Visual colorimetric sensor, preparation method thereof and method for detecting nitrite

Abstract

The invention provides a visual colorimetric sensor, a preparation method thereof and a method for detecting nitrite, the visual colorimetric sensor comprises a substrate and a color developing functional layer, the color developing functional layer is loaded on the substrate through a hydrogen bond, the substrate comprises a cellulose membrane, and the color developing functional layer comprises a TMB material. According to the invention, the cellulose membrane is used as the substrate and the hydrogen bond is used for loading the TMB to form the color developing functional layer, so that the contact area between the TMB and nitrite ions is increased due to the porous characteristic of the cellulose membrane, and the TMB is reduced in loss due to stable loading, so that the reaction is more sufficient to realize high-sensitivity detection; by means of the specific chromogenic reaction of TMB and nitrite ions and the extremely strong anti-interference ability, it is ensured that detection is accurate and high in selectivity in a complex sample, meanwhile, color change is visual and distinguishable, visual detection easy and convenient to operate is achieved, and finally the detection requirements of the World Health Organization and the national standard for nitrite in food are met.

Description

Technical Field

[0001] This invention relates to the interdisciplinary fields of new materials, sensors and ion detection, and in particular to a visual colorimetric sensor and its preparation method, as well as a method for detecting nitrite. Background Technology

[0002] Nitrite is a chemical formula of NO2 - Inorganic salts, NO2, are widely found in food, drinking water, soil, and other natural and human activities. - It possesses strong oxidizing and reducing properties, and can react with various organic and inorganic substances through nitration. In the environment, NO2... - NO2 is a significant water pollutant that is highly harmful to aquatic life, causing fish to die from oxygen depletion. - It can also react with other pollutants to form more toxic substances, such as nitrobenzene. In the field of food safety, NO2... - It is also an important indicator. Many foods produce NO2 during processing and preservation. - The World Health Organization has classified NO2 as a contaminant. - Listed as a carcinogen. Excessive intake of NO2. - It can react with amino compounds in the body or external environment to form nitrosamines, which can induce cancer and cause digestive system diseases. Therefore, accurate and rapid detection of NO2 is crucial. - It is of great significance to ensure human food safety and maintain environmental health. However, currently common techniques for detecting nitrite, such as spectrophotometry, chemiluminescence, electrochemical detection, chromatography, and capillary electrophoresis, are often limited by complex procedures, low selectivity, and poor portability.

[0003] Therefore, there is an urgent need for a visual colorimetric sensor and its preparation method, as well as a method for detecting nitrite, to solve the above-mentioned technical problems. Summary of the Invention

[0004] The purpose of this invention is to provide a visual colorimetric sensor and its preparation method, as well as a method for detecting nitrite, to solve the technical problems of high cost, low accuracy and poor selectivity of existing methods for detecting nitrite.

[0005] This invention provides a visual colorimetric sensor based on a TMB-immobilized cellulose membrane. Based on the color reaction between TMB and nitrite ions, and combined with the porous properties of the cellulose membrane, it achieves high sensitivity, high selectivity, and visual detection of nitrite. When nitrite ions are present in the sample, NO... 2-The nitrite sensor reacts with TMB to generate oxTMB, resulting in a significant colorless to green transition in the thin film, thus establishing a visual detection system for nitrite. This sensor exhibits strong anti-interference capabilities and meets the detection requirements for nitrite in food set by the World Health Organization and national standards.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention first provides a visual colorimetric sensor, including a substrate and a color-developing functional layer, wherein the color-developing functional layer is loaded on the substrate via hydrogen bonds; The substrate includes a cellulose membrane, and the color-developing functional layer includes TMB material.

[0007] Furthermore, the visual colorimetric sensor and NO 2- Upon contact, the color will change noticeably, from colorless to green.

[0008] Accordingly, the present invention also provides a method for preparing a visual colorimetric sensor, comprising the following steps: TMB material was mixed with cellulose solution and stirred. After sufficient adsorption, a TMB-cellulose mixed solution was obtained. The TMB-cellulose mixed solution was degassed and then solidified in deionized water. After washing with water until neutral, a visual colorimetric sensor was obtained.

[0009] Preferably, in the step of mixing TMB material with cellulose solution and then stirring to obtain a TMB-cellulose mixed solution after sufficient adsorption, the method for preparing the cellulose solution includes: dissolving sodium hydroxide, urea and cotton linters in water at a mass ratio of (10~20):(20~30):(5~15) to prepare a cellulose solution with a mass concentration of 1wt%~15wt%.

[0010] Preferably, in the step of mixing TMB material with cellulose solution and then stirring to obtain a TMB-cellulose mixed solution after sufficient adsorption, the stirring step specifically includes: Mix 0.1~0.3g of TMB material with 80~120ml of cellulose solution and stir until homogeneous.

[0011] Preferably, in the step of mixing TMB material with cellulose solution and then stirring to obtain a TMB-cellulose mixed solution after sufficient adsorption, the temperature during the mixing and adsorption of TMB material and cellulose solution is 10~30℃, and the adsorption time is 1~2h.

[0012] Accordingly, the present invention also provides a method for detecting nitrite using the above-described visual colorimetric sensor or a visual colorimetric sensor prepared by the above method, comprising the following steps: The visual colorimetric sensor and acidic buffer solution were added to the nitrite sample to be tested, and then allowed to stand to allow the redox reaction to occur.

[0013] Preferably, in the step of adding the visual colorimetric sensor and the acidic buffer solution to the nitrite sample to be tested and allowing it to stand to allow the redox reaction to occur, the acidic buffer solution is a disodium hydrogen phosphate-citric acid buffer solution with a pH of 2.0~3.0.

[0014] Preferably, in the step of adding the visual colorimetric sensor and the acidic buffer solution to the nitrite sample to be tested, and allowing it to stand to allow the redox reaction to occur: the volume ratio of the nitrite solution to the acidic buffer solution required for the reaction is 1:1, the reaction temperature is room temperature, and the reaction time is 10~60 min.

[0015] Preferably, in the step of adding the visual colorimetric sensor and the acidic buffer solution to the nitrite solution to be tested, and allowing it to stand to allow the redox reaction to occur, the detection limit of the visual colorimetric sensor for nitrite is 10 μM.

[0016] Preferably, in the step of adding the visual colorimetric sensor and the acidic buffer solution to the nitrite solution to be tested, and allowing it to stand to allow the redox reaction to occur, the nitrite sample to be tested is derived from any one of drinking water, ambient water, milk, and pickled food.

[0017] The beneficial effects of this invention are as follows: Unlike existing technologies, this invention provides a visual colorimetric sensor and its preparation method, as well as a method for detecting nitrite. The visual colorimetric sensor includes a substrate and a colorimetric functional layer. The colorimetric functional layer is loaded onto the substrate via hydrogen bonds. The substrate includes a cellulose membrane, and the colorimetric functional layer includes TMB material. This visual colorimetric sensor uses a cellulose membrane as the substrate and utilizes hydrogen bonds to load TMB to form the colorimetric functional layer. This increases the contact area between TMB and nitrite ions due to the porous nature of the cellulose membrane, and the stable loading of TMB reduces loss, allowing for a more complete reaction and achieving high-sensitivity detection. Furthermore, thanks to the specific colorimetric reaction between TMB and nitrite ions and its strong anti-interference ability, it ensures accurate detection and high selectivity in complex samples. Simultaneously, the color change is intuitively discernible, enabling easy-to-operate visual detection. Ultimately, it meets the detection requirements of the World Health Organization and national standards for nitrite in food. Attached Figure Description

[0018] Figure 1 A flowchart illustrating the fabrication method of the visual colorimetric sensor provided in this embodiment of the invention; Figure 2 This is a flowchart of a method for detecting nitrite provided in an embodiment of the present invention; Figure 3 This is a digital image of the colorimetric response of the visual colorimetric sensor prepared in Example 1 after interacting with samples of different concentrations of nitrite. Figure 4 This is a digital image of the colorimetric response of the visual colorimetric sensor obtained in Example 1 under the presence of different metal ions and common interfering substances; Figure 5 The images show the colorimetric response of the visualized colorimetric sensor obtained in Example 1 at different storage days. Figure 6 This is a digital image of the colorimetric response of the visualized colorimetric sensor obtained in Example 1 after reacting with a real sample; Figure 7 The images show the colorimetric response digital images of the cellulose membrane-based colorimetric sensor (a) obtained in Comparative Example 1 and the cellulose membrane-based colorimetric sensor (b) obtained in Example 1 after interacting with the nitrite sample. Detailed Implementation The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0019] Currently, colorimetric sensors have garnered widespread attention in the scientific community due to their economical nature, improved sensitivity and specificity, and obvious visibility without the need for optical components, enabling the detection of various analytes. Visual colorimetric sensors generally consist of a receptor with a molecular recognition structure possessing a chemically sensitive layer and a transducer capable of signal conversion, detecting color changes caused by specific chemical reactions or molecular interactions between the analyte and the sensing material.

[0020] To address the shortcomings of existing technologies, the inventors discovered that the performance of visual colorimetric sensors is highly dependent on the type of solid support within the sensor. Solid supports used to develop chemical sensors must be chemically inert, possess high surface area, and exhibit excellent surface properties. Cellulose, with its regular molecular chain structure and no significant absorption in the visible light range, can be dissolved and regenerated to create a uniform, defect-free, and highly optically transparent membrane material. The intermolecular hydrogen bonds and semi-crystalline structure of cellulose endow it with significant stability, enabling it to remain stable in various chemical environments. It is not easily damaged or altered in properties by strong acids, strong alkalis, water, and most organic solutions, providing an ideal substrate material for optoelectronic devices, biomedical materials, and other fields. Furthermore, cellulose possesses a highly loose interpenetrating porous structure and abundant active hydroxyl groups, making it an ideal immobilization platform. Therefore, the inventors believe that cellulose membranes are an excellent substrate material for colorimetric sensors and are attempting to fabricate them into a visual colorimetric sensor for detecting nitrite.

[0021] This invention provides a visual colorimetric sensor and its preparation method, as well as a method for detecting nitrite. The visual colorimetric sensor uses a cellulose membrane as a substrate, and TMB is fixed onto the surface of a porous cellulose membrane via a blending method to obtain a TMB-loaded cellulose membrane sensor. When NO2... - When present, the sensor changes color from colorless to green, enabling visual qualitative and semi-quantitative detection, providing a portable and efficient solution for nitrite monitoring. The visual colorimetric sensor provided by this invention has advantages such as high selectivity (resistant to common ion interference), rapid response (qualitative analysis within 10 minutes), high sensitivity (visual detection limit (VLOD) 10 μM), and long-term stability (performance unchanged after 120 days of storage). It can perform real-time visual detection of nitrite in samples such as drinking water, ambient water, milk, and pickled foods.

[0022] To achieve the above technical objectives, this application adopts the following technical solution: In a first aspect, the present invention provides a visual colorimetric sensor, comprising a substrate and a colorimetric functional layer, wherein the colorimetric functional layer is loaded onto the substrate via hydrogen bonds; wherein the substrate comprises a cellulose membrane, and the colorimetric functional layer comprises TMB (3,3',5,5'-tetramethylbenzidine) material; the visual colorimetric sensor (TMB@CM sensor) and NO 2- Upon contact, the color will change noticeably, from colorless to green.

[0023] Specifically, this invention first loads a color-developing functional layer (containing TMB material) onto a cellulose membrane substrate via hydrogen bonds. This allows TMB to adhere stably and, thanks to the properties of the cellulose membrane, to fully contact nitrite ions, ensuring high efficiency and stability of the reaction. The sensor then produces a clear color change from colorless to green upon contact with nitrite ions, exhibiting excellent response performance. This intuitive color change enables convenient and visual detection of nitrite ions, allowing for rapid identification without complex equipment. Ultimately, due to its reasonable structural design and clear reaction signal, this sensor meets the basic requirements for nitrite ion detection, providing a practical and effective tool for detection in related fields and possessing practical application value.

[0024] Furthermore, when nitrite ions are present in the sample to be tested, NO 2- The nitrite reacts with TMB (reduced state, colorless) to generate oxTMB (oxidized state, green), and the film exhibits a significant colorless to green transition, thus constructing a visual detection system for nitrite.

[0025] Please see Figure 1 The present invention also provides a method for preparing a visual colorimetric sensor, comprising the following steps: S10: TMB material is mixed with cellulose solution and then stirred. After sufficient adsorption, a TMB-cellulose mixed solution is obtained.

[0026] Specifically, step S10 also includes: First, sodium hydroxide, urea, and cotton linters are dissolved in water at a mass ratio of (10~20):(20~30):(5~15) to prepare a cellulose solution (alkaline cellulose sol) with a mass concentration of 1wt%~15wt%. Second, 0.1~0.3g of TMB material is mixed with 80~120ml of cellulose solution and stirred until uniformly mixed. After sufficient adsorption, a TMB-cellulose mixed solution is obtained. The temperature during the adsorption of TMB material and cellulose solution is 10~30℃, and the adsorption time is 1~2h.

[0027] Furthermore, this application first prepares a cellulose solution of 1wt%~15wt% by mixing sodium hydroxide, urea, and cotton linters in a specific mass ratio of (10~20):(20~30):(5~15), providing a stable and suitable matrix environment for subsequent TMB loading and ensuring that cellulose molecules can fully dissolve and form a homogeneous solution. Secondly, the ratio of TMB material dosage (0.1~0.3g) to cellulose solution volume (80~120ml) is controlled, while the temperature (10~30℃) and time (1~2h) during mixing and adsorption are limited to ensure optimal dispersion of TMB in the cellulose solution. The adsorption state avoids waste or aggregation caused by excessive TMB, while also preventing insufficient dosage from affecting the color development effect. Furthermore, suitable temperature and time promote the formation of intermolecular forces, enhancing the binding stability of TMB and cellulose. This results in a more uniform composition and more stable performance of the TMB-cellulose mixed solution, laying the foundation for the subsequent preparation of high-quality visual colorimetric sensors. This, in turn, improves the batch consistency and detection sensitivity of the sensor. The specific ratios and conditions facilitate standardized production and reduce operational difficulty. Ultimately, this makes the preparation process more controllable and practical, further ensuring the sensor's detection performance and facilitating large-scale applications.

[0028] S20, the TMB-cellulose mixed solution is degassed and then placed in deionized water for solidification. After being washed with water until neutral, a visual colorimetric sensor is obtained.

[0029] Specifically, step S20 also includes: Because the TMB-cellulose mixed solution contains a large number of bubbles (mainly originating from bubbles in the alkaline cellulose sol), centrifugation at 4000 rpm for 10 minutes is required to remove the bubbles. After degassing, the alkaline cellulose sol is uniformly dispersed on the glass surface using a delayed flow method. The glass is then immersed in deionized water to allow the sol to solidify into a film. After removing the film, it is repeatedly rinsed with deionized water until neutral to obtain a visual colorimetric sensor.

[0030] Specifically, the centrifugal degassing process described above effectively removes a large number of air bubbles present in the mixed solution, preventing structural defects such as pores and wrinkles in the film during subsequent film formation and ensuring uniform film texture. After degassing, a delayed flow method is used to uniformly disperse the alkaline cellulose sol on the glass surface, ensuring consistent sol spreading and laying the foundation for forming a film of uniform thickness. Next, the glass is immersed in deionized water to solidify the sol into a film. This solidification method is gentle and controllable, reducing TMB loss while ensuring the orderly arrangement of cellulose molecules and enhancing the structural stability of the film. After removing the film, it is repeatedly rinsed with deionized water until neutral to remove residual sodium hydroxide and other impurities, preventing them from affecting TMB and NO2. - The colorimetric reaction can cause interference, thus ensuring the accuracy of sensor detection.

[0031] Please see Figure 2 The present invention also provides a method for detecting nitrite using the above-described visual colorimetric sensor or a visual colorimetric sensor prepared by the above method, comprising the following steps: SS10 involves adding the visual colorimetric sensor and acidic buffer solution to the nitrite sample to be tested, and allowing it to stand to allow the redox reaction to occur.

[0032] Specifically, the SS10 steps also include: The visual colorimetric sensor and a pH 2.0–3.0 disodium hydrogen phosphate-citric acid buffer solution were mixed at a 1:1 volume ratio and added to the nitrite sample to be tested. After standing, the sample was allowed to undergo a redox reaction (reaction temperature: room temperature, reaction time: 10–60 min).

[0033] Furthermore, the aforementioned buffer solution with a specific pH provides a suitable acidic environment for the redox reaction of TMB and nitrite, and acidic conditions promote the reaction of NO2. - The key to generating oxTMB (resulting in a color change) through the oxidation of TMB enhances the specificity and efficiency of the reaction. A 1:1 volume ratio balances the proportions of the sensor, buffer, and sample, preventing excessive sample dilution or insufficient buffer from failing to maintain a stable pH, ensuring consistent reaction conditions. Simultaneously, setting the reaction temperature to room temperature eliminates the need for additional temperature control equipment, simplifying the operation process, reducing detection costs, and facilitating convenient detection in various scenarios. The reaction time is controlled between 10 and 60 minutes, ensuring both sufficient reaction time (avoiding insufficient signal due to insufficient time) and improved detection efficiency (preventing excessive time from affecting detection timeliness). This time can be flexibly adjusted according to the actual nitrite concentration in the sample, balancing sensitivity and efficiency.

[0034] Specifically, the visual colorimetric sensor has a detection limit of 10 μM for nitrite. When its trigger color changes from colorless to green, it indicates that the sample contains nitrite. The 10 μM detection limit means that the sensor can capture low concentrations of nitrite in the sample, meeting the needs for detecting trace amounts of nitrite. This is especially true for the stringent requirements of nitrite limits in the food and other fields, effectively avoiding missed detections due to excessively high detection limits. The intuitive color change signal is clear and easy to identify, allowing for qualitative judgment without the need for complex instruments, thus reducing the professional skills required of the testing personnel. Specifically, the 10μM detection limit ensures the sensor's high sensitivity, enabling accurate response to low concentrations of nitrite and providing strong support for the reliability of detection results. The significant color change from colorless to green enhances the intuitiveness and convenience of the detection, making the process fast and efficient, allowing for immediate on-site assessment. This design allows the sensor to possess both high sensitivity and ease of operation, thereby improving its practicality. It meets the needs of both precise detection and rapid screening scenarios, ultimately making the sensor more competitive in nitrite detection in food and other fields, providing a reliable and convenient technical means to ensure food safety.

[0035] Specifically, the nitrite samples to be tested can originate from any one of drinking water, ambient water, milk, and pickled foods. This means that the visual colorimetric sensor has a wide range of applications. For drinking water and ambient water, the matrix is ​​relatively simple, and the sensor can quickly screen for excessive nitrite with a low detection limit of 10 μM and intuitive color changes, ensuring water quality safety. Although the milk matrix contains components such as proteins, the sensor's anti-interference ability ensures detection accuracy and meets the needs of dairy product safety testing. Pickled foods are a high source of nitrite, and the sensor can sensitively detect the nitrite content in them, safeguarding food processing and consumption safety.

[0036] The technical solution of the present invention will now be further described with reference to specific embodiments.

[0037] Example 1: This embodiment 1 provides a visual colorimetric sensor (TMB@CM sensor), including a substrate and a colorimetric functional layer, wherein the colorimetric functional layer is loaded onto the substrate via hydrogen bonds; wherein, the substrate includes a cellulose membrane, and the colorimetric functional layer includes TMB (3,3',5,5'-tetramethylbenzidine) material; the visual colorimetric sensor and NO 2- Upon contact, the color will change noticeably, from colorless to green.

[0038] This embodiment 1 also provides a method for preparing a visual colorimetric sensor, including the following steps: Step (a): After mixing sodium hydroxide / urea / water solution (where the mass ratio of sodium hydroxide, urea and water is 7:12:81) evenly, freeze to -12.5°C, then add cotton linters and stir rapidly for 10 min to obtain an alkaline cellulose sol with a concentration of 3 wt%. Step (b): Add 0.2 g of TMB powder to 3 wt% alkaline cellulose sol and continue stirring vigorously for 1 h to obtain a TMB-cellulose mixed solution. Step (c): Centrifuge the TMB-cellulose mixture at 4000 rpm for 10 min to remove bubbles; Step (d): After degassing, the TMB-cellulose mixed solution is evenly dispersed on the glass surface using a delayed flow method. Then, the glass is immersed in deionized water to solidify the alkaline cellulose sol in the TMB-cellulose mixed solution into a film. After removing the film, it is repeatedly rinsed with deionized water until neutral. Then, it is cut into 1cm×1cm pieces and immersed in pure water for later use to obtain the TMB@CM sensor.

[0039] Specifically, a series of performance tests were conducted on the TMB@CM sensor prepared in Example 1, as follows: Using sodium nitrite and water as raw materials, test solutions with sodium nitrite concentrations of 0, 10, 15, 20, 30, 40, 50, 60, 80, 100, 120, 200, 300, 400, 500, and 600 μM were prepared using a gradient dilution method. The TMB@CM sensor prepared in Example 1 was then added to each test solution, along with a disodium hydrogen phosphate-citric acid buffer solution at pH 2.4. After standing for 60 minutes, the solutions were retrieved for colorimetric detection, yielding the results shown below. Figure 3 The digital image shown. From Figure 3 As can be seen, as the sodium nitrite concentration gradually increases from 0 to 600 μM, the color of the TMB@CM sensor gradually changes from colorless to green, which provides a possibility for the quantitative detection of nitrite. Figure 3 It can also be found that the visual detection limit (VLOD) of the TMB@CM chemical colorimetric sensor for nitrite is 10 μM.

[0040] Furthermore, to verify that the TMB@CM sensor prepared in Example 1 can trigger a significant color change upon reaction with nitrite and is unaffected by other chemical components, the color change of the TMB@CM sensor in solutions of different metal ions and several common interfering substances (300 μM) was tested. The results are as follows: Figure 4 As shown. By Figure 4 It can be seen that the TMB@CM sensor is effective against 10 common cations (NH4+). + Ag + Mg 2+ Ca 2+ Cu 2+ Li + Ba 2+ Pb 2+ Al 3+ K + ), 9 common anions (I - Cl - HCO3 - SO3 2- CO3 2- H2PO4- CH3COO - B4O7 2- NO3 - There was no significant change in H2O2; when sodium nitrite was added to these solutions, the color of the TMB@CM sensor quickly changed from colorless to green, thus proving the specific selectivity of the TMB@CM sensor for nitrite.

[0041] Furthermore, to determine whether the TMB@CM sensor possesses long-term stable performance, the TMB@CM prepared in Example 1 was stored in an aqueous solution at room temperature for 1, 7, 10, 30, 60, 90, and 120 days. It was then immersed in a 300 μM sodium nitrite solution, with the addition of a disodium hydrogen phosphate-citric acid buffer solution at pH 2.4. After standing for 60 minutes, it was retrieved for colorimetric detection. The results are as follows: Figure 5 As shown. By Figure 5 It can be seen that, under the same detection conditions, the difference in color intensity observed by the naked eye after the TMB@CM sensor reaction is not significant as the storage days increase. Even after storage for more than 120 days, the detection effect of the TMB@CM sensor on nitrite remains unaffected.

[0042] Furthermore, to verify the actual performance of the TMB@CM sensor, the inventors applied it to the detection of nitrite in ham sausages, cured meat, and luncheon meat. The specific process is as follows: Weigh 5 grams of the actual sample (luncheon meat) and grind it using a mortar and pestle. Then add 12.5 mL of Na₂B₄O₇ (50 g / L) and 150 mL of hot water (approximately 70°C), and stir in a boiling water bath for 15 minutes. After cooling to room temperature, add 5 mL of K₃[Fe(CN)₆] (106 g / L) and 5 mL of (CH₃COO)₂Zn (220 g / L). Add deionized water and let stand for 30 minutes. Then remove the residue and filter the resulting aqueous phase through a 0.45 μm membrane. First, quantify the nitrite content in the collected filtrate. Subsequently, recovery experiments were conducted by adding different concentrations of nitrite to the filtrate. The detection method for nitrite in the three food samples was the same as that for the water sample.

[0043] Depend on Figure 6 As can be seen, the green color of the TMB@CM sensor gradually deepens as the nitrite concentration in the sample increases; compared with the addition of standard sodium nitrite solution, the color intensity is not significantly different to the naked eye, indicating that the TMB@CM sensor has the potential to detect nitrite in real samples.

[0044] Example 2: The TMB@CM sensor and its preparation method provided in this embodiment 2 are exactly the same as those provided in this embodiment 1. The difference lies in the detection object: referring to the method for detecting nitrite in luncheon meat in this embodiment 1, this embodiment 2 uses cured meat (as a substitute for luncheon meat) for testing. The results show that the TMB@CM sensor provided in this embodiment 2 can indeed detect nitrite in the real sample (cured meat).

[0045] Example 3: The TMB@CM sensor and its preparation method provided in Example 3 are exactly the same as those provided in Example 1. The difference lies in the detection object: referring to the method for detecting nitrite in luncheon meat in Example 1, this Example 3 uses sausage (as a substitute for luncheon meat) for testing. The results show that the TMB@CM sensor provided in Example 3 can indeed detect nitrite in the real sample (sausage).

[0046] Example 4: The TMB@CM sensor and its preparation method provided in Example 4 are exactly the same as the TMB@CM sensor and its preparation method provided in Example 1, except that the detection object is different: Referring to the method for detecting nitrite in luncheon meat in Example 1, Example 4 used drinking water (instead of luncheon meat) for testing. The results showed that the TMB@CM sensor provided in Example 4 could indeed detect nitrite in drinking water.

[0047] Example 5: The TMB@CM sensor and its preparation method provided in Example 5 are exactly the same as the TMB@CM sensor and its preparation method provided in Example 1, except that the detection object is different: Referring to the method for detecting nitrite in luncheon meat in Example 1, Example 5 used lake water (instead of luncheon meat) for testing. The results showed that the TMB@CM sensor provided in Example 5 could indeed detect nitrite in drinking water.

[0048] Example 6: The TMB@CM sensor and its preparation method provided in Example 6 are exactly the same as the TMB@CM sensor and its preparation method provided in Example 1, except that the detection object is different: Referring to the method for detecting nitrite in luncheon meat in Example 1, Example 6 used milk (instead of luncheon meat) for testing. The results showed that the TMB@CM sensor in Example 6 could indeed detect nitrite in milk.

[0049] Comparative Example 1: Comparative Example 1 provides a TMB@CM sensor with TMB fixed by an impregnation method. Its preparation method is largely the same as that of Example 1, except that the regenerated cellulose membrane is prepared using an impregnation method. The specific preparation method is as follows: Step (a): After mixing sodium hydroxide / urea / water solution (where the mass ratio of sodium hydroxide, urea and water is 7:12:81) evenly, freeze to -12.5°C, then add cotton linters and stir rapidly for 10 min to obtain an alkaline cellulose sol with a concentration of 3 wt%. Step (b): A cellulose membrane with a thickness of approximately 1 mm is prepared by a molding method; Step (c): Rinse the cellulose membrane with distilled water until the pH of the washing solution is neutral, then cut it into 1cm×1cm pieces to obtain a usable cellulose membrane. Step (d) involves immersing the cellulose membrane in a 1M TMB solution at room temperature for 24 hours, followed by rinsing it three times with deionized water to obtain the TMB@CM sensor prepared by the impregnation method.

[0050] Specifically, under the same experimental conditions, the same concentrations of sodium nitrite and buffer solution were added to the TMB@CM sensor obtained by the impregnation method in Comparative Example 1 and the TMB@CM sensor obtained in Example 1, respectively, and the differences in their color development effects were compared. The results are shown in [Figure 1]. Figure 7 ;Depend on Figure 7 As can be seen, compared with the TMB@CM sensor with TMB fixed by the impregnation method, the TMB@CM sensor prepared by the blending method provided in Example 1 can achieve a higher content and more uniform distribution of TMB loading effect, which is conducive to obtaining higher detection sensitivity and better color development effect, thereby realizing rapid and efficient detection of nitrite.

[0051] Furthermore, the blending method is superior to the impregnation method, mainly because there are fundamental differences in the loading method and distribution of TMB: From the perspective of loading mechanism, the blending method involves uniformly incorporating TMB during the cellulose solution preparation stage. TMB molecules directly embed into the three-dimensional network structure of cellulose through hydrogen bonding with cellulose molecules. This "in-situ blending" mode allows TMB to form a tighter bond with the cellulose matrix, and the loading amount is not limited by the adsorption capacity of the cellulose membrane surface, enabling higher TMB content loading. In contrast, the impregnation method involves immersing the formed cellulose membrane in a TMB solution, relying on physical adsorption or weak interactions on the membrane surface and pores to fix TMB. Limited by the number of adsorption sites on the membrane and the diffusion efficiency of TMB in the solution, the loading amount is often lower, and it is prone to loss due to elution.

[0052] In terms of distribution uniformity, in the blending method, TMB is uniformly dispersed throughout the entire membrane during the stirring and film-forming process of the cellulose solution, exhibiting a stable distribution from the surface to the interior. When reacting with nitrite, it generates a comprehensive and synchronous colorimetric signal, avoiding the problem of uneven local reactions. In contrast, in the impregnation method, TMB is mainly adsorbed on the surface and shallow pores of the cellulose membrane, with very little loading in the deeper layers. This results in only significant color development on the surface during the reaction, with limited TMB participating in the reaction internally, leading to poor color intensity and uniformity.

[0053] Therefore, a higher TMB loading ensures a more sufficient "signal reserve" when reacting with nitrite, allowing even low concentrations of nitrite to trigger significant color changes, thus improving detection sensitivity. A more uniform distribution ensures consistency in the color development area, preventing uneven local color intensity from affecting judgment and resulting in superior color development. Therefore, the TMB@CM sensor prepared by the blending method can achieve nitrite detection more quickly and efficiently.

[0054] This invention uses a cellulose membrane as a substrate and employs a blending method to immobilize TMB on the surface of a porous cellulose membrane to form TMB@CM, successfully fabricating a visual colorimetric sensor for nitrite detection. When the sample contains or is present with nitrite, the TMB in the sensor reacts with NO2. - An oxidation reaction occurs under acidic conditions, causing the trigger membrane to change from colorless to green.

[0055] Compared with existing similar products or technologies, the advancements of this invention are mainly reflected in the following aspects: (1) The preparation method is simple, easy to implement, and low in cost; (2) The present invention uses a blending method to adsorb and fix TMB onto a cellulose membrane through hydrogen bonding; the blending method uses solution blending to make TMB and cellulose uniformly dispersed at the molecular scale to form a homogeneous composite system. Hydrogen bonds can spontaneously form between the hydroxyl groups of cellulose chains and the amino groups of TMB, avoiding local concentration gradients or uneven surface adsorption caused by solvent evaporation. This improves the stability and repeatability of hydrogen bond adsorption. In colorimetric reactions, blending allows TMB to be uniformly loaded on the membrane surface, reducing differences in color spots and improving detection sensitivity. Blending directly achieves physical mixing of TMB and cellulose in a solvent-free system, preserving the active groups of cellulose and ensuring efficient utilization of hydrogen bond adsorption sites, thus enhancing adsorption strength. Blending can directly prepare TMB-loaded cellulose membranes in a one-step mixing process, eliminating multiple steps (such as impregnation → drying → solvent removal steps in the impregnation method), significantly shortening the production cycle and reducing energy consumption. Blending does not require the use of large amounts of organic solvents (such as DMSO or ethanol), reducing VOC (Volatile Organic Compounds Emissions) emissions and subsequent solvent recovery costs, which aligns with green chemistry principles.

[0056] (3) The present invention uses the TMB@CM sensor as the core element for target recognition and signal transduction, and constructs a visual colorimetric sensor with high sensitivity, good stability and excellent signal resolution, and uses it for the detection of nitrite in various real samples.

[0057] (4) This invention utilizes TMB and NO2 - The significant color change during the oxidation reaction under acidic conditions is used to detect nitrite. When the sample contains nitrite, the sensor color changes from colorless to green. This process enables the visual qualitative and semi-quantitative detection of nitrite. (5) The TMB@CM sensor prepared by this invention has many advantages such as high selectivity (resistant to common ion interference), fast response (qualitative analysis within 10 minutes), high sensitivity (visual detection limit (VLOD) 10 μM) and long-term stability (performance unchanged after 120 days of storage). It can realize the real-time visual detection of nitrite in actual samples such as drinking water, environmental water, milk and pickled food, providing a portable and efficient solution for nitrite detection and meeting the safety standard requirements for nitrite detection in food.

[0058] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0059] The above embodiments merely illustrate implementation methods of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A visual colorimetric sensor, characterized in that, It includes a substrate and a color-developing functional layer, wherein the color-developing functional layer is loaded onto the substrate via hydrogen bonds; The substrate includes a cellulose membrane, and the color-developing functional layer includes TMB material.

2. A method for fabricating a visual colorimetric sensor as described in claim 1, characterized in that, Includes the following steps: TMB material was mixed with cellulose solution and stirred. After sufficient adsorption, a TMB-cellulose mixed solution was obtained. The TMB-cellulose mixed solution was degassed and then solidified in deionized water. After being washed with water until neutral, the visual colorimetric sensor was obtained.

3. The method for preparing a visual colorimetric sensor according to claim 2, characterized in that, In the step of mixing TMB material with cellulose solution and then stirring to obtain a TMB-cellulose mixed solution after sufficient adsorption, the method for preparing the cellulose solution includes: dissolving sodium hydroxide, urea and cotton linters in water at a mass ratio of (10~20):(20~30):(5~15) to prepare the cellulose solution with a mass concentration of 1wt%~15wt%.

4. The method for preparing a visual colorimetric sensor according to claim 3, characterized in that, In the step of mixing TMB material with cellulose solution and then stirring to obtain a TMB-cellulose mixed solution after sufficient adsorption, the stirring step specifically includes: Mix 0.1-0.3g of TMB material with 80-120ml of the cellulose solution and stir until homogeneous.

5. The method for preparing a visual colorimetric sensor according to claim 2, characterized in that, In the step of mixing TMB material with cellulose solution and then stirring to obtain a TMB-cellulose mixed solution after sufficient adsorption, the temperature during the mixing and adsorption of TMB material with the cellulose solution is 10~30℃, and the adsorption time is 1~2h.

6. A method for detecting nitrite using a visual colorimetric sensor as described in claim 1 or a visual colorimetric sensor prepared by the method described in any one of claims 2 to 5, characterized in that, Includes the following steps: The visual colorimetric sensor and the acidic buffer solution were added to the nitrite sample to be tested, and allowed to stand to allow the redox reaction to occur.

7. The method for detecting nitrite according to claim 6, characterized in that, In the step of adding the visual colorimetric sensor and the acidic buffer solution to the nitrite sample to be tested, and allowing it to stand to allow the redox reaction to occur: the acidic buffer solution is a disodium hydrogen phosphate-citric acid buffer solution with a pH of 2.0~3.

0.

8. The method for detecting nitrite according to claim 6, characterized in that, In the step of adding the visual colorimetric sensor and the acidic buffer solution to the nitrite sample to be tested, and allowing it to stand to allow the redox reaction to occur: the volume ratio of the nitrite solution to the acidic buffer solution required for the reaction is 1:1, the reaction temperature is room temperature, and the reaction time is 10~60 min.

9. The method for detecting nitrite according to claim 6, characterized in that, In the step of adding the visual colorimetric sensor and the acidic buffer solution to the nitrite solution to be tested, and allowing it to stand to allow the redox reaction to occur: the detection limit of the visual colorimetric sensor for nitrite is 10 μM.

10. The method for detecting nitrite according to claim 6, characterized in that, In the step of adding the visual colorimetric sensor and the acidic buffer solution to the nitrite solution to be tested, and allowing it to stand to allow for a redox reaction to occur: the nitrite sample to be tested is derived from any one of drinking water, ambient water, milk, and pickled food.

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