Cellulose membrane-based fluorescence colorimetric sensor based on Tb-AMP compound as well as preparation method and application of cellulose membrane-based fluorescence colorimetric sensor

Abstract

The invention discloses a cellulose membrane-based fluorescence colorimetric sensor based on a Tb-AMP compound as well as a preparation method and application thereof, and belongs to the cross technical fields of new materials, fluorescence sensors, inspection and detection and the like. The fluorescent colorimetric sensor takes a cellulose membrane as a substrate, and the cellulose membrane is loaded with a Tb < 3 + > and AMP compound after carboxymethylation. Through the synergistic effect of the lanthanide series metal Tb-AMP compound and in combination with the porous characteristic of the cellulose membrane, high sensitivity, high selectivity and visual detection of FQs are achieved, meanwhile, the performance is not changed after the cellulose membrane is stored for 60 days, the cellulose membrane has long-term stability, the cellulose membrane serves as a substrate, the raw material cost is low, and the method is suitable for industrial production. The method can be widely applied to real-time visual detection of FQs in practical samples such as drinking water, environmental water and milk.

Description

Technical Field

[0001] This invention relates to the interdisciplinary fields of new materials, fluorescence sensors, and testing and detection, specifically to a cellulose membrane-based fluorescence colorimetric sensor based on a Tb-AMP complex, its preparation method, and its application in the detection of fluoroquinolone drugs. Background Technology

[0002] Fluoroquinolones (FQs) are a class of synthetic broad-spectrum antibacterial drugs widely used in clinical medicine, animal husbandry, aquaculture, and agriculture due to their potent antibacterial efficacy and broad therapeutic spectrum. However, increasing consumption of FQs and improper disposal have led to their widespread presence in surface water, groundwater, and even food. FQs are chemically stable, poorly biodegradable, and easily accumulate in the environment. Residual FQs not only disrupt microbial ecosystems but also accelerate the evolution and spread of antibiotic resistance genes, posing a serious threat to public health and environmental safety.

[0003] Currently, the detection of fluoroquinolones mainly relies on instrumental analytical methods, such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and enzyme-linked immunosorbent assay (ELISA). However, these methods typically require expensive, large-scale instruments, cumbersome sample pretreatment processes, specialized testing personnel, and long detection times, making it difficult to meet the needs of on-site, rapid detection. Summary of the Invention

[0004] This invention provides a cellulose membrane-based fluorescence colorimetric sensor based on a Tb-AMP complex, its preparation method, and its application. The resulting sensor has the advantages of low cost, high selectivity, high sensitivity, fast response speed (visual), and no need for large instruments, thus solving the problem that existing technologies cannot achieve low-cost, rapid on-site detection.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: One objective of this invention is to provide a cellulose membrane-based fluorescence colorimetric sensor based on a Tb-AMP complex, using a cellulose membrane as a substrate, wherein the cellulose membrane is carboxymethylated and then loaded with Tb. 3+ The complex with AMP (adenosine 5-phosphate); in which Tb 3+ Loaded in a covalent manner.

[0006] A second objective of this invention is to provide a method for preparing the aforementioned cellulose membrane-based fluorescence colorimetric sensor, comprising the following steps: 1) The cellulose membrane is first mixed with sodium hydroxide solution and reacted, then sodium chloroacetate is added, and the reaction is carried out at 40-100℃ for 5-24 hours to obtain carboxymethyl cellulose membrane; wherein the mass ratio of cellulose membrane to sodium chloroacetate is 1:0.2~0.8; 2) The carboxymethyl cellulose membrane obtained in step 1) is mixed with Tb 3+ The solutions were mixed and reacted to obtain loaded Tb. 3+ Carboxymethyl cellulose membrane; 3) The load Tb obtained in step 2) 3+ The carboxymethyl cellulose membrane was reacted in AMP solution to obtain a cellulose membrane-based fluorescent colorimetric sensor.

[0007] According to the above scheme, in step 1), the preparation method of cellulose membrane is as follows: cellulose is dissolved in sodium hydroxide / urea aqueous solution to prepare cellulose solution, the cellulose solution is degassed to form a membrane, and then the membrane is placed in an acidic solution to solidify, thereby obtaining cellulose membrane.

[0008] Preferably, in the sodium hydroxide / urea aqueous solution, the mass ratio of sodium hydroxide, urea, and water is 6-8:10-14:81.

[0009] Preferably, the concentration of the cellulose solution is 0.5-20 wt%.

[0010] Preferably, the acidic solution is dilute sulfuric acid with a concentration of 2-5 wt%.

[0011] According to the above scheme, in step 1), the mass ratio of cellulose membrane to sodium hydroxide is 1:0.02-2; preferably 1:0.1~1, more preferably 1:0.1-0.3.

[0012] According to the above scheme, in step 1), the concentration of sodium hydroxide solution is 3~10wt%.

[0013] According to the above scheme, in step 1), the reaction time between the cellulose membrane and the sodium hydroxide solution is 0.5-3 hours.

[0014] According to the above scheme, in step 1), the mass ratio of cellulose membrane to sodium chloroacetate is 1:0.3~0.6.

[0015] According to the above scheme, in step 1), when sodium chloroacetate is added, the reaction temperature is 40-80℃ and the reaction time is 10-18h.

[0016] According to the above scheme, in step 2), the cellulose membrane and Tb 3+ The mass-to-volume ratio of the solution is 15g:40-80mL; Tb 3+ The concentration of the solution is 1-10 mM.

[0017] According to the above scheme, in step 2), the reaction time is 1-5 hours.

[0018] According to the above scheme, in step 3), the AMP solution is prepared as follows: AMP is prepared using HEPES buffer solution with a concentration of 0.01-1M, and the concentration of the prepared AMP solution is 0.01-0.5mM.

[0019] According to the above scheme, in step 3), the reaction time is 0.5-10h.

[0020] A third objective of this invention is to provide the application of the aforementioned cellulose membrane-based fluorescence colorimetric sensor in the detection of fluoroquinolone drugs. When the sample to be tested contains or is present with fluoroquinolone drugs (FQs), the Tb in the sensor... 3+ It combines with FQs and AMP to form a luminescent complex, which changes the triggered fluorescence color from essentially colorless to green.

[0021] According to the above scheme, the objects of the application include edible products such as drinking water, ambient water, milk, and egg whites.

[0022] According to the above scheme, the detection limit of the application is 0.1 μM. When the triggered fluorescence color changes from basically colorless to green, it indicates that the sample contains fluoroquinolone antibiotics.

[0023] Preferably, standard solutions of fluoroquinolone drugs at different concentrations are prepared using a gradient dilution method. These solutions are then added to the aforementioned cellulose membrane-based fluorescence colorimetric sensor. After standing, the solutions are retrieved and subjected to fluorescence detection to obtain digital color images. A standard card based on the concentration and color information of the fluoroquinolone drugs is constructed, and rapid detection of unknown fluoroquinolone drug concentrations is achieved based on the color information in the standard card. More preferably, the standing time is 20-40 minutes.

[0024] The beneficial effects of this invention are as follows: 1. This invention provides a cellulose membrane-based fluorescence colorimetric sensor, using a cellulose membrane as a substrate, wherein the cellulose membrane is carboxymethylated and then loaded with Tb. 3+ The lanthanide metal Tb-AMP complex, combined with the porous properties of cellulose membranes, achieves high sensitivity (detection limit 0.1 μM), high selectivity (unique green fluorescence signal, resistant to various metal ions and interferences), and rapid response (qualitative analysis within 5 seconds) for flavonoids (FQs). Furthermore, its performance remains unchanged after 60 days of storage, demonstrating long-term stability. Using cellulose membranes as a substrate results in low raw material costs, making it widely applicable for real-time visual detection of FQs in practical samples such as drinking water, environmental water, and milk.

[0025] 2. This invention provides a method for preparing the above-mentioned cellulose membrane-based fluorescent colorimetric sensor, which involves carboxymethylating the cellulose membrane and then reacting it with lanthanide metal terbium (Tb). 3+It is composed of cellulose membrane and sodium chloroacetate; wherein: the present invention first controls the ratio of cellulose membrane and sodium chloroacetate, reaction temperature and time to control the appropriate degree of substitution, while retaining the macroscopic solid morphology and microscopic porous structure of cellulose membrane, and introduces high-density carboxyl groups as Tb. 3+ The specific anchoring points not only prevent the membrane from dissolving in the detection solution, but also form the key technological basis for achieving subsequent high-efficiency loading; then, Tb is covalently attached... 3+ The AMP is stably loaded onto a carboxymethyl cellulose membrane and then composited with AMP to obtain a fluorescent colorimetric sensor. The preparation process of this invention is simple and the conditions are mild. The resulting sensor has excellent long-term stability, high sensitivity and selectivity, and has promising prospects for industrial application. Attached Figure Description

[0026] Figure 1 Digital images of the Tb-AMP@CCMs sensor prepared in Example 1 after interaction with CIP samples of different concentrations.

[0027] Figure 2 These are digital color images of the Tb-AMP@CCMs sensor prepared in Example 1 in the presence of different metal ions and common interfering substances.

[0028] Figure 3 Digital images of the colorimetric response of the Tb-AMP@CCMs sensor prepared in Example 1 at different storage days.

[0029] Figure 4 The image shows the color change of the Tb-AMP@CCMs sensor prepared in Example 1 after reaction with CIP solutions of different concentrations in milk under ultraviolet light.

[0030] Figure 5 The Tb-AMP@CCMs prepared in Example 1, the Tb-AMP@CMs prepared in Comparative Example 1, and the Tb-AMP@CMs prepared in Comparative Example 2 are compared with those prepared in Comparative Example 2. 3+ @CCM is compared with digital images of CIP after the reaction at the same concentration. Detailed Implementation

[0031] To enable those skilled in the art to fully understand the technical solution and beneficial effects of the present invention, the following detailed description is provided in conjunction with specific embodiments and accompanying drawings. It should be emphasized that the following embodiments are merely preferred embodiments of the present invention and do not constitute any limitation on the present invention. The present invention can have many other embodiments, all of which fall within the protection scope of the present invention.

[0032] Example 1 A method for preparing a cellulose membrane-based fluorescence colorimetric sensor based on a Tb-AMP complex is provided, comprising the following steps: 1) Pre-cool the sodium hydroxide / urea aqueous solution (where the mass ratio of sodium hydroxide, urea, and water is 7:12:81) to... At 12.5℃, add cellulose and stir until completely dissolved to obtain a cellulose solution with a concentration of 3wt%. Place the cellulose solution in a centrifuge and centrifuge at 4000 rpm for 10 minutes to remove bubbles.

[0033] 2) Pour the degassed cellulose solution onto a glass plate, spread it into a uniform film with a glass rod, place the film into a dilute sulfuric acid solution (concentration: 3wt%), and solidify it into a film by casting.

[0034] 3) Cut the cellulose membrane into 1cm×1cm pieces and then immerse them in pure water for later use.

[0035] 4) Take 15g of the cellulose membrane from step 3) and add it to 3g of sodium hydroxide and 60mL of water to prepare a solution. React at room temperature for 1 hour.

[0036] 5) Weigh 6g of sodium chloroacetate and add it to the mixed solution in step 4). Heat the mixture in a water bath at 50°C for 14 hours. After the reaction, remove the membrane and wash it until it is neutral to obtain a carboxymethyl cellulose membrane.

[0037] 6) Add the carboxymethyl cellulose membrane obtained in step 5) to 50 mL of a 5 mM TbCl3·6H2O solution, mix and stir for 1 hour to obtain the Tb-loaded membrane. 3+ Carboxymethyl cellulose membrane (Tb 3+ @CCM).

[0038] 7) Prepare 50 mL of AMP (adenosine 5-phosphate) solution with a concentration of 0.05 mM using 0.1 M 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer.

[0039] 8) Take the Tb obtained in step 6) 3+ @CCM was placed in AMP solution and gently stirred for 1 hour. The membrane was then removed and stored in deionized water to obtain Tb-AMP@CCM.

[0040] To fully understand the performance of the Tb-AMP@CCM sensor prepared in Example 1, a series of tests were conducted on samples, as follows: Using ciprofloxacin (CIP) and water as raw materials, test solutions with CIP concentrations of 0, 0.1, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 80, 90, 100, 200, and 500 μM were prepared using a gradient dilution method. The TB-AMP@CCMs sensor prepared in Example 1 was then added to each test solution. After standing for 30 minutes, the sensor was retrieved for fluorescence detection, yielding the results shown below. Figure 1 The digital image shown. From Figure 1 As can be seen, as the CIP concentration gradually increases from 0-500 μM, the fluorescence of Tb-AMP@CCMs gradually changes from orange-red to green, which provides a possibility for the quantitative detection of FQs. Figure 1 It can also be found that the visual detection limit (VLOD) of Tb-AMP@CCM for the chemical colorimetric sensor of FQs is 0.1 μM.

[0041] To verify that the Tb-AMP@CCMs sensor prepared in Example 1 can trigger significant fluorescence upon interaction with fluoroquinolone drugs and is unaffected by interference from other chemical components, the color changes of the Tb-AMP@CCM sensor in solutions of different metal ions and several common interfering substances (2 mM, 5 mL) were tested. The results are as follows: Figure 2 As shown. By Figure 2 It can be seen that the Tb-AMP@CCMs sensor is effective against five metal ions (K+, K ... + Ca 2+ Na + Mg 2+ Ba 2+ No significant changes were observed in the Tb-AMP@CCMs sensor in eight common interfering solutions (Melamine, Sucrose, Glu, VA, AZM, Urea, IBU, GSH) and H2O; however, when CIP was added to these solutions, the color of the Tb-AMP@CCMs sensor rapidly changed from orange to green. This demonstrates the specific selectivity of the Tb-AMP@CCMs sensor for FQs.

[0042] To determine whether the Tb-AMP@CCMs sensor possesses long-term stable performance, the Tb-AMP@CCMs prepared in Example 1 were stored in an aqueous solution at room temperature for 1 day, 10 days, 30 days, 50 days, and 60 days. They were then immersed in a test solution with an FQs concentration of 100 μM, allowed to stand for 30 minutes, and then retrieved for fluorescence detection. The results are as follows: Figure 3 As shown. By Figure 3 It can be seen that, under the same detection conditions, the difference in fluorescence intensity observed by the naked eye after the reaction of the Tb-AMP@CCMs sensor is not significant as the storage days increase. Even after storage for more than 60 days, the detection effect of the TB-AMP@CCM sensor on TC remains unaffected.

[0043] To verify the actual performance of the TB-AMP@CCMs sensor, we applied it to the detection of ciprofloxacin in milk. The specific process is as follows: (1) Dilute the milk sample to be tested by 10 times, and then add CIP standard solution to prepare a series of CIP solutions of different concentrations (5, 10, 50µM); (2) Take 5mL of CIP solution and place it in a reaction bottle. After washing the Tb-AMP@CCMs sensor with water, contact it with each sample for detection and analysis; (3) Place the Tb-AMP@CCMs sensor under a UV lamp for irradiation and record the color change results. The specific results are as follows: Figure 4 As shown.

[0044] Depend on Figure 4 It can be seen that as the CIP solution in the milk sample gradually increases from 5-50 μM, the green fluorescence of the Tb-AMP@CCMs sensor gradually increases; compared with the addition of standard CIP solution, the fluorescence intensity is not significantly different by the naked eye, indicating that the Tb-AMP@CCMs sensor has the potential to detect FQs in real samples.

[0045] Example 2 The Tb-AMP@CCM sensor prepared in Example 1 was used to detect the lake water (instead of milk) of Jingsi Lake. The results showed that the Tb-AMP@CCM sensor could indeed detect CIP in the lake water.

[0046] Example 3 The Tb-AMP@CCM sensor prepared in Example 1 was used to test tap water (instead of milk) in the laboratory. The results showed that the Tb-AMP@CCM sensor could indeed detect CIP in tap water.

[0047] Comparative Example 1 A method for preparing Tb-AMP@CM is provided, wherein the cellulose membrane is not carboxylated, and the method specifically includes the following steps: 1) Pre-cool the sodium hydroxide / urea aqueous solution (where the mass ratio of sodium hydroxide, urea, and water is 7:12:81) to... At 12.5℃, add cellulose and stir until completely dissolved to obtain a cellulose solution with a concentration of 3wt%. Place the cellulose solution in a centrifuge and centrifuge at 4000 rpm for 10 minutes to remove bubbles.

[0048] 2) Pour the degassed cellulose solution onto a glass plate, spread it into a uniform film with a glass rod, place the film into a dilute sulfuric acid solution (concentration: 3wt%), and solidify it into a film by casting.

[0049] 3) Cut the cellulose membrane into 1cm×1cm pieces and then immerse them in pure water for later use.

[0050] 4) Add the pure cellulose membrane obtained in step 3) to 50 mL of a 5 mM TbCl3·6H2O solution, mix and stir for 1 hour to obtain the Tb-loaded membrane. 3+ cellulose membrane (Tb 3+ @CM).

[0051] 5) Prepare 50 mL of AMP solution with a concentration of 0.05 mM using 0.1 M 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer.

[0052] 6) Take the Tb obtained in step 4) 3+ @CM was placed in AMP solution and gently stirred for 1 hour. The membrane was then removed and stored in deionized water to obtain Tb-AMP@CM.

[0053] Comparative Example 2 Provide a Tb 3+ The preparation method of @CCM, without composite AMP, specifically includes the following steps: 1) Pre-cool the sodium hydroxide / urea aqueous solution (where the mass ratio of sodium hydroxide, urea, and water is 7:12:81) to... At 12.5℃, add cellulose and stir until completely dissolved to obtain a cellulose solution with a concentration of 3wt%. Place the cellulose solution in a centrifuge and centrifuge at 4000 rpm for 10 minutes to remove bubbles.

[0054] 2) Pour the degassed cellulose solution onto a glass plate, spread it into a uniform film with a glass rod, place the film into a dilute sulfuric acid solution (concentration: 3wt%), and solidify it into a film by casting.

[0055] 3) Cut the cellulose membrane into 1cm×1cm pieces and then immerse them in pure water for later use.

[0056] 4) Take 15g of the cellulose membrane from step 3) and add it to 3g of sodium hydroxide and 60mL of water to prepare a solution. React at room temperature for 1 hour.

[0057] 5) Weigh 6g of sodium chloroacetate and add it to the mixed solution in step 4). Heat the mixture in a water bath at 50°C for 14 hours. After the reaction, remove the membrane and wash it until it is neutral to obtain a carboxymethyl cellulose membrane.

[0058] 6) Add the carboxymethyl cellulose membrane obtained in step 5) to 50 mL of a 5 mM TbCl3·6H2O solution, mix and stir for 1 hour to obtain the Tb-loaded membrane. 3+Carboxymethyl cellulose membrane (Tb 3+ @CCM).

[0059] The Tb-AMP@CM obtained in Comparative Example 1 and the Tb obtained in Comparative Example 2 were compared. 3+ The reaction of @CCM and Tb-AMP@CCM obtained in Example 1 with 100 μM CIP solution yielded the following results: Figure 5 As shown: This indicates that the membrane after carboxymethyl oxidation can better absorb Tb. 3+ Grafting is necessary to better integrate with Tb, especially with AMP. 3+ It forms a rigid structure, and after reacting with fluoroquinolone drugs, it forms an antenna effect film with enhanced fluorescence.

[0060] It should be understood that those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A cellulose membrane-based fluorescence colorimetric sensor based on a Tb-AMP complex, characterized in that, Using cellulose membrane as a substrate, the cellulose membrane was carboxymethylated and then loaded with Tb. 3+ and AMP complex; in which Tb 3+ Loaded in a covalent manner.

2. A method for preparing the cellulose membrane-based fluorescence colorimetric sensor according to claim 1, characterized in that, Includes the following steps: 1) The cellulose membrane is first mixed with sodium hydroxide solution and reacted, then sodium chloroacetate is added, and the reaction is carried out at 40-100℃ for 5-24 hours to obtain carboxymethyl cellulose membrane; wherein the mass ratio of cellulose membrane to sodium chloroacetate is 1:0.2~0.8; 2) The carboxymethyl cellulose membrane obtained in step 1) is mixed with Tb 3+ The aqueous solution was mixed and reacted to obtain loaded Tb. 3+ Carboxymethyl cellulose membrane; 3) The load Tb obtained in step 2) 3+ The carboxymethyl cellulose membrane was reacted in AMP solution to obtain a cellulose membrane-based fluorescent colorimetric sensor.

3. The preparation method according to claim 2, characterized in that, In step 1), the mass ratio of cellulose membrane to sodium hydroxide is 1:0.02-2; the concentration of sodium hydroxide solution is 3~10wt%.

4. The preparation method according to claim 2, characterized in that, In step 1), the reaction time between the cellulose membrane and the sodium hydroxide solution is 0.5-3 hours.

5. The preparation method according to claim 2, characterized in that, In step 1), when sodium chloroacetate is added, the reaction temperature is 40-80℃ and the reaction time is 10-18h.

6. The preparation method according to claim 2, characterized in that, In step 2), the cellulose membrane and Tb 3+ The mass-to-volume ratio of the solution is 15g:40-80mL; Tb 3+ The concentration of the solution is 1-10 mM.

7. The preparation method according to claim 2, characterized in that, In step 2), the reaction time is 1-5 hours.

8. The preparation method according to claim 2, characterized in that, In step 3), the reaction time is 0.5-10 hours.

9. The preparation method according to claim 2, characterized in that, In step 3), the AMP solution is prepared as follows: AMP is prepared using HEPES buffer solution with a concentration of 0.01-1M, and the concentration of the prepared AMP solution is 0.01-0.5mM.

10. The use of a cellulose membrane-based fluorescence colorimetric sensor according to any one of claims 1-9 in the detection of fluoroquinolone drugs.

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