Nitrite (NO₂⁻), a food additive, is widely found in drinking water and food [1]. However, N-nitrosamine molecules, which are extremely carcinogenic, can be produced after amines, amides or proteins interact with nitrite. Human health is seriously threatened by long-term excessive nitrite consumption [2,3]. Thus, it is desired to develop a simple and fast method for nitrite detection in food. Currently, different methods including electrochemistry [4], fluorometry [5–7], gas chromatography [8] have been developed to detect NO₂⁻. However, these conventional methods usually face some shortcomings, including requirements of bulky and expensive equipment or expert skills, complex operation and time-consuming procedures. Compared with the abovementioned methods, colorimetric assay based on the measurement of colored compounds has attracted increasing attention by taking advantage of its low cost, easy operation, rapid visual detection and potential for on-site application. However, constructing a catalyst with excellent efficiency is a key step to effectively improve colorimetric changes and achieve excellent detection performance in colorimetric assay.

Nanozyme is a kind of nanomaterial with intrinsic enzymatic activities, possessing splendid advantages such as good stability, high catalytic activity, low cost, desired reusability and fine adaptability to environment [9,10]. With these features, nanozyme has been extensively used in the aspect of biochemical sensing, biomedicine, food industry and environmental science [11–13]. Notably, nanozymes have been widely utilized to catalyze the chromogenic substrates in various colorimetric sensing applications, and provide catalytic amplification signals for highly sensitive detection [14–16]. Currently, nanoceria (CeO₂) has been confirmed to have a wide variety of enzymatic activities, such as oxidase (OXD), peroxidase (POD), phosphatase, nuclease, and urease owing to the abundance of oxygen vacancies and the conversion between the Ce(Ⅲ)/Ce(Ⅳ) valence states [17–21]. Thus, CeO₂ as a fascinating sensor material has been extensively used to detection various analytes [17,22]. Generally, the catalytic performance of CeO₂ is the core of the colorimetric probe. Thus, the rational design of CeO₂ with high catalytic performance is significant to meet the demand of detection sensitivity in practical application. Recently, the strategy of doping metal ions has played an irreplaceable role in regulating the catalytic activity of nanoceria [23,24]. Numerous studies have shown that doping pure CeO₂ with other metal ions can realize the enhancement of multi-enzyme-like activities through increasing the concentration and migration of oxygen vacancies [25–28]. Mn has obtained great attention due to its changeable oxidation state and better redox behavior. The doping of Mn²⁺ can cause the reconstruction of CeO₂ structure, accelerate the production of oxygen vacancies and the conversion of Ce⁴⁺ to Ce³⁺ [26]. Furthermore, lanthanide ions with ionic radius and electronegativity close to those of cerium are thought to be the most appropriate modifiers of ceria, which facilitates its easy incorporation into the crystal lattice of CeO₂. Usually, when Ce⁴⁺ ions are substituted with lanthanide ions, introducing stress into ceria lattice to execute charge neutrality and nonstoichiometry compensation, enhances oxygen storage capacity from lattice to its surface and decreases the activation energy for the formation of oxygen vacancy [29]. A novel Er and Yb codoped CeO_2−x showed higher enzyme-like catalytic activity than that of CeO_2−x, because the doping of Yb and Er ions can generate extra oxygen vacancies [30]. Co-doping is an effective method for increasing the catalytic performance of CeO₂. Metal ions such as Mn²⁺, Yb³⁺ and Er³⁺ can be chosen as possible co-dopants to improve the structural, surface, and redox properties of CeO₂. A synergistic effect can be expected, such as the redox cycles Ce³⁺/Ce⁴⁺ and Mn⁴⁺/Mn³⁺/Mn²⁺ ions, the increased conversion process of Ce⁴⁺ to Ce³⁺, and the enhanced number of defective sites (e.g., oxygen vacancies), which is required for the catalytic activity. Additionally, previous metal doping CeO₂ nanozymes are usually prepared by rigorous methods and complex synthetic processes, such as high temperature and high pressure, which restricts their practical applications. Therefore, constructing a high-performance codoped CeO₂ nanozyme via a facile and efficient strategy is highly desirable to extent their practical applications.

Herein, via a simple and feasible strategy, Mn/Yb/Er triple-doped CeO₂ nanozyme (Mn/Yb/Er/CeO₂) was synthesized for highly sensitive ratiometric detection of nitrite. By doping Mn, Yb, Er into CeO₂ lattice structure, Mn/Yb/Er/CeO₂ showed enhanced oxidase-like activity, obtaining a higher density of oxygen vacancy and a higher ratio of Ce³⁺ to Ce⁴⁺ than that of CeO₂. The 3,3′,5,5′-tetramethylbenzidine (TMB) can be effectively oxidized by Mn/Yb/Er/CeO₂ to produce the oxidized TMB (oxTMB), showing a significant absorption signal at 652 nm. Additionally, nitrite can react with oxTMB to produce yellow diazotized oxTMB, which is accompanied by an elevated absorption signal at 445 nm and a decreased absorption signal at 652 nm. Thus, based on the oxidase-mimetic activity of Mn/Yb/Er/CeO₂ and the diazotization reaction between NO₂⁻ and oxTMB, a ratiometric colorimetric assay was established for NO₂⁻ detection in food (Scheme 1). Furthermore, by integrating Mn/Yb/Er/CeO₂ with a smartphone, a colorimetric platform was successfully fabricated for visualization and quantitative detection of NO₂⁻. Notably, this two-detection mode (colorimetric spectrophotometer mode and smartphone-sensing platform) showed excellent sensitivity, selectivity, reliability and practicability in monitoring the NO₂⁻ in real samples, implying its great potential for food safety.

To improve the oxidase mimicking activity of CeO₂, Mn/Yb/Er were utilized to doping into the lattice structure of CeO₂. First, via low-temperature hydrothermal method, CeO₂ and doped CeO₂ nanozymes (Mn/CeO₂ and Mn/Yb/Er/CeO₂) were prepared. Then, the as-prepared nanozymes were modified with citrate to improve their water-solubility and stability. As illustrated in Fig. S1 (Supporting information), zeta-potential of the citrate modified Mn/Yb/Er/CeO₂ was −40 mV, and significantly negative-shifted compared with that of the bared Mn/Yb/Er/CeO₂ (15 mV). Similarly, other nanozymes with the modifying of citrate also showed electronegative.

Next, the crystal structures of CeO₂, Mn/CeO₂, and Mn/Yb/Er/CeO₂ were identified by X-ray diffraction (XRD). The characteristic peaks at 28.8°, 33.3°, 47.7°, 56.5°, 69.6°, and 76.7° shown in Fig. S2 (Supporting information) can be ascribed respectively to the diffraction planes of (111), (200), (220), (311), (400), and (331) of cubic fluorite structured of CeO₂ (PDF# 43–1002), indicative of the CeO₂ structure of the prepared nanozymes. Notably, owing to the incorporation of smaller atomic radius ions (Mn, Yb, and Er) (Table S1 in Supporting information) into CeO₂, a shift and broadening of the diffraction peak ascribed to (111) were observed in the sample of Mn/CeO₂ and Mn/Yb/Er/CeO₂. This phenomenon was consistent with the results reported in the literatures [24,31]. Furthermore, the transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) images of CeO₂, Mn/CeO₂, and Mn/Yb/Er/CeO₂ were conducted. Fig. 1a revealed that the lattice spacing of the (111) plane of CeO₂ (0.328 nm) was larger than that of doped CeO₂ (Mn/CeO₂: 0.320 nm, and Mn/Yb/Er/CeO₂: 0.310 nm), which was consistent with XRD data. This may be explained by the incorporation of smaller atomic radius ions (Mn, Yb, and Er) into the lattice structure of CeO₂. Additionally, elemental mapping analysis of Mn/Yb/Er/CeO₂ indicated that Ce, Mn, Yb, Er, and O were evenly distributed in the sample (Fig. 1b).

To distinguish crystallization or defects of samples and provide more information on the surface of samples, Raman spectra were obtained and illustrated in Fig. S3 (Supporting information). A sharp Raman peak around 460 cm⁻¹ related to the F_2g characteristic vibration peak of CeO₂ were observed in all three samples, which was attributed to the symmetrical stretching vibration of oxygen atoms around cerium ion (Ce-8O) in ceria lattice. Additionally, F_2g peaks of doped CeO₂ was shifted and widened compared with that of CeO₂, indicating that O atoms around Ce ion have been altered. In general, an obvious Raman band around 600–615 cm⁻¹ usually indicated the formation of oxygen vacancies. The oxygen vacancy Raman peak of CeO₂ was found at 603 cm⁻¹, while that of Mn/Yb/Er/CeO₂ and Mn/CeO₂ was 615 cm⁻¹. The peak area ratio of the oxygen vacancy Raman peak (615 cm⁻¹ or 603 cm⁻¹) to the F_2g characteristic vibration peak (460 cm⁻¹), defined as A₆₁₅/A₄₆₀ or A₆₀₃/A₆₁₅, is used to represent the oxygen vacancy relative concentration. It can be found from Fig. S3 that the ratio gradually increases with metal ion doping (Mn/Yb/Er/CeO₂ > Mn/CeO₂ > CeO₂). This result suggested that Mn/Yb/Er/CeO₂ has the highest concentration of oxygen vacancies.

To further elucidate the chemical composition and valence state of material in detail, the X-ray photoelectron spectroscopy (XPS) tests were conducted. The full XPS of Mn/Yb/Er/CeO₂ showed that Ce, Mn, Yb, Er, and O were found present in the sample (Fig. S4 in Supporting information). Fig. S5a (Supporting information) depicts the Ce XPS spectra of three samples. The binding energies at 900.7, 906.7, 916.2, 883.0, 887.6, and 897.8 eV can be ascribed to surface Ce⁴⁺, while binding energies at 903.3, 899.5, 881.5 and 885.2 eV are assigned to Ce³⁺. Compared to CeO₂ and Mn/CeO₂, Mn/Yb/Er/CeO₂ has the highest Ce³⁺/Ce⁴⁺ concentration ratio on the surface, indicating that the introduction of Mn/Yb/Er can increase the amount of Ce³⁺ species and improve the catalytic activities of Mn/Yb/Er/CeO₂. Oxygen vacancy is a typical anionic defect that plays a very important role in surface catalytic reactions [32]. Then, the peak deconvolutions of the O 1s XPS spectra of three samples were investigated (Fig. S5b in Supporting information). The surface oxygen species (529.9–531.5 eV; O_α) and lattice oxygen (528.5–529.5 eV; O_β) are all present in the O 1s XPS spectrum. The surface oxygen vacancy change can be represented by using the percentage of O_α [31]. Mn/Yb/Er/CeO₂ possesses the higher ratio of O_α/O_β (1.74) than that of CeO₂ (1.36) and Mn/CeO₂ (1.04), indicating that the surface of Mn/Yb/Er/CeO₂ has higher density of oxygen vacancies. The O 1s XPS spectra results were consistent with the Raman data.

As a chromogenic substrate, TMB was employed to conduct a typical chromogenic reaction. Then, the oxidase-like activity of Mn/Yb/Er/CeO₂ was evaluated. Fig. 2a showed that neither UV–vis absorbance nor apparent color changes were observed in the solution with only Mn/Yb/Er/CeO₂ or TMB. When Mn/Yb/Er/CeO₂ was added into TMB solution with dissolved O₂, apparent color changes from colorless to blue were observed, and a maximum absorbance at 652 nm appeared, owing to the production of oxTMB. This result implied that Mn/Yb/Er/CeO₂ is important in the redox reaction between TMB and O₂. Notably, the production of oxTMB was partially inhibited in N₂-saturated buffer, accompanied by an obvious reduce of the absorbance intensity at 652 nm. The result confirmed that the TMB oxidation reaction is dependent on dissolved O₂, and Mn/Yb/Er/CeO₂ acts as an oxidase-like nanozyme in the catalytic oxidation of TMB. In general, reactive oxygen species (ROS) produced by dissolved O₂ plays an important role in the oxidative reaction. Thus, the influence of ROS on this reaction was investigated to confirm the catalytic mechanism of Mn/Yb/Er/CeO₂. ROS like O₂^•−, ^•OH, ¹O₂ can be captured by p-benzoquinone (PBQ), isopropanol alcohol (IPA), and tryptophan (Trp), respectively. As depicted in Fig. 2b, the presence of PBQ reduced the production of oxTMB, reducing its absorbance intensity at 652 nm, while IPA and Trp had less effect on this process. This phenomenon suggested that O₂^•− radicals were generated during oxidative reaction. Based on above mentioned analysis, it is reasonable to infer that Mn/Yb/Er/CeO₂ may first catalyzes O₂ to generate O₂^•− radicals, thus oxidizing TMB to oxTMB.

Generally, reaction duration, nanozyme concentration, buffer pH, and temperature have a significant impact on the catalytic activity of nanozymes. To maximally present the oxidase-like activity of Mn/Yb/Er/CeO₂ in TMB solution with dissolved O₂, reaction conditions as above mentioned were optimized. As depicted in Fig. S6a (Supporting information), a plateau and steady state of the absorbance intensity at 652 nm was reached after 10 min. Thus, 10 min was utilized as the TMB oxidation time in the following study. Fig. S6b (Supporting information) demonstrated the absorbance intensity of oxTMB increases with the increasing amounts of Mn/Yb/Er/CeO₂. To ensure the assay results more accurate, 25 µg/mL was chosen as the amount of Mn/Yb/Er/CeO₂ in the following study. As shown in Fig. S6c (Supporting information), the catalytic activity of Mn/Yb/Er/CeO₂ changed with altered buffer pH, with maximum activity at pH 3.0. When the temperature ranged from 25 ℃ to 65 ℃, Mn/Yb/Er/CeO₂ consistently showed excellent catalytic activity (Fig. S6d in Supporting information). Given the convenience of operation, 25 ℃ was selected.

To further evaluate the oxidase-mimicking ability of Mn/Yb/Er/CeO₂, we conducted the steady-state kinetic analysis under optimal conditions. Correlation of initial reaction velocity with different concentrations of TMB was shown in Fig. 2c. According to the Lineweaver-Burk double reciprocal graph, the maximum velocity (V_max) and the apparent Michaelis-Menten constant (K_m) were calculated to 0.2068 µmol L⁻¹ s⁻¹ and 45.54 µmol/L (Fig. 2d). Meanwhile, CeO₂ and Mn/CeO₂ was also investigated under optimal conditions (Fig. S7 in Supporting information). As compared in Fig. S8 (Supporting information), the apparent K_m value obtained by Mn/Yb/Er/CeO₂ was lower than that of CeO₂ and Mn/CeO₂, indicating that Mn/Yb/Er/CeO₂ has a stronger affinity toward TMB. Moreover, Fig. S9 (Supporting information) revealed that Mn/Yb/Er/CeO₂ exhibits a higher activity in catalyzing the TMB oxidation reaction than that of CeO₂ and Mn/CeO₂. This may be attributed to Mn/Yb/Er/CeO₂ has higher density of oxygen vacancies and Ce³⁺/Ce⁴⁺ content ratio. In addition, Mn/Yb/Er/CeO₂ showed excellent oxidase-like catalytic stability even after a prolonged storage, which facilitates its application in the field of analysis (Fig. S10 in Supporting information).

Interestingly, the UV-vis absorption spectra of the Mn/Yb/Er/CeO₂ + TMB solution was significantly altered when the nitrite was added. As exhibited in Fig. S11a (Supporting information), a new UV-vis absorption peak appeared at 445 nm when nitrite was present, while the peak at 652 nm was reduced. Additionally, the solution’s color was changed from blue to green (inset in Fig. S11a). This phenomenon may be ascribed to the occurrence of the diazidation reaction between the nitrite and oxTMB intermediate under the acidic condition. As illustrated in Fig. S11b (Supporting information), a diazonium salt (diazotizated oxTMB) can be formed by the reaction between nitrite with the aromatic primary amine group of the oxTMB under the acidic condition (pH 3.0). Next, an ascorbic acid (AA) reduction test was conducted to verify this conclusion. As demonstrated in Fig. S12 (Supporting information), AA can reduce oxTMB, overoxidized oxTMB, and the mixture of oxTMB and overoxidized oxTMB to colorless TMB [33]. However, when NO₂⁻ was present in Mn/Yb/Er/CeO₂ + TMB solution, the diazotized oxTMB was produced by the diazotization reaction. Notably, AA cannot reduce the produced diazotized oxTMB to colorless TMB. This phenomenon demonstrated the occurrence of diazozation reaction.

Based on the above mechanisms, Mn/Yb/Er/CeO₂ + TMB solution system was developed for nitrite detection. Diazozation reaction time was optimized firstly to better detect nitrite. Fig. S13 (Supporting information) displayed that the ratio of the absorbance intensity of reaction solution at 445 nm to that at 652 nm (A₄₄₅/A₆₅₂) obtained a plateau and steady state after 30 min. Thus, 30 min was utilized as diazozation reaction time in the following study. The detection sensitivity of Mn/Yb/Er/CeO₂ + TMB solution system to nitrite was investigated. With gradually increased concentration of nitrite, the absorbance intensity increased significantly at 445 nm. Simultaneously, the absorbance intensity decreased at 652 nm during the experiments (Fig. 3a). Accordingly, the color of solution was also changed obviously as shown in the inset in Fig. 3a, which demonstrated the detection feasibility of nitrite. In Fig. 3b, the absorption ratio A₄₄₅/A₆₅₂ increased gradually with the increase of NO₂⁻ concentration. A₄₄₅/A₆₅₂ displayed a good linearity for nitrite at 2.0–100 µmol/L (R² = 0.9869), and the limit of detection (LOD) was 0.29 µmol/L. Additionally, TMB can directly react with nitrite under acidic solution to produce diazonium salts with an obvious absorption signal at 445 nm [34]. Thus, single-signal colorimetric detection mode based on TMB + NO₂⁻ solution system was investigated. A₄₄₅ displayed a good linearity for nitrite at 35–150 µmol/L (R² = 0.9963), and the LOD was 1.3 µmol/L (Fig. S14 in Supporting information). In contrast, the sensitivity and LOD of the ratiometric colorimetric detection mode is superior to that of the single-signal colorimetric detection mode. Next, the selectivity and anti-interference ability of this ratiometric colorimetric assay were investigated by detecting nitrite (100 µmol/L) with or without other interferences species (1 mmol/L). Interferences such as K⁺, Na⁺, Ca²⁺, Mn²⁺, Mg²⁺, Ag⁺, Cl⁻, Ac⁻, NO₃⁻, CO₃²⁻, PO₄³⁻, urea, and glucose interfere very little on the nitrite analysis, illustrating this method has excellent selectivity and anti-interference ability for nitrite detection (Fig. 3c). These excellent performances of this method may result from the cascade of oxidase-like catalysis and diazidation.

Recently, smartphone-based portable sensing platforms have been widely developed due to their advantages of accessibility and portability [35,36]. Additionally, the accuracy and resolution of smartphone-sensing platforms exceed visual detection. Thus, by integrating Mn/Yb/Er/CeO₂ with a smartphone, a colorimetric smartphone-sensing platform was fabricated for visualization and quantitative detection of NO₂⁻. Smartphone was employed as the signal readout to enable portable nitrite detection. The photos of samples with nitrite concentration from 0 to 200 µmol/L were taken by a smartphone, showing the color transition from blue to yellow (Fig. 4a). Then, these photos were analyzed by the color recognition APP, and further converted into RGB (red, green and blue) values to achieve portable nitrite detection (Fig. 4b). The ratio of R and G (R/G) showed a good linearity for nitrite at 5–200 µmol/L (R/G = 0.004190 [NO₂⁻] + 0.5513, R² = 0.9948) with a LOD of 3.3 µmol/L (Fig. 4c). Compared with the ratiometric colorimetric assay on the absorption spectrometer, the smartphone-sensing platform has the merits of low cost, flexibility, portability, and wider detection ranges. Furthermore, the performance of our assays (colorimetric spectrophotometer mode and smartphone-sensing platform) are comparable or better than those of reported methods for nitrite detection (Table S2 in Supporting information). The above results revealed that this smartphone-sensing platform has the potential application for the on-site of nitrite detection.

To evaluate the reliability, feasibility and practicality of our method, real samples including Tap water, Mei Lake water, pickle water and sausage were selected to detect nitrite. Samples were pretreated according to previous literature reports [3]. As shown in Table S3 (Supporting information), the spiked recoveries ranged from 95.3% to 101.0% for the colorimetric spectrophotometer mode, and 94.3% to 108.1% for the smartphone-sensing platform, implying the wonderful feasibility and practicality of the two-detection mode for nitrite detection in real samples. Furthermore, to check the accuracy of our method, all samples were simultaneously determined by a commercial detection kit. The results showed that the nitrite concentration detected by colorimetric spectrophotometer mode and smartphone-sensing platform were almost consistent with that measured by the commercial detection kit, indicating that our two-detection mode has the reliable and potential applications in food safety.

In this work, Mn/Yb/Er/CeO₂ with enhanced oxidase-like activity was successfully prepared by a low-temperature hydrothermal method. The oxygen vacancies and Ce³⁺ concentration of Mn/Yb/Er/CeO₂ were efficiently improved by doping Mn/Yb/Er. Take advantage of the excellent oxidase-mimetic activity of Mn/Yb/Er/CeO₂ and the diazotization reaction between NO₂⁻ and oxTMB, a ratiometric colorimetric assay was established for NO₂⁻ detection in food. Beside the fabrication of colorimetric spectrophotometer mode, a smartphone-sensing platform based on this detection system and smartphone was successfully fabricated for visualization and quantitative detection of NO₂⁻. Notably, this two-detection mode showed excellent reliability and practicability in monitoring the NO₂⁻ in real samples. This work not only provides a method for preparing nanozymes with enhanced oxidase mimic activity, but also implies its great potential for food safety.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The work was supported by the National Natural Science Foundation of China (Nos. 22004111 and 21974125), the Tackle Key Problems in Science and Technology Project of Henan Province, China (No. 222102310386), China Postdoctoral Science Foundation (No. 2020M682327), Henan Postdoctoral Foundation (No. 202002009), Program for Innovative Research Team (in Science and Technology) in University of Henan Province (No. 22TRTSTHN002), and Excellent Youth Foundation of Henan Scientific Committee (No. 232300421021).

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2023.109114.