggested previously by Sandoval et al.

TableS1 summarizes the bands identified for each copolymer. Copolymer 0.9 - 0.1 shows an excellent coincidence with some bands obtained for homopolymers, which means that there are contributions from both chemical structures to the polymeric skeleton, consistent with what was reported by Bruno et al. [24] In elemental terms, this suggests the successful formation of the copolymer from the chemical point of view where the catalyst does not exert a significant effect. The molar ratios (0.7 - 0.3, 0.5 - 0.5, and 0.3 - 0.7) show bands of both PEDOT and PPY that confirm the presence of the copolymers. Thus, FTIR spectroscopy allows us to conclude that neither the catalyst nor the type of dopant exerts a significant change in the chemical structure of the copolymer EDOT-PY, which is only modified when the monomer molar ratio changes.

Figure 1(b) presents the deposition of the biomimetically synthesized copolymer EDOT-PY 0.9 - 0.1 and 0.7 - 0.3 carried out on stainless steel surfaces assisted by electrophoresis. The coatings exhibit intense bands at 72:1000 and 1130 cm⁻¹ associated with stretching and deformation of SOR and SO₂ bonds at 1250 cm⁻¹ with CN bonds. The bands between 1330 and 1450 cm⁻¹ are associated with stretching and deformation of the CC sp³ and sp² of the heterocycle, and at 1640 cm⁻¹ the presence of carbonyl groups. It is reasonable to conclude that the methods work for coating stainless steel because the EDOT-PY PSS/PO₄ was deposited cathodically and not anodically as would be expected. Also, the coating shows a wide and intense band centered at 1023 cm⁻¹, which could be associated with the co-deposition of phosphate on the surface [25], which masks or overlaps the vibrational contributions of the copolymer.

Figure 1(c) shows the PEDOT, PPY and EDOT-PY diffractograms. In the case of PEDOT-PSS (2:1) the diffractions become wider because the PSS, being a semicrystalline polymer, introduces a higher level of amorphousness to the structure. Despite this, extremely intense and narrow diffraction maxima can be observed at 2θ = 4.9˚, which could indicate a significant increase in the order between adjacent segments on the XY plane [26]. In the case of PPY:PSS (2:1) the diffractions are extremely wide. However, diffraction at approximately 2θ = 30˚ does not appear when doped only with TSA, so it could be associated with chain stacking of PSS [26] [27] [28] [29]. The homopolymer and copolymer diffractograms show four widened diffractions, which indicate an increase in the disorder of the structure due to both, the loss of planarity introduced by the copolymer, and the presence of PSS [30]. In the case of the polyanion PSS and TSA, an interesting effect occurs in the plane associated with chain stacking (2θ = 26.2˚), since it narrows the diffraction maxima, which indicates higher order in the stacking of chains, and this could possibly increase the electrical conductivity of the material. This is likely either due to the shape and size of the polyanion nanoparticles that can promote a higher order, or that serves as nucleation and growth points for crystalline nanodomains [31]. In general, CCBS diffractograms present four wide signals centered on: 2θ = 5, 16.7, 28.6 and 41.1˚ [32] [33] [34]. These diffractions are very broad and rounded, which indicate that the PSS introduces a high degree of amorphousness to the structure. However, the 2θ = 28.6˚ diffraction maxima is sharper compared with the homopolymers probably because the structure of the copolymer generates well defined crystalline stacking microdomains.

Figure 1(d) shows the cyclic voltammograms obtained for PEDOT:PSS and EDOT-PY 0.9 - 0.1:PSS electrophoretically deposited on stainless-steel using a 50 mM PBS electrolyte. In orderto check, if it was functional to generate electrophoretic coatings under conditions of very low toxicity, we proposed the use of PBS to favor electrophoretic deposition. This is due to reports in the literature on the use of organic and inorganic phosphates [35] that act as fixatives in the electrophoretic deposit of colloids. The PEDOT:PSS electrophoretic deposited voltammogram (Figure 1(d)) exhibits a similar shape compared with PEDOT:PSS in bulk. The use of the PBS electrolyte probably promotes in situ FePO₄ electrosynthesis that helps to entrap the polymer cathodically. There is a tendency for copolymer mobilization to the cathode even when the particles have a negative Z potential. One hypothesis for this is due to adsorption and ionic exchange phenomena induced by protons, acid phosphate ions and Fe²⁺ cations on the surface of the polymer; nanoparticles modify the surface potential [36] and transport the polymer to the cathode surface, generating a coalescence and layer growth.

Figure 2(a) shows the electrophoretic deposition kinetics of EDOT-PY 0.9 - 0.1:PSS on stainless-steel electrode. In the presence of PBS, a quasi-linear increase in the mass can be observed over time, reaching up to 8 mg/cm² after 60 minutes. However, an increase in the resistance of the electrode with an increase in the mass deposit was observed, therefore deposit times of less than 60 minutes are desirable, ensuring the maximum amount of deposited mass with the least possible electrical resistivity. The EDOT-PY 0.9 - 0.1:PSS/PO₄ voltammogram (Figure 1(d)) has a typical quasi-rectangular shape of highly capacitive surfaces with low resistivity, or in other words, non-faradaic (ECDL) plus faradaic process kinetics that contributes to the net’s electrochemical response of the material [37]. When compared to the voltametric curve of the uncoated

stainless-steel electrode (blue curve, Figure 1(d)) with the voltammogram of the copolymer deposit, the minimum capacitance contributed by the metallic surface of the steel is evident. Therefore, the majority of the additional surface capacitance is attributed to the electrophoretically deposited copolymer [38]. The data above shows that the electrophoretic deposition of the EDOT-PY copolymer with PBS as an electrolyte and fixative was successful. The voltammogram obtained from a conventional Ag/AgCl electrode exhibits a capacitive behavior (ECDL), which is consistent with a high surface area and consequently a satisfactory signal-to-noise ratio (SNR). However, the voltametric evaluation shows that its potential window is very small with the PBS electrolyte (−0.045 to 0.2 V vs. Ag/AgCl), which is detrimental, since Ag/AgCl electrodes have been shown to release reactive oxygen species [39] [40] when used as a stimulation electrode in cells or microorganisms due to a narrow zone of electrochemical stability [41]. Polymer-coated electrodes should not present such problems since their operating window is wide (−0.7 to 0.7 V). In general, the specific capacitance per unit area of all the electrodes analyzed in Table 1 and Figure 2(b) are higher than those reported by various authors in the field [38] [42] [43].

Figure3(a) presents the SEM micrograph of the PEDOT:PSS/PO₄ coating by electrophoretic deposit. The micromorphology is densely porous and homogeneous. Amorphous reliefs appear as well as racemic clusters of polymer nanoparticle coalescence that give rise to supra structures with spheroidal shapes. The pores are nano-sized, the EDX mapping shows a uniform distribution of the elements on the surface, and the content of S, C and O are lower concerning the bulk polymer (FigureS1). Figure3(b) shows the SEM micrograph of the EDOT-PY 0.9 - 0.1:PSS/PO₄ coating. A microporous and homogeneous surface is appreciated, no signs of delamination were seen, the copolymer produces porosities smaller in size than those observed in homopolymers, the surface area of the

electrode increases drastically, the EDX mapping shows a uniform distribution of the elements on the surface, and the elemental ratio is similar to the one found for PEDOT:PSS/PO₄. However, there is an increase in sulfur content from 0.1% to 1.3%, as well as a decrease in the content of P and Na (Figure 3(c)) concerning PEDOT, which indicates an increase in the number of polymer particles deposited on the surface and a decrease in phosphorous-sodium complexes associated with carry-over and coalescence on the electrode. The reason fundamentally may be due to the size of the copolymer particle that is smaller regarding PEDOT (~30 nm vs. 200 nm, see TEM characterization). In general, it is homogeneous, although micrometric grooves are observed which generate laminar segments along with the electrode, which could generate long-term delamination. In the cycles evaluated in this study, no loss of electrode integrity or variations in resistance and signal-to-noise ratio were observed (see Conductivity and SNR below), which indicate that it is only a morphological effect of the macro-relief and obeys the mobility kinetics.

Figure 3(d) corresponds to the TEM micrograph obtained from the PEDOT:PSS; 500 nm spheroidal particles were observed. Figure 3(e) shows the TEM micrograph of the EDOT-PY:PSS 0.9 - 0.1 copolymer. It is possible to observe a change in morphology and particle size relative to PEDOT:PSS, which had already been observed in previous studies of potential Z and DLS, where it is shown that the copolymer with a small molar fraction of pyrrole drastically modifies its colloidal properties. The effect is corroborated by the decrease in particle size and the racemic morphology observed of multiple nanoparticles between 10 and 20 nm that form micrometric clusters. In Figure 3(b), we can see morphological characteristics that could be related to the nanometric detail of Figure 3(e). The electrophoretic deposit drives the massive coalescence and agglomeration of cathodically transported nanoparticles which eventually becomes a highly porous coating.

Electrocardiogram recordings were performed using the methodology described in the experimental section, which consists of placing active electrodes on the right and left forearm and a ground electrode on the test subject’s ankle. The electrodes available for the electrophysiological test were flat stainless-steel sheets (1.5 × 0.5 × 0.01 cm) covered by electrophoretic deposit with EDOT-PY 0.9 - 0.1:PSS/PO₄. This electrode presented a voltametric profile close to the ideal in an EDLC, approximately 8 mg/cm² of deposited mass, high capacitance per unit area, and an acceptable resistance (49 ohms/cm) as discussed above.

Figure 4 (blue ECG plot) shows the electrocardiogram obtained with EDOT-PY 0.9 - 0.1:PSS/PO₄ electrode. In this plot, it is possible to observe typical bioelectric signals of the myocardium. With adequate precision it has been

distinguished that [44]: the P wave corresponds to atrial depolarization, the QRS complex is associated with massive ventricular depolarization. The peak constitutes the highest signal intensity due to the massive contribution of ventricular fibers that reflect millions of action potentials of individual cardiomyocytes. After this event, the T wave is distinguished, which corresponds to ventricular repolarization. The frequencies between the P and Q waves are within 180 - 190 ms, while the S-T frequency is for less than 400 ms, which indicates that there are no AV blocks. In general, the quality of the electrocardiogram is sufficiently satisfactory for diagnostic purposes [7]. The upper red plot of Figure 4 corresponds to the electrocardiogram recorded simultaneously with Ag/AgCl Biopac contact electrodes. The recorded signals are less wide, the amplitude of the QRS complex wave is lower, and the sinusoidal shape of the P and T waves was observed. This is verified when performing the electronic analysis of the signals; both electrodes have a noise RMS close to 6 µV (Figure 6(c)). However, the SNR of the EDOT-PY electrode is equal to 36 dB while the Ag/AgCl electrode is close to 24 dB (Figure 6(b)).

This implies a better signal-to-noise ratio for the conductive copolymer electrode, which is strongly associated with surface conductivity and electrode capacitance. The copolymer coated electrode exhibits a surface conductivity of 0.08 S*sq and capacitance of 2.02 F/cm² against a surface conductivity of 0.25 S*sq and a specific capacitance of 0.5 F/cm² for the Ag/AgCl electrode. In a very general sense, it can be argued that CCBS copolymer electrodes have lower skin contact impedance than the commercial electrodes mostly used in commercial assays (Figure 6(a)). The results reflect differences in surface conductivity and capacitance per unit area. It has a significant effect on electrophysiological recordings and paves the way for future tests for stimulation and recording of electrical activity in neurons.

Figure 5 shows the ECG recordings obtained for the II, III and AVR leads. The same protocol described above was followed in this case. The only difference was the position of the electrodes on the test subject. The ECG segment shows typical signals for all recordings: P wave, QRS complex, and T90 wave. The recording is satisfactory for all leads. Not much thermal noise is observed (Figure 6(c)), and neither is the signal amplitude; for all of them is similar concerning the first lead signal amplitude. The electroactive area is the same for all electrodes and therefore the density of signals collected per unit area [45]. Any changes of the bioelectric signal amplitude are according to the electrical organ behavior and electrode positions, but not to the electrode sensitivity. Electrophysiological ECG tests revealed that the EDOT-PY 0.9 - 0.1:PSS/PO₄ electrode exhibits excellent properties for the collection of bioelectrical signals in humans, and of even higher quality than the commercial Ag/AgCl electrodes. These results constitute a solid basis to continue the research and development of electrodes made with CCBS for electrophysiological and neurophysiological applications, as well as for the candidates to replace even the most expensive types of metallic electrodes that can be found in the market.

Figure 6(c) shows how thermal noise voltage varies depending on the electrode positions according to Einthoven’s triangle. In the plot, it is possible to observe that the first, second, and third ECG leads exhibit similar skin impedance, but the AVR lead is lower than the other recordings. This implies that the position of the electrodes is highly dependent on the skin impedances obtained by CCBS electrodes as well as by Ag/AgCl electrodes. However, the thermal noise voltage remains low for all ECG recordings.

We believe that both kinds of electrodes are sufficiently accurate for low noise electrocardiogram recording which would imply that the CCBS electrodes are as useful and effective as commercial Ag/AgCl ECG as discussed above.

Figure 6(d) represents the results related to the SNR electrodes after several times reused. Briefly, it consisted of placing three CCBS electrodes according to Einthoven’s lead I and recording one ECG for two hours with normal subject test mobility throughout the assay. After that, the electrode was washed with water and ethanol several times and dried, then the same protocol was repeated two more times. We found that the SNR decreased in a monotonical way after each cycle. The electrode held 80% of the initial SNR value after being reused three times, however the SNR value was still high and acceptable for ECG recording with low noise. This implies that the CCBS electrodes probably work for long-term recordings or wearable applications.