**1. Introduction**

For the food industry and for the consumers, it is very important to monitor the quality and freshness of raw meat. Different factors are a sign of meat alteration (e.g., discoloration, rancidity, alteration of flavor) [1–3]. One pathway of alteration is the scavenging activity of myoglobin toward nitro-oxidative species (such as peroxynitrite, PON). For example, the formation of metmyoglobin can alter the flavor due to lipid and protein oxidation [4]. The lack of metmyoglobin (MbFe3+OH2 or metMb) reducing enzymatic systems in meat

**Citation:** Hosu, I.S.;

Constantinescu-Aruxandei, D.; Oancea, F.; Doni, M. The Scavenging Effect of Myoglobin from Meat Extracts toward Peroxynitrite Studied with a Flow Injection System Based on Electrochemical Reduction over a Screen-Printed Carbon Electrode Modified with Cobalt Phthalocyanine: Quantification and Kinetics. *Biosensors* **2021**, *11*, 220. https:// doi.org/10.3390/bios11070220

Received: 13 June 2021 Accepted: 29 June 2021 Published: 2 July 2021

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after slaughter determines the irreversibility of the oxidation processes of myoglobin [5]. The color changes are a sign of these processes, and some possible oxidation pathways are described in Figure 1 [6]. The aspect of meat, by itself, has a grea<sup>t</sup> impact on consumers, and the impact on the food industry is huge. Adding nitrites to the raw meat helps keeping the pink color of the meat, as NO (nitric oxide) can bind to the iron ion in a similar way as oxygen molecule does. Nitrites and nitrates are also two of the decomposition compounds of peroxynitrite. Distinguishing between these species is important for meat quality.

The detection of peroxynitrite, being a short-living ROS in biological samples, is a big challenge that scientists still try to solve nowadays. Even if there are different methods of detection presented in the literature, most of them rely on indirect methods after the formation of secondary species. Forming different species, such as 3-nitrotyrosine, that can be detected by immunochemical or chromatographic techniques, or oxidizing different probes with peroxynitrite, further to be detected with fluorescent and chemiluminescent methods, are the usual methods [7]. The problem is that the selectivity toward peroxynitrie is not assured using these methods, as other ROS/RNS species could give the same response. Other usually used methods are high-performance liquid chromatography, UV-Vis absorbance spectroscopy, electron spin resonance, and electrochemistry [8]. These methods usually use antioxidants such as resveratrol, polyphenols, or catechins [3], especially in batch analysis, but also using flow injection analysis, for example by injecting antioxidants that can quench the peroxynitrite [9]. The microfluidic injection analysis presents different advantages such as the presence of laminar flow with no dilution effects, necessity of low volume of analyte (lower than 150 μL), miniaturization, the possibility of real-time continuous monitoring (process control), faster and more sensitive response, or the possibility of automated processes [10].

Electrochemistry uses usually low-cost instrumentation, has fast response time, and can be coupled with online analysis. Electrochemical methods are a better alternative than the usually used methods as they can assure direct, label-free, specific, real-time measurements. Different electrochemically active matrices are described in the literature, such as polymeric films (based on porphyrins, metal phthalocyanine, and/or conducting polymers) hybridized or not with graphene [7,11–16]. Only very few reports present the batch reduction (or oxidation at low potential) of peroxynitrite using a chemically modified electrode (presented in Table 1). Ligands based on extended π conjugated systems can create coordinative chemical bonds with different metals and act as good electrochemical mediators for different redox processes, even nowadays. Phthalocyanines (PCs) are part of this class, and due to different oxidation states of various metallic centers and high conductivity, they are a good platform for the detection of oxygen/nitrogen reactive species [11] or other molecules [17]. PCs are not toxic and have high thermal resistance and are quite stable at room temperature, assuring the stability of the biosensors in time. Except for the metallic centers, the ring-based redox processes may also influence the catalytical activity.


**Table 1.** Literature study of the developed sensors used to detect PON via electroreduction.

The sensitivity of chemically modified electrodes is greatly improved using a flow analysis system (FIA) compared to batch [18]. FIA is easier to use in comparison with batch; it increases the reproducibility and simplifies quantification. In addition, the optimization of a method is more rapid, and testing electrodes is more efficient [10]. Understanding the mechanism of reaction is very important, and FIA provides several advantages in electrochemistry that could be useful for this purpose. For example, by using microreactors, one can narrow the diffusion layers of the electrodes that could also "overlap", which helps to optimize the reaction conditions and facilitates the determination of the mechanism. Channon et al. achieved pharmaceutical detection limits with an FIA electrochemical method for hydrazine detection [19]. They described that convective mass transport enhances the electrochemical signal by comparison with diffusive stationary experiments (batch). Recently, nanomolar levels were achieved for the detection of acetaminophen and codeine, using an FIA system combined with multiple pulse amperometry, and the analytes were also quantified in urine and human serum with excellent recoveries [20].

Herein, we describe the electrochemical reduction of peroxynitrite at 0.1 V, using a commercial cobalt (II) phthalocyanine complex (CoPc) and screen-printed carbon electrodes (SPCE). This electrochemical sensor is able to select between the most important interfering species of peroxynitrite (nitrite, nitrate, and hydrogen peroxide) and other molecules (e.g., ascorbic acid and dopamine), due to a specific, but simple design, combined with the advantages of a micro-fluidic system.

In the last part, we show that our proposed method could be used to further study the decay kinetics of PON in the absence and presence of myoglobin. Our method is both a detection and quantification method and a further tool for kinetic studies, as the RSDs values between the classical static UV-Vis method and our method are low (less than 10%).

**Figure 1.** Graphical abstract. ferrylmyoglobin: MbFe4+ = O, oxymyoglobin: MbFe2+ O2, deoxymyoglobin: MbFe2+(OH2), metmyoglobin: MbFe3+(OH2), nitrosylmetmyoglobin: MbFe3+NO, nitrosylmyoglobin: MbFe2+NO. This is a schematic representation of the chemical reactions of different forms of myoglobin with peroxynitrite and other interfering species/decomposition products. This scheme is not exhaustive and was inspired from data from different literature references [2,21–26].

#### **2. Materials and Methods**

Sodium nitrite, hydrogen peroxide (30%), manganese dioxide (MnO2), myoglobin from equine skeletal muscle, sodium hydroxide (NaOH), sodium phosphate dibasic dihydrate (Na2HPO4 2H2O), cobalt (II) phthalocyanine (CoPc), phthalocyanine (H2Pc), DMF (dimethylformamide), and TBATBF4 (tetrabutylammonium tetrafluoroborate 99%), hydrochloric acid (HCl), hydrogen peroxide 30% (H2O2), sodium nitrite (NaNO2), sodium nitrate (NaNO3),

ascorbic acid, and dopamine were acquired from Sigma-Aldrich. Screen-printed carbon electrodes were acquired from DropSens, Spain.

### *2.1. Peroxynitrite Synthesis*

Peroxynitrite (PON) was synthesized following a slightly modified procedure [27]. Briefly, a solution of 0.7 M HCl + 0.6 M H2O2 was added over an ice-cooled stirring solution of 0.6 M NaNO2, and almost simultaneously, a solution of 3M NaOH was added over the mixture to quench the decomposition of peroxynitrite (a yellow solution). After several reaction minutes, a few grams of MnO2 (0.1 g/mL) were added to the mixture, to catalyze the decomposition of hydrogen peroxide. After the gas liberation was finished (approximatively 15 min), the MnO2 was filtered under vacuum, and the solution was divided into small aliquots (1 mL) and stored in the freezer (−20 ◦C).

#### *2.2. Electrode Chemical Modification*

The SPCE electrodes were modified by drop casting 2 μL of a (cobalt phthalocyanine) CoPc solution (1 mg/mL in DMF). The CoPc solution was prepared by dissolving CoPc in DMF (1 mg/mL), using ultrasonication over 1 h (power 100%, frequency 37 Hz). After the drop-cast, the electrodes were dried at 60 ◦C in the oven (for 15 min). Before different dropcasting steps, the electrodes were rinsed with DMF and dried with nitrogen. The process was repeated 3 times, without rinsing. The electrode was stabilized by cycling between −0.6 and 0.6 V (in PBS pH 12). Two reduction pre-treatments were proposed for the optimization of the SPCE/CoPc for PON detection: amperometry at −0.3 V for different time periods and chemical reduction with 25 mM sodium borohydride, during 20 min, followed by rinsing with deionized water.

#### *2.3. Meat Extracts and Myoglobin Solutions*

Manz meat (veal under the age of 2) was achieved from a local store. Yellow filtering paper (Filtrak n. 389), ROTI®Spin MINI-3 25 units CL12.1 (gel ultrafiltration, 1.5 Eppendorf tubes, for 3 kDa), and Sephadex G-25 in PD-10 Desalting Columns were used to remove the strong reducer (sodium borohydride) from the metmyoglobin (metMb) reduced system (redMb). The separation systems were bought from Sigma Aldrich. For oxymyoglobin (redMb) synthesis, a solution of 75 mM of sodium borohydride (NaBH4) in PBS pH 9 was added to a solution of 25 μM metMb.

The meat extraction was done according to the procedure from [28]. Briefly, 200 g of meat were cut in small pieces and blended with 100 mL of PBS pH 9. In addition, to the mixture, 400 mL of 0.1 M PBS (pH 9) were added, and the solution was stirred during 30 min, on an ice bath. After stirring, the mixtures were centrifuged 20 min, at 15 ◦C, at 9000 RPM, and the supernatant was centrifuged in the same conditions. Filtration on yellow filter paper was performed under vacuum and the pH was adjusted to 9, using sodium hydroxide. The desired pH values of the solutions were 12, 9, and 7.4, and the concentration of the phosphate was 100 mM.

For PBS pH 9, Na2HPO4·2H2O (0.1 M) and KCl (0.1 M) were dissolved in 500 mL of ultra-pure water. The solution of pH 12 was prepared in the same way, but NaOH was added: 450 mL solution was titrated with NaOH (approximately 35 mL of 1.4 M NaOH) until pH 12 and brought to 500 mL at the end. For PBS, pH 7.4 prepared tablets were used. As we designed the synthesis of PON to obtain high-concentration stock solutions and only added very small amounts of alkaline PON solutions to PBS pH 9 buffer, the pH 9 was practically constant [29].
