**4. Conclusions**

Our label-free electrochemical method proposes a cobalt phthalocyanine deposition on the screen-printed carbon electrode (SPCE/CoPc) for the direct detection of peroxynitrite via electrocatalyzed reduction. This method is simple, sensitive, highly selective, rapid, and cost-effective. A simple flow injection system based on amperometric detection at potentials near +0.1 V was designed for PON determination, bringing the possibility for the automation and fast-response measurements. CA (chronoamperometry) was used to establish that changing the applied potential, the sensing mechanism of the electrode is changing/becomes easier to distinguish: the oxidation of PON occurs near 0.07 V and the reduction occurs near 0.05 V. At those low redox potentials (60 mV), the most important interfering species are less likely to appear, especially for the reduction potential.

We optimized different parameters (flow rate, reduction potential, the quantity of deposited CoPc on the surface of the electrode, determined surface coverage etc.), and we suggested a mechanism that described the electro-reduction of PON, involving two electrons, based on the electrochemical characterization of the electrodes. We also determined that the electrochemical reactions taking place at the surface of the electrode are not simply diffusion-controlled ones, and other processes such as adsorption, ligand-based processes may take place. Interfering species were studied at a 5-fold concentration compared to PON, and only hydrogen peroxide responded with a very low sensitivity and under an electro-oxidation catalyzed mechanism, not influencing our PON detection.

Compared to the literature, this is one of the few articles based on the electro-catalyzed reduction of PON. Even though we obtained good sensitivity (with an LOD of 0.4 μM compared to 1.9 nM [14] and 1.0 nM [15], but higher than 5 μM [16]), we achieved a much better selectivity against all the important interfering species (ascorbic acid, hydrogen peroxide, nitrate, and nitrite) at biological concentrations.

The optimized SPCE/CoPc calibration helped us monitor and quantify PON and the reaction between peroxynitrite and myoglobin. The literature presents the fact that the PON to redMb ratio must be 1:10 for efficient scavenging and that metMb is less efficient in scavenging PON (as the metal center is already oxidized) [24,52]. The scavenging of PON with myoglobin (in the form of metMb and redMb) decreases the amount of PON, and this decrease was measured/quantified with our SPCE/CoPc electrode. As the UV-Vis technique cannot be used to study the interaction with myoglobin (as the protein peak at 280 nm interferes with the PON peak at 302 nm), our FIA-EC method is an alternative technique that is able to study the scavenging effect of myoglobin from meat extracts toward peroxynitrite. The similarity of absorption spectra, especially in the UV zone, corresponding to redMb and meat extracts after incubation with PON, proving once again that PON is decomposed during the irreversible oxidation of redMb to metMb.

We propose a simple method that has grea<sup>t</sup> impact on the PON sensor choice, as it can be used to quantify PON in complex media (such as meat samples) but also to study the kinetics of PON decay. We have also demonstrated that one can study the interaction of PON with different forms of myoglobin. Kinetic studies were also performed and correlated with the literature to study the scavenging effect. Meat extracts scavenged better PON, which was probably due to different forms of Mb and the possibility of another scavenger. The scavenging effect is a second-order decay rate (when PON is not in excess, when pseudo first orders apply), and the rate constants were determined.

Our study for the kinetics should be regarded more as an alternative possible method ("proof of concep<sup>t</sup>") for studying the interaction between PON and myoglobin using the FIA-EC method than a finalized kinetic study. The kinetic parameters obtained with both proposed kinetic methods (A and B) were in good correlation with each other. We can conclude that our FIA-EC method is precise enough for further studying the interaction of PON with scavengers. Evaluation of decay order for the interactions is impossible with the classical static UV-Vis method (that also has a low sensitivity toward PON, as an absorbance of 0.08 a.u. corresponds to 50 μM PON).

A further research direction is to use the already described hybrid materials (CoPcgraphene) to increase the solubility of the hybrid material in aqueous solvents and electron transfer at the surface of the electrode. The electroactive film could be also deposited on micro-electrodes to be used for the detection of PON in physiological conditions.

**Author Contributions:** Conceptualization, M.D. and I.S.H.; methodology I.S.H., investigation, I.S.H. and D.C.-A.; project administration, M.D. and F.O.; supervision, M.D. and F.O.; validation, M.D., visualization, M.D. and I.S.H., writing—original draft preparation, I.S.H.; writing—review and editing, M.D., I.S.H., D.C.-A. and F.O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by Romanian National Authority for Scientific Research and Innovation, CCDI-UEFISCDI, Project PN-II-ID-PCE-2011-3-1076 and project ERANET-MANUNET-NITRISENS, contract no. 216/2020 within PNCDI III. The authors also acknowledge the financial support to Ministry of Research and Innovation, Nucleu Programme ChemEmergent, Project PN.19.23.01.01—Smart-Bi.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** The authors are grateful to Sanda Doncea for FTIR measurements and Alina Vasilescu for all the advices and the cobalt phthalocyanine.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
