Double-signal sensor based on metal-free carbon dot nano-enzyme ratio, preparation method of double-signal sensor and application of double-signal sensor in visual detection of nitrite

Abstract

The invention belongs to the technical field of nano-enzyme probes, and provides a dual-signal sensor based on a metal-free carbon dot nano-enzyme ratio, a preparation method of the dual-signal sensor and application of the dual-signal sensor in visual detection of nitrite. The preparation method comprises the following steps: by taking tea polyphenol as a carbon precursor and ethidene diamine as a nitrogen doping agent, carrying out one-step hydrothermal synthesis to obtain N-CDs nano-enzyme, and then dialyzing, filtering and freeze-drying to obtain metal-free carbon dot nano-enzyme N-CDs powder; the ratio type N-CDs / OPD fluorescence sensor or the N-CDs / TMB ratio colorimetric sensor is constructed by using the dual characteristics of photoluminescence and enzyme-like activity of the N-CDs nano enzyme and the diazotization of a nitrite-mediated substrate OPD or TMB. The obvious color change of the dual-wavelength sensor is easier to identify by naked eyes, and a foundation is laid for constructing a more sensitive intelligent mobile phone auxiliary sensing platform. And in combination with intelligent mobile phone equipment, optical double-signal output and a ratio sensing strategy, not only is on-site real-time detection of a target object realized, but also the accuracy of measured data is greatly improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanozyme probes, and in particular relates to a metal-free carbon dot nanozyme ratio dual-signal sensor, a preparation method thereof, and an application thereof in visual detection of nitrite. Background Art

[0002] Nitrite is used as a food additive in food processing and has both beneficial and adverse effects. Studies have found a certain correlation between the incidence of colorectal cancer / gastric cancer and the dietary intake of processed foods, so the role of such additives has been explored in depth. Although nitrite is not directly related to such cancers, it can react with amides to form carcinogenic N-nitrosamines. Excessive intake of nitrite can cause symptoms such as gastrointestinal reactions and systemic hypoxia. For these reasons, it is necessary to accurately measure nitrite to ensure food safety.

[0003] Methods for nitrite detection have been developed, including chromatography, electrochemistry, and spectroscopic sensing techniques. Among all these methods, fluorescence and colorimetry are commonly used as optical dual-signal outputs for detecting nitrite. However, most optical dual-signal sensing strategies rely on a single wavelength and are limited by environmental factors and instrument efficiency. Therefore, there is an urgent need to develop a new method for rapid, efficient, and quantitative detection of nitrite.

[0004] In order to make up for the above shortcomings, the dual-wavelength ratiometric sensing strategy is favored. Because this strategy can effectively eliminate adverse interference and improve the reliability of analytical data, it is attributed to its inherent self-calibration. At present, dual-signal sensors based on nanozymes with enzyme-like activity and fluorescence have been widely studied. However, the optical signals of these sensors are usually output in the form of a single wavelength. Therefore, it is challenging to explore dual-wavelength-dual-signal sensors for nitrite detection.

[0005] As an emerging carbon nanomaterial, carbon dots are widely used not only as fluorescent probes but also as colorimetric nanozymes due to their easy synthesis, low toxicity, high solubility, excellent optical properties and tunable catalytic activity. Summary of the invention

[0006] In order to solve the problem that the current dual-signal sensor based on carbon dot nanozymes relies on a single wavelength and is easily limited by environmental factors and instrument efficiency, the present invention provides a metal-free carbon dot nanozyme ratio dual-signal sensor and its preparation method and application in visual detection of nitrite.

[0007] The present invention is realized by the following technical scheme: a ratio dual-signal sensor based on metal-free carbon dot nanozyme, using tea polyphenols as a carbon precursor and ethylenediamine as a nitrogen doping agent, obtaining nitrogen-doped carbon dot N-CDs nanozyme by a one-step hydrothermal synthesis method, and then dialyzing, filtering, and freeze-drying to obtain metal-free carbon dot nanozyme N-CDs powder; utilizing the dual characteristics of photoluminescence and enzyme-like activity of N-CDs nanozyme and nitrite-mediated diazotization of substrates o-phenylenediamine (OPD) or 3,3′,5,5′-tetramethylbenzidine (TMB) to construct a nitrite-mediated ratio N-CDs / OPD fluorescence sensor or N-CDs / TMB ratio colorimetric sensor.

[0008] The preparation method of the nitrogen-doped carbon dot N-CDs nanozyme comprises: (1) Weigh 0.1 g of tea polyphenols and 1.5 mmol of ethylenediamine, dissolve in 10 mL of ultrapure water, and treat with ultrasound at 15 kHz and 25°C for 5 min. Transfer the resulting clear solution to an autoclave lined with polytetrafluoroethylene, heat the temperature to 200°C at a heating rate of 10°C / min, and keep the temperature for 8 h. After the reaction is completed, cool the temperature naturally to room temperature to obtain a dark brown solution. (2) The obtained dark brown solution was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da and placed in water for 24 h. The water was changed every 6 h. The dialyzed liquid was passed through a 0.22 μm filter membrane. The filtrate was freeze-dried at -60 °C for 36 h and the brown powder was collected, which was the N-CDs nanozyme.

[0009] The preparation method of the ratiometric N-CDs / OPD fluorescence sensor comprises: (1) Preparation of stock solutions: N-CDs powder was dissolved in ultrapure water to obtain a 10 mg / mL N-CDs stock solution; OPD powder was dissolved in ultrapure water to obtain a 50 mmol / L OPD stock solution; sodium nitrite powder was dissolved in ultrapure water to obtain 10 mmol / L and 1 mmol / L nitrite stock solutions; (2) Construction of ratiometric N-CDs / OPD fluorescent probe: 0.5-10 μL of 1 mmol / L nitrite stock solution or 1-55 μL of 10 mmol / L nitrite stock solution and 20 μL of OPD stock solution were sequentially introduced into 0.1 mol / L HCl solution to generate benzotriazole BTA. After 5 min of reaction, the pH of the reaction solution was adjusted to 7.0-9.0 with 2 mol / L NaOH to activate the yellow fluorescence of BTA. Subsequently, 2 μL of blue-luminescent N-CDs stock solution was thoroughly mixed with the above solution and diluted to 1.0 mL with ultrapure water to obtain a ratiometric N-CDs / OPD fluorescent sensor.

[0010] Furthermore, the concentration ratio of the N-CDs to OPD is 20 μg / mL:1 mmol / L; and the final volume of the ratiometric N-CDs / OPD fluorescence sensor is 1.0 mL.

[0011] The preparation method of the N-CDs / TMB ratio colorimetric sensor comprises: (1) Preparation of stock solutions: N-CDs powder was dissolved in ultrapure water to obtain a 10 mg / mL N-CDs stock solution; TMB powder was dissolved in ultrapure water to obtain a 10 mmol / L TMB stock solution; sodium nitrite powder was dissolved in ultrapure water to obtain 10 mmol / L and 1 mmol / L nitrite stock solutions; (2) Construction of N-CDs / TMB ratiometric sensor: 10 μL TMB stock solution and 10 μL N-CDs stock solution were added to 500 μL HAc-NaAc buffer solution in sequence; the colorimetric sensor was detected at 365 nm and 20 μW / cm 2 After irradiation with ultraviolet light for 15 min, TMB was catalyzed to oxidized TMB, namely TMB ox ; Subsequently, 0.5-10 μL of 1 mmol / L nitrite stock solution or 1-17.5 μL of 10 mmol / L nitrite stock solution was added to the above reaction solution according to a gradient and incubated for 15 min. The solution was diluted to 1.0 mL with ultrapure water to obtain the N-CDs / TMB ratio colorimetric sensor.

[0012] Furthermore, the concentration of the HAc-NaAc buffer solution is 0.2 M and the pH is 3.5.

[0013] The concentration ratio of the N-CDs to TMB is 100 μg / mL:100 μmol / L; and the final volume of the ratiometric colorimetric sensor is 1.0 mL.

[0014] The application of the metal-free carbon dot nanoenzyme ratio dual signal sensor in visual detection of nitrite is specifically described as follows: (1) Building a smartphone-assisted sensing platform: incubate the N-CDs / OPD fluorescent sensor solution or the N-CDs / TMB ratiometric colorimetric sensor solution with different concentrations of nitrite in a test tube; then, place the test tube in a dark box with an ultraviolet lamp or a fluorescent lamp on the top, turn on the ultraviolet lamp or the fluorescent lamp, and use the smartphone to take pictures through the monitoring window on the front of the dark box; (2) Acquisition of color information: The color of the image obtained is recognized by the smartphone and converted into R, G, and B values; (3) Establishment of standard curve: Through data processing, a standard curve of R / B or R / G ratio and nitrite concentration was established; through linear fitting of Origin software, the linear equation between the R / B ratio of the ratiometric N-CDs / OPD fluorescence sensor and the nitrite concentration was: R / B = 0.0048C （亚硝酸盐） + 0.3544, R 2 = 0.993, the linear range is: 2~150 μmol / L, the minimum detection limit is 1.92 μmol / L; the linear equation between the R / G ratio of the ratiometric N-CDs / TMB colorimetric sensor and the nitrite concentration is: R / G = 0.0048C （亚硝酸盐） + 0.5836 (R 2 = 0.995), the linear range is: 5~100 μmol / L, and the minimum detection limit is 1.36 μmol / L; (4) Determination of nitrite in actual samples: Replace the nitrite standard solution with the actual sample solution, carry out the reaction according to the steps, obtain the color information of the actual sample, and use a smartphone calculator to substitute the R / B or R / G value into the linear equation to calculate the nitrite content in the actual sample.

[0015] The UV lamp in step (1) is 365 nm.

[0016] Furthermore, the actual sample described in step (4) is pre-treated before detection. The specific treatment method is as follows: the actual sample is mixed with 50 g / L sodium tetraborate solution, ground into slurry with a blender, immersed in ultrapure water, and heated at 70°C for 15 minutes; then 106 g / L potassium ferrocyanide solution and 220 g / L zinc acetate solution are added to the above solution, stirred to precipitate protein, and diluted with ultrapure water to obtain a uniform solution.

[0017] The actual sample in step (4) is pickled pork, salted fish or beef jerky.

[0018] The present invention selects metal-free carbon dot nanozymes as candidates for dual-signal-dual-wavelength sensors to detect nitrite. Most importantly, the obvious color change of the dual-wavelength sensor is easier to be recognized by the naked eye compared to the single-wavelength sensor, which lays the foundation for building a more sensitive smartphone-assisted sensing platform. Combining smartphone devices, optical dual-signal output and ratio sensing strategies, not only can on-site real-time detection of targets be achieved, but also the accuracy of measurement data can be greatly improved.

[0019] Compared with the prior art, the present invention integrates smart phones, optical dual signal output and ratio sensing strategies in the same carbon dot nanozyme system. SThe N-CDs / OPD system produces the diazo compound benzotriazole (BTA), which produces an inner filter effect between N-CD and BTA and presents a ratiometric fluorescence response. Therefore, its ratiometric fluorescence signal can be further used for mobile phone-assisted visual fluorescence detection. Similarly, the N-CDs / TMB system first generates oxidized TMB (TMB) under the catalysis of N-CDs. ox ), which is then converted to diazotized TMB after the introduction of nitrite ox , and presents a ratiometric colorimetric response, so its ratiometric colorimetric signal can be further used for mobile phone-assisted visual colorimetric detection. In summary, compared with other methods for detecting nitrite, this method has the advantages of on-site visualization, high sensitivity, and good selectivity, providing a new method for nitrite detection. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a transmission electron microscopy image of the N-CD prepared in Example 1; Figure 2 The UV absorption and fluorescence spectra of N-CDs prepared in Example 1; Figure 3 is the UV absorption spectrum of the N-CDs prepared in Example 1 oxidizing TMB under light activation; Figure 4 The result diagram of the selectivity experiment of the mobile phone sensing platform based on N-CDs / OPD to different ions in Example 2; Figure 5 is a graph showing the change in fluorescence signal of the N-CDs / OPD ratio mediated by nitrite in Example 3; Figure 6 The fluorescence color change diagram of the N-CDs / OPD system in Example 3 under different concentrations of nitrite; Figure 7 This is a standard curve diagram for detecting nitrite using the mobile phone sensing platform based on N-CDs / OPD in Example 3; Figure 8 The result diagram of the selectivity experiment of the mobile phone sensing platform based on N-CDs / TMB to different ions in Example 5; Fig. 9 is a graph showing the colorimetric signal change of the N-CDs / TMB ratio mediated by nitrite in Example 6; Fig.10 This is a graph showing the visible light color change of N-CDs / TMB under different nitrite concentrations in Example 6; Fig.11 This is the standard curve diagram for detecting nitrite using the mobile phone sensing platform constructed based on N-CDs / TMB in Example 6. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0022] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0023] The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0024] Example 1: Preparation and characterization of N-CDs nanozymes Step 1: Weigh 0.1 g of tea polyphenols and 1.5 mmol of ethylenediamine, dissolve in 10 mL of ultrapure water, and perform ultrasonic treatment at 15 KHz and 25°C for 5 minutes; transfer the obtained clear solution to an autoclave containing a polytetrafluoroethylene liner, and heat it in an oven at 200°C for 8 hours; after the reaction is completed, the temperature is naturally cooled to room temperature to obtain a dark brown solution; Step 2: The obtained dark brown solution was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da and placed in water for 24 h. The water was changed every 6 h. The dialyzed liquid was passed through a 0.22 μm filter membrane. The filtrate was freeze-dried and the brown powder was collected, which was N-CDs. Step 3: Accurately weigh 0.1 g of N-CDs powder and dissolve it in 10 mL of ultrapure water to obtain a 10 mg / mL N-CDs stock solution.

[0025] For morphology and properties, see Figure 1 , Figure 2 and Figure 3 . Figure 1 It shows that the prepared N-CDs are spherical and the particle size is relatively uniform; Figure 2 The optical properties were demonstrated, with two broad absorption peaks of N-CDs at 280 nm and 340 nm, and the optimal excitation and emission wavelengths at 360 nm and 430 nm, respectively, indicating that N-CDs have photoluminescence properties; Figure 3 The enzyme-like properties of N-CDs were demonstrated, which can oxidize the substrate TMB to TMB under light. ox , indicating that N-CDs have photoactivated oxidase-like properties.

[0026] Example 2: Anti-interference experiment of detecting nitrite using a mobile phone sensing platform based on N-CDs / OPD Step 1: Dissolve sodium nitrite powder in ultrapure water to obtain a 0.01 mol / L nitrite standard solution. Step 2: Weigh different masses of chloride salts (NaCl, KCl, CaCl2, MgCl2, CuCl2, HgCl2, CdCl2) or sodium salts (NaF, NaCl, Na2S, NaNO3, NaHCO3, Na2SO4, Na3PO4), add 10 mL of ultrapure water, and prepare a 0.1 mol / L chloride or sodium salt stock solution; Step 3: Add 10 μL of chloride or sodium salt stock solution or nitrite standard solution and 20 μL of OPD (50 mmol / L) to 0.1 mol / L HCl solution. After reacting for 5 min, adjust the pH of the reaction solution to slightly alkaline (7.0-9.0) with 2 mol / L NaOH, then add 2 μL of blue luminescent N-CDs (10 mg / mL) and dilute to 1.0 mL with ultrapure water. At this time, the concentration ratio of N-CDs to OPD is 20 μg / mL:1 mmol / L. Step 4: Place the final solution obtained above in a dark box, and take a picture with a mobile phone under ultraviolet light. Then use the mobile phone software to perform color recognition and convert it into color information RGB. Use R / B as the vertical axis parameter to obtain a bar graph. The experimental results are shown in Figure 4 .

[0027] like Figure 4 As shown, after introducing nitrite into the N-CD / OPD system, the R / B ratio increased significantly, while in the presence of other metal ions, the R / B ratio remained constant, indicating that the mobile phone sensing platform constructed based on N-CDs / OPD has good selectivity for nitrite detection.

[0028] Example 3: Establishment of a standard curve for nitrite detection based on a mobile phone sensing platform constructed based on N-CDs / OPD Step 1: Dissolve sodium nitrite powder in ultrapure water to obtain a nitrite standard solution with a concentration of 0.01 mol / L. Step 2: Add different volumes of nitrite standard solution and 20 μL OPD (50 mmol / L) to 400 μL HCl (0.1mol / L) solution. After 5 min, adjust the pH to slightly alkaline (7.0-9.0) with 2 mol / L NaOH solution, then add 2 μL N-CDs (10 mg / mL) and dilute to 1 mL with ultrapure water. At this time, the concentration ratio of N-CDs to OPD is 20 μg / mL: 1mmol / L. Measure the fluorescence intensity respectively, and the fluorescence change is shown in Figure 5 .

[0029] Step 3: Place the final solutions obtained above in a dark box, take pictures with a mobile phone under ultraviolet light, and the picture results at different nitrite concentrations are shown in Figure 6 .

[0030] Step 4: Use mobile phone software to perform color recognition on the obtained image and convert it into color information RGB. Use R / B as the ordinate and nitrite concentration as the abscissa to draw a standard curve. The linear fitting results are shown in Figure 7 .

[0031] Figure 5 It was shown that nitrite can mediate the change of the ratio fluorescence signal of N-CDs / OPD, which makes the fluorescence color change more obvious, which is conducive to the construction of a mobile phone sensing platform for the visual detection of nitrite; Figure 6 The fluorescence color change diagram of N-CDs / OPD under different concentrations of nitrite is presented; Figure 7 It can be seen that the linear equation between the R / B ratio of the N-CDs / OPD ratio fluorescence sensor and the nitrite concentration is: R / B = 0.0048C （亚硝酸盐） + 0.3544, R 2 = 0.993, the linear range is: 2~150 μmol / L, and the minimum detection limit is 1.92 μmol / L.

[0032] Example 4: Detection and spike recovery experiment of nitrite in actual samples based on the mobile phone sensing platform constructed based on N-CDs / OPD Step 1: Mix the pickled pork, salted fish or beef jerky samples with sodium tetraborate solution (50 g / L), grind them into slurry with a blender, immerse them in ultrapure water, and heat them at 70°C for 15 min. Then treat the above solution with potassium ferrocyanide solution (106 g / L) and zinc acetate solution (220 g / L) to precipitate the protein, and dilute with ultrapure water to obtain a uniform solution for later use.

[0033] Step 2: Add 400 μL of the sample solution and 20 μL of OPD (50 mmol / L) to 500 μL of HCl (0.1 mol / L) solution. After 5 min, adjust the pH to slightly alkaline (7.0-9.0) with 2 mol / L NaOH solution, then add 2 μL of N-CDs (10 mg / mL) and dilute to 1.0 mL with ultrapure water. At this time, the concentration ratio of N-CDs to OPD is 20 μg / mL:1 mmol / L. Then, place the final solution in a dark box, take pictures with a mobile phone under ultraviolet light, and then use mobile phone software to perform color recognition on the pictures, convert them into color information RGB, and record them as R1 / B1.

[0034] Step 3: Substitute R1 / B1 into the linear equation and calculate the nitrite content in the corresponding sample, recorded as c1.

[0035] Step 4: Add 10 μL of nitrite standard solution (0.001 mol / L), 400 μL of the sample solution to be tested and 20 μL of OPD (50 mmol / L) to 500 μL of HCl (0.1 mol / L) solution. After 5 min, adjust the pH to slightly alkaline (7.0-9.0) with 2 mol / L NaOH solution, then add 2 μL of N-CDs (10 mg / mL) and dilute to 1.0 mL with ultrapure water. Then, place the final solution in a dark box, take pictures with a mobile phone under ultraviolet light, and then use mobile phone software to perform color recognition on the pictures, convert them into color information RGB, and record them as R2 / B2.

[0036] Step 5: Substitute R2 / B2 into the linear equation to calculate the content of nitrite in the spiked sample, recorded as c2.

[0037] Step six, calculate the spike recovery rate according to the spike recovery formula (c2-c1) / c, where c is the known concentration of nitrite added.

[0038] The results are shown in Table 1. Table 1 shows that the spiked recovery rate of nitrite in actual samples is between 92.3% and 108.5%, indicating that the mobile phone sensing platform based on N-CDs / OPD can be used for the detection of nitrite in actual samples, and the method has good reproducibility.

[0039] Table 1: Nitrous acid content in actual samples and spike recovery results Example 5: Selective experiment of detecting nitrite based on the mobile phone sensing platform constructed by N-CDs / TMB Step 1: Dissolve sodium nitrite powder in ultrapure water to obtain a nitrite standard solution with a concentration of 0.01 mol / L.

[0040] Step 2: Weigh different masses of chloride salts (NaCl, KCl, CaCl2, MgCl2, CuCl2, HgCl2, CdCl2) or sodium salts (NaF, NaCl, Na2S, NaNO3, NaHCO3, Na2SO4, Na3PO4), add 10 mL of ultrapure water, and prepare 0.1 mol / L chloride or sodium salt stock solution.

[0041] Step 3: 10 μL TMB solution (10 mM) and 10 μL N-CD solution (10 mg / mL) were added to 500 μL HAc-NaAc buffer solution (0.2 M, pH 3.5) in sequence. The samples were irradiated under ultraviolet light for 15 min (365 nm, 20 μW / cm 2 ). Add 10 μL of chloride or sodium salt stock solution or nitrite standard and incubate for 15 min, dilute to 1.0 mL with ultrapure water, at which point the concentration ratio of N-CDs to TMB is 100 μg / mL: 100 μmol / L.

[0042] Step 4: Place the final solution obtained above in a dark box, and take a picture with a mobile phone under the illumination of a fluorescent lamp. Then use the mobile phone software to perform color recognition and convert it into color information RGB. A bar graph is obtained with R / G as the ordinate parameter. The experimental results are shown in Figure 8 .

[0043] like Figure 8 As shown, after introducing nitrite into the N-CD / TMB system, the R / G ratio increased significantly, while in the presence of other metal ions, the R / G ratio remained constant, indicating that the mobile phone sensing platform constructed based on N-CDs / TMB has good selectivity for nitrite detection.

[0044] Example 6: Establishment of a standard curve for nitrite detection based on the mobile phone sensor platform constructed based on N-CDs / TMB Step 1: Dissolve sodium nitrite powder in ultrapure water to obtain a nitrite standard solution with a concentration of 0.01 mol / L.

[0045] Step 2: Add 10 μL of TMB solution (10 mM) and 10 μL of N-CD solution (10 mg / mL) to 500 μL of HAc-NaAc buffer solution (0.2 mmol / L, pH 3.5) and irradiate under ultraviolet light for 15 min (365 nm, 20 μW / cm 2), then add different volumes of nitrite standard solution and incubate for 15 min, dilute to 1.0 mL with ultrapure water, at this time, the concentration ratio of N-CDs to TMB is 100 μg / mL: 100 μmol / L, measure the absorption intensity respectively, and the absorption change is shown in Fig. 9 .

[0046] Step 3: Place the final solutions obtained above in a dark box, take pictures with a mobile phone under fluorescent light, and the picture results at different nitrite concentrations are shown in Fig.10 .

[0047] Step 4: Use mobile phone software to perform color recognition on the obtained image and convert it into color information RGB. Use R / G as the ordinate and nitrite concentration as the abscissa to draw a standard curve. The linear fitting results are shown in Fig.11 .

[0048] Fig. 9 It was shown that nitrite can mediate the change of the ratiometric colorimetric signal of N-CDs / TMB, which makes the visible light color change more obvious, which is conducive to the construction of a mobile phone sensing platform for the visual detection of nitrite; Fig.10 The visible light color change graph of N-CDs / OPD under different concentrations of nitrite is presented; Fig.11 It can be seen that the linear equation between the R / G ratio of the N-CDs / TMB ratiometric sensor and the nitrite concentration is: R / G = 0.0048C （亚硝酸盐） + 0.5836 (R 2 = 0.995), the linear range was 5-100 μmol / L, and the minimum detection limit was 1.36 μmol / L.

[0049] Example 7: Detection and spike recovery experiment of nitrite in actual samples based on the mobile phone sensing platform constructed based on N-CDs / TMB Step 1: Mix the pickled pork, salted fish or beef jerky samples with sodium tetraborate solution (50 g / L), grind them into slurry with a blender, immerse them in ultrapure water, and heat them at 70°C for 15 min. Then treat the above solution with potassium ferrocyanide solution (106 g / L) and zinc acetate solution (220 g / L) to precipitate the protein, and dilute with ultrapure water to obtain a uniform solution for later use.

[0050] Step 2: Add 10 μL of TMB solution (10 mM) and 10 μL of N-CD solution (10 mg / mL) to 500 μL of HAc-NaAc buffer solution (0.2 mmol / L, pH 3.5) and irradiate under ultraviolet light for 15 min (365 nm, 20 μW / cm 2), add 400 μL of the sample solution to be tested, incubate for 15 min, and dilute to 1.0 mL with ultrapure water. At this time, the concentration ratio of N-CDs to TMB is 100 μg / mL:100 μmol / L; then, place the final solution in a dark box, take pictures with a mobile phone under fluorescent light, and then use mobile phone software to identify the color of the picture, convert it into color information RGB, and record it as R1 / G1.

[0051] Step 3: Substitute R1 / G1 into the linear equation and calculate the nitrite content in the corresponding sample, recorded as c1.

[0052] Step 4: Add 10 μL of TMB solution (10 mM) and 10 μL of N-CD solution (10 mg / mL) to 500 μL of HAc-NaAc buffer solution (0.2 mmol / L, pH 3.5) and irradiate under ultraviolet light for 15 min (365 nm, 20 μW / cm 2 ), add 400 μL of the sample solution to be tested and 10 μL of nitrite standard solution (0.001 mol / L), incubate for 15 min, and dilute to 1.0 mL with ultrapure water; then, place the final solution in a dark box, take pictures with a mobile phone under fluorescent light, and then use mobile phone software to perform color recognition on the pictures, convert them into color information RGB, and record them as R2 / G2.

[0053] Step 5: Substitute R2 / G2 into the linear equation to calculate the nitrite content in the spiked sample, recorded as c2.

[0054] Step six, calculate the spike recovery rate according to the spike recovery formula (c2-c1) / c, where c is the known concentration of nitrite added.

[0055] The results are shown in Table 2. Table 2 shows that the spike recovery rate of nitrite in actual samples is between 92.2% and 98.8%, indicating that the mobile phone sensing platform based on N-CDs / TMB can be used for the detection of nitrite in actual samples, and the method has good reproducibility.

[0056] Table 2: Nitrous acid content in actual samples and spike recovery results Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A dual-signal sensor based on metal-free carbon dot nanozyme ratio, Features: Using tea polyphenols as carbon precursor and ethylenediamine as nitrogen doping agent, nitrogen-doped carbon dot N-CDs nanozyme was obtained by a one-step hydrothermal synthesis method, and then dialyzed, filtered and freeze-dried to obtain metal-free carbon dot nanozyme N-CDs powder; utilizing the dual properties of photoluminescence and enzyme-like activity of N-CDs nanozyme and nitrite-mediated diazotization of substrates o-phenylenediamine OPD or 3,3′,5,5′-tetramethylbenzidine TMB, a nitrite-mediated ratiometric N-CDs / OPD fluorescence sensor or N-CDs / TMB ratiometric colorimetric sensor was constructed.

2. A dual-signal sensor based on metal-free carbon dot nanozyme ratio according to claim 1, Features: The preparation method of nitrogen-doped carbon dot N-CDs nanozyme includes: (1) Weigh 0.1 g of tea polyphenols and 1.5 mmol of ethylenediamine, dissolve in 10 mL of ultrapure water, and treat with ultrasound at 15 kHz and 25°C for 5 min. Transfer the resulting clear solution to an autoclave containing a polytetrafluoroethylene liner, heat the temperature to 200°C at a heating rate of 10°C / min, and keep the temperature for 8 h. After the reaction is completed, cool the temperature naturally to room temperature to obtain a dark brown solution. (2) The obtained dark brown solution was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da and placed in water for 24 h. The water was changed every 6 h. The dialyzed liquid was passed through a 0.22 μm filter membrane. The filtrate was freeze-dried at -60 °C for 36 h and the brown powder was collected, which was the N-CDs nanozyme.

3. A dual-signal sensor based on metal-free carbon dot nanozyme ratio according to claim 1, Features: The preparation method of the ratiometric N-CDs / OPD fluorescence sensor comprises: (1) Preparation of stock solutions: N-CDs powder was dissolved in ultrapure water to obtain a 10 mg / mL N-CDs stock solution; OPD powder was dissolved in ultrapure water to obtain a 50 mmol / L OPD stock solution; sodium nitrite powder was dissolved in ultrapure water to obtain 10 mmol / L and 1 mmol / L nitrite stock solutions; (2) Construction of ratiometric N-CDs / OPD fluorescent probe: 0.5-10 μL of 1 mmol / L nitrite stock solution or 1-55 μL of 10 mmol / L nitrite stock solution and 20 μL of OPD stock solution were sequentially introduced into 0.1 mol / L HCl solution to generate benzotriazole BTA. After 5 min of reaction, the pH of the reaction solution was adjusted to 7.0-9.0 with 2 mol / L NaOH to activate the yellow fluorescence of BTA. Subsequently, 2 μL of blue-luminescent N-CDs stock solution was thoroughly mixed with the above solution and diluted to 1.0 mL with ultrapure water to obtain a ratiometric N-CDs / OPD fluorescent sensor.

4. A dual-signal sensor based on metal-free carbon dot nanozyme ratio according to claim 3, Features: The concentration ratio of the N-CDs to OPD is 20 μg / mL:1 mmol / L; and the final volume of the ratiometric N-CDs / OPD fluorescent sensor is 1.0 mL.

5. A dual-signal sensor based on metal-free carbon dot nanozyme ratio according to claim 1, Features: The preparation method of the N-CDs / TMB ratio colorimetric sensor comprises: (1) Preparation of stock solutions: N-CDs powder was dissolved in ultrapure water to obtain a 10 mg / mL N-CDs stock solution; TMB powder was dissolved in ultrapure water to obtain a 10 mmol / L TMB stock solution; sodium nitrite powder was dissolved in ultrapure water to obtain 10 mmol / L and 1 mmol / L nitrite stock solutions; (2) Construction of N-CDs / TMB ratiometric sensor: 10 μL TMB stock solution and 10 μL N-CDs stock solution were added to 500 μL HAc-NaAc buffer solution in sequence; the colorimetric sensor was detected at 365 nm and 20 μW / cm 2 After irradiation with ultraviolet light for 15 min, TMB was catalyzed to oxidized TMB, namely TMB ox ; Subsequently, 0.5-10 μL of 1 mmol / L nitrite stock solution or 1-17.5 μL of 10 mmol / L nitrite stock solution was added to the above reaction solution according to a gradient and incubated for 15 min, and diluted to 1.0 mL with ultrapure water to obtain the N-CDs / TMB ratio colorimetric sensor.

6. A dual-signal sensor based on metal-free carbon dot nanozyme ratio according to claim 5, Features: The concentration of the HAc-NaAc buffer solution is 0.2 M, and the pH is 3.5; the concentration ratio of the N-CDs to TMB is 100 μg / mL:100 μmol / L; and the final volume of the ratiometric colorimetric sensor is 1.0 mL.

7. Application of the metal-free carbon dot nanozyme ratio dual signal sensor according to claim 1 in visual detection of nitrite, Features: The specific application method is: (1) Building a smartphone-assisted sensing platform: incubate the N-CDs / OPD fluorescent sensor solution or the N-CDs / TMB ratiometric colorimetric sensor solution with different concentrations of nitrite in a test tube; then, place the test tube in a dark box with an ultraviolet lamp or a fluorescent lamp on the top, turn on the ultraviolet lamp or the fluorescent lamp, and use the smartphone to take pictures through the monitoring window on the front of the dark box; (2) Acquisition of color information: The color of the image obtained is recognized by the smartphone and converted into R, G, and B values; (3) Establishment of standard curve: Through data processing, a standard curve of R / B or R / G ratio and nitrite concentration was established; through linear fitting of Origin software, the linear equation between the R / B ratio of the ratiometric N-CDs / OPD fluorescence sensor and the nitrite concentration was: R / B = 0.0048C （亚硝酸盐） + 0.3544, R 2 = 0.993, the linear range is: 2~150 μmol / L, the minimum detection limit is 1.92 μmol / L; the linear equation between the R / G ratio of the ratiometric N-CDs / TMB colorimetric sensor and the nitrite concentration is: R / G = 0.0048C （亚硝酸盐） + 0.5836 (R 2 = 0.995), the linear range is: 5~100 μmol / L, and the minimum detection limit is 1.36 μmol / L; (4) Determination of nitrite in actual samples: Replace the nitrite standard solution with the actual sample solution, carry out the reaction according to the steps, obtain the color information of the actual sample, and use a smartphone calculator to substitute the R / B or R / G value into the linear equation to calculate the nitrite content in the actual sample.

8. The use according to claim 7, Features: The UV lamp in step (1) is 365 nm.

9. The use according to claim 7, Features: The actual sample described in step (4) is pre-treated before detection. The specific treatment method is as follows: the actual sample is mixed with 50 g / L sodium tetraborate solution, ground into slurry with a blender, immersed in ultrapure water, and heated at 70°C for 15 min; then 106 g / L potassium ferrocyanide solution and 220 g / L zinc acetate solution are added to the above solution, stirred to precipitate protein, and diluted with ultrapure water to obtain a uniform solution.

10. The use according to claim 7, Features: The actual sample in step (4) is pickled pork, salted fish or beef jerky.

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