Colorimetric / electrochemical dual-mode sensor for detecting nitrite as well as preparation and application of colorimetric / electrochemical dual-mode sensor

Abstract

The invention discloses a colorimetric / electrochemical dual-mode sensor for detecting nitrite as well as preparation and application of the colorimetric / electrochemical dual-mode sensor. Vanadium is doped in a ferroferric oxide nano-enzyme structure through a post-doping method, so that the vanadium-doped ferroferric oxide nano-enzyme with high-performance peroxidase activity is obtained. Methylene blue is used as a bifunctional signal molecule, and a colorimetric-electrochemical dual-mode sensing platform is constructed for high-sensitivity detection of nitrite. The synthesized nano enzyme has high catalytic activity and strong affinity with a substrate, and the constructed ratio type colorimetric-electrochemical dual-mode sensor has the characteristics of high sensitivity, wide detection linear range and low detection limit, and has great application potential in the field of nitrite content analysis.

Description

Technical Field

[0001] The present invention relates to the technical field of nanozyme synthesis and detection, and in particular to a colorimetric / electrochemical dual-mode sensor for detecting nitrite, and the preparation and application thereof. Background Art

[0002] Nanozyme catalysts have similar chemical properties to biological enzymes and offer advantages over biological enzymes, such as higher activity, greater stability, and easier structural control. Since Fe3O4 nanoparticles were reported to possess excellent catalytic activity in 2007, Fe3O4 nanozymes have been widely used in biomedicine, environmental monitoring, and food safety monitoring (Adv. Colloid Interface, 2020, 281, 102165). However, Fe3O4 nanozymes have problems such as low catalytic activity and weak affinity. In recent years, researchers have used a one-step hydrothermal / solvothermal method to dope transition metals such as manganese (Appl. Surf. Sci., 2022, 590, 153-120), cobalt (J. Environ. Sci., 2025, 148, 198-209), copper (Adv. Powder Technol., 2018, 29, 796-803), and nickel (Biosens. Bioelectron., 2015, 63, 384-391) into the Fe3O4 nanozyme structure, thereby increasing the number of oxygen vacancies and enhancing its mimetic enzyme activity. It should be noted that this one-step preparation method requires careful control of the synthesis process to maintain the original structure of the Fe3O4 nanozyme.

[0003] The transition metal element vanadium (V) has multivalent characteristics (V 3+ 、V 4+ and V 5+ ), can participate in redox reactions to simulate natural enzyme activity. 5+ with Fe 3+ The ionic radius of the two is similar. Based on the ion exchange process, the high-valence V (V 5+ ) can be doped into the Fe3O4 nanozyme structure by post-doping strategy without changing its original morphology. To the best of our knowledge, there is no method to prepare vanadium-doped Fe3O4 (VF e3 O4) nanozymes and further developed a dual-mode sensing platform based on their enzyme-mimicking activity.

[0004] Detection platforms built on a single principle have their own limitations in terms of sensitivity, selectivity, and detection range, making it difficult to meet complex and diverse detection needs. Dual-mode detection combines two sensing modes based on different chemical recognition mechanisms and signal conversion principles. The detection results of the two modes verify each other, which not only makes up for the inherent defects of a single sensing mode but also significantly improves the accuracy and reliability of the detection results, providing strong technical support for the precise analysis of trace substances in complex samples. Among them, colorimetric detection has the advantages of simple operation, intuitive and visual results, and low cost; the electrochemical detection method is to perform quantitative analysis by recording the Faraday current signal generated by the redox reaction of electroactive substances at the electrode interface, which can effectively avoid the interference of colored matrices on the detection results, improve the accuracy of the detection results, and has high sensitivity.

[0005] 3,3',5,5'-Tetramethylbenzidine (TMB), a commonly used substrate for evaluating the catalytic activity of nanozymes, exhibits both colorimetric and electrochemical response properties and shows great potential in the development of dual-mode sensors. However, research on the construction of colorimetric / electrochemical dual-mode sensing platforms based on Fe3O4 nanozymes and bifunctional TMB signaling molecules is still rare. Summary of the Invention

[0006] This invention addresses the low catalytic activity of existing Fe₃O₄ nanozymes. By manipulating their electronic structure through vanadium doping, this method synthesizes V-Fe₃O₄ nanozymes with enhanced peroxidase (POD) activity. This allows for the construction of a ratiometric colorimetric-electrochemical dual-mode sensing platform for highly sensitive nitrite detection. This dual-mode sensor exhibits a wide linear range and a low detection limit, holding great promise for food safety supervision and environmental monitoring.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] A colorimetric / electrochemical dual-mode sensor for nitrite detection, comprising vanadium-doped ferroferric oxide nanozyme, 3,3',5,5'-tetramethylbenzidine, and hydrogen peroxide;

[0009] The method for preparing the vanadium-doped ferroferric oxide nanozyme comprises: dissolving a raw material containing ferroferric oxide nanozyme and a vanadium salt in water, adding urea and reacting the mixture, and washing and drying the product to obtain the vanadium-doped ferroferric oxide nanozyme.

[0010] This study modulates the electronic structure of vanadium-doped Fe3O4 nanozymes, overcoming the kinetic limitations of the traditional Fenton reaction. The researchers synthesized V-Fe3O4 nanozymes with enhanced peroxidase (POD) activity. Using 3,3',5,5'-tetramethylbenzidine (also known as methylene blue, TMB) as a bifunctional signaling molecule, the researchers constructed a ratiometric colorimetric-electrochemical dual-mode sensing platform for highly sensitive nitrite detection based on the pseudo-peroxidase nanozyme activity of the vanadium-doped Fe3O4 nanozyme and the diazotization reaction between nitrite and methylene blue cations. This dual-mode sensor exhibits a wide linear range and a low detection limit, eliminating the need for complex pretreatment. It can be used for the accurate analysis of nitrite content in seawater and food samples, and holds great promise for food safety supervision and environmental monitoring.

[0011] The concentration of the ferroferric oxide nanozyme is 0.1 to 25 mg / mL, more preferably 1 to 5 mg / mL; the concentration of the vanadium salt is 0.1 to 25 mg / mL, more preferably 1 to 5 mg / mL; the mass ratio of the ferroferric oxide nanozyme to the vanadium salt is 5:1 to 1:5, more preferably 2:1 to 1:2;

[0012] The urea concentration is 0.1 to 30 mg / mL, more preferably 0.8 to 5 mg / mL. The reaction temperature is 80 to 150°C, more preferably 110 to 130°C. The reaction time is 6 to 18 hours, more preferably 8 to 12 hours. The drying temperature is 25 to 80°C, more preferably 50 to 70°C.

[0013] The preparation method of the ferroferric oxide nanozyme includes any one of a hydrothermal method, a coprecipitation method, a solvent thermal method or a calcination method; the vanadium salt includes one or more supplementary substitutes selected from vanadium chloride, vanadium bromide, vanadium fluoride, ammonium metavanadate, vanadyl sulfate, vanadium pentoxide, vanadyl acetylacetonate or vanadyl nitrate.

[0014] The present invention also provides a method for preparing the colorimetric / electrochemical dual-mode sensor for detecting nitrite, comprising the steps of:

[0015] Step 1, dissolving a raw material containing ferroferric oxide nanozyme and a vanadium salt in water, adding urea to react, and washing and drying the product to obtain the vanadium-doped ferroferric oxide nanozyme;

[0016] Step 2: Mix vanadium-doped ferroferric oxide nanozyme, 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide to obtain the colorimetric / electrochemical dual-mode sensor.

[0017] The present invention also provides a method for detecting nitrite, comprising the following steps:

[0018] Step 1, adding the colorimetric / electrochemical dual-mode sensor to the test solution, adding a buffer solution to adjust the pH of the solution, and after the reaction, separating the vanadium-doped ferroferric oxide nanozyme from the reaction solution by applying an external magnetic field to obtain a test solution;

[0019] Step 2: Detect the ultraviolet absorption signal of the test solution at 445 nm and 652 nm in the colorimetric mode; or, in the electrochemical mode, use a silica nanoporous membrane modified electrode as the working electrode to measure the electrochemical signal of the test solution to determine the content of nitrite in the test solution.

[0020] The detection principle is as follows: V-Fe3O4 nanozyme has POD activity, which can catalyze hydrogen peroxide (H2O2) to produce highly active reactive oxygen species (ROS), which can oxidize colorless TMB into blue TMB. + , produces a strong UV absorption signal at 652nm. Under acidic conditions, NO2 – With TMB + The amino group of the molecule undergoes a diazotization reaction, and the blue TMB + It is converted into a yellow diazotized product and a new UV absorption peak is generated at 445nm. – With the increase of concentration, the visible light absorption intensity at 652nm decreases, while the absorbance at 445nm increases.

[0021] In the electrochemical mode, an indium tin oxide (ITO) electrode modified with a silicon dioxide nanochannel film (SNF) was used as the working electrode. Through electrostatic interaction, the TMB + Selective detection and enrichment of TMB in coexisting solutions + , while TMB + The diazotization product does not produce an electrochemical response, thus achieving a multifunctional integrated NO2 separation, enrichment and detection system. – Sensing platform. Essentially, TMB + The reduction of NO2 – Therefore, the absorbance ratio at 652 nm and 445 nm, or TMB + The reduction of electrochemical signals can realize ratiometric colorimetric-electrochemical dual mode detection of NO2 – .

[0022] The concentration of the vanadium-doped ferroferric oxide nanozyme in the detection solution is 1-100 μg / mL, preferably 20-100 μg / mL, more preferably 30-50 μg / mL, and more preferably 30-40 μg / mL;

[0023] The concentration of 3,3',5,5'-tetramethylbenzidine (TMB) is 0.05 to 20 mM, more preferably 0.1 to 2 mM;

[0024] The concentration of the hydrogen peroxide is 0.01 to 50 mM, more preferably 0.05 to 2 mM;

[0025] Preferably, the buffer solution comprises any one or more of a sodium chloride aqueous solution, a sodium sulfate aqueous solution, a potassium chloride aqueous solution or a phosphate buffer solution, and the pH of the buffer solution is 2-6.

[0026] Preferably, the pH of the reaction solution is 3-4, more preferably pH 4. This is because a lower pH value (2.0-4.0) is conducive to H2O2 capturing protons to form more stable oxonium ions (H2O2 + ) and then form ROS.

[0027] The reaction time in step 2 is 5 to 25 minutes, preferably 10 to 20 minutes, more preferably 10 to 15 minutes; the reaction temperature is required to be 20 to 60°C, more preferably 30 to 50°C.

[0028] The method for preparing the silicon dioxide nanoporous membrane modified electrode includes an electrochemical assisted method, Any one of solution growth method, two-phase layered growth method, evaporation-induced self-assembly method, π-π interaction induced method, epitaxial growth method, strong magnetic field method, electric field method, and organic solvent-induced self-assembly method; the electrode used to prepare the working electrode includes any one of indium tin oxide electrode, fluorine-doped tin oxide electrode, glassy carbon electrode, gold electrode, screen printing electrode, and carbon fiber electrode.

[0029] During the electrochemical test, the reference electrode includes any one or more of an Ag / AgCl electrode, a saturated calomel electrode, and a Hg / HgO electrode, and the counter electrode includes any one or more of a platinum wire and a platinum sheet.

[0030] Preferably, the nitrite concentration range in the test solution is 3 μM to 350 μM in the colorimetric mode, and 50 nM to 500 μM in the electrochemical mode. The detection limits in these two modes are 2.22 μM and 18.8 nM, respectively. This detection method has a wide linear detection range, high selectivity, and a low detection limit. It also eliminates the need for complex sample pretreatment and separation, reducing detection costs, simplifying operation, and making it suitable for large-scale applications.

[0031] In some embodiments, the test solution contains any one or more interfering substances selected from potassium ions, sodium ions, calcium ions, magnesium ions, sulfate ions, chloride ions, starch, and glucose, and the concentration of any interfering substance is below 1 mM.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The V-Fe3O4 nanozyme prepared by the present invention has enhanced pseudoperoxidase activity and can catalyze H2O2 to produce highly active ROS, oxidizing colorless TMB to blue TMB. + .TMB + It produces a strong UV-visible absorption signal at 652 nm and can also generate an electrochemical signal, providing sufficient background signal for colorimetric and electrochemical detection modes.

[0034] (2) The colorimetric detection method of the present invention is based on NO2 – With TMB + The diazotization reaction between the two groups was determined by measuring the reaction solution at 445 nm (corresponding to TMB + diazotized product) and 652 nm (corresponding to TMB + ) to construct a ratiometric colorimetric sensing platform. Compared to single-signal output modes, the ratiometric colorimetric detection mode in this invention offers advantages such as a wide detection range, high sensitivity, high accuracy, and a low detection limit.

[0035] (3) The silica nanoporous membrane of the present invention has ultra-small nanochannels (2-3 nm), and the pore surface is rich in silanol groups. Its pK a ~2, in the environment of pH>2, ionization makes the film negatively charged, on the one hand, it can be attracted by electrostatic attraction from TMB and TMB + Selective determination of TMB in coexisting fluid + It can also achieve signal enhancement and improve detection sensitivity; on the other hand, it can hinder the influence of large-sized negatively charged pollutants (such as cells, proteins and other biological macromolecules) in complex samples on the base electrode through electrostatic repulsion, hydrophilic interaction and size screening effect, and has good anti-fouling and anti-interference capabilities.

[0036] (4) Colorimetric-electrochemical dual-mode NO2 of the present invention – The sensor has the characteristics of self-calibration and high accuracy. The colorimetric mode is easy to operate, low-cost, and the results are intuitive and visual, while the electrochemical mode has higher detection sensitivity and lower detection limit. The combination of the two is suitable for a wider range of practical application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1Schematic diagram of the synthesis process of V-Fe3O4 nanozyme in Example 1 (A), SEM scanning electron microscopy (B) and TEM transmission spectrum (CD) images of V-Fe3O4 nanozyme at different magnifications, (E) nitrogen BET adsorption-desorption curve of V-Fe3O4 nanozyme, and the inset is the pore size distribution diagram of V-Fe3O4 nanozyme; (F) element distribution diagram of O element, Fe element and V element of V-Fe3O4 nanozyme, high-resolution TEM image of V-Fe3O4 nanozyme (the yellow circle area indicates local lattice disorder) (G) and lattice fringe image (H); (I) magnetization curves of Fe3O4 and V-Fe3O4 nanozyme, and the inset is a photo of V-Fe3O4 nanozyme in the presence (right) and absence (left) of a magnet.

[0038] Figure 2 XRD diffraction patterns of V-Fe3O4 and Fe3O4 nanozymes prepared in Example 1 (A), with the inset being a local magnified view of the (311) crystal plane; (B) Fourier transform infrared (FT-IR) images of V-Fe3O4 and Fe3O4 nanozymes; (C) XPS spectrum of V2p of V-Fe3O4, and XPS spectra of Fe 2p (D) and O1s (E) of V-Fe3O4 and Fe3O4; (F) Zeta potential diagram of V-Fe3O4 and Fe3O4 nanozymes.

[0039] Figure 3 UV-visible absorption spectra of Fe3O4 nanozymes after reaction with TMB and H2O2 after different metal doping in Example 1 (A); UV-visible absorption spectra of different systems after 30 minutes of reaction (B); (CD) is the steady-state kinetic analysis of V-Fe3O4, and the inset is the double reciprocal plot of V-Fe3O4 activity obtained by changing the TMB (C) or H2O2 (D) concentration under the condition of fixed H2O2 (C) or TMB (D) concentration by the Michaelis-Menten model; (E) OH, O2 during the reaction of V-Fe3O4+TMB+H2O2 system ·- and 1 Electron paramagnetic resonance (EPR) spectrum of O2; (F) Schematic diagram of the mechanism by which V-Fe3O4 nanozyme catalyzes H2O2 to produce ROS.

[0040] Figure 4 TEM top view of SNF in Example 1 (A); SEM cross-sectional view of SNF / ITO electrode (B); TEM cross-sectional view of SNF (C); CV graphs of ITO, SM@SNF / ITO and SNF / ITO electrodes in 50 mM potassium hydrogen phthalate solution containing 0.5 mM K3[Fe(CN)6] (D), FcMeOH (E) and [Ru(NH3)6]Cl3 (F).

[0041] Figure 5 These are the electrochemical signals obtained when V-Fe3O4 nanozyme catalyzed TMB at different pH (A), V-Fe3O4 nanozyme concentrations (B), reaction times (C) and reaction temperatures (D) in Application Example 2.

[0042] Figure 6 For the detection of NO2 by the V-Fe3O4+TMB+H2O2 system in Application Example 3 – Mechanism diagram (A), V-Fe3O4+TMB+H2O2 system (B) and different concentrations of NO2 – UV absorption spectrum of the solution after the reaction, the inset shows the solution containing different concentrations of NO2 – Photo of the reaction solution; (C) Colorimetric detection of NO2 – The linear calibration curve, A 652 / A 445 is the ratio of the absorbance intensity at 652nm and 445nm; (D) A of different reaction solutions 652 / A 445 ; (E) Anti-interference ability of V-Fe3O4+TMB+H2O2 system to different substances under colorimetric detection mode.

[0043] Figure 7 For the SNF / ITO electrode detection of NO2 in Application Example 4 – Mechanism diagram (A), V-Fe3O4+TMB+H2O2 system (B) and different concentrations of NO2 – DPV signal after reaction; (C) Electrochemical mode detection of NO2 – (D) Effect of simulated colored system on electrochemical and colorimetric detection. I and I0 as well as A and A0 represent the current signal and absorbance in the presence and absence of colored matrix, respectively. The insets are photos of blank solution and solutions with different colored matrices added. (E) DPV response obtained after SNF / ITO electrode measurement-elution repeated 5 times. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiment. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Those skilled in the art will make modifications or equivalent substitutions based on understanding the technical solution of the present invention, without departing from the spirit and scope of the technical solution of the present invention, and all should be encompassed within the protection scope of the present invention.

[0045] Unless otherwise specified, the raw materials used in the following specific embodiments were purchased from commercial channels and used directly without special treatment. The main raw materials used in the examples are as follows:

[0046] Ferric chloride hexahydrate: Shanghai MacLean Biochemical Technology Co., Ltd.

[0047] Ethylene glycol: Shanghai MacLean Biochemical Technology Co., Ltd.;

[0048] Sodium acetate: Shanghai MacLean Biochemical Technology Co., Ltd.;

[0049] 1,2-Ethylenediamine: Shanghai MacLean Biochemical Technology Co., Ltd.;

[0050] Vanadium chloride: Shanghai MacLean Biochemical Technology Co., Ltd.;

[0051] Urea: Shanghai MacLean Biochemical Technology Co., Ltd.;

[0052] Sodium nitrite: Shanghai MacLean Biochemical Technology Co., Ltd.;

[0053] Hexadecyltrimethylammonium bromide: Shanghai MacLean Biochemical Technology Co., Ltd.;

[0054] Tetraethyl orthosilicate: Shanghai Aladdin Biochemical Technology Co., Ltd.

[0055] Ammonia: Shanghai Aladdin Biochemical Technology Co., Ltd.

[0056] Sodium nitrate: Hangzhou Gaojing Fine Chemical Co., Ltd.;

[0057] Ethanol: Hangzhou Gaojing Fine Chemical Co., Ltd.;

[0058] Potassium ferrocyanide: Shanghai Aladdin Biotechnology Co., Ltd.;

[0059] Hexaammineruthenium(III) trichloride: Shanghai Aladdin Biotechnology Co., Ltd.;

[0060] Potassium hydrogen phthalate: Shanghai Aladdin Biotechnology Co., Ltd.

[0061] Example 1

[0062] The preparation method of V-Fe3O4 nanozyme comprises the following steps:

[0063] (1) Preparation of Fe3O4 nanozyme by solvothermal method: 1 g of FeCl3·6H2O was weighed and added to 20 mL of ethylene glycol. The mixture was stirred magnetically for 30 min to obtain a uniformly mixed yellow transparent solution. 3 g of sodium acetate solid powder was quickly added and mixed well. After that, 10 mL of 1,2-ethylenediamine was added and the reaction time was 30 min to obtain a brownish-yellow solution. The solution was transferred to a polytetrafluoroethylene-lined container for solvothermal reaction at 200 °C for 8 h. After magnetic separation of the black solid, the solid was repeatedly washed with distilled water and anhydrous ethanol and dried to obtain Fe3O4 nanozyme.

[0064] (2) The Fe3O4 nanozyme and vanadium chloride obtained in step (1) were dissolved in 40 mL of deionized water in a mass ratio of 1:1 and a concentration of 2.5 mg / mL. Then, 120 mg of urea was added, mixed evenly, and transferred to a hydrothermal reactor for reaction for 10 h at a reaction temperature of 120°C. After cooling, the precipitate was repeatedly washed three times with deionized water and ethanol, and dried in a vacuum oven at 60°C to obtain V-Fe3O4 nanozyme.

[0065] The preparation method of SNF / ITO electrode includes the following steps:

[0066] use The solution growth method was used to grow SNF on the surface of ITO electrode (2.5 cm × 5 cm): 160 mg of hexadecyltrimethylammonium bromide was added to 100 mL of ethanol-water mixed solution (V 乙醇 :V 水 =3:7), stirred for 5 minutes until completely dissolved, then quickly added ammonia (10%, 100μL) and tetraethoxysilane (80μL), and stirred for 5 minutes to obtain a clear and transparent precursor solution. A clean ITO electrode was immersed in the above precursor solution and reacted in a constant temperature water bath at 60°C for 24 hours. After the reaction is complete, the electrode surface is thoroughly rinsed with a large amount of deionized water to remove the residual solution, blown dry with N2, and placed in a 100°C oven for aging for 10 hours to prepare a SNF / ITO electrode containing a micelle template (SM), namely SM@SNF / ITO. Finally, the SM@SNF / ITO electrode was immersed in a hydrochloric acid-ethanol solution (0.1M) and stirred for 5 minutes to remove the SM in the pores, obtaining an electrode with open pores, namely the SNF / ITO electrode.

[0067] Application Example 1

[0068] Using V-Fe3O4 nanozyme with POD activity and TMB as the signal molecule, NO2 was detected by colorimetric and electrochemical methods. – The process:

[0069] (1) First, NO2 –Dissolve in water to form NO2 – The stock solution was then added with 30 μg / mL V-Fe3O4 nanozyme, 0.2 mM TMB, 0.1 mM H2O2 and different volumes of NO2 in acetic acid-sodium acetate buffer solution (0.2 mol / L, pH = 4.0). – Stock solution (NO2 in colorimetric mode – The concentrations of NO2 in electrochemical mode were 3μM, 5μM, 20μM, 50μM, 100μM, 150μM, 200μM, 225μM, 350μM and 350μM respectively. – The concentrations of the V-Fe₃O₄ nanozyme were 50 nM, 100 nM, 500 nM, 1 μM, 3 μM, 4 μM, 5 μM, 10 μM, 50 μM, 100 μM, 200 μM, 300 μM, 400 μM, and 500 μM, respectively. After reacting at 40°C for 10 min, the V-Fe₃O₄ nanozyme was separated from the reaction solution by applying an external magnetic field to obtain a series of test solutions.

[0070] (2) In the colorimetric mode, the absorbance at 652 nm and 445 nm (A 652 and A 445 ), by recording A 652 / A 445 Changes in NO2 – In electrochemical mode, the test solution was drop-coated on the surface of the SNF / ITO electrode. After 5 minutes, the surface solution was gently rinsed with a buffer solution. Finally, the enriched SNF / ITO electrode was placed in an acetic acid-sodium acetate buffer solution (0.2 mol / L, pH = 4.0) for electrochemical testing.

[0071] Performance observation test

[0072] 1. V-Fe3O4 morphology observation

[0073] Figure 1 (A) Schematic diagram of the preparation process and microstructure of V-Fe3O4. Figure 1 The SEM image in (B) shows that the V-Fe3O4 particles are about 100 nm in diameter and have uniform size. Figure 1 (CD) shows that V-Fe3O4 has a mesoporous structure. Figure 1 As shown in (E), the nitrogen adsorption-desorption isotherm of V-Fe3O4 is type IV and exhibits an H3 hysteresis loop. The strong adsorption at a relative pressure (P / P0) of 1.0 is attributed to the macroporous cavities of V-Fe3O4. The BET specific surface area is 15.7 m 2 / g, and its high specific surface area enables it to fully contact and react with other substances. The average diameters of V-Fe3O4 measured using the Barrett-Joyner-Halenda (BJH) method were approximately 3.67nm and 34.5nm. This confirms that V-Fe3O4 still has a mesoporous structure similar to that of Fe3O4.

[0074] Figure 1 (F) is the element distribution diagram of V-Fe3O4. O, Fe and V elements are evenly distributed in V-Fe3O4, indicating that vanadium is evenly doped in V-Fe3O4. In addition, Figure 1 The high-resolution TEM image of (GH)V-Fe3O4 shows that the order of its lattice fringes is slightly reduced and the (311) crystal plane belonging to Fe3O4 still exists, which confirms that V-Fe3O4 still presents the Fe3O4 crystal structure. Figure 1 (I) shows the magnetization curves of Fe3O4 and V-Fe3O4 nanozymes. The insets show photos of V-Fe3O4 nanozymes in the presence (right) and absence (left) of a magnet. It can be seen that the saturation magnetization intensity of V-Fe3O4 nanozymes is slightly lower than that of Fe3O4, and under the action of an external magnetic field, the V-Fe3O4 nanozyme can be quickly separated from the reaction solution, effectively preventing its own color from affecting the detection results during colorimetric detection.

[0075] 2. V-Fe3O4 structural characterization

[0076] Figure 2 (A) XRD spectra corresponding to Fe₃O₄ and V-Fe₃O₄. All diffraction peaks match well with the standard XRD pattern (PDF#99-0073), and no diffraction peaks from other phases are detected, indicating that V-Fe₃O₄ and Fe₃O₄ have consistent structures and good crystallinity. Compared with Fe₃O₄, the diffraction peak intensity of V-Fe₃O₄ is slightly reduced and significantly shifted toward lower binding energies, indicating that vanadium is incorporated into the Fe₃O₄ structure via substitutional rather than interstitial doping. Figure 2 (B) FT-IR is located at 600 cm -1 The characteristic peak of Fe-O bond at 584 cm-1 is slightly red-shifted to 584 cm-1. -1 , indicating that Fe-OV bonds are formed in V-Fe3O4, which is consistent with the XRD test results. -1 and 1450cm -1 The absorption peaks at 1600 cm-1 are attributed to the stretching vibration and bending vibration of OH, respectively. -1 and 1178cm -1 The peaks at are attributed to the stretching vibrations of the C=O group and the CO group, respectively.

[0077] Figure 2 (CE) High-resolution XPS spectra of V 2p, Fe 2p, and O1s. Figure 2 (C) The characteristic peaks at 515.1eV, 515.9eV, and 517.1eV correspond to V 3+ 、V 4+ and V 5+ . Figure 2 (D) shows the high-resolution XPS spectra of Fe 2p of Fe3O4 and V-Fe3O4. The two peaks at binding energies of 710.1eV and 724.5eV correspond to Fe 2p 3 / 2 and Fe 2p 1 / 2 Comparing the valence of Fe in V-Fe3O4 and Fe3O4, we found no significant difference, but the binding energy of Fe in V-Fe3O4 increased slightly from 710.12eV to 710.64eV. This is because V 5+ It has strong electronegativity, which attracts surrounding electrons and causes the electron cloud density of Fe atoms to decrease. Figure 2 The valence distribution of V element in (C) is consistent with the results [ . Figure 2 (E) The peaks of oxygen at 530.30 eV, 531.90 eV and 533.20 eV are attributed to metallic oxygen (MO), oxygen vacancy (O V ) and surface oxygen (OH).

[0078] Figure 2 (F) Zeta potential diagram of V-Fe3O4 and Fe3O4 nanozymes. Compared to Fe3O4, the proportion of oxygen vacancies in V-Fe3O4 increases from 21.7% to 33.7%, and the surface has a stronger negative charge. This change facilitates the adsorption of reactants, thereby improving the catalytic efficiency of V-Fe3O4 on substrates.

[0079] 3. Study on the activity of V-Fe3O4 enzyme

[0080] Fe3O4 is a nanozyme with good POD activity. However, Fe3O4 nanozyme still has the disadvantages of low catalytic activity and poor substrate affinity. Transition metal elements and their compounds have been reported many times for their excellent POD activity. Based on this, the present invention attempts to improve the POD activity of Fe3O4 nanozyme by doping with multiple transition metal elements. The preparation method is as in Example 1, except that vanadium chloride is replaced with an equal amount of other metal salts, including cerium nitrate hexahydrate, cobalt nitrate hexahydrate, anhydrous copper chloride, manganese chloride tetrahydrate and nickel hydroxide.

[0081] like Figure 3As shown in (A), the POD activity of V-Fe₃O₄ is significantly stronger than that of Fe₃O₄ nanozymes and other metal-doped Fe₃O₄ nanozymes. This enhanced effect is presumably due to the valence state of the V atom. The high-resolution V 2p XPS spectrum indicates that V exists primarily in a +5 valence state, which makes it highly oxidizing and facilitates the production of ROS through the oxidation of H₂O₂.

[0082] Figure 3 (B) The enzymatic activity of V-Fe₃O₄ was further investigated using TMB as a chromogenic substrate. When V-Fe₃O₄, TMB, and H₂O₂ were coexisting, the solution exhibited a deep blue color after 30 minutes of reaction, with a distinct UV-visible absorption peak at 652 nm. This indicates that V-Fe₃O₄ enhances POD activity. Figure 3 The concentrations of TMB, H2O2, and V-Fe3O4 in (A) and (B) were 0.2 mM, 0.1 mM, and 10 μg / mL, respectively.

[0083] Figure 3 (CD) The steady-state kinetic constants of V-Fe3O4 were analyzed using the Michaelis-Menten equation and the Lineweaver-Burk curve using TMB and H2O2 as substrates, respectively. The K constants of V-Fe3O4 were calculated based on the experimental results when TMB was used as the substrate. m and V max The values ​​were 0.194 mM and 4.12 × 10 -8 M / s. K of V-Fe3O4 when H2O2 is used as substrate m and V max 0.015 mM and 2.62 × 10 -8 M / s. The nanozymes synthesized in this patent have better affinity with the substrate and faster reaction speed.

[0084] In order to further explore the types and mechanisms of ROS generated by the V-Fe3O4 catalytic oxidation of H2O2 system, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 2,2,6,6-tetramethylpiperidine (TEMP) were used as capture agents to conduct electron paramagnetic resonance (EPR) tests. The results are shown in Figure 2. Figure 3 (E) shows a typical DMPO-·OH four-line EPR spectrum was monitored in the DMPO+H2O2 system, and the relative intensities of the peaks were 1:2:2:1. ·- and 1 No obvious DMPO-O2 was detected ·- and TEMP- 1 O2 signal. Based on the above test results, it is proved that the main free radicals produced in the catalytic process are· OH.

[0085] Based on this, the mechanism of V-Fe3O4 catalyzing H2O2 to produce ROS is speculated: Figure 3 (F) Schematic diagram of the mechanism of V-Fe3O4 nanozyme catalyzing H2O2 to produce ROS. Since Fe3O4 itself has a certain POD activity, some · OH comes from Fe 2+ / Fe 3+ Fenton and Fenton-like reactions occur (Equation 1-2). However, due to Fe 3+ to Fe 2+ The reduction kinetics are much slower than that of Fe 2+ to Fe 3+ The oxidation process seriously affects the degree of Fenton reaction and · The rate of OH production. 5+ The doping of Fe3O4 prolongs the Fe-O bond, optimizes the electronic structure of Fe3O4 and reduces the FeOH 2+ Reaction energy barrier, indirect reduction of Fe via surface electron transfer 3+ , accelerating Fe 3+ to Fe 2 + The reduction process makes more Fe 2+ Through the Fenton reaction · OH (Equation 3). V 5+ The empty d orbital of H2O2 polarizes the OO bond by adsorbing the lone pair electrons of H2O2, causing it to split and generate · OH (Equation 4). Through the reaction process (Equation 3), part of V 5+ Restored to V 4+ . V 4+ It has excellent POD activity and can react with H2O2 to produce a large amount of · OH (Equation 5). V 4+ The reducing ability is stronger than Fe 3+ , the two undergo redox reaction, further accelerating electron transfer, through Fe 2+ / Fe 3+ With V 5+ / V 4+ The synergistic effect breaks through the kinetic limitations of the traditional Fenton reaction and greatly improves the catalytic performance and · The generation rate of OH (Eq. 6).

[0086] The specific equation is as follows:

[0087] Fe 2+ +H2O2+H + →Fe 3+ +·OH+H2O equation (1)

[0088] Fe 3+ +H2O2→Fe 2+ +·OOH+H + Equation (2)

[0089] V 5+ +Fe 3+ +H2O2→V 4+ +Fe 2+ +·OOH Equation (3)

[0090] V 5+ +H2O2→V 5+ +·OOH Equation (4)

[0091] V 4+ +H2O2+H + →V 5+ +·OH+H2O Equation (5)

[0092] V 4+ +Fe 3+ →V 5+ +Fe 2+ Equation (6)

[0093] 4. Characterization of SNF / ITO electrodes

[0094] Figure 4 Characterization of the morphology and charge permselectivity of SNF. Figure 4 (A) is the TEM top view, Figure 4 (B) SEM cross-sectional image of SNF / ITO electrode; Figure 4 (C) TEM cross-section of the SNF. The top and cross-sectional TEM images show that the SNF is uniform and crack-free, with numerous nanopores with pore sizes of 2 to 3 nm uniformly distributed in a worm-like pattern. These nanoscale pores endow the SNF with excellent size exclusion capabilities, preventing nonspecific adsorption of large molecules in complex samples on the electrode surface, which could reduce sensitivity. TEM was then used to further characterize the morphology and structure of the SNF cross-section. The pores are long-range ordered, with a nanochannel length (film thickness) of approximately 86 nm. This unique morphology endows the SNF with excellent mass transfer capabilities.

[0095] Figure 4(D-F) CV curves of ITO, SM@SNF / ITO, and SNF / ITO electrodes measured in different probe solutions. ITO exhibits distinct redox peaks in probe solutions of varying electrical properties. When SM@SNF / ITO is used as the working electrode, almost no Radaic current is generated. This is because the ITO electrode surface is covered by non-conductive SNFs, and the SNF nanochannels are filled with SM, rendering them closed and preventing redox probe molecules from passing through the pores to reach the electrode surface. This demonstrates the integrity of the SNFs modified on the ITO surface. SM@SNF / ITO was stirred and eluted in a hydrochloric acid-ethanol solution to remove the SM, yielding an SNF / ITO electrode. Because SNFs are rich in silanol groups (pKa ≈ 3), deprotonation of the silanol groups at a buffer pH > 3 renders the pores negatively charged. Consequently, the SNF / ITO electrode exhibits varying permeability to probe solutions of varying electrical properties.

[0096] The SNF / ITO electrode was exposed to negative probe solution (Fe(CN)6 3– ) is significantly smaller than that of ITO, and in the positive probe solution (Ru(NH3)6 3+ ) is significantly greater than that of ITO. This is because the negatively charged pores are more sensitive to the negatively charged Fe(CN)6 3– The probe showed obvious electrostatic repulsion to the positively charged Ru(NH3)6 3+ The probe exhibited significant electrostatic attraction. Furthermore, the reduction peak current of the SNF / ITO electrode in a neutral probe solution (ferrocenium methanol, FcMeOH) was greater than the oxidation peak current. This is because the positively charged oxidation products of FcMeOH molecules can be enriched in the pores, resulting in a higher reduction signal. These results demonstrate the excellent integrity and permselectivity of SNF / ITO.

[0097] Application Example 2

[0098] Optimization of detection conditions

[0099] To maximize the sensitivity of the detection mode, Figure 5 A series of optimizations were performed on the detection conditions, including the pH of the supporting electrolyte, V-Fe3O4 concentration, reaction time, and reaction temperature. Figure 5(A) The pH optimization results of Tong as the supporting electrolyte. Five buffer solutions with different pH values ​​(2.0-6.0) were used as the reaction medium in step (1) of Application Example 1. TMB, H2O2, and V-Fe3O4 nanozymes of the same concentration were added to react for 10 minutes. After the reaction was completed, 50 μL of the solution was drop-coated on the surface of the SNF / ITO electrode and statically enriched for 5 minutes. The electrode surface solution was then gently rinsed with a buffer solution. The enriched SNF / ITO electrode was placed in a blank buffer solution for electrochemical testing.

[0100] The results are as follows Figure 5 (A) When the pH of the reaction medium is 4.0, the highest electrochemical oxidation signal is obtained, proving that the catalytic effect of V-Fe3O4 is the best at this time. This is because H2O2 can capture protons at a lower pH value (2.0-4.0) to form more stable oxonium ions (H2O2 + ) and thus form ROS. When the pH value is higher than 5.0, H2O2 gradually hydrolyzes and loses its ability to oxidize TMB. Therefore, pH = 4.0 is selected as the optimal reaction pH value.

[0101] The concentration of V-Fe3O4 nanozyme was then optimized under the condition of pH=4.0. Figure 5 As shown in (B), when the V-Fe₃O₄ concentration is low, the catalytic effect increases with increasing V-Fe₃O₄ concentration. However, when the concentration reaches 40 μg / mL, the catalytic effect does not change significantly. Therefore, the V-Fe₃O₄ dosage was set at 40 μg / mL for subsequent applications.

[0102] In the color reaction, the reaction time and reaction temperature significantly affect the color intensity. Figure 5 (CD) The reaction time and temperature were optimized. As the reaction time increased, the electrochemical signal gradually increased, indicating that sufficient TMB + This provides a comparable background signal, increasing the signal-to-noise ratio. However, the ΔI / I0 ratio decreases with increasing reaction time, affecting sensor sensitivity. Therefore, considering both the base signal intensity and sensitivity, a 10-minute reaction time was selected as the optimal reaction time for subsequent work. Similarly, the reaction temperature was optimized and continued at 40°C.

[0103] Application Example 3

[0104] Colorimetric detection of NO2 -

[0105] Figure 6 (A) is the colorimetric mode for detecting NO2 – Schematic diagram. In the acetate buffer solution, V-Fe3O4 with excellent POD activity acts on H2O2 to produce a large amount of· OH oxidizes the colorless TMB molecule to blue TMB + , with different concentrations of NO2 – With the addition of some TMB + The diazotized product was oxidized to yellow. The absorbance ratio (A 652 / A 445 ) changes, build for NO2 – Sensitive detection ratio colorimetric sensing platform, to achieve different concentrations of NO2 – Quantitative detection of .

[0106] Figure 6 (BC) in NO2 – In the concentration range of 3μM~350μM, as NO2 – With the increase of concentration, the absorbance of the reaction solution at 652nm gradually decreased, and the absorbance at 445nm gradually increased. 652 / A 445 Ratio and NO2 – There is a good linear relationship between the concentrations of (R 2 =0.996), with a detection limit of 2.2 μM.

[0107] Figure 6 (D) The reaction solutions obtained under the same reaction conditions were tested. The RSD of the absorbance intensity of the five solutions was only 0.3%, indicating that the method has good parallelism. Considering the complex interference in the actual samples, the anti-interference performance of the sensing mode was investigated. The concentration and dosage of the interference were K + 、Na + , Ca 2+ Mg 2+ 、SO4 2– and Cl – The concentration of ions is 1mM, the concentration of glucose (Glu) and starch (Star) is 0.1mM, such as Figure 6 As shown in (E), different types of interfering substances did not significantly affect the detection results, proving that the sensing platform has good anti-interference performance.

[0108] Application Example 4

[0109] Electrochemical detection of NO2 -

[0110] Figure 7 (A) Electrochemical mode for detecting NO2 – Schematic diagram of target substance NO2 – Will work with TMB+ The diazotization product is generated, leading to TMB + The SNF / ITO electrode is used to selectively enrich and detect TMB in the reaction solution through electrostatic interaction. + The amount of change can achieve NO2 – Detection. Figure 7 (B) SNF / ITO with different concentrations of NO2 – Overlay of DPV signals from electrochemical detection of the reaction solution. Figure 7 (C) Further analysis of the electrochemical signal -NO2 – The concentration was fitted and it was found that in the concentration range of 50nM to 500μM, TMB + Oxidation current and NO2 – The concentration shows a two-stage linear relationship. The linear equation in the low concentration range (50nM~5μM) is expressed as R 2 =0.994; the linear equation in the high concentration range (5-500 μM) is R 2 =0.998, and the detection limit was 18.8 nM.

[0111] In addition, crystal violet, riboflavin, malachite green and Congo red were selected to simulate colored matrices to further verify the electrochemical sensor constructed by the present invention for NO2 in colored samples. – Advantages of detection. Figure 7 (D) The presence of colored substances has a significant impact on the colorimetric detection results, but has a weak effect on the electrochemical detection results. This proves that the electrochemical detection model constructed in this chapter is suitable for NO2 in colored samples. – Detection. At the same time, one SNF / ITO electrode was tested and then the test was repeated 5 times. Figure 7 (E) The obtained DPV signal is almost the same as that of the initial detection, proving that the electrode has good regenerative properties and greatly saves detection costs in the actual detection process.

Claims

1. A colorimetric / electrochemical dual-mode sensor for detecting nitrite, characterized in that: These include vanadium-doped ferroferric oxide nanozymes, 3,3',5,5'-tetramethylbenzidine, and hydrogen peroxide; The method for preparing the vanadium-doped ferroferric oxide nanozyme comprises: dissolving a raw material containing ferroferric oxide nanozyme and a vanadium salt in water, adding urea and reacting the mixture, and washing and drying the product to obtain the vanadium-doped ferroferric oxide nanozyme.

2. The colorimetric / electrochemical dual-mode sensor for detecting nitrite according to claim 1, characterized in that: The concentration of the ferroferric oxide nanozyme is 0.1-25 mg / mL, the concentration of the vanadium salt is 0.1-25 mg / mL, the mass ratio of the ferroferric oxide nanozyme to the vanadium salt is 5:1-1:5; the concentration of urea is 0.1-30 mg / mL; the reaction temperature is 80-150° C., and the reaction time is 6-18 hours.

3. The colorimetric / electrochemical dual-mode sensor for detecting nitrite according to claim 1, characterized in that: The preparation method of the ferroferric oxide nanozyme includes any one of a hydrothermal method, a coprecipitation method, a solvothermal method or a calcination method; The vanadium salt includes one or more of vanadium chloride, vanadium bromide, vanadium fluoride, ammonium metavanadate, vanadyl sulfate, vanadium pentoxide, vanadyl acetylacetonate or vanadyl nitrate.

4. A method for preparing a colorimetric / electrochemical dual-mode sensor for detecting nitrite according to any one of claims 1 to 3, characterized in that: Including steps: Step 1, dissolving a raw material containing ferroferric oxide nanozyme and a vanadium salt in water, adding urea to react, and washing and drying the product to obtain the vanadium-doped ferroferric oxide nanozyme; Step 2: Mix vanadium-doped ferroferric oxide nanozyme, 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide to obtain the colorimetric / electrochemical dual-mode sensor.

5. A method for detecting nitrite, characterized in that, The following steps are involved: Step 1, adding the colorimetric / electrochemical dual-mode sensor according to any one of claims 1 to 3 to a test solution, adding a buffer solution to adjust the pH of the solution, and after the reaction, separating the vanadium-doped ferroferric oxide nanozyme from the reaction solution by applying an external magnetic field to obtain a test solution; Step 2: Detect the ultraviolet absorption signal of the test solution at 445 nm and 652 nm in the colorimetric mode; or, in the electrochemical mode, use a silica nanoporous membrane modified electrode as the working electrode to measure the electrochemical signal of the test solution to determine the content of nitrite in the test solution.

6. The method for detecting nitrite according to claim 5, wherein The concentration of the vanadium-doped ferroferric oxide nanozyme in the detection solution is 1 to 100 μg / mL, the concentration of 3,3',5,5'-tetramethylbenzidine is 0.05 to 20 mM, and the concentration of hydrogen peroxide is 0.01 to 50 mM; And / or, the buffer solution includes any one or more of a sodium chloride aqueous solution, a sodium sulfate aqueous solution, a potassium chloride aqueous solution or a phosphate buffer solution, and the pH of the buffer solution is 2-6.

7. The method for detecting nitrite according to claim 5, wherein The reaction time in step 2 is 5 to 25 minutes, and the reaction temperature is 20 to 60°C.

8. The method for detecting nitrite according to claim 5, wherein The method for preparing the silicon dioxide nanoporous membrane modified electrode includes an electrochemical assisted method, Any one of solution growth method, two-phase layered growth method, evaporation-induced self-assembly method, π-π interaction induced method, epitaxial growth method, strong magnetic field method, electric field method, and organic solvent-induced self-assembly method; During the electrochemical test, the reference electrode includes any one or more of an Ag / AgCl electrode, a saturated calomel electrode, and a Hg / HgO electrode, and the counter electrode includes any one or more of a platinum wire and a platinum sheet.

9. The method for detecting nitrite according to claim 5, wherein In the colorimetric mode, the nitrite concentration in the test solution ranges from 3 μM to 350 μM, and in the electrochemical mode, the nitrite concentration in the test solution ranges from 50 nM to 500 μM.

10. The method for detecting nitrite according to claim 5, wherein In the colorimetric mode, the test solution contains any one or more interfering substances selected from potassium ions, sodium ions, calcium ions, magnesium ions, sulfate ions, chloride ions, starch and glucose, and the concentration of any interfering substance is below 1 mM.

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