Mesoporous CoO x /C Nanocomposites Functionalized Electrochemical Sensor for Rapid and Continuous Detection of Nitrite

: Nitrite is widespread in the environment, and is frequently used as an additive to extend the shelf life of meat products. However, the excess intake of nitrite can be harmful to human health. Hence, it is very important to know and control the content of nitrite in foodstuffs. In this work, by the means of self-assembly induced by solvent evaporation, we used the amphiphilic PEO- b PS diblock copolymers resol and cobalt nitrate as a template to synthesize ordered mesoporous CoO x /C nanocomposites. Then, the CoO x /C nanocomposites were modiﬁed on a glassy carbon electrode (GCE), which showed excellent sensitivity, good selectivity, and a wide detection range for nitrite. Through cyclic voltammetry and current–time techniques, the electrochemical performance of the GCE modiﬁed with CoO x /C nanocomposites was analyzed. Under the optimized conditions, we found that anodic currents were linearly related to nitrite concentrations with a regression equation of lp ( µ A) = 0.36388 + 0.01616C (R 2 = 0.9987) from 0.2 µ M to 2500 µ M, and the detection limit was 0.05 µ M. Furthermore, the electrochemical sensor behaved with high reproducibility and anti-interference ability towards various organic and inorganic ions, such as NO 3 − , SO 42 − , Cl − , COOH − (Ac − ), Na + , K + , Mg 2+ , and NH 4+ . Our results indicated that these CoO x /C nanocomposites could be applied in electrochemical sensors for the rapid and sensitive detection of the food preservative nitrite.


Introduction
Nitrite is widespread in environmental, food, and life science, and is used as a preservative in food and in agricultural preservation to inhibit damage caused by foodborne pathogens, especially Clostridium botulinum [1]. However, the excess intake of nitrite is harmful to human health. In blood, nitrite can react with hemoglobin and cause hemoglobin oxidation, which brings about a disease known as "blue baby syndrome" [2]. In addition, nitrite can combine with amines to form nitrosamines, which have been shown to cause cancer as well as hypertension [3]. The reduction of the residual nitrite levels could be an acceptable alternative to reduce the intake of nitrite. At present, an increasing number of effective methods such as physical processing, chemical processing, and bio-enzymatic degradation have been applied for the abatement of nitrite compounds [4][5][6][7]. Due to its potentially hazardous nature, the determination of nitrite is very important for human health and in the quality control of foods. Several common methods have been used to determine the content of nitrite, including spectrophotometry [8], chemiluminescence [9], chromatography [10], capillary electrophoresis [11], and so on. These methods can achieve good detection results for nitrite. However, they may have some shortcomings, such as complicated sample preparation processes, relatively low sensitivity and selectivity, high cost, and the requirement of specialized operations. Therefore, finding a simple, low-cost, and sensitive method for nitrite detection is essential to ensure food safety.
Owing to their advantages, such as low cost and direct on-site measurements, microfluidic paper-based analytical devices (µPADs) and glass-based devices are also applied in the detection of nitrite [12][13][14]. Compared to the above-mentioned analytical methods, electrochemical techniques have many advantages, such as simple operation, low cost, good selectivity, fast response, excellent sensitivity, and no need for additional chemical reagents [15,16]. Nitrite is an electroactive substance with redox reaction at the surface of carbon electrodes, but the electron exchange rate is usually poor, which causes great limitations in the detection sensitivity [17]. It is reported that many nanostructured materials are excellent electrode materials with high conductivity [18][19][20]. Therefore, it is a good alternative approach to modify electrodes with these nanomaterials. For example, Wang et al. reported a paper-based disposable electrode for the detection of nitrite in environmental and food samples, taking advantage of the physicochemical properties of graphene nanosheets and gold nanoparticles [21]. Chen et al. constructed an electrochemical sensing platform for nitrite based on a Cu-based metal-organic framework (Cu-MOF) decorated with gold nanoparticles, which could ensure the stability of the platform and enhance its conductivity and catalytic activity [22]. However, these proposed methods failed to achieve either a relatively high sensitivity or a wide detection range. Therefore, it is urgent to develop other strategies to improve the electrochemical performance. It is well known that metal oxide nanoparticles/carbon nanomaterial hybrids can significantly enhance the performance of electrochemical sensors [23][24][25][26]. Metal oxides are often used as electrode materials due to their excellent electron transport rate. In order to achieve good electrocatalytic ability and chemical stability, cobalt oxides have often been used as modifiers of electrodes. For example, to detect nitrite sensitively, Haldorai et al. [25] proposed an electrochemical sensor modified with cobalt oxide (Co 3 O 4 ) nanospindles-decorated reduced graphene oxide (RGO). Based on a cobalt monoxide (CoO)-nanoparticles-modified glassy carbon electrode (CS-CoO/GCE), Huang et al. [26] developed a new method which provided good catalysis and could improve the oxidation of the nitrite. Liu [27] synthesized a Co 3 O 4 /RGO composite film immobilizing horseradish peroxidase, which exhibited excellent electrocatalytic activity toward nitrite. However, few studies have focused on metal oxide nanoparticles/ordered mesoporous carbon nanocomposites for nitrite detection. Owing to its large specific surface area, considerable pore volume, and adjustable pore size, ordered mesoporous carbon can make metal oxides evenly dispersed and highly active [28]. Furthermore, the physicochemical properties of the system can be enhanced by combining ordered mesoporous carbon with metal oxides, due to their synergistic effect.
There are many methods to synthesize ordered mesoporous carbon/metal oxides, including the post-loading method [29,30] and the direct synthesis method [31]. The postloading method is carried out by preparing carbon materials and metal oxides separately, and then mixing them to obtain the nanocomposites. Meanwhile, the direct method uses a single process that can directly obtain the nanocomposites. Obviously, the direct synthesis method is easier and faster, which usually adopts a triblock copolymer as a template, combining a phenolic resin and metal salt precursors (metal nanoparticles) in the system, and then the target product of ordered mesoporous carbon/metal oxide is directly obtained by baking the template in an inert atmosphere [32]. Amphiphilic copolymers such as PEO-b-PS, PEO-b-PMMA, PEO-b-PHB, which have high molecular weight, have recently seen wide use as templates for the synthesis of ordered mesoporous materials with different compositions [33][34][35][36][37].
Hence, in this work, ordered mesoporous CoO x /C nanocomposites were synthesized via a self-assembly method induced by solvent evaporation. After modifying an electrode with CoO x /C nanocomposites, the glassy carbon electrode (GCE) showed excellent electrocatalytic activity for nitrite, which indicated that these nanocomposites could be applied in electrochemical sensors for the assessment of nitrite. When applying the sensor in experimental sausage samples, it obtained recoveries from 99.8% to 101.8%, further demonstrating the feasibility of the proposed method in practical samples.

Synthesis of CoO x /C Nanocomposites
According to a previously studied method [38], we synthesized the amphiphilic PEO-b-PS copolymers and soluble low-molecular-weight phenolic resin. The preparation method of the mesoporous CoO x /C nanocomposites is depicted in Figure 1. In Step 1, the nanocomposites were synthesized in one step with the template method. To be specific, 0.08 g of Co (NO 3 ) 2 ·6H 2 O was dissolved in 5 mL tetrahydrofuran (THF) which contained 0.4 g resol molecules. Then, the mixture was agitated for 0.5 h to obtain a transparent darkred solution. After that, 0.1 g PEO-b-PS was dissolved in another 5 mL tetrahydrofuran (THF). Then, it was added dropwise into the above dark-red solution, with 2 h further stirring. To evaporate the tetrahydrofuran (THF), the solution was dropped on Petri dishes at 25 • C for 0.5 h. Following this, it was placed at 50 • C for 24 h to evaporate the solvent completely, and to fix the nanostructure via the thermal polymerization of resol, obtaining a yellow film as shown in Step 2. In Step 3, the yellow film was scraped off, and then ground into a powder in a tube furnace under N 2 atmosphere at 600 • C. Then, the final ordered mesoporous CoO x /C nanocomposites could be obtained after the pyrolysis treatment. By adjusting the ratios, we obtained a series of samples and designated them as CoO x /C-n, wherein n is the weight ratio of cobalt precursor to resol.

Preparation of the Modified Electrode
Before modification, we polished the glassy carbon electrode (GCE) with alumina powder (grain size: 0.3 and 0.05 µ M) successively. Next, the electrodes were sonicated with absolute alcohol and distilled water for 10~15 min respectively, and then dried in nitrogen environment. After that, a certain amount of the above CoOx/C nanocomposite was dispersed in 1 mL water by ultrasonic stirring for 30 min, and then a homogenous suspension was obtained. Finally, a certain amount of the suspension was dropped on the surface of a GCE and dried in nitrogen environment.

Characterization and Measurements
Powder X-ray diffraction (XRD) patterns were recorded on a Bruker D4 X-ray diffractometer (Bruker, Karlsruhe, Germany) with Ni-filtered Cu Kα radiation (40 kV, 40 mA). Transmission electron microscopy (TEM) images were taken on a JEM-2100 F microscope (JEOL, Tokyo, Japan) operated at 200 kV. The samples for TEM measurements were ultrasonically dispersed in absolute ethanol. Small-angle X-ray scattering (SAXS) measurements were employed using a Nanostar U small-angle X-ray scattering system (Bruker, Karlsruhe, Germany) with Cu Kα radiation (40 kV, 35 mA).
All electrochemical measurements were performed using CHI 660D electrochemical workstation (Chenhua, Shanghai, China) with a conventional three-electrode system. The reference, working, and counter electrodes of the electrochemical sensor were modified by saturated calomel electrode (SCE), glassy carbon electrode (GCE), and a platinum foil, respectively.

Characterization
According to the scheme in Figure 1, through the solvent-evaporation-induced selfassembly method, we first synthesized a series of samples with different weight ratios (cobalt precursor/resol). SAXS and XRD were used to investigate their properties. As can be seen in Figure 2a, the samples of CoOx/C-n-600 all had excellent diffraction peaks, suggesting that the ordered mesoporous structure can be formed at cobalt precursor/resol ratios from 0.1 to 0.3. In addition, CoOx/C-0.2-600 exhibited the strongest diffraction peak,

Preparation of the Modified Electrode
Before modification, we polished the glassy carbon electrode (GCE) with alumina powder (grain size: 0.3 and 0.05 µM) successively. Next, the electrodes were sonicated with absolute alcohol and distilled water for 10~15 min respectively, and then dried in nitrogen environment. After that, a certain amount of the above CoO x /C nanocomposite was dispersed in 1 mL water by ultrasonic stirring for 30 min, and then a homogenous suspension was obtained. Finally, a certain amount of the suspension was dropped on the surface of a GCE and dried in nitrogen environment.

Characterization and Measurements
Powder X-ray diffraction (XRD) patterns were recorded on a Bruker D4 X-ray diffractometer (Bruker, Karlsruhe, Germany) with Ni-filtered Cu Kα radiation (40 kV, 40 mA). Transmission electron microscopy (TEM) images were taken on a JEM-2100 F microscope (JEOL, Tokyo, Japan) operated at 200 kV. The samples for TEM measurements were ultrasonically dispersed in absolute ethanol. Small-angle X-ray scattering (SAXS) measurements were employed using a Nanostar U small-angle X-ray scattering system (Bruker, Karlsruhe, Germany) with Cu Kα radiation (40 kV, 35 mA).
All electrochemical measurements were performed using CHI 660D electrochemical workstation (Chenhua, Shanghai, China) with a conventional three-electrode system. The reference, working, and counter electrodes of the electrochemical sensor were modified by saturated calomel electrode (SCE), glassy carbon electrode (GCE), and a platinum foil, respectively.

Characterization
According to the scheme in Figure 1, through the solvent-evaporation-induced selfassembly method, we first synthesized a series of samples with different weight ratios (cobalt precursor/resol). SAXS and XRD were used to investigate their properties. As can be seen in Figure 2a, the samples of CoO x /C-n-600 all had excellent diffraction peaks, suggesting that the ordered mesoporous structure can be formed at cobalt precursor/resol ratios from 0.1 to 0.3. In addition, CoO x /C-0.2-600 exhibited the strongest diffraction peak, revealing that the ordered mesoporous structure was the best. However, the diffraction peaks were weaker for two other ratios of cobalt precursor to resol (CoO x /C-0.1, CoO x /C-0.3), indicating that excessive or insufficient addition of cobalt precursor may affect the ordered structure. Figure 2b shows XRD measurements of the nanocomposites with different ratios of cobalt precursor/resol treated under N 2 atmosphere at 600 • C. As observed from the three curves, the distinct diffraction peaks appeared at 31.3, 36.9, 44.9, 59.6, and 65.4, corresponding to the Co 3 O 4 cubic phase (JCPDS card no. 65-3103). The corresponding crystal planes were (220), (311), (400), and (440), respectively. This confirmed the presence of cobalt oxide on the ordered mesoporous carbon. Based on the pyrolysis treatment at 600 • C, cobalt valence changes were likely to happen [39][40][41].
Coatings 2021, 11, x FOR PEER REVIEW 5 of 11 revealing that the ordered mesoporous structure was the best. However, the diffraction peaks were weaker for two other ratios of cobalt precursor to resol (CoOx/C-0.1, CoOx/C-0.3), indicating that excessive or insufficient addition of cobalt precursor may affect the ordered structure. Figure 2b shows XRD measurements of the nanocomposites with different ratios of cobalt precursor/resol treated under N2 atmosphere at 600 °C. As observed from the three curves, the distinct diffraction peaks appeared at 31.  The appearances of the nanocomposites with different cobalt precursor/resol ratios were further examined by TEM. In Figure 3, it can be seen that the CoOx nanoparticles were evenly distributed on the ordered mesoporous carbon nanocomposites. As the relative amount of cobalt precursor was increased, the size of the cobalt nanoparticles showed negligible changes, but the ordered pores in the mesoporous carbon were deteriorated. Especially, the ordered pores became the worst as excess CoOx nanoparticles distributed on the ordered mesoporous carbon nanocomposites and the cobalt precursor/resol ratio reached 0.3. Considering the ordering of the material pores and the loading of cobalt precursor, we chose CoOx/C-0.2 as the modifier on the electrode. The appearances of the nanocomposites with different cobalt precursor/resol ratios were further examined by TEM. In Figure 3, it can be seen that the CoO x nanoparticles were evenly distributed on the ordered mesoporous carbon nanocomposites. As the relative amount of cobalt precursor was increased, the size of the cobalt nanoparticles showed negligible changes, but the ordered pores in the mesoporous carbon were deteriorated. Especially, the ordered pores became the worst as excess CoO x nanoparticles distributed on the ordered mesoporous carbon nanocomposites and the cobalt precursor/resol ratio reached 0.3. Considering the ordering of the material pores and the loading of cobalt precursor, we chose CoO x /C-0.2 as the modifier on the electrode.

Electrochemical and Catalytic Activity
The catalytic activity of a GCE modified with the ordered mesoporous CoOx/C-0.2 nanocomposites was investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Figure 4a shows that the CVs of the bare electrode and the GCE modified with the CoOx/C-0.2 nanocomposites in 0.1 M PBS (pH = 3.5) showed responses to nitrite

Electrochemical and Catalytic Activity
The catalytic activity of a GCE modified with the ordered mesoporous CoO x /C-0.2 nanocomposites was investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Figure 4a shows that the CVs of the bare electrode and the GCE modified with the CoO x /C-0.2 nanocomposites in 0.1 M PBS (pH = 3.5) showed responses to nitrite at the potential of 0.8 V. Furthermore, the bare electrode (curve 1) and the GCE modified with ordered mesoporous CoO x /C-0.2 nanocomposites (CoO x /C@GCE, curve 3) had no current of the oxidation peak in the 0.1 M PBS solution without nitrite. Additionally, the bare electrode showed an insignificant current of the oxidation peak in 0.1 M PBS containing 0.5 mM nitrite (curve 2). However, after modifying with ordered mesoporous CoO x /C-0.2 nanocomposites, the GCE displayed an obvious current, with the oxidation peak located at 0.8 V (curve 4). These results indicate that nitrite oxidation may be catalyzed by the electron transport activity of the CoO x /C-0.2 nanocomposites, which had excellent electrochemical and catalytic activity for nitrite.  Experimental parameters can have a great influence on electrochemical sensor performance. Thus, the experimental conditions were optimized, including the applied potential, the volume of modifier, and the pH of the buffer solution. The optimized experimental conditions for applied potential, volume of modifier, and PBS pH were as follows: 0.8 V, 10 µ L, pH = 3.5 of 0.1 M PBS (as shown in Figure S1).
With successive addition of nitrite, the amperometric responses were detected by the CoOx/C@GCE in 0.1 M PBS (pH = 3.5) at 0.8 V potential, under the optimal conditions. Figure 5 shows the typical amperometric response curves for different concentrations of nitrite. The currents responded to a wide detection range of nitrite concentrations, demonstrating the efficient catalytic property of the CoOx/C-0.2 nanocomposites toward nitrite oxidation. Currents showed linear responses to nitrite concentrations from 0.2 µM to 2500 µ M ( Figure S2), which can be expressed with the regression equation lp (µ A) = 0.36388 + 0.01616C (R 2 = 0.9987). When the signal-to-noise ratio was 3, the detection limit was 0.05 µM. Further, Figure 4b shows the DPVs of CoO x /C@GCE in 1 mM nitrite and 0.1 M PBS (pH = 3.5) at 0.8 V potential with a scan rate of 0.05~0.2 V·s −1 . There is a good linear relationship between the responding current and the square root of the scan rate. The magnitude of the current was proportional to the square root of the scan rate indicating that the electrode process was a diffusion-controlled process.
Experimental parameters can have a great influence on electrochemical sensor performance. Thus, the experimental conditions were optimized, including the applied potential, the volume of modifier, and the pH of the buffer solution. The optimized experimental conditions for applied potential, volume of modifier, and PBS pH were as follows: 0.8 V, 10 µL, pH = 3.5 of 0.1 M PBS (as shown in Figure S1).
With successive addition of nitrite, the amperometric responses were detected by the CoO x /C@GCE in 0.1 M PBS (pH = 3.5) at 0.8 V potential, under the optimal conditions. Figure 5 shows the typical amperometric response curves for different concentrations of nitrite. The currents responded to a wide detection range of nitrite concentrations, demonstrating the efficient catalytic property of the CoO x /C-0.2 nanocomposites toward nitrite oxidation. Currents showed linear responses to nitrite concentrations from 0.2 µM to 2500 µM ( Figure S2), which can be expressed with the regression equation lp (µA) = 0.36388 + 0.01616C (R 2 = 0.9987). When the signal-to-noise ratio was 3, the detection limit was 0.05 µM. Figure 5 shows the typical amperometric response curves for different concentrations of nitrite. The currents responded to a wide detection range of nitrite concentrations, demonstrating the efficient catalytic property of the CoOx/C-0.2 nanocomposites toward nitrite oxidation. Currents showed linear responses to nitrite concentrations from 0.2 µM to 2500 µ M (Figure S2), which can be expressed with the regression equation lp (µ A) = 0.36388 + 0.01616C (R 2 = 0.9987). When the signal-to-noise ratio was 3, the detection limit was 0.05 µM.

Stability and Selectivity
Stability and selectivity are crucial indicators to evaluate the performance of electrochemical sensors. In the presence of 1 mM nitrite, the stability of the sensor was investigated by running 10 scans at 0.8 V in 0.1 M PBS (pH = 3.5). As shown in Figure 6a, the anodic peak current showed weak fluctuation after 10 scans, illustrating that the sensor had good stability within a certain range of detection. Additionally, we demonstrated the reproducibility of the sensor. To better reveal the reproducibility, five kinds of CoO x /C-0.2 nanocomposites were synthesized and modified glassy carbon electrodes. The current signals of the five different electrodes gave a relative standard deviation of 3.86%, which exhibited good reproducibility. Furthermore, various interferences was added into 0.1 M PBS (pH = 3.5) containing 20 µM nitrite to verify the selectivity of the nitrite sensor. As shown in Figure 6b, a significant increase of current was observed when 20 µM nitrite was introduced, but no obvious current responses appeared when 2 mM NaNO 3 , Na 2 SO 4 , KCl, MgSO 4 , NH 4 NO 3 , or AcNa were introduced into the system at regular intervals. Therefore, this revealed that the nitrite sensor had an acceptable stability and reproducibility for nitrite detection, as well as an excellent anti-interference ability towards various organic and inorganic ions such as NO 3 − , SO 4 2− , Cl − , COOH − and Na + , K + , Mg 2+ , NH 4+ .

Stability and Selectivity
Stability and selectivity are crucial indicators to evaluate the performance of electrochemical sensors. In the presence of 1 mM nitrite, the stability of the sensor was investigated by running 10 scans at 0.8 V in 0.1 M PBS (pH = 3.5). As shown in Figure 6a, the anodic peak current showed weak fluctuation after 10 scans, illustrating that the sensor had good stability within a certain range of detection. Additionally, we demonstrated the reproducibility of the sensor. To better reveal the reproducibility, five kinds of CoOx/C-0.2 nanocomposites were synthesized and modified glassy carbon electrodes. The current signals of the five different electrodes gave a relative standard deviation of 3.86%, which exhibited good reproducibility. Furthermore, various interferences was added into 0.1 M PBS (pH = 3.5) containing 20 µ M nitrite to verify the selectivity of the nitrite sensor. As shown in Figure 6b, a significant increase of current was observed when 20 µ M nitrite was introduced, but no obvious current responses appeared when 2 mM NaNO3, Na2SO4, KCl, MgSO4, NH4NO3, or AcNa were introduced into the system at regular intervals. Therefore, this revealed that the nitrite sensor had an acceptable stability and reproducibility for nitrite detection, as well as an excellent anti-interference ability towards various organic and inorganic ions such as NO3 − , SO4 2− , Cl − , COOH − and Na + , K + , Mg 2+ , NH 4+ .

Application of the Sensor in Practical Food Samples
In order to evaluate the fabricated sensor's performance in real food samples, the sensor was used to detect the nitrite concentration in sausage samples. The sausage samples were obtained from a local market, and the pretreated samples were obtained accord-

Application of the Sensor in Practical Food Samples
In order to evaluate the fabricated sensor's performance in real food samples, the sensor was used to detect the nitrite concentration in sausage samples. The sausage samples were obtained from a local market, and the pretreated samples were obtained according to the national standard [42] of operation. The accuracy of this proposed sensor was evaluated by a standard addition method. All the measurements were conducted under the optimal conditions of 0.1 M PBS (pH = 3.5) at 0.8 V using the current-time technique. The nitrite content in the sausage was measured to be 12.66 mg/kg, which is under the limited concentration set by national standard for food safety (20 mg/kg). As shown in Table 1, recoveries of the standard addition were 101.8%, 97.7%, and 99.8% for 5, 10, and 15 mg/kg added nitrite, respectively, which indicated that the proposed sensor had good performance for nitrite detection in sausage samples. The results also confirmed that the CoO x /C-0.2 nanocomposites are very suitable for the construction of electrochemical sensors for the detection of nitrite in foods.

Conclusions
In this work, we used amphiphilic PEO-b-PS, resol, and cobalt nitrate as templates to synthesize ordered mesoporous CoO x /C nanocomposites in one step. With these CoO x /C-0.2 nanocomposites, we fabricated a nitrite electrochemical sensor, which showed magnificent electrocatalytic activity for the nitrite. For nitrite, the sensor exhibited high selectivity, excellent sensitivity, and a wide detection range (from 0.2 µM to 2500 µM). The current responses to different nitrite concentrations showed a linear relationship, with the regression equation lp (µA) = 0.36388 + 0.01616C (R 2 = 0.9987). When the signal-to-noise ratio was 3, the detection limit was 0.05 µM. Moreover, the electrochemical sensor demonstrated good stability and reproducibility, which indicated that the ordered mesoporous CoO x /C nanocomposites could be applied in electrochemical sensors for the detection of nitrite in food samples. Although we managed to develop a high-performance electrochemical sensor based on CoO x /C nanocomposites, the electrochemical method is sensitive to the environment. Great attention should be paid to the influence of the nanomaterials and substrates on the signal outputs. Despite their various advantages, electrochemical methods suffer from shortcomings such as relatively low reproducibility and selectivity, short working life, and difficult reuse of the electrode and electrolyte. Therefore, there are still many remaining challenges to be solved in developing ideal electrochemical methods.