A method for detecting nitrite ions using a sensitive fluorescent sensor

Abstract

This invention discloses a sensitive fluorescence sensor for detecting NO in ham sausage samples. 2‑ The method belongs to the field of analytical detection. The steps are as follows: 1. Prepare carbon dots using citric acid and cauline as precursors; 2. NO in the carbon dots 2‑ The addition of [a specific substance] will quench fluorescence through dynamic quenching and diazotization effects, based on which NO [a specific substance] can be constructed. 2‑ Standard curves of NO concentration and fluorescence quenching degree were obtained, and the NO concentration in ham sausage samples was determined. 2‑ The concentration of NO. This invention is the first to use a sensitive fluorescence sensor to sequentially detect the concentration of NO. 2‑ It enables quantitative detection of substances and is simple, safe, and efficient, making it suitable for routine analysis.

Description

Technical Field

[0001] This invention relates to a sensitive fluorescence sensor for detecting NO2. - This method belongs to the field of analytical testing. Background Technology

[0002] Nitrite ions (NO2) - NO2 is one of the most common nitrogenous compounds in daily life. Before the invention of refrigeration technology, foods with high moisture content were generally not easy to preserve, leading to spoilage and unusable food. However, around the 10th century AD, the addition of saltpeter to meat products improved their shelf life. - Nitrites are widely used in people's daily lives, particularly in modern food processing. In prepared foods, they are extensively used as a food additive to enhance color, improve flavor, and inhibit bacterial growth. However, excessive nitrite intake can lead to reactions with amides in the body to form harmful nitrosamines, which can then bind to hemoglobin, inducing the production of methemoglobin and reducing the body's oxygen-carrying capacity. Under normal circumstances, the amount of nitrite used should not exceed 30 mg / kg. Therefore, to protect human health and prevent accidental ingestion of excessive nitrites, the amount of NO2 in food should be controlled. - Conducting this test has significant research value.

[0003] In recent years, various methods for detecting NO2 have been developed. - Analytical methods for NO2 mainly include high-performance liquid chromatography (HPLC), electrochemical methods, and chemiluminescence methods. While these methods offer high precision and reliability, they suffer from drawbacks such as poor selectivity, high toxicity, low sensitivity, and complex operation. Therefore, there is an urgent need to find a simpler, faster, and more sensitive method for NO2 analysis. - Detection methods.

[0004] Fluorescence-based detection has attracted increasing attention due to its low cost, ease of use, and high sensitivity and specificity. Among available fluorescent materials, carbon dots are an excellent choice due to their ease of synthesis and surface modification, excellent biocompatibility, tunable emission wavelength, excellent photostability, and high fluorescence quantum yield. The interaction between carbon dots and analytes induces changes in the fluorescence properties of carbon dots, such as intensity, wavelength, and lifetime, thereby generating multiple signals for selective and sensitive analyte monitoring. Therefore, the synthesis of carbon dots and the construction of green fluorescent probes still have significant research potential and require continuous exploration. Summary of the Invention

[0005] Technical issues

[0006] Currently, various analytical methods for detecting nitrite ions have been developed, mainly including high-performance liquid chromatography (HPLC), electrochemical methods, and spectrophotometry. These methods suffer from problems such as poor selectivity, high toxicity, low sensitivity, and complex operation. Meanwhile, fluorescence sensing technology is being used to detect NO2 in food. - There are few reports on this method, and its sensitivity and detection limit are not superior to traditional methods.

[0007] Technical solution

[0008] This invention provides a method for preparing a sensitive fluorescence sensor, comprising the following steps:

[0009] Citric acid, caustic soda, and water were mixed and reacted at high temperature to prepare a crude carbon dot solution. The crude carbon dot solution was then centrifuged, filtered to remove unreacted particles, and finally purified by dialysis to obtain a fluorescent sensor.

[0010] Furthermore, the mass ratio of citric acid to caustic acid is 8–12:1.

[0011] Furthermore, the mass ratio of citric acid to water is 1:8 to 12.

[0012] Furthermore, the high-temperature reaction is carried out at 180–200°C for 6–8 hours.

[0013] Furthermore, a 0.22 μM microporous membrane was used for centrifugation.

[0014] Furthermore, the molecular weight cutoff for dialysis is 1000 Da.

[0015] This invention provides a sensitive fluorescence sensor prepared by the above method.

[0016] The application of the sensitive fluorescence sensor provided by this invention in the field of nitrite ion detection.

[0017] Furthermore, the application refers to the detection of nitrite ions in aquatic environments and processed meat products.

[0018] This invention provides a method for preparing a sensitive fluorescent sensor for detecting nitrite ions, comprising the following steps:

[0019] Citric acid, caustic soda, and water were mixed and reacted at high temperature to prepare a crude carbon dot solution. The crude carbon dot solution was then centrifuged, filtered to remove unreacted particles, and finally purified by dialysis to obtain a fluorescent sensor.

[0020] Furthermore, the mass ratio of citric acid to caustic acid is 8–12:1.

[0021] Furthermore, the mass ratio of citric acid to water is 1:8 to 12.

[0022] Furthermore, the high-temperature reaction is carried out at 180–200°C for 6–8 hours.

[0023] Furthermore, a 0.22 μM microporous membrane was used for centrifugation.

[0024] Furthermore, the molecular weight cutoff for dialysis is 1000 Da.

[0025] This invention provides a sensitive fluorescent sensor for detecting nitrite ions prepared by the above method.

[0026] This invention provides a sensitive fluorescence sensor for detecting NO2. - The method includes the following steps:

[0027] (1) Prepare NO2 at different concentrations - Standard solution, NO2 - The standard solution and the fluorescence sensor solution were mixed evenly and added to an HCl / NaOH buffer solution with pH=3 to obtain the sample solution. After incubation for a period of time, fluorescence spectroscopy was performed.

[0028] (2) According to NO2 - The changes in fluorescence intensity before and after addition were used to construct the fluorescence quenching degree F0 / F and NO2. - Linear model of concentration;

[0029] (3) Mix the sample solution to be tested and the fluorescence sensor solution, add HCl / NaOH buffer, incubate for a period of time, and then perform fluorescence spectroscopy detection. Based on the linear model in step (2), the NO2 in the sample is obtained. - concentration.

[0030] In one embodiment of the present invention, the concentration of the fluorescence sensor solution in step (1) is 0.001 to 0.005 g / mL.

[0031] In one embodiment of the present invention, in step (1) the fluorescence sensor solution and NO2 - The volume ratio of the standard solution is 1:0.8 to 1.2.

[0032] In one embodiment of the present invention, the volume ratio of the fluorescence sensor solution to the HCl / NaOH buffer solution in step (1) is 1:8 to 12.

[0033] In one embodiment of the present invention, the incubation time in step (1) is 5 to 8 hours.

[0034] In one embodiment of the present invention, the conditions for fluorescence spectroscopy detection in step (1) are as follows: the fluorescence spectrum is measured using a fluorescence spectrometer, the width of the excitation slit and the emission slit of the spectrometer are both 3.0 nm, the integration time is 0.1 s; the excitation wavelength of the fluorescence spectrometer is 402 nm, the emission wavelength range is 420 nm-600 nm, and the step size is 1 nm.

[0035] In one embodiment of the present invention, the linear relationship described in step (2) is:

[0036] In an aquatic environment, I0 / I = 0.14914c(NO2) - )+0.94375 (0.5-10 μM) and I0 / I=0.36651c (NO2) - ) ±0.83801 (10-30 μM), where F0 and F represent NO2. - Fluorescence intensity of the system before and after addition, c(NO2) - ) represents NO2 - The concentration;

[0037] In the testing of processed meat products, I0 / I = 0.0261c (NO2) - +0.97536 (1-5 μM) and

[0038] I0 / I = 0.10364c(NO2) - )+0.5906(5-15μM), where F0 and F represent NO2, respectively. - Fluorescence intensity of the system before and after addition, c(NO2) - ) represents NO2 - The concentration.

[0039] In one embodiment of the present invention, the sample to be tested in step (3) includes processed meat products and environmental water.

[0040] In one embodiment of the present invention, when the sample to be tested is a processed meat product, the preparation of the sample solution in step (3) includes the following steps:

[0041] Meat products are mashed into a paste, then water and KOH solution (0.5-1M) are added and mixed. The mixture is then placed in a water bath at 60-80℃ for 3-5 minutes, and finally centrifuged and filtered. The supernatant is then collected to obtain the sample solution to be tested.

[0042] Furthermore, the ratio of the mass of processed meat products to the volume of water is 5g:50-100mL.

[0043] Furthermore, the ratio of the mass of processed meat products to the volume of KOH solution is 5g:1-2mL.

[0044] In one embodiment of the present invention, the volume ratio of the fluorescence sensor solution to the sample solution to be tested in step (3) is 1:0.8 to 1.2.

[0045] In one embodiment of the present invention, the volume ratio of the fluorescence sensor solution to the HCl / NaOH buffer solution in step (3) is 1:8 to 12.

[0046] In one embodiment of the present invention, the incubation time in step (3) is 5 to 8 hours.

[0047] In one embodiment of the present invention, the conditions for fluorescence spectroscopy detection in step (3) are as follows: the fluorescence spectrum is measured using a fluorescence spectrometer, the width of the excitation slit and the emission slit of the spectrometer are both 3.0 nm, the integration time is 0.1 s; the excitation wavelength of the fluorescence spectrometer is 402 nm, the emission wavelength range is 420 nm-600 nm, and the step size is 1 nm.

[0048] Beneficial effects

[0049] 1. This invention applies a sensitive fluorescence sensor to NO2. - The detection showed that the carbon dots were spherical or near-spherical in appearance and contained abundant functional groups on their surface, which improved their water solubility and affinity for NO2. - The ability to combine.

[0050] 2. This invention is the first to use carbon dots synthesized from citric acid and caustic soda as fluorescent sensors to detect NO2 in ham sausage samples. - This invention provides a quantitative detection method for NO2, which is simple, rapid, and safe, making it suitable for routine analysis. - The dynamic quenching effect caused by the interaction with the functional groups on the carbon dot surface and the diazotization effect has a significant impact on NO2. - It has good selectivity.

[0051] 3. The detection limit of this invention can reach 5.2 × 10⁻⁶ in a pure water testing environment. -8 With a concentration of mol / L, the detection limit in actual ham sausage samples can be as low as 6.7 × 10⁻⁶ mol / L. -7 With a sensitivity of mol / L, the product of this invention has extremely high sensitivity and is of great significance in the field of food testing.

[0052] 4. The present invention is simple to prepare, uses harmless raw materials, and has a simple and reliable experimental method. It can be used for measurement in relatively complex food environments and has practical application value. Attached Figure Description

[0053] Figure 1 To detect NO2 using a sensitive fluorescence sensor - The flowchart.

[0054] Figure 2 Different concentrations of NO2 were added to the system in Example 2. - Fluorescence spectrum at that time.

[0055] Figure 3 The fluorescence quenching degree and NO in Example 2 2- Concentration-relative curve.

[0056] Figure 4 For NO2 in Example 2, the fluorescence quenching level and concentration range are 0.5-10 μM. - The linear fitting curve.

[0057] Figure 5 For NO2 in Example 2, the fluorescence quenching level and concentration range are 10-30 μM. - The linear fitting curve.

[0058] Figure 6 The fluorescence quenching degree and NO in Example 3 2- Concentration-relative curve.

[0059] Figure 7 For NO2 in Example 3, the fluorescence quenching level and concentration range are 1-5 μM. - The linear fitting curve.

[0060] Figure 8 For NO2 in Example 3, the fluorescence quenching level and concentration range are 5-15 μM. - The linear fitting curve.

[0061] Figure 9 For example 4, NO2 is detected. - Selective test results graph.

[0062] Figure 10 For example 4, NO2 is detected. - The test results of anti-interference capability are shown in the figure.

[0063] Figure 11 Comparative Example 1 and Example 1 were compared by adding different concentrations of NO2. - Comparison of fluorescence intensity change rates before and after

[0064] Figure 12 For the detection of NO2 in Comparative Example 2 - Selective test results graph. Detailed Implementation

[0065] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention.

[0066] Example 1: Preparation of Carbon Dot (CDs) Solution

[0067] Weigh 1.5 g of citric acid and 0.15 g of methyl methacrylate into a beaker, add 15 mL of deionized water, and sonicate for 10 min to fully dissolve the mixture. Then react at 180 °C for 8 h. Centrifuge the synthesized crude carbon dot solution at 10,000 rpm for 10 min, then filter through a 0.22 μM microporous membrane to remove unreacted particles. Finally, dialyze the carbon dots through a dialysis membrane with a molecular weight cutoff of 1000 Da for 5 h to obtain the final carbon dots.

[0068] Example 2: Constructing a linear model for the determination of nitrite ions in a pure water environment

[0069] Prepare the following sample solutions: carbon dot solution (diluted 1000 times, i.e., concentration 0.001 g / mL), HCl / NaOH buffer (pH=3), and nitrite ion standard solutions with concentrations of 0 (blank control), 0.5 μM, 0.7 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, and 50 μM.

[0070] Take 0.3 mL of carbon dot solution and 0.3 mL of nitrite ion solution of different concentrations, then make up to 3 mL with HCl / NaOH buffer, let stand and incubate for 6 h to obtain solutions of different nitrite ions and perform fluorescence spectroscopy detection. The reaction temperature is 20 °C.

[0071] The fluorescence spectrum of the system was measured under the following conditions: excitation wavelength was 402 nm, emission wavelength scanning range was 420-600 nm, scanning was performed every 1 nm, and the slit width was set to 3.0 nm / 3.0 nm (excitation slit / emission slit). The fluorescence intensity peak I was obtained at 477 nm. A sample solution with a nitrite ion concentration of 0 was added for fluorescence spectroscopy detection, and the fluorescence intensity peak I0 was obtained at 477 nm. The fluorescence quenching degree I0 / I was recorded.

[0072] Plot the relationship between the fluorescence quenching degree of the sample solution and the nitrite ion concentration, such as... Figure 3 As shown, the fitting curves for the quenching degree and nitrite ions are as follows: Figure 4 , Figure 5 ,from Figure 4 , Figure 5 It can be seen that when the nitrite ion concentration is 0.5-10 μM and 10-30 μM, the degree of fluorescence quenching of the solution is linearly related to the nitrite ion concentration, with the linear equations being I0 / I = 0.14914c(NO2). -)+0.94375 and I0 / I=0.36651c(NO2) - The correlation coefficients were ±0.83801, and the correlation coefficients were R0.83801. 2 =0.9912 and R 2 =0.9952, detection limit is 5.2×10 -8 mol / L.

[0073] Example 3: Constructing a linear model for the determination of nitrite ions in ham sausage samples.

[0074] Sample solution preparation: carbon dot solution (diluted 1000 times, i.e., concentration of 0.001 g / mL), HCl / NaOH buffer (pH=3), ham sausage sample solution; Preparation of ham sausage sample solution: First, obtain 5 g of ham sausage sample, mash it into meat paste, then place the sample in a 250 ml Erlenmeyer flask, add 70 ml of water and 1 ml of KOH solution (1M), then sonicate the Erlenmeyer flask for 40 minutes, shaking the Erlenmeyer flask every 5 minutes. Afterwards, the solution was in a water bath at 75℃ for 5 minutes. After cooling to room temperature, the solution was centrifuged for 10 minutes and then filtered through a 0.22 μm microporous organic filter membrane to remove proteins and biomolecules. The supernatant was then collected and nitrite ions were added at concentrations of 0 (blank control), 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, and 15 μM to obtain standard solutions of ham sausage samples with different concentrations of nitrite ions.

[0075] Take 0.3 mL of carbon dot solution and standard solutions of ham sausage samples with different concentrations of nitrite ions, then make up to 3 mL with HCl / NaOH buffer, let stand and incubate for 6 h to obtain spiked ham sausage sample solutions with different nitrite ions and perform fluorescence spectroscopy detection. The reaction temperature is 20℃.

[0076] The fluorescence spectrum of the system was measured under the following conditions: excitation wavelength was 402 nm, emission wavelength scanning range was 420-600 nm, scanning was performed every 1 nm, and the slit width was set to 3.0 nm / 3.0 nm (excitation slit / emission slit). The fluorescence intensity peak I was obtained at 477 nm. A sample solution with a nitrite ion concentration of 0 was added for fluorescence spectroscopy detection, and the fluorescence intensity peak I0 was obtained at 477 nm. The fluorescence quenching degree I0 / I was recorded.

[0077] Plot the relationship between the fluorescence quenching degree of the sample solution and the nitrite ion concentration, such as... Figure 6 As shown, the fitting curves for the quenching degree and nitrite ions are as follows: Figure 7 , Figure 8 .from Figure 7 , Figure 8 It can be seen that when the nitrite ion concentration is 1-5 μM and 5-15 μM, the degree of fluorescence quenching of the solution is linearly related to the nitrite ion concentration, with the linear equations being I0 / I = 0.0261c(NO2). - )+0.97536 and I0 / I=0.10364c(NO2) - The correlation coefficients were R0.5906 and R1, respectively. 2 =0.9931 and R 2 =0.9966, detection limit is 6.7×10 -7 mol / L.

[0078] Example 4 investigates the selectivity and anti-interference ability of fluorescence "off" process in detecting nitrite ions.

[0079] Referring to Example 2, ultrapure water was used as the solution, and 17 different common metal ions (K+, K ... + Cu 2+ Zn 2+ Mg 2+ Co 2+ Fe 2+ Fe 3+ Pb 3+ Sn 2+ SO3 2- SO4 2- HCO3 - S 2- NO3 - HPO4 2- H2PO4 - F-) as interfering substances, such as Figure 9 As shown, I0 and I represent the fluorescence intensity before and after the addition of metal ions, respectively. Figure 7 As shown, the fluorescence intensity represents the fluorescence intensity after nitrite ions are added simultaneously with other ions. All ion concentrations were 10 μM, and all fluorescence detections were performed under the same conditions.

[0080] The test results show that although some ions interfere with the fluorescence intensity of carbon dots, the impact is not as significant as that of nitrite ions. In addition, the interfering substances do not show a significant change in fluorescence intensity for the detection of nitrite ions. Therefore, all factors are taken into account and they will not cause significant interference to the detection of nitrite ions.

[0081] Example 5: Detection of nitrite ions in the environment of ham sausage samples

[0082] Referring to Example 3, nitrite ions at concentrations of 4.5, 5.5, and 6.5 μM were measured, and the results are shown in Table 1.

[0083] Table 1 Test results of Example 4

[0084]

[0085] Comparative Example 1

[0086] The carbon dots were prepared according to the method described in "Method for the Detection of Nitrite with Orange Fluorescent Carbon Dots". The specific method is as follows:

[0087] 0.42 g of citric acid and 0.16 g of p-phenylenediamine were dissolved in 5 mL of deionized water and sonicated until completely dissolved. The solution was then transferred to a 25 mL polytetrafluoroethylene-lined container and heated at 180 °C for 4 h. The resulting CDs solution was centrifuged at 10000 rpm for 10 min, followed by continuous dialysis (dialysis bag molecular weight cutoff 500 Da) for 24 h to further purify the N-CDs aqueous solution and remove other small molecules. The dialyzed CDs were diluted for later use.

[0088] Take the solution from Comparative Example 1 and mix it with 1 mM NO at a volume ratio of 10:1. 2- The solutions were mixed, and after a five-minute reaction, a sample solution was obtained, and its fluorescence spectrum was measured.

[0089] like Figure 11 As shown, the fluorescence change rates before and after the addition of nitrite ions were compared between Comparative Example 1 and Example 1. The fluorescence change rate of Example 1 before and after the addition of 1 mmol / L nitrite ion solution was lower than that of Example 1 before and after the addition of 10 μmol / L nitrite ion solution. It can be seen that Example 1 has better sensitivity to nitrite ions and a more sensitive detection capability.

[0090] Comparative Example 2

[0091] Take the solution obtained from Comparative Example 1 and mix it with different types of ions at the same concentration as in Example 4. After reacting for 5 minutes, obtain the sample solution and measure its fluorescence spectrum.

[0092] like Figure 12 As shown, F0 and F represent the fluorescence intensity before and after the addition of metal ions, respectively. Some ions have a significant impact on the fluorescence intensity of the carbon dots obtained in Comparative Example 1. Figure 9 The effect of different ions on the fluorescence intensity of the carbon dots obtained in Example 1 is shown. The results indicate that the carbon dots obtained in Example 1 have better selectivity and can exhibit superior detection capability and anti-interference ability in more complex detection systems. These two points demonstrate the superiority of the carbon dots obtained in Example 1 in detecting nitrite ions.

[0093] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A fluorescent sensor for detecting nitrite ions, characterized in that, The fluorescence sensor is prepared according to the following steps: Citric acid, caustic soda, and water were mixed and reacted at high temperature to prepare a crude carbon dot solution. The crude carbon dot solution was then centrifuged, filtered to remove unreacted particles, and finally purified by dialysis to obtain a fluorescent sensor.

2. The fluorescence sensor according to claim 1, characterized in that, The mass ratio of citric acid to caustic soda is 8~12:1; the mass ratio of citric acid to water is 1:8~12.

3. The fluorescence sensor according to claim 1, characterized in that, The high-temperature reaction is carried out at 180~200℃ for 6~8 hours.

4. The fluorescence sensor according to claim 1, characterized in that, The detection of nitrite ions refers to the detection of the concentration of nitrite ions in the water environment or processed meat products.

5. A fluorescence sensor for detecting NO2 - The method is characterized by, Includes the following steps: (1) Prepare NO2 at different concentrations - Standard solution, NO2 - The standard solution and the fluorescence sensor solution were mixed evenly and added to an HCl / NaOH buffer solution with a pH of 2.5-3.5 to obtain a sample solution. After incubation for a period of time, fluorescence spectroscopy was performed. The fluorescence sensor solution was obtained by diluting the fluorescence sensor described in any one of claims 1-3. The concentration of the fluorescence sensor solution was 0.001-0.005 g / mL. The fluorescence sensor solution and NO2... - The volume ratio of the standard solution was 1:0.8~1.2; the volume ratio of the fluorescence sensor solution to the HCl / NaOH buffer solution was 1:8~12. (2) According to NO2 - The changes in fluorescence intensity before and after addition were used to construct the fluorescence quenching degree F0 / F and NO2. - Linear model of concentration; (3) Mix the sample solution to be tested and the fluorescence sensor solution and add them to an HCl / NaOH buffer solution with pH=2.5~3.

5. After incubation for a period of time, perform fluorescence spectroscopy detection and obtain the NO2 in the sample to be tested according to the linear model in step (2). - concentration.

6. The method according to claim 5, characterized in that, In steps (1) and (3), the incubation time is 5 to 8 hours.

7. The method according to claim 5, characterized in that, In steps (1) and (3), the conditions for fluorescence spectroscopy detection are as follows: the fluorescence spectrum is measured using a fluorescence spectrometer, the width of the excitation slit and the emission slit of the spectrometer are both 3.0 nm, the integration time is 0.1 s; the excitation wavelength of the fluorescence spectrometer is 402 nm, the emission wavelength range is 420 nm-600 nm, and the step size is 1 nm.

8. The method according to claim 5, characterized in that, In step (3), the samples to be tested include processed meat products and environmental water; When the sample to be tested is processed meat, the preparation of the sample solution includes the following steps: the processed meat is mashed into a paste, then water and 0.5~1M KOH solution are added and mixed, then the mixture is placed in a water bath at 60~80℃ for 3~5 minutes, and finally centrifuged and filtered to obtain the supernatant and the sample solution to be tested.

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