Nitrite detection reagent, nitrite detection method and application

Abstract

The invention relates to a nitrite detection reagent, a nitrite detection method and application. The nitrite detection reagent comprises TMB and iron alkoxide. In the nitrite detection reagent provided by the invention, colorless TMB reacts with oxygen by taking IA as a catalyst, so that an oxidation product TMB < + > of the TMB is formed, and the TMB < + > is blue; on the basis, when the TMB < + > is in contact with the nitrite, the TMB < + > reacts with the nitrite, so that a diazotization reaction is carried out, and green diazotized TMB < + > is formed. Therefore, the nitrite detection reagent disclosed by the invention can realize detection of nitrite by observing the color change of the nitrite detection reagent through naked eyes under sunlight irradiation. The detection mode does not need to depend on instruments, and low-cost rapid detection of nitrite can be realized.

Description

Technical Field

[0001] The present invention relates to the field of nitrite detection, in particular to a nitrite detection reagent, a nitrite detection method and applications. Background Art

[0002] Nitrite, a key form of nitrogen in nature, is widely used for its remarkable effectiveness in food preservation and microbial growth inhibition. However, long-term and excessive intake of nitrite can cause food poisoning and cancer, posing a significant threat to human health. Therefore, nitrite detection is of vital importance.

[0003] At present, the detection of nitrite mainly relies on chromatography, colorimetry and other methods. These methods are more dependent on professional instruments, have relatively high costs, and are not conducive to large-scale promotion. Summary of the Invention

[0004] Based on this, it is necessary to provide a nitrite detection reagent, a nitrite detection method and an application to address the problem of high cost of nitrite detection.

[0005] The technical solution provided by the present invention is: A nitrite detection reagent comprises TMB and iron alkoxide.

[0006] In one embodiment, the nitrite detection reagent further includes a solvent, the pH of the nitrite detection reagent is between 3-5, the concentration of iron alkoxide is not less than 80 μM, and the concentration of TMB is not less than 3 μM.

[0007] In one embodiment, carbon quantum dots are further included. The preparation method of the carbon quantum dots comprises: dissolving melamine and o-phenylenediamine in a solvent and transferring the solvent to a reactor, and reacting at 150° C.-170° C.

[0008] In one embodiment, the nitrite detection reagent further includes a solvent, the pH of the nitrite detection reagent is between 3-5, and the concentration of the carbon quantum dots is 1.8 mg / L-2.4 mg / L.

[0009] An application of the nitrite detection reagent in detecting nitrite.

[0010] A method for detecting nitrite, comprising: Obtain the UV-vis spectrum of the nitrite detection reagent and determine the absorption peak intensity a at 457 nm. 457 And the absorption peak intensity at 652nm a 652 ; The nitrite detection reagent is added to the test solution and the UV-vis spectrum of the nitrite detection reagent is obtained again, and the absorption peak intensity A at 457 nm is determined from the spectrum. 457 And the absorption peak intensity A at 652nm 652 ; At least based on a 457 、a 652 、A 457 、A 652 Determine the presence and concentration of nitrite in the test solution.

[0011] A nitrite detection method comprises: adding the nitrite detection reagent into a solution to be tested, and determining the presence and concentration of nitrite in the solution to be tested according to the color of the nitrite detection reagent.

[0012] A method for detecting nitrite, comprising: Obtain the PLE spectrum of the nitrite detection reagent and determine the fluorescence intensity i at 371 nm from it. 371 and the fluorescence intensity i at 470 nm 470 ; The nitrite detection reagent is added to the test solution and the PLE spectrum of the nitrite detection reagent is obtained again, and the fluorescence intensity I at 371 nm is determined from the spectrum. 371 and the fluorescence intensity I at 470 nm 470 ; At least based on i 371 、i 470 , I 371 , I 470 Determine the presence and concentration of nitrite in the test solution.

[0013] A method for preparing carbon quantum dots comprises the following steps: dissolving melamine and o-phenylenediamine in a solvent, transferring the solvent to a reactor, and reacting the solvent at a temperature of 150° C. to 170° C.

[0014] Catalytic application of iron alkoxides in TMB oxidation.

[0015] The beneficial effects of the present invention are: In the nitrite detection reagent provided by the present invention, colorless TMB will react with oxygen using IA as a catalyst to form TMB oxidation product TMB + , TMB + In blue; on this basis, when TMB + When it comes into contact with nitrite, it will react with nitrite, resulting in a diazotization reaction, thereby forming green diazotized TMB + .

[0016] Therefore, the nitrite detection reagent of the present invention can detect nitrite by observing its color change with the naked eye under sunlight. This detection method does not require instrumentation and can achieve low-cost and rapid detection of nitrite. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 UV-vis spectrum of IA in Example 1 of the present invention; UV-vis spectrum of TMB; UV-vis spectrum of the mixture of IA and TMB; UV-vis spectrum of IA, TMB and NO2 - UV-vis spectrum of the mixture; Figure 2 is the absorption peak intensity at 652 nm in the UV-vis spectrum of the nitrite detection reagent in Example 1 of the present invention under different pH conditions; Figure 3 is the absorption peak intensity at 652 nm in the UV-vis spectrum of the nitrite detection reagent under different concentration conditions of IA in Example 1 of the present invention; Figure 4 is the absorption peak intensity at 652 nm in the UV-vis spectrum of the nitrite detection reagent under different concentration conditions of TMP in Example 1 of the present invention; Figure 5 The UV-vis spectra of the nitrite detection reagent after mixing with nitrite of different concentrations in Example 1 of the present invention; Figure 6 After the nitrite detection reagent in Example 1 of the present invention is mixed with nitrite of different concentrations, A 457 / A 652 and nitrite concentration; Figure 7 The UV-vis spectrum, PL spectrum and PLE spectrum of the carbon quantum dots in Example 2 of the present invention; Figure 8 is the three-dimensional fluorescence spectrum of the carbon quantum dots in Example 2 of the present invention; Figure 9 The PLE spectra of carbon quantum dots mixed with different substances in Example 2 of the present invention; Figure 10 The UV-vis spectrum of the carbon quantum dots in Example 2 of the present invention, the diazotized TMB + UV-vis spectra, carbon quantum dots and diazotized TMB + Theoretical UV-vis spectra of mixed carbon quantum dots and diazotized TMB + The actual UV-vis spectrum after mixing; Figure 11 The three-dimensional fluorescence spectrum of the carbon quantum dots and the diazotized TMB in Example 2 of the present invention +UV-vis spectrum; Figure 12 The fluorescence quenching efficiency of carbon quantum dots at different temperatures after the nitrite detection reagent in Example 2 of the present invention is mixed with nitrite; Figure 13 The fluorescence quenching efficiency of carbon quantum dots at different pH values after the nitrite detection reagent in Example 2 of the present invention is mixed with nitrite; Figure 14 The fluorescence quenching efficiency of the carbon quantum dots after the nitrite detection reagent in Example 2 of the present invention is mixed with nitrite for different times; Figure 15 The fluorescence quenching efficiency of carbon quantum dots at different concentrations after the nitrite detection reagent in Example 2 of the present invention is mixed with nitrite; Figure 16 The PLE spectra of carbon quantum dots after the nitrite detection reagent in Example 2 of the present invention is mixed with nitrite at different concentrations; Figure 17 For the nitrite concentration and I in Example 2 of the present invention 371 / I 470 The functional relationship between them. DETAILED DESCRIPTION

[0018] To make the above-mentioned objects, features, and advantages of the present invention more readily apparent, specific embodiments of the present invention are described in detail below. The following description sets forth numerous specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0019] Example 1

[0020] This embodiment provides a nitrite detection reagent, including 3,3',5,5'-tetramethylbenzidine (TMB) and iron alkoxide (IA).

[0021] The preparation method of IA includes the following steps: 180mL of ethylene glycol, 1.2g of FeCl3·6H2O, and 5.4g of urea are mechanically stirred for 20 minutes, and then the above mixture is reacted at 195°C for 30 minutes. After that, the IA particles are recovered by centrifugation, washed three times with alcohol and deionized water, and then dried at 60°C.

[0022] The nitrite detection reagent was prepared as follows: an aqueous solution of NaAc-HAc, 20 μL of an aqueous solution of TMB (3 μM), and 1 mg of IA were mixed and stirred uniformly.

[0023] The NaAc-HAc aqueous solution was prepared as follows: 0.2 M NaAc aqueous solution and 0.2 M HAc aqueous solution were mixed at a volume ratio of 1:1, and then the pH was adjusted to 4.

[0024] The mechanism of the nitrite detection reagent in this embodiment for detecting nitrite includes two parts. First, TMB will react with oxygen using IA as a catalyst to form TMB oxidation product TMB. + On this basis, TMB + It will react with nitrite to form diazotization TMB + .

[0025] Specific as Figure 1 As shown in the figure, the UV-vis spectra of IA alone and TMB alone do not have absorption peaks at 457 nm and 652 nm. However, when IA and TMB are mixed, IA can act as an oxidase mimic, converting the dissolved oxygen in the mixture into superoxide radicals, thereby oxidizing TMB to TMB. + , which ultimately leads to the mixture of IA and TMB producing two absorption peaks at 457nm and 652nm, among which the absorption peak intensity at 652nm is relatively strong, and the absorption peak intensity at 457nm is relatively weak.

[0026] On this basis, when nitrite is further added to the mixture of IA and TMB, TMB + The aromatic primary amine group in the nitrite reacts with the nitrite to produce diazotization and convert into diazotized TMB. + , diazotized TMB + While increasing the absorption peak intensity at 457 nm, the absorption peak intensity at 652 nm is also weakened, thereby causing the ratio between the absorption peak intensity at 457 nm and the absorption peak intensity at 652 nm to change. In other words, after adding the nitrite detection reagent of this embodiment to the test solution, based on the absorption peak intensity at at least one of 457 nm and 652 nm, and / or the ratio of the two absorption peak intensities, it is possible to determine whether nitrite is present in the test solution and to detect the concentration of nitrite.

[0027] Not only that, from Figure 1 As can be seen from the three photos taken in daylight, TMB solution is colorless. When TMB is oxidized to form TMB + After TMB +The solution turns from transparent to blue. Therefore, based on the solution color of the nitrite detection reagent of this embodiment, the degree of oxidation of TMB can be judged by the naked eye. + After diazotization, diazotized TMB + The color of the solution changes from blue to green. Therefore, by observing the color change of the solution with the naked eye under daylight, it is possible to determine whether nitrite is present in the test solution and, to a certain extent, to determine the concentration of nitrite. Thus, the nitrite detection reagent of this embodiment can achieve rapid detection of nitrite without relying on an instrument.

[0028] In order to improve the sensitivity of the nitrite detection reagent of this embodiment to nitrite detection, this embodiment conducted several investigations on its detection conditions.

[0029] like Figure 2 As shown, when pH=4, the absorption peak intensity of the nitrite detection reagent (TMB+IA) of this embodiment is the largest at 652nm. Accordingly, the color of the final nitrite detection reagent solution is the bluest, and the color change will be the most obvious when nitrite is detected later. The detection sensitivity of nitrite is also the highest. Therefore, pH = 4 is the optimal pH value for nitrite detection by nitrite detection reagent. Figure 3 and Figure 4 When the IA concentration is not less than 80 μM and the TMB concentration is not less than 3 μM, the color of the nitrite detection reagent solution is bluer and the detection sensitivity of nitrite is the highest. Finally, the solution of the nitrite detection reagent of this embodiment is used to detect nitrite at a TMB concentration of 3 μM, an IA concentration of 80 μM, and a pH of 4.

[0030] See also Figure 5 and Figure 6 As shown, after the nitrite detection reagent is mixed with different concentrations of nitrite, the higher the concentration of nitrite, the stronger the absorption peak intensity A at 457nm in the UV-vis spectrum of the nitrite detection reagent. 457 The stronger the absorption peak intensity A at 652nm 652 The weaker, in other words, A 457 / A 652 There is a positive correlation between nitrite concentration and A. 457 / A 652 There is a high linear relationship between them, based on A 457 / A 652 The detection limit of nitrite is 73 nM.

[0031] Example 2

[0032] The difference between this embodiment and embodiment 1 is that the nitrite detection reagent includes carbon quantum dots in addition to 3,3',5,5'-tetramethylbenzidine (TMB) and iron alkoxide (IA).

[0033] The synthesis method of carbon quantum dots is as follows: 1 mmol of melamine and 1 mmol of o-phenylenediamine (OPD) are dissolved in 20 mL of N,N-dimethylformamide (DMF) and ultrasonicated for 15 minutes; the ultrasonicated mixture is transferred to a reactor and reacted at 160°C for 10 hours; the reaction product is cooled to room temperature and then filtered with a 0.22 μm filter mesh, and the filtrate is transferred to a vacuum evaporation to obtain the carbon quantum dots.

[0034] The nitrite detection reagent of this example was prepared as follows: an aqueous solution of NaAc-HAc, 20 μL of an aqueous solution of TMB (3 μM), and 1 mg of IA were mixed, followed by the addition of 60 μL of an aqueous solution of carbon quantum dots and stirring. The NaAc-HAc aqueous solution was prepared in the same manner as in Example 1 and will not be further described in this example.

[0035] In addition, the basic morphological and structural characteristics of the carbon quantum dots in this embodiment have been disclosed in the prior application 202310467205.X and are therefore not further described in this embodiment. Due to experimental errors, changes in test conditions, and other factors, the optical properties of the carbon quantum dots in this embodiment may deviate to a certain extent from those in the prior application. To ensure the accuracy of the final experimental results of this embodiment, this embodiment retested the optical properties of the carbon quantum dots. For any conflicts or significant deviations between the optical properties of the carbon quantum dots in this embodiment and those in the prior application, the actual test results of this embodiment shall prevail.

[0036] like Figure 7 As shown, when the concentration of carbon quantum dots is 2.4 mg / ml, its UV-vis spectrum has an absorption peak near 287 nm, corresponding to the aromatic sp 2 The π-π* transition in the structural domain has an absorption peak in the UV-vis spectrum within the range of 350-460nm, which corresponds to the n-π* transition of the surface state of the carbon quantum dots. Figure 7 PL and PLE spectra in the Figure 8 It can be seen that the emission wavelength λ of the carbon quantum dots in this embodiment is em is 533 nm, and the corresponding two excitation wavelengths λ ex1 =371nm, λ ex2 =470nm.

[0037] The detection mechanism of nitrite detection reagent in this embodiment is different from that in embodiment 1. In this embodiment, the nitrite detection reagent is detected by diazotization of TMB.+ The fluorescence of carbon quantum dots is quenched to achieve the detection of nitrite.

[0038] Specific as Figure 9 As shown, carbon quantum dots alone mixed with TMB, carbon quantum dots alone mixed with IA, carbon quantum dots alone mixed with NO2 - In the case of , none of them could significantly change the excitation wavelength and fluorescence intensity of carbon quantum dots, which indicates that TMB, IA, NO2 - Neither of them reacts with carbon quantum dots, and almost does not cause fluorescence quenching of carbon quantum dots. This ensures the stability of carbon quantum dots in nitrite detection reagents and is not easily oxidized. On the other hand, it also proves that carbon quantum dots do not directly react with NO2 - Conduct testing.

[0039] Furthermore, after the carbon quantum dots were mixed with TMB and IA, the excitation wavelength and fluorescence intensity were almost unchanged, indicating that TMB + It also cannot react directly with carbon quantum dots, nor will it cause fluorescence quenching of carbon quantum dots. - After that, the excitation wavelength and fluorescence intensity hardly changed, indicating that NO2 - The oxidized products cannot cause fluorescence quenching of carbon quantum dots.

[0040] However, carbon quantum dots mixed with TMB, IA and NO2 - After that, the fluorescence intensity of carbon quantum dots decreased significantly, indicating that the diazotized TMB + It can cause the fluorescence quenching of carbon quantum dots, based on which the carbon quantum dots can detect nitrite. In addition, carbon quantum dots can also mix TMB and NO2 - In the absence of IA, the fluorescence intensity of carbon quantum dots did not change significantly, which indirectly proved that TMB could not be effectively converted into TMB without IA assistance. + , and further diazotization cannot be achieved, which also proves the indispensability of IA in the nitrite detection reagent system of Example 1 and Example 2.

[0041] To explore the diazotization of TMB + In this example, several experiments were conducted on the fluorescence quenching mechanism of carbon quantum dots.

[0042] First, as Figure 10 As shown, the UV-vis spectra of carbon quantum dots (N-CDs) and diazotized TMB + (diazotized TMB + ) UV-vis spectrum (N-CDs+diazotized TMB)+ ), compared with carbon quantum dots and diazotized TMB + mixture (N-CDs@diazotized TMB + ) of the UV-vis spectrum, no peak shift occurs, which firstly indicates that the carbon quantum dots and diazotized TMB + No new compounds were produced.

[0043] Further, see Figure 11 The PL and PLE spectra of carbon quantum dots are consistent with those of diazotized TMB. + The absorption spectra of diazotized TMB + The fluorescence quenching mechanism of carbon quantum dots includes at least the inner filter effect (IFE) and fluorescence resonance energy transfer (FRET).

[0044] Furthermore, when the excitation wavelength is 371 nm, diazotized TMB + The fluorescence lifetime of carbon quantum dots will increase from 3.20ns to 3.26s. When the excitation wavelength is 470nm, the diazotized TMB + The fluorescence lifetime of carbon quantum dots will increase from 3.07ns to 3.13s, which proves that diazotized TMB + The fluorescence quenching mechanism of carbon quantum dots is mainly based on the inner filtering effect, supplemented by fluorescence resonance energy transfer.

[0045] It is not difficult to understand that due to the diazotization of TMB + The carbon quantum dots are simultaneously subjected to fluorescence quenching based on the inner filter effect and fluorescence resonance energy transfer. Therefore, after the nitrite detection reagent of this embodiment is mixed with nitrite, not only the PLE spectrum of the carbon quantum dots will undergo fluorescence quenching, but the PL spectrum will also undergo quenching. In other words, the ratio of the fluorescence intensity at 533 nm in the PL spectrum of the carbon quantum dots before and after the nitrite detection reagent is mixed with nitrite can be used to determine the presence of diazotized TMB. + The specific fluorescence quenching efficiency of carbon quantum dots. The greater the fluorescence quenching efficiency, the stronger the carbon quantum dots' ability to detect nitrite and the higher the accuracy.

[0046] This example further conducted several experiments to determine the optimal detection conditions for carbon quantum dots to nitrite. Figure 12-15 The fluorescence quenching efficiency in the middle vertical axis is obtained through the PL spectrum of carbon quantum dots.

[0047] See also Figure 12 After the nitrite detection reagent is mixed with nitrite, even if it is heated to 60°C, the fluorescence quenching efficiency of the carbon quantum dots remains almost unchanged. Therefore, when the nitrite detection reagent is used to detect nitrite, the temperature can be any temperature between 15°C and 60°C.

[0048] See also Figure 13 After the nitrite detection reagent is mixed with nitrite, the fluorescence quenching efficiency of the carbon quantum dots gradually increases as the pH rises from 1 to 4, and then the fluorescence quenching efficiency of the carbon quantum dots gradually decreases as the pH rises from 4 to 11. As the pH rises from 11 to 14, the fluorescence quenching efficiency remains almost unchanged. Based on this, the optimal pH for the nitrite detection reagent to detect nitrite is 4. It is worth noting that this condition is exactly the same as Figure 2 The results shown in are consistent, which confirms the reliability and accuracy of the experimental results of this embodiment.

[0049] See also Figure 14 After the nitrite detection reagent is mixed with nitrite, the fluorescence quenching efficiency of the carbon quantum dots gradually increases with the increase of mixing time. After 120 seconds of mixing, the fluorescence quenching efficiency is roughly stable. Therefore, after the nitrite detection reagent is added to the test solution, it is preferably 120 seconds before the nitrite detection is performed.

[0050] See also Figure 15 After the nitrite detection reagent is mixed with nitrite, as the concentration of carbon quantum dots increases, the fluorescence quenching efficiency first increases and then decreases. When the concentration of carbon quantum dots reaches 2.4 mg / ml, the fluorescence quenching efficiency reaches the maximum. Therefore, when the nitrite detection reagent is used to detect nitrite, the concentration of carbon quantum dots should be maintained at 2.4 mg / ml.

[0051] Further Figure 16 and Figure 17 As shown in the figure, with the increase of nitrite concentration, the fluorescence intensity I at 371 nm in the PLE spectrum of carbon quantum dots increases. 371 The fluorescence intensity at 470 nm decreases slowly. 470 Rapidly reduce. Eventually make the nitrite concentration and I 371 / I 470 There is a positive correlation between the concentration of nitrite and I. 371 / I 470 A good linear relationship can be maintained between the two, and the corresponding lower limit of nitrite detection is 49 nM. Therefore, the nitrite detection reagent of this embodiment can achieve quantitative detection of nitrite concentration.

[0052] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0053] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A nitrite detection reagent, characterized in that, Includes TMB and iron alkoxide.

2. The nitrite detection reagent according to claim 1, wherein The nitrite detection reagent further includes a solvent. The pH of the nitrite detection reagent is between 3 and 5. The concentration of the iron alkoxide is not less than 80 μM, and the concentration of TMB is not less than 3 μM.

3. The nitrite detection reagent according to claim 1, wherein The invention also includes carbon quantum dots. The preparation method of the carbon quantum dots includes: dissolving melamine and o-phenylenediamine in a solvent and transferring the solvent to a reactor, and reacting at 150-170°C.

4. The nitrite detection reagent according to claim 3, wherein The nitrite detection reagent also includes a solvent, the pH of the nitrite detection reagent is between 3 and 5, and the concentration of the carbon quantum dots is 1.8 mg / L-2.4 mg / L.

5. Use of the nitrite detection reagent according to any one of claims 1 to 4 in detecting nitrite.

6. A method for detecting nitrite, characterized in that: include: Obtain the UV-vis spectrum of the nitrite detection reagent as claimed in claim 1 or 2, and determine the absorption peak intensity a at 457 nm therefrom. 457 And the absorption peak intensity at 652nm a 652 ; The nitrite detection reagent according to claim 1 or 2 is added to the test solution and the UV-vis spectrum of the nitrite detection reagent is obtained again, and the absorption peak intensity A at 457 nm is determined therefrom. 457 And the absorption peak intensity A at 652nm 652 ; At least based on a 457 、a 652 、A 457 、A 652 Determine the presence and concentration of nitrite in the test solution.

7. A method for detecting nitrite, characterized in that: include: The nitrite detection reagent according to claim 1 or 2 is added to a solution to be tested, and the presence and concentration of nitrite in the solution to be tested are determined according to the color of the nitrite detection reagent.

8. A method for detecting nitrite, characterized in that: include: Obtain the PLE spectrum of the nitrite detection reagent as claimed in claim 3 or 4, and determine the fluorescence intensity i at 371 nm therefrom. 371 And the fluorescence intensity i at 470 nm 470 ; The nitrite detection reagent as claimed in claim 3 or 4 is added to the test solution and the PLE spectrum of the nitrite detection reagent is obtained again, and the fluorescence intensity I at 371 nm is determined therefrom. 371 And the fluorescence intensity I at 470 nm 470 ; At least based on i 371 、i 470 , I 371 , I 470 Determine the presence and concentration of nitrite in the test solution.

9. An application of carbon quantum dots in detecting nitrite, characterized in that: The preparation method of the carbon quantum dots comprises: dissolving melamine and o-phenylenediamine in a solvent, transferring the solvent to a reaction kettle, and reacting at 150° C.-170° C.

10. Catalytic application of iron alkoxide in TMB oxidation process.

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