Hydrogel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of gel preparation and nitrite detection, and particularly relates to hydrogel as well as a preparation method and application thereof. The invention develops a pretreatment-free colorimetric detection method for rapid determination of nitrite. A diazotization oxidation reaction of nitrite and TMB under an acidic condition enables the solution to generate color change from colorless to orange, and the color change process can be observed by naked eyes. And the absorbance value and the image gray value at 450nm can be used for quantitative detection. Based on the principle, hydrogel is introduced to construct the nitrite sensor. By utilizing the inherent three-dimensional grid structure of the hydrogel, macromolecules such as protein and fat compounds are blocked outside the gel and only can be retained on the surface layer of the gel; and nitrite permeates into the gel along with water to initiate a chromogenic reaction. The bottom image of the gel is collected through the smart phone, and the gray value of the gel is analyzed, so that the nitrite concentration can be quantified.

Description

Technical Field

[0001] This invention belongs to the field of gel preparation and nitrite detection technology, and specifically relates to a hydrogel, its preparation method and application. Background Technology

[0002] Nitrites are common nitrogenous compounds that naturally exist in water bodies and various foods, including meat products, fruits, and vegetables. They are used as preservatives and antibacterial agents; however, excessive intake of nitrites can lead to serious health risks, including methemoglobinemia, gastrointestinal cancers, and even fatal poisoning at high doses. The World Health Organization (WHO) and national food safety standards have set strict limits for nitrite content in food. Therefore, developing rapid and highly selective methods for detecting nitrites is of great significance for ensuring food safety and public health.

[0003] In recent years, various methods for nitrite detection have been proposed. Current detection mainly relies on chromatography, mass spectrometry, electrochemical methods, capillary electrophoresis, and surface-enhanced Raman scattering (SERS) sensing technology. While these methods offer high sensitivity, they require expensive equipment, skilled operators, and cumbersome sample pretreatment (such as precipitation, filtration, or centrifugation). Although colorimetric methods such as the Griess reagent colorimetric method simplify the detection process, they are still often limited by complex sample purification or dilution steps. These limitations hinder on-site detection applications, especially for complex matrices such as milk and meat, necessitating the development of rapid, pretreatment-free detection solutions.

[0004] Hydrogel materials, with their three-dimensional network structure, offer innovative approaches for the direct detection of target analytes in complex samples. By controlling the pore size of hydrogels, large molecular interferences can be effectively blocked, avoiding the need for pretreatment of complex samples. For example, surface-enhanced Raman scattering (SERS) based hydrogel microspheres have been successfully applied to the pretreatment-free detection of drugs in biological samples and the detection of glucose in whole blood. Furthermore, hydrogel microspheres can serve as solid-phase carriers, enabling rapid on-site detection of nitrite in food (within 35 minutes) through solid-phase colorimetric reactions, exhibiting good tolerance to complex food matrices. These studies demonstrate that hydrogel materials provide an efficient and reliable analytical platform for the pretreatment-free detection of target molecules in complex samples.

[0005] Inspired by the size exclusion effect of gels, this patent develops a pretreatment-free colorimetric detection method for rapid determination of nitrite. Under acidic conditions, the diazotization oxidation reaction of nitrite with TMB causes a color change from colorless to orange in the solution, a process observable to the naked eye. The absorbance value at 450 nm and the image grayscale value can be used for quantitative detection. Based on this principle, a hydrogel is introduced to construct a nitrite sensor. Utilizing the inherent three-dimensional mesh structure of the hydrogel, large molecules such as proteins and lipid complexes are blocked outside the gel, remaining only on the gel surface; while nitrite permeates into the gel interior with water, triggering a colorimetric reaction. By capturing an image of the bottom of the gel with a smartphone and analyzing its grayscale value, the nitrite concentration can be quantified. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention provides a hydrogel, its preparation method, and its application.

[0007] A method for preparing a hydrogel includes the following steps: dispersing gelatin in a mixed solution of deionized water, HAc-NaAc buffer and TMB solution, and heating and stirring at 40 °C.

[0008] Furthermore, the HAc-NaAc buffer solution had a concentration of 100 mM and a pH of 2.5, and the HAc-NaAc concentration in the mixed solution was 50 mM.

[0009] Furthermore, the concentration of TMB in the TMB solution is 10 mM, and the concentration of TMB in the mixed solution is 2 mM.

[0010] Furthermore, the volume ratio of deionized water, HAc-NaAc buffer, and TMB solution is 3:5:2.

[0011] The present invention also provides a hydrogel prepared by the aforementioned method. The hydrogel has a three-dimensional mesh structure, preventing protein and fat complexes from penetrating its interior.

[0012] The present invention also provides the application of the hydrogel in the detection of nitrite.

[0013] Furthermore, the testing time is 6 minutes.

[0014] Furthermore, the hydrogel is used in the detection of nitrite in milk.

[0015] The beneficial technical effects of this invention are as follows: Based on the principle that TMB can be oxidized by nitrite under acidic conditions to generate an orange diazotized product, this patent proposes a simple and rapid colorimetric sensing method for nitrite. This method exhibits a clear color change, allowing for both intuitive judgment and quantitative analysis using instruments. Under optimal reaction conditions, the detection limit for nitrite in water is as low as 6.0 μM, with a dynamic detection range of 0.02 mM to 0.8 mM, and detection can be completed within 6 minutes. By introducing hydrogel materials and utilizing the hydrogel's own three-dimensional mesh structure, an automatic filtering function for biomolecules in complex milk samples is achieved. This effectively blocks interference from large molecules such as fats and proteins, while allowing the target nitrite molecule to freely diffuse into the gel interior. This eliminates the cumbersome sample pretreatment steps such as precipitation, centrifugation, and filtration required in traditional detection methods, significantly simplifying the operation process and improving detection efficiency. A sensor for the rapid detection of nitrite in milk samples has been successfully developed. This study fully validated the feasibility of using a hydrogel sensor to directly detect nitrite in food. The detection limit for nitrite in milk was as low as 45.5 μM, with a detection range of 0.1 to 1.0 mM. This sensor integrates sample pretreatment and detection processes, reducing reagent consumption and human error, as well as lowering detection costs and time. It is particularly suitable for rapid on-site screening and real-time monitoring of large batches of samples. Therefore, the TMB colorimetric reaction-based hydrogel nitrite sensor represents an economical, rapid, and convenient on-site detection strategy with broad application prospects in the field of food safety. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the TMB-based colorimetric detection principle for nitrite. Figure 2 (A) UV-Vis absorption spectra of different systems; (B) UV-Vis absorption spectra of nitrite from different sources. Figure 3 (A) pH value of buffer solution, (B) TMB concentration and (C) reaction time effect on absorbance at 450 nm; Figure 4 (A) The absorption spectra of nitrites of different concentrations and (B) the absorbance at 450 nm, (C) the linear relationship between the absorbance at 450 nm and the concentration of nitrites, (D) the absorption spectra of different types of sodium salts. Figure 5 (A) UV-Vis absorption spectra at different times; 5(B) and 5(C) are absorption spectra of the changes in absorbance at 450 nm and 650 nm. Figure 6 To ensure consistency of absorbance at 450 nm in parallel experiments; Figure 7This is a schematic diagram illustrating the preparation of a hydrogel sensor and the colorimetric detection of nitrite in milk. Figure 8 The FTIR spectrum of the hydrogel; Figure 9 (A) Absorbance of different concentrations of nitrite in milk at 450 nm, (B) Linear relationship between gray value and nitrite concentration in milk. Detailed Implementation

[0017] Example 1: Exploration of Nitrite Detection (1) Working principle of the rapid colorimetric nitrite sensor system: like Figure 1 As shown, a nitrite colorimetric sensing system using TMB was designed. In the absence of nitrite, TMB is colorless in acidic sodium acetate buffer. In the presence of nitrite, the diazotization oxidation reaction between nitrite and TMB under acidic conditions causes the solution color to change from colorless to orange. This color change can be directly observed with the naked eye. The UV absorption spectrum shows a maximum absorption peak at 450 nm. Therefore, the absorbance and grayscale value at 450 nm will be further used for quantitative analysis.

[0018] (2) Principle verification To confirm the validity of the proposed principle, the absorption spectra of different experimental groups were first measured. For example... Figure 2 As shown in Figure A, a significant absorption peak appeared at 450 nm in the presence of nitrite, TMB, and HAc-NaAc buffer, while a weaker absorption peak appeared at 650 nm. The two absorption peaks at 650 nm and 450 nm are attributed to the oxidation and diazotization oxidation of TMB, respectively. The intensity of the absorption peak at 450 nm was 4.7 times that at 650 nm, indicating a more sensitive response to nitrite. Furthermore, the solution color after the reaction was orange, consistent with the absorbance at 450 nm. Therefore, the absorbance value at 450 nm was used as the signal in subsequent detections. Simultaneously, a TMB negative control group, a TMB / nitrite group, or a TMB / HAc-NaAc group (as negative controls) were designed and tested. No absorption peaks were observed at either 650 nm or 450 nm.

[0019] To further verify the feasibility, experiments were conducted using different nitrites. The results are as follows: Figure 2 As shown in Figure B, regardless of the type of nitrite used, a significant absorption peak appears at 450 nm. The absorbance intensities at 450 nm are similar for different nitrites, with a relative standard deviation (RSD) of 5.8%. This indicates that different nitrites can induce diazotization oxidation of TMB, and that this reaction is independent of the type of cation.

[0020] (3) Experimental conditions to investigate the effect of pH value of buffer solution: See results Figure 3 A. When the pH value decreases from 4.5 to 2.5, the absorbance increases as the pH value decreases. When the pH value is below 2.5, the change in absorbance tends to level off. Therefore, pH 2.5 is chosen as the optimal pH value for the buffer solution.

[0021] Effect of TMB concentration: See results Figure 3 B. When the TMB concentration increased from 1 mM to 2 mM, the absorbance increased from 2.5 to 3.2, an increase of 28%; while when the TMB concentration increased from 2 mM to 3 mM, the absorbance value increased from 3.2 to 3.2, an increase of only 3%. Considering both reagent dosage and signal enhancement, 2 mM was selected as the optimal concentration of TMB.

[0022] The effect of reaction time: Reaction time affects the intensity of color; therefore, the effect of reaction time on absorbance is also a factor that needs to be considered. For example... Figure 3 As shown in Figure C, the effect of reaction time on the absorbance of different nitrite concentrations (low, medium, and high) was investigated. When the reaction time was 2 minutes, the absorbance of the high concentration group was close to 87% of the maximum value, indicating that the reaction rate of this principle is fast. Considering the three different concentrations (low, medium, and high), the optimal reaction time was finally determined to be 6 minutes.

[0023] (4) Under the above optimal conditions, study the variation of absorbance with nitrite concentration.

[0024] The experimental steps are as follows: To detect nitrite, 20 μL of TMB (10 mM), 50 μL of HAc-NaAc buffer (100 mM, pH 2.5), and 30 μL of H2O were added to each well of a 96-well microplate. Then, 100 μL of sodium nitrite solutions of different concentrations (0.02 mM, 0.05 mM, 0.10 mM, 0.20 mM, 0.40 mM, 0.60 mM, 0.80 mM, 1.00 mM) were added to the mixture. Finally, the total volume of the mixture (200 μL) was gently agitated in a microplate reader. The absorption spectra from 300 nm to 800 nm and the absorbance value at 450 nm were then acquired using the microplate reader. The results are shown below. Figure 4 .

[0025] like Figure 4 As shown in A and 4B, when the nitrite concentration is in the range of 0.02-1 mM, the absorbance at 450 nm gradually increases with increasing concentration. Figure 4C shows the linear relationship between nitrite concentration and absorbance at 450 nm. When the nitrite concentration is in the range of 0.02-0.8 mM, the absorbance and concentration show a good linear relationship, with the linear equation being y = 3.23x + 0.08 and a correlation coefficient of 0.999. The detection limit is calculated to be 6.0 μM based on the 3σ principle.

[0026] The specificity of the sensing mechanism is a key factor affecting practical applications. By examining the effects of different sodium salts, it was found that only sodium nitrite produced significant absorbance at 450 nm, while the reaction solutions of other sodium salts remained colorless and showed no absorption. Figure 4 D). This indicates that only sodium nitrite can oxidize TMB under given conditions, verifying the high specificity of this sensing mechanism.

[0027] Besides specificity, stability is another key factor for reliable detection. This is achieved by monitoring the absorption spectra of the reaction solution at different time points (measured every 10 minutes). Figure 5 A) found that the absorbance at 450 nm decreased slowly over 50 minutes. Figure 5 B), while the absorbance at 650 nm increases slowly (B). Figure 5 C). The relative standard deviation (RSD) of the absorbance at 450 nm over 50 minutes was 1.8%, indicating that the reaction was stable during this period. Furthermore, the RSD of eight parallel measurements was 3.9% (…). Figure 6 This demonstrates that the reaction has good reproducibility.

[0028] Example 2 Preparation of hydrogel 0.2 g of gelatin was dispersed in a mixture of 300 μL H₂O, 500 μL HAc-NaAc buffer (100 mM, pH 2.5), and 200 μL TMB (10 mM). The mixture was then heated and stirred at 40 °C using a magnetic stirrer to obtain a hydrogel. 100 μL of the hydrogel was transferred to each well of a 96-well plate. Finally, the hydrogel was sealed tightly with a silicone stopper and stored at 4 °C for subsequent analysis.

[0029] Example 3: Detection of Nitrite in Milk Nitrites may be added to milk as a preservative, but excessive use can have negative health effects; therefore, it is essential to ensure their use within safe limits. Existing methods for detecting nitrites in milk typically require precipitation and filtration steps to eliminate protein or fat interference, which are cumbersome pretreatment processes. Developing pretreatment-free methods for detecting nitrites is of great significance.

[0030] Inspired by the size exclusion properties of gels, a method such as... Figure 7The illustrated method is a colorimetric assay without pretreatment. TMB and sodium acetate buffer are prepared in a gelatin hydrogel, and nitrite-containing milk is directly added to the wells of the plate. Utilizing the three-dimensional network structure of the gel, biomacromolecules such as proteins and lipid complexes are completely blocked from the gel exterior. Nitrite, as a small molecule, can freely permeate into the gel interior like water molecules. Based on... Figure 7 The sensing principle involves the formation of orange diazotized TMB products within the gel. Since the top of the hydrogel is covered by a white solid, quantitative detection can be achieved by analyzing the grayscale value at the bottom of the gel. Surface tension prevents the solid material from detaching from the top of the gel, thus allowing direct image capture of the bottom simply by inverting the well plate, eliminating the need for additional cleaning steps.

[0031] Fourier transform infrared (FTIR) characterization of the hydrogel: First, the hydrogel was sliced ​​into thin sections using a sharp blade. Then, the surface of the hydrogel sections was non-destructively inspected using the attenuated total reflectance (ATR) mode of a Fourier transform infrared spectroscopy (FTIR) instrument.

[0032] The hydrogel was characterized by FTIR: gelatin showed characteristic peaks for NH stretching vibration and C=O stretching vibration at 3300 cm⁻¹ and 1633 cm⁻¹, respectively. Figure 8 This confirms its polymer properties; the characteristic peaks of the gelatin and TMB mixture are consistent with those of pure gelatin. When using this hydrogel sensor to detect nitrite concentration in fresh milk, the grayscale value gradually increases with increasing concentration. Figure 9 A), exhibiting a linear relationship in the range of 0.1-1.0 mM ( Figure 9 The detection limit was 45.5 μM (3σ). Recovery rates were calculated using grayscale measurements (Table 1), showing recoveries ranging from 98.71% to 110.72%. These results indicate that the hydrogel sensor can accurately detect nitrite in fresh milk samples.

[0033] Table 1. Standard Addition Method for Detection of Nitrite in Milk sample Dosage (mM) Measured quantity (mM) Recovery rate (%) RSD (%) 1 0.1 0.10 101.85 7.44 2 0.4 0.44 110.72 5.43 3 0.8 0.79 98.71 3.57

Claims

1. A method for preparing a hydrogel, characterized in that: The process includes the following steps: dispersing gelatin in a mixed solution of deionized water, HAc-NaAc buffer and TMB solution, and heating and stirring at 40 °C.

2. The method for preparing the hydrogel according to claim 1, characterized in that: The HAc-NaAc buffer solution has a concentration of 100 mM and a pH of 2.

5. The concentration of HAc-NaAc in the mixed solution is 50 mM.

3. The method for preparing the hydrogel according to claim 1, characterized in that: The concentration of TMB in the TMB solution is 10 mM, and the concentration of TMB in the mixed solution is 2 mM.

4. The method for preparing the hydrogel according to claim 1, characterized in that: The volume ratio of deionized water, HAc-NaAc buffer, and TMB solution was 3:5:

2.

5. A hydrogel prepared by the preparation method according to any one of claims 1-4.

6. The hydrogel according to claim 5, characterized in that: The hydrogel has a three-dimensional mesh structure, preventing protein and fat complexes from entering its interior.

7. The application of a hydrogel prepared by any one of claims 1-4 in the detection of nitrite.

8. The application of the hydrogel according to claim 7 in nitrite detection, characterized in that: The testing time is 6 minutes.

9. The application of the hydrogel according to claim 7 in nitrite detection, characterized in that: The hydrogel is used for the detection of nitrite in milk.

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