Nitrite detection is of profound significance for guaranteeing food safety and avoiding poisoning incidents. Gold nanoclusters decorated hollow ZIF-8 encapsulating iron-catecholates (Fe-HHTP@HZIF-8@ AuNCs) was formed through self-assembly of Fe3+ and 2,3,6,7,10,11 -hexahydroxytriphenylene (HHTP), in situ embedding of ZIF-8, and Au3+ -Zn2+ exchange reaction. Its morphology and structure were fully characterized by HRTEM, XRD, TEM element mapping and XPS. Additionally, its oxidase-like activity was explored with Km of 0.21 mM and Vmax of 1.74×10− 6 M·s− 1 towards 3,3',5,5'-tetramethylbenzidine (TMB). Due to its excellent catalytic activity and nitrite mediated diazotization of oxTMB, a ratiometric colorimetric method for nitrite detection was established and validated with wide linear range (2.0-400.0 µM), low LOD (0.12 µM), high accuracy (recovery of 95.11-102.14%) and fine selectivity. This method was then utilized to analyze nitrite content in sausages and tap water. This study provided a new idea for developing efficient nanozymes, and offered an accurate approach for nitrite determination.
Research Article
Gold Nanoclusters Decorated Hollow ZIF-8 Encapsulating Iron-catecholates as Oxidase Mimetics for Ratiometric Colorimetric Detection of Nitrite
https://doi.org/10.21203/rs.3.rs-5249009/v1
This work is licensed under a CC BY 4.0 License
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Nitrite
Nanozymes
Ratiometric colorimetric detection
Oxidase-like activity
Diazotization reaction
Nitrite is a common nitrogen-containing compound in nature, which is regard as a food preservative and widely used in the processing and production of various meat products. It can inhibit the growth and reproduction of microorganisms, maintain the good appearance of meat products, reduce the decay rate of food, extend the shelf life of food and so on [1]. However, excessive nitrite in food can quickly combine with hemoglobin, resulting in significant methemoglobin accumulation. In addition, prolonged consumption of excessive nitrite in human body can lead to carcinogenesis in digestive system, posing a serious threat to human health [2]. Thus, World Health Organization (WHO) has set the daily intake limit for nitrite at 0.07 mg/kg and the maximum allowable level of nitrite in drinking water at 3.0 mg/L (65.2 µM) [3]. Therefore, it is of profound significance to monitor nitrite content in food with analytical methods for guaranteeing its safety and avoiding poisoning incidents.
At present, a variety of analytical methods have been developed for the detection of nitrite in food samples, including chromatography [4], capillary electrophoresis [5], fluorescence probe [6], electrochemical sensor [7] and colorimetric methods [8]. Compared with traditional instrumental methods and biosensors with other signal modes, colorimetric method with naked eyes has attracted great attention due to its advantages of simple operation, low cost and rapid response. Common strategy is relied on Griess reaction [8–10], which refers to the reaction among sulfanilamide, N-(1-naphthyl)-ethylenediamine and nitrite under acidic condition to form red azo dye. However, the requirement of strong acidic reaction condition and the susceptibility of sulfanilamide to oxygen in air influence its accuracy and limit its application prospects [11]. Developing colorimetric method under mild reaction condition with high accuracy is desired in the research area of food safety and analytical chemistry.
Recently, accumulating evidence has suggested that the introduction of nanomaterials with tailored physico-chemical property in the construction of colorimetric biosensor can improve its analytical performance [12]. Typical example is nanozyme, which is defined as nanomaterial with enzyme-mimicking characteristic and firstly reported by Yan’s research group in 2007 [13]. Till now, several kinds of redox enzymes, including peroxidase, oxidase, catalase, superoxide dismutase, hydrolase and so on, have been mimicked by various nanomaterials [14, 15]. For nitrite detection, nanozymes with oxidase-mimetics activity have been utilized in the construction of colorimetric sensors. The principle is that oxidase-like nanozymes catalyze colorless 3,3,5,5-tetramethyl-benzidine (TMB) to form blue oxidized TMB (oxTMB), which is further oxidized by nitrite inducing diazotization reaction to obtain yellow derivative products. P-N-C nonmetal nanozymes and Mn-doped N-rich carbon dots have been reported to possess high oxidase-like activity and utilize in developing colorimetric sensing systems for nitrite sensing [16, 17]. However, owing to limited numbers of reported nanozymes, research about colorimetric biosenosrs for nitrite detection has been in the initial stage with only several reports. Designing novel nanozymes with highly efficient oxidase-mimicking activity has great significance in the construction of nitrite sensing platform.
Numerous reports have demonstrated that conjugating several kinds of reported nanozymes to form hybrid nanomaterials would improve the catalytic activity as a result of synergistic effect. In our former study [18], metalloporphyrin and gold nanoparticles (AuNPs) was utilized to modify hollow ZIF-8 (HZIF-8) to obtain nanozymes with excellent peroxidase like activity for colorimetric determination of choline. Herein, iron-catecholates@HZIF-8@AuNCs composite with highly efficient oxidase-mimetics activity was designed for ratiometric colorimetric detection of nitrite. This composite was constructed using iron-catecholates (Fe-HHTP) as a core and ZIF-8 as a shell to form a hybrid, followed by anchoring AuNCs onto the surface of iron-catecholates@HZIF-8 through ion exchange reaction and in-situ reduction. Then, its morphology, structure and catalytic property were fully studied. Based on its outstanding oxidase-mimetics activity, a ratiometric colorimetric biosensor of nitrite detection was established, validated and applied to the analysis of nitrite content in different sausages samples.
2.1 Materials and reagents
FeCl3∙6H2O and NaAuCl4∙2H2O were both bought from Macklin Biochemical Technology Co., Ltd. (Shanghai, China). 2, 3, 6, 7, 10, 11-
Hexahydroxytriphenylene (HHTP) was purchased from D&B Biological Science and Technology Co., Ltd. (Shanghai, China). 2-Methylimidazole was obtained from Aladdin Biochemical Technology Co., Ltd (Shanghai, China). Hydrogen peroxide solution (30%, w/w) was supplied from Tianjin Tianli Chemical Reagent Co., Ltd (Tianjin, China). TMB was supplied by Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China). Tris(hydroxymethyl) aminoethane [Tris] was available from Guangzhou Sopo Biological Technology Co., Ltd (Guangzhou, China). DMSO, methanol and some inorganic salts (Zn(NO3)2∙6H2O, NaNO2 and so on) were all obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Food nitrite content assay kit was bought from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). For real samples analysis, three kinds of commercial sausages were all bought from local supermarket and tap water was obtained from Wuhan Polytechnic University laboratory. All reagents and chemicals used here were analytical grade. Deionized water (DW) was employed in the entire study.
2.2 Instruments
The morphology and structure of Fe-HHTP@ZIF-8@AuNCs were comprehensively characterized using a bunch of different instruments. TEM images were observed by using high-resolution transmission electron microscope (HRTEM, JEOL 2100F). Crystal structures were recorded by X-ray powder diffraction (XRD, Rigaku SmartLab). Six elements (C, N, O, Fe, Zn and Au) were detected visually by EDS element mapping (INCA X-Max 80 TEM). Chemical composition and valence electronic states of synthetic materials were revealed by X-ray photoelectron spectroscopy (XPS) analysis using a K-Alpha spectrometer (Thermo SCIENTIFIC ESCALAB 250Xi). UV absorbance was measured using a UV-Vis spectrophotometer (EVOLUTION 220). Electron paramagnetic resonance spectra (EPR, EMXPLU-12) was used for the detection of ·O2−, ·OH and 1O2.
2.3 Synthesis of Fe-HHTP@HZIF-8@AuNCs
2.3.1 Synthesis of Fe-HHTP
Fe-HHTP was prepared according to previous literatures with slight modification [19]. Firstly, 202.73 mg FeCl3⋅6H2O (10.0 mM) was dissolved in 75.0 mL deionized water while 81.08 mg HHTP (10.0 mM) was dissolved in 25.0 mL DMSO. Then, HHTP solution was added to Fe3+ solution and the resulting mixed solution was stirred for 10 min (800 rpm). Subsequently, pH value of the above mixed solution was adjusted to 7.4 with Tris-HCl buffer (20.0 mM, pH 8.0). After stirring at room temperature for 1h, the solution was centrifugated at 8000 rpm for 10 min to obtain Fe-HHTP nanoparticles, which was then washed with methanol and water for three times, and finally dried under vacuum.
2.3.2 Synthesis of Fe-HHTP@ZIF-8
Briefly, 30.0 mg Fe-HHTP and 740.0 mg 2-methylimidazole (36.0 mM) were dispersed in 30.0 mL methanol while 445.0 mg Zn(NO3)2·6H2O (6.0 mM) was dissolved in 30.0 mL methanol. Then, the stirred Zn2+ solution was mixed with the solution of 2-methylimidazole and Fe-HHTP. The obtained solution was stirred at room temperature for another 3 h (800 rpm). After that, the products were centrifuged (8000 rpm, 5 min), washed with methanol for three times and dried at 70℃ for 5 h to get Fe-HHTP@HZIF-8 nanoparticles.
2.3.3 Synthesis of Fe-HHTP@HZIF-8@AuNCs
Through cation exchange between Au3+ and Zn2+, Au element was modified on Fe-HHTP@ZIF-8 to form Fe-HHTP@HZIF-8@AuNCs. In detail, 100.0 mg of Fe-HHTP@ZIF-8 nanoparticles were dispersed in 100.0 mL methanol and stirred for 5 min vigorously. Then, 50.0 mg of NaAuCl4⋅2H2O was added and the obtained solution was stirred at room temperature for 8 h. At last, the solution was centrifuged to obtain Fe-HHTP@ZIF-8@AuNCs, which were washed and finally dried (the same steps as that of Fe-HHTP).
2.4 Oxidase-like activity of Fe-HHTP@HZIF-8@AuNCs
By using TMB as chromogenic substrate, the oxidase-mimicking activity of Fe-HHTP@HZIF-8@AuNCs was fully studied. Firstly, the effects of pH, temperature and incubation time on the catalytic activity of this nanozyme were explored. For studying the influence of pH, 50.0 µL Fe-HHTP@HZIF-8@AuNCs (1.0 mg·mL− 1), 40.0 µL TMB (20.0 mM) and 910.0 µL acetate buffer (0.2 M) at different pH (3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0) were all mixed and incubated at 37℃ for 5 min. After the reaction, the absorbance of obtained solution at 652 nm was measured. Similarly, with the pH of acetate buffer set at 4.0 and incubation time set for 5 min, the influence of temperature (25, 30, 37, 45, 55, 60, 70 ℃) was studied. In addition, by using acetate buffer at pH 4.0 as reaction solvent, the colorimetric reaction was conducted at 37℃ for different times (1–9 min) to explore the influence of incubation time. Under optimal condition, oxidase-like activity of ZIF-8, Fe-HHTP, Fe-HHTP@ZIF-8 and Fe-HHTP@HZIF-8@AuNCs were comparatively studied.
Moreover, the steady-state kinetic parameters of Fe-HHTP@HZIF-8@AuNCs, including Michaelis-Menten constant (Km) and maximum reaction rate (Vmax), were evaluated by changing TMB concentration. Specifically, 50.0 µL Fe-HHTP@HZIF-8@AuNCs (1.0 mg·mL− 1), 40.0 µL TMB solution (0.1–0.9 mM) and 910.0 µL acetate buffer (0.2 M, pH 4.0) were all mixed and incubated at 37℃ for 5 min. After that, the absorbance at 652 nm was measured. Km and Vmax of Fe-HHTP@HZIF-8@AuNCs towards TMB were determined according to Michaelis-Menten equation and Lineweaver-Burk reciprocal equation:
Where Km and Vmax are Michaelis constant and maximum reaction rate respectively, [S] is substrate concentration and v is reaction rate.
2.5 Colorimetric detection of nitrite
Owing to excellent oxidase-mimicking activity of Fe-HHTP@HZIF-8@AuNCs, colorless TMB can be oxidized to the corresponding blue product oxTMB. With the presence of nitrite, the blue substance can be changed into blue-green or even yellow diazotized oxTMB. Thus, a ratio colorimetric biosensor for nitrite detection could be constructed based on the induced product color changes.
50.0 µL Fe-HHTP@HZIF-8@AuNCs (1.0 mg·mL− 1), 40.0 µL TMB solution (20.0 mM) and 870 µL acetate buffer (0.2 M, pH 4.0) were all mixed and incubated at 37℃ for 5 min. Then 40.0 µL nitrite solution with different concentrations (5.0 ~ 3000.0 µM) was added and the obtained mixture was incubated at 37℃ for another 10 min. After the reaction, the absorbance at 652 nm and 445 nm were both measured. The data were processed to calculate the linear relationship and limit of detection (LOD).
For method validation, six repeated analyses were performed on the nitrite standard sample (50.0 µM) to verify reproducibility. Three different concentrations of nitrite solution standard samples (10.0 µM, 50.0 µM, 300.0 µM) were spiked into tap water and different sausages pretreated samples to prepare different spiked samples. Nitrite content in these samples were measured for three times to calculate the recovery rate and then evaluate the accuracy of ratio colorimetric method. The selectivity of this biosensor was verified by measuring some possible interfering compounds present in the sausage. the concentration of all interferes were set at 400.0 µM, which was the upper limit of this ratio colorimetric method for nitrite detection.
2.6 Determination of nitrite in real samples
Real samples included commercially available sausages of different brands (1-Shuanghui square leg lunch sausage; 2-Smithfield lunch sausage; 3-TOPVALU sliced ham) and tap water ( sample 4). According to former study [20, 21], the pretreatment of commercially available sausages was conducted as follows: 5.0 g ground sausage samples were put into 250.0 mL conical bottle, then 12.5 mL saturated borax solution (50.0 g·L− 1) and 150.0 mL water (70℃) were both added and mixed thoroughly. The obtained mixture were heated in boiling water bath for 15 min and then cooled to room temperature. The extracted solution was transferred into 200.0 mL volumetric bottle and 5.0 mL potassium ferricyanide solution (106.0 g·L− 1) was added under vigorous shaking. Subsequently, 5.0 mL zinc acetate solution (220.0 g·L− 1) was added to precipitate protein and then water was added to reach the scale line of volumetric bottle. After standing for 30 min to remove the upper fat, the supernatant was filtered by filter paper (0.45 µm). Finally, the first 30.0 mL filtrate was discarded and remaining filtrate was collected for later use. Tap water was filtered with 0.45 µm filter membrane for reserve use.
In order to evaluate the practicability of the established ratio colorimetric method in food detection, the nitrite content of different brands of commercially available sausages and tap water was determined. Specific procedures were as follows: 50.0 µL Fe-HHTP@HZIF-8@AuNCs (1.0 mg·mL− 1), 40.0 µL TMB solution (20.0 mM) and 870 µL acetic acid buffer solution (pH 4.0, 0.2 M) were all mixed and then incubated at 37℃ for 5 min. Subsequently, 40.0 µL of pretreated real sample was added immediately and incubated at 37℃ for another 10 min. Finally, the absorbance of reacted solution at 652 nm and 445 nm was measured to calculate nitrite content according to standard curve. Meanwhile, the nitrite content in four different real samples were detected by commercial kits to verify the accuracy and applicability of this colorimetric method.
3.1 Characterization of Fe-HHTP@HZIF-8@AuNCs
Hybrid nanozyme Fe-HHTP@HZIF-8@AuNCs was obtained through three mild and easy steps as shown in Scheme. 1A [18, 19]. Firstly, Fe-HHTP was synthesized from one-step self-assembly between Fe3+ and HHTP at room temperature. Then, by using Fe-HHTP as the core, ZIF-8 shell was formed through the coordination between Zn2+ and 2-methylimidazole. Lastly, based on cation exchange between Au3+ and Zn2+, Au element was embellished on the surface of Fe-HHTP@HZIF-8 in the format of AuNCs, forming Fe-HHTP@[email protected] demonstrate successful synthesis of Fe-HHTP@HZIF-8@AuNCs, a battery of integral characterization experiments were executed including TEM, HRTEM, XRD, EDS and XPS. TEM images of Fe-HHTP, Fe-HHTP@ZIF-8 and Fe-HHTP@HZIF-8@AuNCs were showed in Fig. 1A-C. From the comparison of these three graphs, Fe-HHTP displayed a larger lamellar structure, while Fe-HHTP@ZIF-8 presented typical rhombic dodecahedron structure, which was almost the same as that of ZIF-8 [22]. Fe-HHTP@HZIF-8@AuNCs generally maintained the rhombic dodecahedron structure of ZIF-8. However, some obvious hollow nanoparticles arisen from Kirkendall effect could be easily observed in Fig. 1C and Fig. S1. From HRTEM image in Fig. 1D, clear streaks on the surface of AuNCs were showed with lattice spacing of 0.215 nm [23]. From particle size distribution chart in Fig. 1E, the particle size range of AuNCs on the surface of Fe-HHTP@HZIF-8 was between 1.1–2.3 nm and was mainly distributed in the range of 1.3–1.9 nm, accounting for 77.8%. Their average diameter were measured to be 1.60 nm,which was much lower than AuNPs distributed on the surface of other MOFs [24, 25]. From XRD images in Fig. 1F, the diffraction peaks of Fe-HHTP@HZIF-8@AuNCs were basically consistent with that of ZIF-8, indicating its certain crystallinity resulting from MOF structure [18]. EDS element mapping in Fig. 1G showed the uniform distribution of six elements (C, N, O, Fe, Zn and Au) on the obtained nanoparticles, indicating its successful synthesis.
Moreover, to confirm the chemical composition of Fe-HHTP@HZIF-8@AuNCs, high-resolution XPS was conducted with overall spectrum shown in Fig. 1H. Six peaks at 88.1, 284.8, 399.5, 531.4, 723.9 and 1021.9 eV were ascribed to the characteristic peaks of Au 4f, C 1s, N 1s, O 1s, Fe 2p and Zn 2p, respectively. In detail, XPS spectra of Au element in Fig. 1I showed four distinct peaks. Two peaks at 87.7 and 91.3 eV corresponded to Au (III) ions, while the other two peaks at 84.8 and 88.6 eV indicated the presence of Au (0) nanoclusters, which might be produced from a partial reduction reaction between Au3+ and N atoms in ZIF-8 [26–28]. From high-resolution Fe 2p spectrum in Fig. 1J, four peaks at 710.8, 712.4, 723.9 and 727.1 eV corresponded to FeII 2p3/2, FeIII 2p3/2, FeII 2p1/2 and FeIII 2p1/2, respectively. In addition, two small satellite peaks was observed at 718.1 and 731.3 eV. These peaks illustrated the coexistence of Fe2+ and Fe3+ in Fe-HHTP@HZIF-8@AuNCs [20, 27, 29, 30]. Additionally, high-resolution XPS spectra of Zn 2p, C 1s, N 1s, O 1s was presented and analyzed in Fig. S2.
3.2 Oxidase-like activity of Fe-HHTP@HZIF-8@AuNCs
TMB was selected as a chromogenic substrate in the study about oxidase-like activity of Fe-HHTP@HZIF-8@AuNCs. As shown in Fig. 2A, TMB was oxidized to generate dark blue oxTMB product by Fe-HHTP@HZIF-8@AuNCs under air-saturated condition, which accompanied by an enhancement of the ultraviolet absorption peak at 652 nm. On the contrary, TMB oxidation reaction was dramatically inhibited in N2-saturated solution [20]. This phenomenon indicated that O2 in solution played a considerable role in the catalytic reaction, manifesting great oxidase-like activity of Fe-HHTP@HZIF-8@AuNCs. Additionally, the catalytic activities of different nanoparticles, including Fe-HHTP, ZIF-8, Fe-HHTP@ZIF-8 and Fe-HHTP@HZIF-8@AuNCs, were comparatively studied in Fig. 2B. Fe-HHTP@HZIF-8@AuNCs showed the highest oxidase-like catalytic activity, indicating important role of AuNCs in the improvement of oxidase-like activity of hybrid material. Also, the formed hollow structure could accelerate substrate molecules transfer and improve collision efficiency between Fe-HHTP core and TMB.
To maximize the oxidase-like catalytic properties of Fe-HHTP@HZIF-8@AuNCs, influence of pH, temperature and incubating time was fully investigated. As shown in Fig. 2C, Fe-HHTP@HZIF-8@AuNCs exhibited the best catalytic activity at pH 4.0 while its catalytic activity dropped sharply under strong acid or near neutral conditions. This tendency could be explained by the effect of pH value on the surface charge of materials and dissociation state [31–33]. As depicted in Fig. 2D, the catalytic activity of Fe-HHTP@HZIF-8@AuNCs gradually increased at 25 ~ 37℃, reached the highest peak at 37℃, and gradually decreased at 37 ~ 70℃. The influence of temperature on its oxidase like activity were similar to that of some other nanozymes [21, 34, 35]. Then, from the influence of incubation time presented in Fig. 2E, UV signal of oxTMB product showed an obvious upward trend during 1–5 min, and basically remained stable after 5 min. Therefore, optimal conditions for color reaction mediated by Fe-HHTP@HZIF-8@AuNCs were pH 4.0, 37℃ and 5 min’s incubation. To confirm the oxidase like activity of Fe-HHTP@HZIF-8@AuNCs from the view of radical, EPR spectroscopy was utilized to quantify the types of transient free intermediates. As shown in Fig. 2F, by using DMPO as a trapping agent of ·O2− and ·OH while TEMPO as a trapping agent of 1O2, the presence of ·O2−, ·OH and 1O2 was demonstrated in Fe-HHTP@HZIF-8@AuNCs mediated catalytic system. The content of ·O2 − was obviously higher than that of ·OH and 1O2. The production of reactive oxygen species (ROS) could promote the oxidation of TMB to the dark blue TMB complex [21]. Moreover, three types of free radicals enhanced gradually from 0 to 20 min ( Fig. S3).
The catalytic kinetic study of Fe-HHTP@HZIF-8@AuNCs was conducted by varying TMB concentrations in the range of 0.1–0.9 mM. According to reaction kinetic curve in Fig. 3A-B and Michaelis-Menten equation, Km and Vmax of Fe-HHTP@HZIF-8@AuNCs towards TMB were 0.21 mM and 1.74×10− 6 M·s− 1, respectively. This Km value was much lower than that of other nanozymes (Table S1), indicating that Fe-HHTP@HZIF-8@AuNCs has much higher affinity towards TMB and resulted higher catalytic activity. The resulted superior catalytic performance of Fe-HHTP@HZIF-8@AuNCs mighty be attributed to well dispersed AuNCs on the surface and the formed hollow ZIF-8 structure through Kirkendall effect.
3.3 Colorimetric method for nitrite detection
Based on the excellent oxidase-like activity of Fe-HHTP@HZIF-8@AuNCs, a ratiometric colorimetric method was established for the detection of nitrite in food. As shown in Fig. 4A and Scheme 1B-C, Fe-HHTP@HZIF-8@AuNCs catalyzed colorless TMB to produce corresponding blue oxide oxTMB, which subsequently converted to yellowish-brown diazotized oxTMB in the presence of nitrite. Thereby, on the basis of the color changes of reacted solution, highly sensitive detection of nitrite could be realized by measuring of UV signal changes at the wavelength of 652 nm and 445 nm. As illustrated in Fig. 4B, with the increase of nitrite concentration in the range of 0 ~ 700.0 µM, the color of reacted solution mediated by Fe-HHTP@HZIF-8@AuNCs gradually changed from blue to yellowish-brown, accompanied by decreased UV absorbance at 652 nm and increased UV absorbance at 445 nm.
Then, to establish ratiometric colorimetric method for nitrite, the linear relationship between UV absorbance ratio (A652/A445) and nitrite concentration was explored in the linear range of 2.0 ~ 400.0 µM. As presented in Fig. 4C, the calibration curve equation was A652/A445 = 7.049–1.951 log([NO2−]) (R2 = 0.993), where logarithm nitrite concentration (log([NO2−])) was the independent variable while ratio absorbance (A652/A445, A652 and A445 were the UV absorbance of the standard sample at 652 nm and 445 nm, respectively) was the dependent variable. The limit of detection (LOD) was calculated to be 0.12 µM based on S/N = 3. Compared with other reported nitrite sensing detection methods in Table S2, this ratiometric colorimetric method has excellent advantages of low LOD and wide linear range.
Additionally, the reproducibility, accuracy and selectivity of this nitrite visual sensor was validated to demonstrate its feasibility. The relative standard deviation (RSD) of six repeated detection signal of nitrite standard sample (50.0 µM) was 1.8%, indicating good reproducibility. To verify the accuracy of constructed ratiometric colorimetric sensing system, different concentrations of nitrite (10.0, 50.0, 300.0 µM) were added to different real samples of sausage, and then quantitative detection was carried out. As shown in Table 1, the recovery rate of nitrite in all actual sample ranged from 95.11 to 102.14%, indicating qualified accuracy of this colorimetric biosensor. Considering complex composition of food, it was necessary to study the influence of potential interfere on nitrite detection and evaluate the selectivity of this method. Some potential interferes coexisting with nitrite in commercially available sausages, such as Na+, K+, Ca2+, Ba2+, Mg2+, Mn2+, Fe3+, SO42−, CO32−, PO42−, NO3− and glucose (Glu) were analyzed by this colorimetric method. The concentrations of interfering substances were set at 400.0 µM, which was the upper limit of linear range of nitrite detection. Figure 4D showed that only nitrite presented the lowest ratio absorbance while all other interfering substances presented much higher ratio absorbance under the same conditions, showing nice selectivity of this ratiometric colorimetric biosensor for nitrite detection.
| Samples | Spiked concentration (µM) | Detected by this method (µM) | Recovery (%) | RSD (%, n = 3) | Detected by commercial kit (µM) | Recovery (%) | RSD (%, n = 3) |
|---|---|---|---|---|---|---|---|
| 1 | 0 | 4.14 | NA | 1.25 | 4.35 | NA | 1.83 |
| 10 | 13.78 | 97.44 | 3.09 | 14.75 | 102.79 | 2.36 | |
| 50 | 53.85 | 99.45 | 1.35 | 53.88 | 99.14 | 1.05 | |
| 300 | 301.69 | 99.19 | 2.44 | 306.91 | 100.84 | 1.57 | |
| 2 | 0 | 2.73 | NA | 0.76 | 2.53 | NA | 0.89 |
| 10 | 12.92 | 101.55 | 1.32 | 12.46 | 98.64 | 0.76 | |
| 50 | 51.46 | 97.60 | 1.83 | 53.15 | 101.18 | 2.13 | |
| 300 | 298.14 | 98.49 | 2.89 | 311.74 | 103.04 | 1.15 | |
| 3 | 0 | 3.70 | NA | 1.46 | 3.87 | NA | 2.39 |
| 10 | 13.39 | 97.74 | 1.22 | 13.53 | 97.55 | 1.28 | |
| 50 | 54.85 | 102.14 | 3.41 | 54.56 | 101.28 | 2.64 | |
| 300 | 292.69 | 96.38 | 3.17 | 312.92 | 102.98 | 3.55 | |
| 4 | 0 | ND | NA | 1.34 | ND | NA | 0.96 |
| 10 | 9.60 | 96.01 | 1.53 | 10.16 | 101.60 | 1.67 | |
| 50 | 49.45 | 98.89 | 2.05 | 48.76 | 97.52 | 2.02 | |
| 300 | 285.34 | 95.11 | 3.24 | 295.51 | 98.50 | 2.58 | |
| NA = not applicable; ND = not detected. | |||||||
3.4 Detection of nitrite in real samples
By using this ratiometric colorimetric method, nitrite content in three types of sausages and tap water were analyzed. From the results in Table 1, nitrite concentrations in sausage 1–3 were 4.14, 2.73 and 3.70 µM, respectively. Thus, the nitrite content of sausage 1 was the highest, followed by sausage 3, and the nitrite content of sausage 2 was relatively low. These detected concentrations were all far below the limited concentration of nitrite in sausages (30.0 mg/kg, 652.1 µM) by China food and drug administration (GB 2760 − 2014). In addition, nitrite in tap water could not be detected because its concentration was lower than LOD of this method. Moreover, nitrite content in three types of sausages and tap water (Fig. S4) were also detected by commercial kits (GB 5009.33–2016 from CFDA). The obtained detection results were highly consistent with the detection result of nitrite from ratiometric colorimetric method based on Fe-HHTP@HZIF-8@AuNCs. That is to say, this method has good application value for detecting nitrite in food and water environment.
In this study, by using Fe-HHTP as a core and ZIF-8 as a shell, a hybrid Fe-HHTP@ZIF-8 was synthesized. Followed by anchoring AuNCs onto its surface through ion exchange reaction, Fe-HHTP@HZIF-8@AuNCs composite was constructed. Then, its morphology, chemical structure and oxidase-like activity were comprehensively characterized. Based on excellent catalytic activity of Fe-HHTP@HZIF-8@AuNCs and the diazotization between oxTMB and nitrite, a ratiometric colorimetric method for nitrite detection was established and validated with good reproducibility, qualified accuracy and nice selectivity. Additionally, this ratiometric colorimetric method was then applied to the quantitative analysis of nitrite content in sausages and tap water. This study not only provided a simple and reliable method for nitrite determination, but also paved a new way for searching for highly efficient nanozyme materials.
Ethical Approval
Not applicable.
Competing interests
The authors declare no competing interests.
Funding
This work was supported by the National Natural Science Foundation of China (32302220), the Department of Education of Hubei province (grant number T2022023), Research Project Funding from Wuhan Polytechnic University (grant number 2024J01) and Special Project of Central Guide to Local Science and Technology Development (Innovation platform construction for food green processing technology and intelligent equipment), Department of Science and Technology of Hubei Province, Department of Finance of Hubei Province (grant number 2022BGE247).
Author Contribution
Wu: Validation, Data processing, Writing - Original Draft.Dai: Resources.Qin: Supervision.Liu: Project administration.Zhang: Conceptualization, Methodology, Funding acquisition, Writing- Reviewing and Editing.
Availability of data and materials.
The data and materials that support the findings of this study are available on reasonable request.
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Scheme 1 is available in the Supplementary Files section.
No competing interests reported.
- Onlinefloatimage2.png
Scheme 1 (Graphical abstract)
- Supportinginformation.docx
