*2.6. Surface Characterization*

Fourier transform infrared (FTIR) spectra were recorded using a ThermoScientific FTIR instrument (Nicolet 8700, Pleasantville, NY, USA) equipped with a VariGATR accessory (Harrick Scientific Products, Inc., New York, NY, USA). KBr pellets were used as reference, and the powders were ground with KBr to create a solid pellet. For the ATR measurement technique, the Perkin Elmer GX FTIR spectrometer equipped with a Pike MIRacle having a 1.8 mm round diamond crystal was used. Diamond has an intrinsic absorption from approximately 2300 to 1800 cm<sup>−</sup>1, which limits is usefulness in this region.

Micro-Raman spectroscopy measurements were performed on a Horiba Jobin Yvon LabRam HRMicro-Raman system combined with a 473 nm laser diode as an excitation source. Visible light was focused using a 100× objective. The scattered light was collected by the same objective in backscattering configuration, dispersed by an 1800 mm focal length monochromator, and detected using a CCD.

SEM images were obtained using an electron microscope FEI-QUANTA 200 equipped with wolfram filament (W), at the ICECHIM laboratory (Bucharest, Romania). The SEM images were taken at an accelerating voltage of 25–30 kV using a gaseous secondary electron detector (GSED).

Absorption spectra were recorded using Ocean Optics UV-VIS-NIR spectrometer, in the 200–1100 nm range.

The Limit of Detection and the Limit of Quantification were calculated with the following formulas: LOD = 3 σb/m, and LOQ = 10 σb/m, in which <sup>σ</sup>b represents the standard deviation of the background and m is the slope of the calibration graph. The sensitivity of the sensor was calculated from the slope of the calibration curve (plot *concentration* vs. *current*).

#### **3. Results and Discussion**

Detecting reactive oxygen and nitrogen species is of grea<sup>t</sup> importance for many domains. Peroxynitrite, despite being a short half-life oxidative species, induces powerful oxidative stress effects on cells. Scavengers are important tools to eliminate this oxidative stress. Myoglobin is one of the scavengers [35], as it is an oxygen-binding protein from the heme group. Cobalt phthalocyanine was already used in the literature as a bio-mimetic material for biosensors [36]. It has a similar scavenging role when it comes to PON. As the metal cobalt center of the heme, similar to iron, has multiple oxidation states, the reduction of PON seems to be catalyzed by CoPc through redox reactions. We demonstrate here that the Co2+/Co1+ redox couple is more effective than the high potential electrochemical methods reported in the literature for the electrochemical detection of PON, as it offers better selectivity. Cyclic voltammetry as well as other techniques (linear sweep voltammetry or differential pulse voltammetry) were used to determine that this redox reaction of PON is apparently an irreversible process.

Before presenting and discussing the results, several other important factors regarding PON are to be mentioned, which are factors that are also important challenges to overcome in developing a selective and sensitive sensor for PON for meat extracts. (i) PON is most stable in cold alkaline solutions, without any metals and carbonyl compounds [30] and even so, it slowly decomposes, mainly, to nitrite. (ii) Below pH 12, the decomposition rate increases, and at pH 7, the half-life of PON is below 1 s. (iii) Acidic pH favors the decomposition to nitrate. (iv) Temperature, buffers, or several scavengers (e.g., myoglobin in meat as in Figure 1) influence the stability of PON [30,31]. Proteins tend to precipitate at pH values around 12, so such alkaline environment is not suitable for our purpose. As the decay of PON is slower at pH 9 than at neutral pH values, pH 9 is an optimal compromise for the detection of PON. At this pH value, the decay to nitrite is the predominant pathway, and it depends on peroxynitrite concentration. Due to these factors, we needed a fast response technique to help us distinguish between PON, nitrite, and nitrate, and to combat batch problems (mixing of the aliquots added for detection would increase the noise of the measurement, the concentration of the analyte will not be homogenous, etc.).

#### *3.1. Batch Determination of Peroxynitrite Using SPCE/CoPc Electrodes* 3.1.1. Characterization of the Deposited CoPc Films on the SPCE

SEM analysis of the morphology revealed that in accordance with the literature, the CoPc molecules tend to form random agglomerates [37] of various sizes (between 0.4 and 10 μm in length and a few hundred nm in thickness and width) depending on the surface used for the deposition (Figure 2a,b). The thin films were obtained by drop casting CoPc, due to π stacking (as the macrocycle has 18 delocalized π electrons), similar to other materials, such as graphene. This method is deposition is cost-effective and rapid.

**Figure 2.** SEM characterization of the (**a**) unmodified SPCE and (**b**) SPCE/CoPc, (**c**) FTIR spectra of unmodified SPCE and modified SPCE with CoPc (SPCE/CoPc) [1], (**d**) Raman spectra of unmodified and CoPc modified SPCE.

The FTIR analysis (Figure 2c) proves the presence of CoPc on the surface of the electrode, especially due to the 741 cm<sup>−</sup><sup>1</sup> band in the fingerprint region, corresponding to phthalocyanine in plane vibrations [38]. Other vibrations belonging to the graphitic structure of SPCE are also observed: deformation of C-C out of aromatic plane (650 cm<sup>−</sup>1), vibrations of C-H in the aromatic plane (850 and 808 cm<sup>−</sup>1).

In the Raman spectrum of CoPc (Figure 2d), there are active modes of the symmetry A1g at 592 cm<sup>−</sup><sup>1</sup> (benzene radial), 834 cm<sup>−</sup>1, B1g at 684 cm<sup>−</sup><sup>1</sup> (macro breathing and benzene radial), 1542 cm<sup>−</sup><sup>1</sup> (C=N stretching mode), and B2g at 1498 cm<sup>−</sup><sup>1</sup> (pyrrole stretch) [39,40]. The HUMO–LUMO gap energy of CoPc is 1.9 eV.

After the morphological characterization of the surface of the electrode, we also used electrochemistry to extensively characterize the electrode. Cyclic voltammetry gave rise to two anodic and two cathodic peaks (corresponding to Co3+/Co2+ and Co2+/Co1+ redox processes), as previously reported [11]. In this paper, we targeted the exploitation of the Co2+/Co1+ redox couple (Figure 3a), due to the occurrence at more negative potentials (E0 ≈ 0.1 V), than the couple Co3+/Co2+ (E0 ≈ 0.65 V), and because, besides good sensitivity, there is the possibility to reach a remarkable selectivity for peroxynitrite. It can be observed that upon several scans (five cycles), the electrode reaches a steady state. Similar peaks were observed in the literature [41].

**Figure 3.** Cyclic Voltammetry (CV) of (**a**) the screen-printed carbon electrode (SPCE)/cobalt phthalocyanine (CoPc) electrode upon different scans, in PBS pH 12 [1]. The oxido-reduction processes are presented for the cobalt metallic center. (**b**) Cyclic voltammetry was registered using the SPCE/CoPc electrode in the absence (black) and in the presence (red) of 45 μM PON (scan rate 9 mV/s), PBS pH 9. Zoom in of the redox peaks for PON from the same plot.

Cyclic voltammetry (CV), in the −0.6 to 0.6 V range, was used to determine the redox process of PON, using the SPCE/CoPc electrode, at pH 9 and pH 12. A higher pH value (12) presented enhanced anodic and cathodic peaks of the biosensor (Figure 3a) than a lower pH value (pH 9, Figure 3b, both in the presence and absence of PON), but high alkalinity interferes with the integrity of proteins (the final goal of our work) and may interfere with the integrity of the film, so it was not used further for analytical purposes, only for biosensor characterization. The redox process involving peroxynitrite at pH 9 takes place at around 0.1 V, with Ec = 0.047 and Ea = 0.072 (ΔE = 25 mV = Ecathode − Eanode), but there is a small current in the reduction, suggesting that the oxidation might be irreversible. The calculated formal potential (the redox standard potential, E0) is 0.06 V vs. Ag/AgCl pseudoreference electrode. Following the Nernst equation (Equation (3), where ared represents the concentration of reduced species and aox represents the concentration of oxidized species, [32]) described below, we can conclude that two electrons are involved in the redox process (transferred in the cell reaction) at 25 ◦C. This conclusion is consistent with the nature of the peroxynitrite oxidant (usually described as a two-electron oxidant).

$$E\_{\text{cell}} = E\_{\text{cathode}} - E\_{\text{anode}} = E^0 + 0.059 / \text{n} \cdot \lg \text{ } \text{a}\_{\text{red}} / \text{a}\_{\text{ox}} \tag{3}$$

Furthermore, under another probable mechanism, an irreversible reduction takes place around −0.3 V, probably involving also the chemical oxidative reaction of peroxynitrite over the metallic center: cobalt being chemically oxidized by PON, which is electro-reduced, with a higher current, depending on the concentration of PON (Figure 3b).

If the anodic/cathodic current is proportional to the scan rate, the process is an adsorption-controlled process, and if the plot I vs. υ1/2 is linear, the redox processes are more likely diffusion-controlled ones [42]. For this purpose, cyclic voltammetry was used to determine the correlation between the cathodic current and the scan rate for scan rates in the range of 16–800 mV/s (Figure 4a). At lower scan rates, a linear correlation was obtained only for the square root of the scan rate, suggesting a diffusion-controlled process. Deviation from linearity usually means that other processes are involved, processes related to the surface, such as adsorption, ligand–species interaction, etc. At higher scan rates (above 256 mV/s), the correlation between Ipc vs υ1/2 is not linear anymore, suggesting more a mixture of surface and diffusion-controlled processes (Figure 4b).

**Figure 4.** (**a**) Cyclic voltammetry of the SPCE/CoPc electrode for 50 μM PON, PBS pH 12. (**b**) The dependence of the cathodic current (around −0.3 V) with the square root of the scan rate.

The SPCE is a three-electrode electrochemical system. The screen-printed area of the carbon-based working electrode (WE, black circle) of the SPCE is 0.126 cm2, which is surrounded by the Ag/AgCl pseudoreference electrode (silver) and counter/auxiliary electrode (CE, black). The chemical modification should not cover the CE and the reference electrode, but the surface coverage should be optimal. Starting from a solution of classical concentration for CoPc (1 mg/mL), we drop-casted 1 and 2 μL of the solution on the electrodes and calibrated them by DPV, from −0.4 to 0.3 V, step potential 5 mV, amplitude 25 mV, modulation time 50 ms, scan rate 10 mV/s (Figure 5a). The sensitivity for the 2 μL drop-casted CoPc was 0.083 nA μM−1, in comparison to 0.057 nA μM−<sup>1</sup> for the 1 μL drop-casted CoPc. More than 2 μL is difficult to deposit without covering the other electrodes. These sensitivities are very low, but further optimization helps us improve them, especially using chronoamperometry at the optimized potential. The efficiency of different cobalt phthalocyanine layers was studied using Cyclic Voltammetry measurements. By drop-casting different layers of solution of CoPc, the best sensitivity for PON was achieved for three layers (drop-casting 2μL in three successive steps, that also included drying steps). The sensitivity for one layer of 2 μL CoPc was 3.3 nA μM−1, and for three layers of 2 μL CoPc, it was 8.5 nA μM−1, which is already one order and respectively two orders of magnitude improvements from the DPV method, but the LODs remained almost the same (5.45 μM and 5.14 μM, respectively).

Chronoamperometry was used to study the preliminary potential for an improved quantitative detection of PON. Two different potentials were used, and both worked on the catalytical oxidation of PON with sensitivities of 8.75 nA μM−<sup>1</sup> (0.10 V) and 0.91 nA μM−<sup>1</sup> (0.15 V).

Based on the data described above, we propose a simplified mechanism of the catalytic process, involving two electrons as calculated from Nernst equation:

$$\rm Co^{II}PC + e^{-} \rightarrow Co^{I}PC \tag{4}$$

(reduction)

$$2\text{ Co}^{1}\text{PC} + \text{ON}^{\text{V}}\text{CO}^{-} \rightarrow \text{intermediate complex} \rightarrow 2\text{ Co}^{1}\text{PC} + \text{N}^{\text{III}}\text{O}\_{2}^{-} + \frac{1}{2}\text{ O}\_{2} + 2\text{e}^{-} \quad (5)$$

**Figure 5.** (**a**) Calibration curves for 1 μL and 2 μL drop-casted CoPc solution (1 mg/mL in DMF) on the SPCE (from DPV measurements) (**b**) Calibration curves from the amperometric response at +0.15 V (black) and +0.10 V (blue), using a GCE/CoPc electrode.
