Visual double-fluorescent probe composite bacterial cellulose membrane and preparation method thereof

Abstract

The invention discloses a visual double-fluorescent probe composite bacterial cellulose membrane, which comprises a bacterial fiber membrane with a three-dimensional nano network structure, and a cadmium telluride quantum dot fluorescent probe and a rhodamine B fluorescent probe which are loaded on the bacterial fiber membrane, under an ultraviolet lamp, the fluorescence color of the visual double-fluorescence probe composite bacterial cellulose membrane changes along with the acidity value, and the visual double-fluorescence probe composite bacterial cellulose membrane shows green fluorescence under a neutral condition and shows red fluorescence under an acidic condition; wherein in the visual double-fluorescent probe composite bacterial cellulose membrane, the loading capacity of the cadmium telluride quantum dot fluorescent probe is 5wt%-30wt%, and the loading capacity of the rhodamine B fluorescent probe is 5wt%-30wt%. The method disclosed by the invention is simple, convenient and rapid to operate, the acidity result and hydrogen ion distribution of multiple sites in the paper can be rapidly and nondestructively detected, the detection range is relatively wide due to the combination of the two fluorescent probes, and meanwhile, the detection visualization can be well realized due to the strong color contrast of the probes.

Description

Technical Field

[0001] The invention belongs to the field of paper cultural relic protection and polymer materials, and specifically relates to a visualized double-fluorescent probe composite bacterial cellulose membrane and a preparation method thereof, as well as an application in non-destructive visualized detection of the acidification degree of paper cultural relics. Background Art

[0002] Paper artifacts have high cultural and historical value. However, during the long-term preservation process, the paper of paper artifacts will inevitably undergo acidification reaction, resulting in the cleavage of glycosidic bonds of cellulose in the paper, a decrease in polymerization degree, and a decrease in mechanical properties; and the accumulation of acid in the paper will accelerate its aging process. Therefore, acid detection of paper artifacts is particularly important for paper protection.

[0003] At present, the main methods for detecting the acidity of paper cultural relics are hot extraction method, cold extraction method and pH flat-head electrode detection method. The extraction method will cause irreversible damage to the paper sample during sampling, so it cannot be used for pH value detection of precious cultural relics. The pH flat-head electrode detection method cannot achieve rapid detection of the acidity of all areas on the paper surface in a short time. Therefore, it is of great significance to develop a rapid and non-destructive detection method for paper cultural relics.

[0004] Fluorescence analysis has high sensitivity and immediacy for pH detection, and has been tried to be applied to the pH detection of paper artifacts. However, the fluorescence detection results of a single species are greatly affected by its concentration and the detection environment, so it is best to use multi-color fluorescence analysis to improve the detection accuracy by combining multiple fluorescent substances. However, the current multi-color fluorescence analysis method is a paper artifact detection method implemented in the form of a solution, and its residue on the surface of the paper will pose a potential threat to the paper and cause trouble for the preservation of paper artifacts. Therefore, fixing multiple fluorescent substances to the base material can not only improve the accuracy of the detection, but also reduce the residual risk brought by the fluorescence detection method.

[0005] Bacterial cellulose is a polymer material with a similar structure to paper. Due to its high purity and high modifiability, it can be used as a carrier of fluorescent probes. The bacterial cellulose membrane compounded with fluorescent probes can not only retain the fluorescence characteristics of a single probe, but also avoid potential interference between the probe and paper, providing convenience for the visual non-destructive detection of the acidity and hydrogen ion distribution of paper artifacts.

[0006] Patent document CN111808313A discloses a preparation method of in-situ synthesis of cadmium telluride quantum dots on bacterial cellulose and a method for visual detection of pH in paper cultural relics. However, this method only uses cadmium telluride quantum dots as fluorescent probes. Although the fluorescence intensity of the prepared fluorescent film is linearly related to pH in the fluorescence spectrum in the range of pH = 4 to 7, the use of a single fluorescent display for pH measurement, coupled with a slight change in light intensity, may cause distortion of pH detection results and make it difficult to accurately distinguish with the naked eye; especially for the distribution of acidity and the degree of acidification in paper cultural relics, it is even more difficult to accurately visualize.

[0007] In order to solve the problem of limited detection of a single fluorescent probe, the present invention proposes the preparation of a nano fluorescent film loaded with dual fluorescent probes that can be controlled in multiple chromaticities within a specific pH range. Summary of the invention

[0008] The purpose of the present invention is to provide a visualized dual-fluorescence probe composite bacterial cellulose membrane and a preparation method thereof, as well as its application in detecting the acidification degree of paper cultural relics, so as to solve the problems in the prior art such as the interference of single fluorescence method by factors such as concentration and environment, and potential interference between probe and paper.

[0009] In order to solve the above problems, the technical solution of the present invention is as follows:

[0010] The present invention discloses a visualized dual-fluorescence probe composite bacterial cellulose membrane, comprising: a bacterial fiber membrane with a three-dimensional nanostructure, and a cadmium telluride quantum dot fluorescent probe and a rhodamine B fluorescent probe loaded on the bacterial fiber membrane; under ultraviolet light, the fluorescent color of the visualized dual-fluorescence probe composite bacterial cellulose membrane changes with the acidity value, showing green fluorescence under neutral conditions and red fluorescence under acidic conditions;

[0011] Wherein, in the visualized dual-fluorescence probe composite bacterial cellulose membrane, the loading amount of the cadmium telluride quantum dot fluorescent probe is 5wt% to 30wt%, and the loading amount of the rhodamine B fluorescent probe is 5wt% to 30wt%.

[0012] The method for preparing a visualized dual-fluorescence probe composite bacterial cellulose membrane of the present invention comprises the following steps:

[0013] S1, mixing a chromium acetate aqueous solution and a thioglycolic acid aqueous solution in proportion, and adding an alkaline solution dropwise to adjust the pH value to 10.5-12 to obtain a precursor solution; soaking the bacterial cellulose membrane in the precursor solution and standing until adsorption equilibrium; adding a sodium tellurite aqueous solution thereto and stirring evenly, and then adding a sodium borohydride aqueous solution to obtain a reaction solution A;

[0014] S2, heating the reaction solution A to 80-120° C. and refluxing for 10-60 min; filtering and separating the solid, washing with ultrapure water, and removing the surface moisture with filter paper to obtain a bacterial cellulose membrane loaded with cadmium telluride quantum dot fluorescent probes;

[0015] S3, dissolving rhodamine B and hydrazine hydrate in ethanol at a molar ratio of 1:(0.1-0.5), heating to 80-100° C. and reacting under reflux; removing the solvent in the reaction by vacuum distillation, dissolving the residual solid with an acid solution, and adjusting the solution to neutrality with an alkaline solution to obtain rhodamine B hydrazide;

[0016] S4, dissolving rhodamine B hydrazide and 5-sulfosalicylaldehyde in ethanol at a molar ratio of 1:(0.95-1.05), heating to 80-100° C. for reflux reaction; then removing the solvent in the reaction by vacuum distillation, and recrystallizing the reaction product to obtain a rhodamine B fluorescent probe;

[0017] S5, immersing the bacterial cellulose membrane loaded with cadmium telluride quantum dot fluorescent probes in an ethanol solution of rhodamine B fluorescent probes until adsorption equilibrium is reached; taking out the bacterial cellulose membrane and washing it with ultrapure water, and absorbing the surface moisture with filter paper to obtain the visualized dual fluorescent probe composite bacterial cellulose membrane.

[0018] Preferably, in step S1, the molar ratio of chromium acetate (Cd(CH3COO)2), thioglycolic acid (TGA) and sodium tellurite (Na2TeO3) is 1:(0.95-1.05):(0.1-1); the amount of sodium borohydride (NaBH4) added is 5-10 times that of chromium acetate (Cd(CH3COO)2); and the membrane thickness of the bacterial cellulose is 0.05-10 mm.

[0019] Preferably, the concentration of the chromium acetate aqueous solution is 1-20 mmol / L, the concentration of the thioglycolic acid aqueous solution is 1-20 mmol / L, the concentration of the sodium tellurite aqueous solution is 0.1-1 mmol / L; and the concentration of the sodium borohydride aqueous solution is 5-100 mmol / L.

[0020] Preferably, in step S1 and step S3, the alkaline solution is at least one of a sodium hydroxide solution and a potassium hydroxide solution; in step S3, the acid solution is at least one of a hydrochloric acid aqueous solution or a sulfuric acid aqueous solution with a molar concentration of 0.1 to 1.5 mol / L.

[0021] Preferably, in step S3, the concentration of rhodamine B in ethanol is 50-100 mmol / L, the mass concentration of hydrazine hydrate in ethanol is 10%-30%, and the reaction time of heating and reflux is 5-10 h.

[0022] Preferably, in step S4, the concentration of rhodamine B hydrazide in ethanol is 5 to 30 mmol / L, the concentration of 5-sulfonyl salicylaldehyde in ethanol is 5 to 30 mmol / L, and the reaction time of heating under reflux is 5 to 10 h.

[0023] Preferably, in step S5, the mass concentration of the ethanol solvent of the rhodamine B fluorescent probe solution is 0.05 to 2.5 mmol / L, preferably 0.5 to 1.5 mmol / L; and the immersion time of the bacterial cellulose membrane is 30 to 240 min.

[0024] The visualized dual fluorescent probe composite bacterial cellulose membrane of the present invention is used to detect the acidification degree and hydrogen ion distribution of paper cultural relics, and to quickly visualize the surface acidity of paper cultural relics. The detection method includes direct or indirect contact with the paper cultural relics; the detection result is obtained by the fluorescent color change or / and fluorescence spectrum analysis of the bacterial cellulose composite membrane.

[0025] The degree of acidification of paper artifacts can be determined by measuring their pH value: the visualized dual fluorescent probe composite bacterial cellulose film is applied to the surface of the paper artifacts and allowed to stand for at least 5 minutes. The pH value and hydrogen ion distribution of the paper artifacts can be obtained by observing their color changes and calculating the intensity difference of the two fluorescent probes; the fluorescence excitation wavelength is 350-400nm.

[0026] The intensity difference of the two fluorescent probes can be determined by a standard curve method, and the determination method of the standard curve is: the visualized dual fluorescent probe composite bacterial cellulose membrane is placed in a series of buffer solutions with different pH values ​​and soaked for at least 30 seconds, and then its fluorescence spectrum is measured, the intensity difference of the two fluorescent probes at 525nm and 580nm is calculated, and a standard curve of fluorescence difference changing with pH is established. The buffer solution is a phosphate buffer solution with a pH range of 2 to 8.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] (1) The present invention anchors two fluorescent probes on the nano bacterial cellulose membrane. + It is highly selective and sensitive, with complementary pH detection ranges and obvious differences in fluorescence colors, which improves the accuracy and visualization of pH detection of paper cultural relics and realizes visual non-destructive detection of the acidity of common paper cultural relics.

[0029] (2) The dual fluorescent probe composite bacterial cellulose membrane of the present invention anchors the fluorescent probe on the surface of bacterial cellulose through hydrogen bonding interactions, thereby achieving in-situ, non-destructive, and rapid detection of the acidity of paper cultural relics while avoiding the residue of fluorescent probe substances on the paper during the test, thereby reducing the potential threat of detection to the preservation of paper cultural relics.

[0030] (3) The method of the present invention is simple and quick to operate, and can quickly and non-destructively detect the acidity results and hydrogen ion distribution of multiple sites in paper, and obtain the distribution and degree of acidification on the surface of the entire paper cultural relic. The combination of the two fluorescent probes enables it to have a wider detection range, and the strong color contrast of the probes can well realize the visualization of the detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The reaction process of bacterial cellulose membrane loaded with cadmium telluride quantum dot fluorescent probe.

[0032] Figure 2 This is the reaction process of Rhodamine B fluorescent probe.

[0033] Figure 3 This is a scanning electron microscope image of the visualized dual fluorescent probe composite bacterial cellulose membrane in Example 1.

[0034] Figure 4 This is a transmission electron microscopy image of the visualized dual fluorescent probe composite bacterial cellulose membrane in Example 1.

[0035] Figure 5 This is the standard curve of the fluorescence difference of the dual fluorescent probe composite bacterial cellulose membrane at 525nm and 580nm as a function of pH in Example 1. DETAILED DESCRIPTION

[0036] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0037] The raw materials in this embodiment are all commercially available products.

[0038] Example 1

[0039] A method for preparing a visualized dual-fluorescence probe composite bacterial cellulose membrane comprises the following steps:

[0040] S1, 10 mL of 5 mmol / L chromium acetate aqueous solution and 10 mL of 5 mmol / L thioglycolic acid aqueous solution were mixed, and sodium hydroxide solution was added dropwise to adjust the pH value to 10.5 to obtain a precursor solution; a bacterial cellulose membrane with a mass of 2 g and a thickness of 0.2 mm was immersed in the precursor solution, and allowed to stand for 65 min until adsorption equilibrium; 10 mL of 0.2 mmol / L sodium tellurite aqueous solution was added thereto and stirred evenly, and then 10 mL of 30 mmol / L sodium borohydride aqueous solution was added to obtain a reaction solution A;

[0041] S2, heating the reaction solution A to 95°C and refluxing for 50 min, Figure 1 The invention relates to a reaction process of a bacterial cellulose membrane loaded with a cadmium telluride quantum dot fluorescent probe; a solid is obtained by filtering and separating, and then washed with ultrapure water, and surface moisture is absorbed with filter paper to obtain a bacterial cellulose membrane loaded with a cadmium telluride quantum dot fluorescent probe;

[0042] S3, dissolving 2 mmol of rhodamine B and 4 mL of hydrazine hydrate in 30 mL of ethanol, heating to 95° C. and reflux for 6 h; removing the solvent in the reaction by vacuum distillation, then dissolving the residual solid with a 1 mol / L HCl solution, and adjusting the solution to neutrality with a 1 mol / L NaOH solution to obtain rhodamine B hydrazide;

[0043] S4, dissolving 0.1 mmol of rhodamine B hydrazide and 0.1 mmol of 5-sulfonyl salicylaldehyde in 10 mL of ethanol, heating to 85°C and reflux for 6 h; Figure 2 The invention relates to a reaction process of a Rhodamine B fluorescent probe; the solvent in the reaction is removed by vacuum distillation, and the reaction product is recrystallized to obtain a Rhodamine B fluorescent probe;

[0044] S5, immersing the bacterial cellulose membrane loaded with cadmium telluride quantum dot fluorescent probes in an ethanol solution of rhodamine B fluorescent probe at a concentration of 1 mmol / L for 34 minutes until adsorption equilibrium is reached; taking out the bacterial cellulose membrane and washing it with ultrapure water, and absorbing the surface moisture with filter paper to obtain a visualized dual fluorescent probe composite bacterial cellulose membrane.

[0045] Figure 3 This is a scanning electron microscope image of the visualized dual fluorescent probe composite bacterial cellulose membrane in Example 1; Figure 4 This is a transmission electron microscope image of the visual dual fluorescent probe composite bacterial cellulose membrane in Example 1. Figure 3 and Figure 4 It can be seen that the fluorescent probe particles are uniformly loaded on the bacterial cellulose nanofibers.

[0046] The bacterial cellulose membrane loaded with the dual fluorescent probe obtained in the experiment was placed in a phosphate buffer solution with a pH value of 2 to 8, and allowed to stand for a while. Its fluorescence spectrum was measured at an excitation light wavelength of 365 nm, and the intensity difference of the two fluorescent probes at 525 nm and 580 nm was calculated. A standard curve of fluorescence difference versus pH was established (see Appendix). Figure 5 ).

[0047] Determination of pH value of paper artifacts: The obtained visualized dual fluorescent probe composite bacterial cellulose film was applied to the surface of the paper artifact 1 to be tested and allowed to stand for 5 minutes; the composite bacterial cellulose film was then placed under an excitation light wavelength of 365nm to observe its color change. The results showed that the composite bacterial cellulose film was light purple-red, indicating that the paper was acidic; the pH value of sample No. 1 was calculated to be about 5.0 by testing the fluorescence intensity, and its pH was verified to be 4.7 using a pH flat-head electrode.

[0048] Example 2

[0049] A method for preparing a visualized dual-fluorescence probe composite bacterial cellulose membrane comprises the following steps:

[0050] S1, 10 mL of chromium acetate aqueous solution with a concentration of 10 mmol / L and 10 mL of thioglycolic acid aqueous solution with a concentration of 10 mmol / L are mixed, and sodium hydroxide solution is added dropwise to adjust the pH value to 11.0 to obtain a precursor solution; a bacterial cellulose membrane with a mass of 1 g and a film thickness of 0.2 mm is immersed in the precursor solution, and allowed to stand for a certain time for 1 hour until adsorption equilibrium is reached; 10 mL of sodium tellurite aqueous solution with a concentration of 0.5 mmol / L is added thereto and stirred evenly, and then 10 mL of sodium borohydride aqueous solution with a concentration of 50 mmol / L is added to obtain a reaction solution A;

[0051] S2, heating the reaction solution A to 100°C and refluxing for 40 minutes, filtering and separating the solid, washing it with ultrapure water, and removing the surface moisture with filter paper to obtain a bacterial cellulose membrane loaded with cadmium telluride quantum dot fluorescent probes;

[0052] S3, dissolving 2 mmol of rhodamine B and 4 mL of hydrazine hydrate in 30 mL of ethanol, heating to 85° C. and reflux for 8 h; removing the solvent in the reaction by vacuum distillation, then dissolving the residual solid with a 1 mol / L HCl solution, and adjusting the solution to neutrality with a 1 mol / L NaOH solution to obtain rhodamine B hydrazide;

[0053] S4, dissolving 0.1 mmol of rhodamine B hydrazide and 0.1 mmol of 5-sulfonic acid salicylaldehyde in 10 mL of ethanol, heating to 85° C. and reflux for 6 h; removing the solvent in the reaction by vacuum distillation, and recrystallizing the reaction product to obtain a rhodamine B fluorescent probe;

[0054] S5, immersing the bacterial cellulose membrane loaded with cadmium telluride quantum dot fluorescent probes in an ethanol solution of rhodamine B fluorescent probe at a concentration of 1 mmol / L for 58 minutes until adsorption equilibrium is reached; taking out the bacterial cellulose membrane and washing it with ultrapure water, and absorbing the surface moisture with filter paper to obtain a visualized dual fluorescent probe composite bacterial cellulose membrane.

[0055] The bacterial cellulose membrane loaded with the dual fluorescent probe obtained in the experiment was placed in a phosphate buffer solution with a pH value of 2 to 8, left to stand for a while, and its fluorescence spectrum was measured at an excitation light wavelength of 365nm. The difference in the intensity of the two fluorescent probes was calculated, and a standard curve of the fluorescence difference changing with pH was established.

[0056] Determination of pH value of paper artifacts: The obtained visualized dual-fluorescence probe composite bacterial cellulose film was applied to the surface of the paper artifact 2 to be tested and allowed to stand for 5 minutes; the composite bacterial cellulose film was then placed under an excitation light wavelength of 365nm to observe its color change. The results showed that the composite bacterial cellulose film was light green, indicating that the paper was weakly acidic. The pH value of sample No. 2 was calculated to be about 5.8 by testing the fluorescence intensity. Subsequently, a pH flat-head electrode was used to verify that its pH was 5.6.

[0057] Example 3

[0058] A method for preparing a visualized dual-fluorescence probe composite bacterial cellulose membrane comprises the following steps:

[0059] S1, 10 mL of chromium acetate aqueous solution with a concentration of 10 mmol / L and 10 mL of thioglycolic acid aqueous solution with a concentration of 10 mmol / L are mixed, and sodium hydroxide solution is added dropwise to adjust the pH value to 11.0 to obtain a precursor solution; a bacterial cellulose membrane with a mass of 2 g and a film thickness of 0.2 mm is immersed in the precursor solution, and allowed to stand for a certain time for 1 hour until adsorption equilibrium is reached; 10 mL of sodium tellurite aqueous solution with a concentration of 0.5 mmol / L is added thereto and stirred evenly, and then 10 mL of sodium borohydride aqueous solution with a concentration of 50 mmol / L is added to obtain a reaction solution A;

[0060] S2, heating the reaction solution A to 100°C and refluxing for 40 minutes, filtering and separating the solid, washing it with ultrapure water, and removing the surface moisture with filter paper to obtain a bacterial cellulose membrane loaded with cadmium telluride quantum dot fluorescent probes;

[0061] S3, dissolving 2.5 mmol of rhodamine B and 6 mL of hydrazine hydrate in 30 mL of ethanol, heating to 85° C. and reflux for 8 h; removing the solvent in the reaction by distillation under reduced pressure, then dissolving the residual solid with a 1 mol / L HCl solution, and adjusting the solution to neutrality with a 1 mol / L NaOH solution to obtain rhodamine B hydrazide;

[0062] S4, dissolving 0.2 mmol of rhodamine B hydrazide and 0.2 mmol of 5-sulfonic acid salicylaldehyde in 10 mL of ethanol, heating to 85° C. and reflux for 6 h; removing the solvent in the reaction by vacuum distillation, and recrystallizing the reaction product to obtain a rhodamine B fluorescent probe;

[0063] S5, immersing the bacterial cellulose membrane loaded with cadmium telluride quantum dot fluorescent probes in an ethanol solution of rhodamine B fluorescent probe at a concentration of 1 mmol / L for 60 minutes until adsorption equilibrium is reached; taking out the bacterial cellulose membrane and washing it with ultrapure water, and absorbing the surface moisture with filter paper to obtain a visualized dual fluorescent probe composite bacterial cellulose membrane.

[0064] The bacterial cellulose membrane loaded with the dual fluorescent probe obtained in the experiment was placed in a phosphate buffer solution with a pH value of 2 to 8, left to stand for a while, and its fluorescence spectrum was measured at an excitation light wavelength of 365nm. The difference in the intensity of the two fluorescent probes was calculated, and a standard curve of the fluorescence difference changing with pH was established.

[0065] Determination of pH value of paper artifacts: The obtained visualized dual fluorescent probe composite bacterial cellulose film was applied to the surface of the paper artifact 3 to be tested and allowed to stand for 5 minutes; the composite bacterial cellulose film was then placed under an excitation light wavelength of 365nm to observe its color change. The results showed that the bacterial cellulose composite film was green, indicating that the paper was weakly acidic. The pH value of sample No. 3 was calculated to be about 6.4 by testing the fluorescence intensity. Subsequently, a pH flat-head electrode was used to verify that its pH was 6.5.

[0066] Example 4

[0067] A method for preparing a visualized dual-fluorescence probe composite bacterial cellulose membrane comprises the following steps:

[0068] S1, 10 mL of 1 mmol / L chromium acetate aqueous solution and 10 mL of 1 mmol / L thioglycolic acid aqueous solution are mixed, and sodium hydroxide solution is added dropwise to adjust the pH value to 11.0 to obtain a precursor solution; a bacterial cellulose membrane with a mass of 2 g and a film thickness of 0.2 mm is immersed in the precursor solution, and allowed to stand for a certain time for 1 hour until adsorption equilibrium is reached; 10 mL of 0.2 mmol / L sodium tellurite aqueous solution is added thereto and stirred evenly, and then 10 mL of 5 mmol / L sodium borohydride aqueous solution is added to obtain a reaction solution A;

[0069] S2, heating the reaction solution A to 110°C and refluxing for 35 minutes, filtering and separating the solid, washing it with ultrapure water, and absorbing the surface moisture with filter paper to obtain a bacterial cellulose membrane loaded with cadmium telluride quantum dot fluorescent probes;

[0070] S3, dissolving 3 mmol of rhodamine B and 6 mL of hydrazine hydrate in 30 mL of ethanol, heating to 82° C. and reflux for 10 h; removing the solvent in the reaction by distillation under reduced pressure, then dissolving the residual solid with a 1 mol / L HCl solution, and adjusting the solution to neutrality with a 1 mol / L NaOH solution to obtain rhodamine B hydrazide;

[0071] S4, dissolving 0.1 mmol of rhodamine B hydrazide and 0.1 mmol of 5-sulfonic acid salicylaldehyde in 10 mL of ethanol, heating to 85° C. and reflux for 8 h; removing the solvent in the reaction by vacuum distillation, and recrystallizing the reaction product to obtain a rhodamine B fluorescent probe;

[0072] S5, immersing the bacterial cellulose membrane loaded with cadmium telluride quantum dot fluorescent probes in an ethanol solution of rhodamine B fluorescent probe at a concentration of 1 mmol / L for 125 min until adsorption equilibrium is reached; taking out the bacterial cellulose membrane and washing it with ultrapure water, and absorbing the surface moisture with filter paper to obtain a visualized dual fluorescent probe composite bacterial cellulose membrane.

[0073] The bacterial cellulose membrane loaded with the dual fluorescent probe obtained in the experiment was placed in a phosphate buffer solution with a pH value of 2 to 8, left to stand for a while, and its fluorescence spectrum was measured at an excitation light wavelength of 365nm. The difference in the intensity of the two fluorescent probes was calculated, and a standard curve of the fluorescence difference changing with pH was established.

[0074] Determination of pH value of paper artifacts: The obtained visualized dual fluorescent probe composite bacterial cellulose film was applied to the surface of the paper artifact 4 to be tested and allowed to stand for 5 minutes; the composite bacterial cellulose film was then placed under an excitation light wavelength of 365nm to observe its color change. The results showed that the bacterial cellulose composite film was purple-red, indicating that the paper was acidic. The pH value of sample No. 4 was calculated to be about 4.7 by testing the fluorescence intensity. Subsequently, a pH flat-head electrode was used to verify that its pH was 4.5.

[0075] Comparative Example 1

[0076] Comparative Example 1 is a comparative sample prepared by using Example 1 of patent document CN111808313A, and is compared with Example 4:

[0077] (1) Excess water was squeezed out of the bacterial cellulose membrane with filter paper to a thickness of 50 μm; the membrane was immersed in an anhydrous toluene solution of 3-aminopropyltriethoxysilane (APTES) at 60°C for 3 h; the treated bacterial cellulose membrane was then rinsed three times with 40 mL of anhydrous toluene and anhydrous acetone in turn to obtain an amino-modified bacterial cellulose membrane.

[0078] (2) 10 mL of chromium acetate solution with a concentration of 1 mmol / L was mixed with 10 mL of thioglycolic acid solution with a concentration of 1 mmol / L, and sodium hydroxide solution was added dropwise to adjust the pH value to 11.5 to obtain a precursor solution; 10 mL of sodium tellurite solution with a concentration of 0.2 mmol / L was added thereto and stirred evenly, and then 10 mL of sodium borohydride solution with a concentration of 0.5 mmol / L was added and stirred evenly; the solution was heated to 100°C and refluxed for 30 min to obtain a CdTe quantum dot fluorescent probe solution. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), CdTe fluorescent probe and amino bacterial cellulose membrane were mixed and reacted for 2 h, the EDC-NHS concentration was 0.05 mmol / L, and EDC / NHS=1:10; after the reaction, the green fluorescent bacterial cellulose membrane was taken out.

[0079] (3) Preparation of standard fluorescence colorimetric card: Prepare a series of different pH buffer solutions (pH = 4-7), immerse the bacterial cellulose loaded with fluorescent probe in them, take out after 1 min and observe in a UV dark box, then test the fluorescence intensity of the bacterial cellulose membrane at each pH to obtain a standard curve (excitation light wavelength 365 nm), take digital photos and organize them to obtain a standard fluorescence colorimetric card.

[0080] (4) pH detection of paper artifacts: The bacterial cellulose membrane was covered on the surface of the paper artifact No. 4 to be tested. After 5 minutes, the changes in the membrane were observed in a UV box. The pH of sample No. 4 was calculated to be 5.0 by testing the fluorescence intensity. A digital photo was taken and compared with the fluorescence colorimetric card, and the pH was 5. Then a pH flat-head electrode was used to verify that the pH was 4.5.

[0081] By comparing the results of Example 4 and Comparative Example 1, it is found that the dual-probe-loaded fluorescent film of Example 4 has higher sensitivity than the single quantum dot probe-loaded fluorescent film of Comparative Example 1, and is closer to the actual result.

[0082] Example 5

[0083] A method for preparing a visualized dual-fluorescence probe composite bacterial cellulose membrane comprises the following steps:

[0084] S1, 10 mL of 1 mmol / L chromium acetate aqueous solution and 10 mL of 1 mmol / L thioglycolic acid aqueous solution are mixed, and sodium hydroxide solution is added dropwise to adjust the pH value to 11.5 to obtain a precursor solution; a bacterial cellulose membrane with a mass of 2 g and a film thickness of 0.2 mm is immersed in the precursor solution, and allowed to stand for a certain time for 1 hour until adsorption equilibrium is reached; 10 mL of 0.5 mmol / L sodium tellurite aqueous solution is added thereto and stirred evenly, and then 10 mL of 5 mmol / L sodium borohydride aqueous solution is added to obtain a reaction solution A;

[0085] S2, heating the reaction solution A to 95°C and refluxing for 40 minutes, filtering and separating the solid, washing it with ultrapure water, and removing the surface moisture with filter paper to obtain a bacterial cellulose membrane loaded with cadmium telluride quantum dot fluorescent probes;

[0086] S3, dissolving 2 mmol of rhodamine B and 4 mL of hydrazine hydrate in 30 mL of ethanol, heating to 90° C. and reflux for 6 h; removing the solvent in the reaction by vacuum distillation, then dissolving the residual solid with a 1 mol / L HCl solution, and adjusting the solution to neutrality with a 1 mol / L NaOH solution to obtain rhodamine B hydrazide;

[0087] S4, dissolving 0.3 mmol of rhodamine B hydrazide and 0.3 mmol of 5-sulfonic acid salicylaldehyde in 10 mL of ethanol, heating to 95° C. and reflux for 7 h; removing the solvent in the reaction by vacuum distillation, and recrystallizing the reaction product to obtain a rhodamine B fluorescent probe;

[0088] S5, immersing the bacterial cellulose membrane loaded with cadmium telluride quantum dot fluorescent probe in an ethanol solution of rhodamine B fluorescent probe with a concentration of 1 mmol / L for 165 minutes until adsorption equilibrium is reached; taking out the bacterial cellulose membrane and washing it with ultrapure water, and absorbing the surface moisture with filter paper to obtain a visualized dual fluorescent probe composite bacterial cellulose membrane.

[0089] The bacterial cellulose membrane loaded with the dual fluorescent probe obtained in the experiment was placed in a phosphate buffer solution with a pH value of 2 to 8, left to stand for a while, and its fluorescence spectrum was measured at an excitation light wavelength of 365nm. The difference in the intensity of the two fluorescent probes was calculated, and a standard curve of the fluorescence difference changing with pH was established.

[0090] Determination of pH value of paper artifacts: The obtained visualized dual fluorescent probe composite bacterial cellulose film was applied to the surface of the paper artifact 5 to be tested and allowed to stand for 5 minutes; the composite bacterial cellulose film was then placed under an excitation light wavelength of 365nm to observe its color change. The results showed that the bacterial cellulose composite film was bright red, indicating that the paper was acidic. The pH value of sample 5 was calculated to be 3.6 by testing the fluorescence intensity. A pH flat-head electrode was then used to verify that its pH was 3.5.

[0091] Comparative Example 2

[0092] Comparative Example 2 is a comparative sample prepared by using Example 3 of patent document CN111808313A, and is compared with Example 5:

[0093] (1) Excess water was squeezed out of the bacterial cellulose membrane with filter paper to a thickness of 50 μm; the membrane was immersed in anhydrous toluene solution of APTES at 60°C for 5 h; the treated bacterial cellulose membrane was then rinsed three times with 40 mL of anhydrous toluene and anhydrous acetone in turn to obtain an amino-modified bacterial cellulose membrane.

[0094] (2) Mix 10 mL of chromium acetate solution with a concentration of 1 mmol / L and 10 mL of thioglycolic acid solution with a concentration of 1 mmol / L, and add sodium hydroxide solution to adjust the pH value to 11.5 to obtain a precursor solution; add 10 mL of sodium tellurite solution with a concentration of 0.5 mmol / L and stir evenly, then add 10 mL of sodium borohydride solution with a concentration of 0.1 mmol / L and stir evenly; heat the solution to 120°C and reflux for 4 h to obtain a CdTe quantum dot fluorescent probe solution. Mix EDC, NHS, CdTe fluorescent probe and amino bacterial cellulose membrane for 6 h, the EDC-NHS concentration is 0.05 mmol / L, and EDC / NHS=1:10; after the reaction, take out the orange-red fluorescent bacterial cellulose membrane.

[0095] (3) Preparation of standard fluorescence colorimetric card: Prepare a series of different pH buffer solutions (pH = 4-7), immerse the bacterial cellulose loaded with fluorescent probe in them, take out after 1 min and observe in a UV dark box, then test the fluorescence intensity of the bacterial cellulose membrane at each pH to obtain a standard curve (excitation light wavelength 370 nm), take digital photos and organize them to obtain a standard fluorescence colorimetric card.

[0096] (4) pH detection of paper artifacts: The bacterial cellulose membrane was covered on the surface of the paper artifact No. 5 to be tested. After 5 minutes, the changes in the membrane were observed in a UV box. The pH of sample No. 5 was calculated to be 3.1 by testing the fluorescence intensity. A digital photo was taken and compared with the fluorescence colorimetric card, and the pH was 3.2. Then a pH flat-head electrode was used to verify that the pH was 3.5.

[0097] By comparing the results of Example 5 and Comparative Example 2, it is found that the dual-probe-loaded fluorescent film of Example 5 has higher sensitivity than the single quantum dot probe-loaded fluorescent film of Comparative Example 1, and is closer to the actual result.

Claims

1. A visualized dual fluorescent probe composite bacterial cellulose membrane, characterized in that: include: A bacterial fiber membrane with a three-dimensional nano-net structure, and a cadmium telluride quantum dot fluorescent probe and a rhodamine B fluorescent probe loaded on the bacterial fiber membrane; under ultraviolet light, the fluorescent color of the visualized dual-fluorescent probe composite bacterial cellulose membrane changes with the acidity value, showing green fluorescence under neutral conditions and red fluorescence under acidic conditions; Among them, in the visualized dual fluorescent probe composite bacterial cellulose membrane, the loading amount of the cadmium telluride quantum dot fluorescent probe is 5wt% to 30wt%, and the loading amount of the rhodamine B fluorescent probe is 5wt% to 30wt%.

2. The method for preparing the visualized dual fluorescent probe composite bacterial cellulose membrane according to claim 1, characterized in that: The following steps are involved: S1, mixing a chromium acetate aqueous solution and a thioglycolic acid aqueous solution in proportion, and adding an alkaline solution dropwise to adjust the pH value to 10.5-12 to obtain a precursor solution; soaking the bacterial cellulose membrane in the precursor solution and standing until adsorption equilibrium; adding a sodium tellurite aqueous solution thereto and stirring evenly, and then adding a sodium borohydride aqueous solution to obtain a reaction solution A; S2, heating the reaction solution A to 80-120° C. and reflux for 10-60 min; The solid matter was separated by filtration, washed with ultrapure water, and the surface water was absorbed by filter paper to obtain the bacterial cellulose membrane loaded with cadmium telluride quantum dot fluorescent probe; S3, dissolving rhodamine B and hydrazine hydrate in ethanol at a molar ratio of 1:(0.1-0.5), heating to 80-100°C and reflux reaction; removing the solvent in the reaction by vacuum distillation, then dissolving the residual solid with an acid solution, and then adjusting the solution to neutrality with an alkaline solution to obtain rhodamine B hydrazide; S4, dissolving rhodamine B hydrazide and 5-sulfosalicylaldehyde in ethanol at a molar ratio of 1:(0.95-1.05), heating to 80-100° C. and reflux reaction; then removing the solvent in the reaction by vacuum distillation, and recrystallizing the reaction product to obtain a rhodamine B fluorescent probe; S5, immersing the bacterial cellulose membrane loaded with cadmium telluride quantum dot fluorescent probes in an ethanol solution of rhodamine B fluorescent probes until adsorption equilibrium is reached; taking out the bacterial cellulose membrane and washing it with ultrapure water, and absorbing the surface moisture with filter paper to obtain the visualized dual fluorescent probe composite bacterial cellulose membrane.

3. The preparation method according to claim 2, characterized in that In the step S1, the molar ratio of the chromium acetate, thioglycolic acid and sodium tellurite is 1: (0.95-1.05): (0.1-1); the amount of sodium borohydride added is 5-10 times that of the chromium acetate; and the membrane thickness of the bacterial cellulose is 0.05-10 mm.

4. The preparation method according to claim 2, characterized in that The concentration of the chromium acetate aqueous solution is 1-20 mmol / L, the concentration of the thioglycolic acid aqueous solution is 1-20 mmol / L, the concentration of the sodium tellurite aqueous solution is 0.1-1 mmol / L; and the concentration of the sodium borohydride aqueous solution is 5-100 mmol / L.

5. The preparation method according to claim 2, characterized in that: In step S1 and step S3, the alkaline solution is at least one of a sodium hydroxide solution and a potassium hydroxide solution; in step S3, the acid solution is at least one of a hydrochloric acid aqueous solution or a sulfuric acid aqueous solution with a molar concentration of 0.1 to 1.5 mol / L.

6. The preparation method according to claim 2, characterized in that In the step S3, the concentration of rhodamine B in ethanol is 50-100 mmol / L, the mass concentration of hydrazine hydrate in ethanol is 10%-30%, and the reaction time of heating and reflux is 5-10 hours.

7. The preparation method according to claim 2, characterized in that In the step S4, the concentration of the rhodamine B hydrazide in ethanol is 5 to 30 mmol / L, the concentration of 5-sulfonyl salicylaldehyde in ethanol is 5 to 30 mmol / L, and the reaction time of heating under reflux is 5 to 10 hours.

8. The preparation method according to claim 2, characterized in that: In the step S5, the mass concentration of the ethanol solvent of the rhodamine B fluorescent probe solution is 0.05 to 2.5 mmol / L; and the immersion time of the bacterial cellulose membrane is 30 to 240 min.

9. The use of the visualized dual fluorescent probe composite bacterial cellulose membrane according to claim 1, characterized in that: It is used to detect the acidification degree and hydrogen ion distribution of paper cultural relics, and to quickly and visually detect the surface acidity of paper cultural relics.

10. The use of the visualized dual fluorescent probe composite bacterial cellulose membrane according to claim 1, characterized in that: The degree of acidification of paper artifacts can be determined by measuring their pH value: the visualized dual fluorescent probe composite bacterial cellulose film is applied to the surface of the paper artifacts and allowed to stand for at least 5 minutes. The pH value and hydrogen ion distribution of the paper artifacts can be obtained by observing their color changes and calculating the intensity difference of the two fluorescent probes; the fluorescence excitation wavelength is 350-400 nm.

11. The use of the visualized dual fluorescent probe composite bacterial cellulose membrane according to claim 10, characterized in that: The intensity difference of the two fluorescent probes can be determined by a standard curve method, and the determination method of the standard curve is: the visualized dual fluorescent probe composite bacterial cellulose membrane is placed in a series of buffer solutions with different pH values ​​and soaked for at least 30 seconds, then its fluorescence spectrum is measured, the intensity difference of the two fluorescent probes is calculated, and a standard curve of fluorescence difference changing with pH is established. The buffer solution is a phosphate buffer solution with a pH range of 2 to 8.

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