Temperature-sensitive fluorescent composite hydrogel for amino acid fluorescence detection and preparation method thereof

Abstract

The invention relates to the technical field of fluorescence detection, in particular to temperature-sensitive fluorescent composite hydrogel for amino acid fluorescence detection and a preparation method of the temperature-sensitive fluorescent composite hydrogel. Traditional fluorescent hydrogel is not sensitive enough to fluorescence detection signals of amino acid. In order to solve the problems, the invention provides the temperature-sensitive fluorescent composite hydrogel for amino acid fluorescence detection, which is a product obtained by adsorbing and wrapping a phenanthrene-based quaternized fluorescent polymer into a three-dimensional polymer network structure of the hydrogel through a hydrophilic effect in a process of crosslinking temperature-sensitive xerogel in an aqueous solution to form the hydrogel, the hydrogel can generate rapid fluorescence response in the presence of amino acid, the detection time is short, and the detection efficiency is high. The three-dimensional network of the phenanthryl quaternized fluorescent polymer molecules and the temperature-sensitive hydrogel forms a physically wound double-network structure, so that the sensitivity of amino acid fluorescence detection is improved.

Description

Technical Field

[0001] This invention relates to the field of fluorescence detection technology, specifically to a thermosensitive fluorescent composite hydrogel for amino acid fluorescence detection and its preparation method. Background Technology

[0002] Amino acids, as important metabolites and building blocks of proteins in living organisms, play a crucial role in biomedicine, food science, and environmental monitoring. Currently, common methods for amino acid detection include high-performance liquid chromatography (HPLC), capillary electrophoresis, and spectrometry. While these methods offer high accuracy, they typically suffer from limitations such as complex operation, expensive equipment, and long detection cycles, making them unsuitable for rapid, on-site detection. In recent years, fluorescence sensing technology has attracted widespread attention due to its high sensitivity, rapid response, and ease of operation. Among these, hydrogel-based fluorescence sensors have become a research hotspot due to their excellent biocompatibility and tunable physicochemical properties.

[0003] However, existing fluorescent hydrogels still face some challenges in amino acid detection. For example, traditional hydrogels often lack thermosensitive properties, making it impossible to achieve controllable adsorption and release at specific temperatures, thus limiting their flexibility in practical applications. Furthermore, the preparation processes of many fluorescent hydrogels are complex, and the fluorescent groups are prone to leakage or poor stability, leading to unstable detection signals and low reproducibility. Simultaneously, their specific recognition ability for amino acids is insufficient, especially the detection sensitivity for important amino acids such as glutamic acid, which needs improvement. Therefore, developing a composite hydrogel with thermosensitive properties, high fluorescence stability, and rapid response capability is of great significance for achieving efficient fluorescent detection of amino acids. Summary of the Invention

[0004] A problem with existing technologies is that traditional fluorescent hydrogels are not sensitive enough to the fluorescence detection signals of amino acids. To address this issue, this invention provides a thermosensitive fluorescent composite hydrogel for amino acid fluorescence detection. It is a product obtained by adsorbing and encapsulating a phenanthrene-quaternized fluorescent polymer into the three-dimensional polymer network structure of a hydrogel through hydrophilic interaction during the crosslinking of a thermosensitive dry gel in an aqueous solution. The general structural formula of the phenanthrene-quaternized fluorescent polymer is as follows: , In the above general structural formula, x, 1-x represents the proportion of repeating structural units in the entire polymer, with x ranging from 20%. The weight-average molecular weight of the phenanthrene quaternized fluorescent polymer is 2.1 × 10⁻⁶. 4 ~2.2×10 4 .

[0005] Preferably, the thermosensitive dry gel is a product obtained by crosslinking N-isopropylacrylamide with acrylamide and N-N'-methylenebisacrylamide in deionized water to form a hydrogel, followed by drying. The lower critical dissolution temperature of the thermosensitive dry gel after saturation with water is 70°C.

[0006] Preferably, the preparation method of the temperature-sensitive dry gel includes the following steps: (1) Dissolve 11.50 mmol N-isopropylacrylamide and 11.50 mmol acrylamide in 10 mL of deionized water to form a homogeneous precursor solution; (2) Subsequently, 0.07 mmol of crosslinking agent N-N'-methylenebisacrylamide BIS, 0.04 mmol of initiator ammonium persulfate APS and 0.05 mmol of accelerator tetramethylethylenediamine TEMED were dissolved in the precursor solution and magnetically stirred at room temperature to form a homogeneous mixture; (3) Inject the mixture obtained in step (2) into the mold and let it stand at room temperature for at least 10 min for prepolymerization. Then place it in a refrigerator at 0℃~-10℃ and let it stand for at least 3 h to finally obtain the thermosensitive hydrogel P (NIPAm-co-AAm). After drying, the thermosensitive dry gel is obtained and sealed for storage.

[0007] Preferably, the preparation method of the phenanthrene quaternized fluorescent polymer includes the following steps: (1) Phenanthrene, diphenyl ether, and N-methyl-4-piperidinone undergo Friedel-Crafts acylation polymerization under superacid catalysis to obtain polyaryl ketone polymers, wherein the molar ratio of phenanthrene to diphenyl ether and N-methyl-4-piperidinone is 2:8:10.5; the general structural formula of the polyaryl ketone polymers is as follows: ; In the above general formula, x, 1-x represents the proportion of repeating structural units in the entire polymer, and the value of x ranges from 20%. (2) By Menshutkin quaternization reaction, the side chain tertiary amine of polyarylene ketone polymers is converted into quaternary ammonium salt groups to obtain phenanthrene quaternized fluorescent polymers.

[0008] Preferably, the preparation method of the thermosensitive fluorescent composite hydrogel includes the following steps: The thermosensitive dry gel was completely impregnated in an aqueous solution of phenanthrene-quaternized fluorescent polymer and allowed to stand for 18 hours for adsorption. After the adsorption was completed, the solid product was collected by solid-liquid separation to obtain the thermosensitive fluorescent composite hydrogel.

[0009] Preferably, the mass of the thermosensitive dry gel is 0.0472 g, the mass concentration of the phenanthrene quaternized fluorescent polymer aqueous solution is 20 mg / mL, and the volume of the phenanthrene quaternized fluorescent polymer aqueous solution is 10 mL.

[0010] Preferably, the amino acid is glutamic acid.

[0011] A quantitative fluorescence detection method for amino acids includes the following steps: (1) Add a fixed amount of the above thermosensitive fluorescent composite hydrogel to an aqueous solution of at least 5 different concentrations of amino acids of the same volume, allow it to stand for the same amount of time to adsorb, and collect the thermosensitive fluorescent composite hydrogels by solid-liquid separation. Measure the maximum fluorescence intensity of the different thermosensitive fluorescent composite hydrogels at a specific excitation wavelength. (2) Using the maximum fluorescence intensity of the thermosensitive fluorescent composite hydrogels collected separately as the Y value and the amino acid aqueous solutions of the corresponding gradient concentrations as the X value, a standard curve was plotted to obtain the linear relationship. (3) Add the thermosensitive fluorescent composite hydrogel to the amino acid solution to be tested, with the same volume as the amino acid aqueous solution in step (1), according to the amount added in step (1). Allow the hydrogel to stand for the same amount of time as in step (1). Collect the solid product through solid-liquid separation and test the fluorescence intensity of the obtained solid product. When the fluorescence intensity falls within the fluorescence intensity range defined by the linear relationship obtained in step (2), substitute the measured fluorescence intensity into the linear relationship obtained in step (2) to calculate the concentration of amino acids in the corresponding amino acid solution to be tested. When the fluorescence intensity does not fall within the fluorescence intensity range defined by the linear relationship obtained in step (2), add the amino acid solution to be tested... The amino acid solution is continuously diluted with deionized water until the temperature-sensitive fluorescent composite hydrogel is added to the diluted amino acid solution to be tested, which has the same volume as the amino acid aqueous solution in step (1). The static adsorption time is the same as in step (1). Solid products are collected by solid-liquid separation. When the fluorescence intensity of the obtained solid product falls within the fluorescence intensity range defined by the linear relationship obtained in step (3), the fluorescence intensity obtained by the test is substituted into the linear relationship obtained in step (3) to calculate the concentration of amino acids in the diluted amino acid solution to be tested. The concentration of amino acids in the diluted amino acid solution to be tested is multiplied by the dilution factor to obtain the concentration of glutamic acid in the amino acid solution to be tested.

[0012] Preferably, the static adsorption time in step (1) is 5 min.

[0013] Beneficial effects: (1) This invention forms a stable fluorescent composite system by adsorbing and encapsulating a phenanthrene-quaternized fluorescent polymer into a three-dimensional network structure of a thermosensitive hydrogel. This hydrogel exhibits a rapid fluorescence response in the presence of amino acids, resulting in short detection time and high detection efficiency. Furthermore, based on the linear relationship between fluorescence intensity and amino acid concentration, quantitative detection of amino acids can be achieved with high sensitivity; the detection limit (LOD) for glutamic acid fluorescence detection is as low as 9 × 10⁻⁶. -3 μg / mL, which is more sensitive than existing methods such as dual-emission carbon quantum dots (LOD=0.085μM) and enzyme-catalyzed colorimetric method (LOD=0.59 μM); (2) The thermosensitive hydrogel of the present invention is made by copolymerization and crosslinking of N-isopropylacrylamide and acrylamide, etc., and its low critical solution temperature (LCST) is 70°C, which makes the hydrogel exhibit reversible swelling-shrinkage behavior at a specific temperature. Amino acid desorption is achieved by a 75°C hot water bath. The composite hydrogel can be recycled multiple times, which significantly reduces the detection cost and reduces reagent waste; (3) The phenanthrene quaternized fluorescent polymer molecules and the three-dimensional network of the thermosensitive hydrogel form a physically entangled double network structure, which increases the mechanical strength of the obtained composite hydrogel by at least 50% compared with the pure hydrogel. This solves the problem of weak mechanical strength and easy breakage of traditional hydrogel sensing materials, making it convenient for practical operation and repeated use. The phenanthrene quaternized fluorescent polymer is firmly fixed in the hydrogel network through hydrophilic interaction, avoiding leakage of fluorescent groups and ensuring the long-term stability of the fluorescent signal. (4) The hydrogel preparation method of the present invention is simple and easy to implement, the raw materials are readily available, and no complex equipment is required. The synthesis of the thermosensitive dry gel and the adsorption process of the fluorescent polymer are both carried out under mild conditions, which is suitable for large-scale production. At the same time, the detection method is simple to operate. It only requires adding the hydrogel to the test solution and measuring the fluorescence intensity. The concentration can be calculated through the standard curve, which reduces the detection cost and is suitable for rapid on-site detection. Attached Figure Description

[0014] Figure 1 Infrared absorption spectra of 10% phenanthrene, 20% phenanthrene, 30% phenanthrene, and quaternized phenanthrene (10%, 20%, and 30%).

[0015] Figure 2 1H NMR spectra of phenanthrene containing 10%, 20%, 30%, and quaternized phenanthrene containing 10%, 20%, and 30% phenanthrene.

[0016] Figure 3 Infrared spectrum (3a) and SEM image (3b) of the thermosensitive dry gel obtained in Example 2.

[0017] Figure 4Differential thermal scanning chromatograms (a), (b) and thermogravimetric curves (c), (d) of 10%, 20%, and 30% phenanthrene and quaternized phenanthrene.

[0018] Figure 5 Fluorescence intensity test graphs of aqueous solutions containing 10%, 20%, and 30% phenanthrene after quaternization at different mass concentrations, under an excitation wavelength of 430 nm.

[0019] Figure 6 The fluorescence intensity of the thermosensitive fluorescent composite hydrogels obtained in Comparative Examples 3-1 and 3-2 was measured at an excitation wavelength of 430 nm.

[0020] Figure 7 The fluorescence intensity of the thermosensitive fluorescent composite hydrogel obtained in Example 3 was measured at an excitation wavelength of 430 nm.

[0021] Figure 8 Stress-strain curve of P(NIPAm-co-AAm) obtained in Example 2.

[0022] Figure 9 Example 3: Fluorescence response test of the thermosensitive fluorescent composite hydrogel to different amino acids.

[0023] Figure 10 Fluorescence response test of aqueous solution of pure phenanthrene-quaternized fluorescent polymer (containing 20% ​​phenanthrene after quaternization) to aqueous solution of glutamic acid.

[0024] Figure 11 Linearity graph obtained in Application 3 for quantitative detection of glutamic acid in aqueous solution of known concentration using a thermosensitive fluorescent composite hydrogel.

[0025] Figure 12 The composite hydrogel after fluorescence detection in application 3 was subjected to repeated adsorption-desorption-adsorption experiments, and the fluorescence intensity comparison curves of adsorbing glutamic acid and then desorbing and re-adsorbing glutamic acid were obtained. Detailed Implementation

[0026] The present invention will be described in detail below with reference to embodiments. However, it should be understood that the following embodiments are merely illustrative examples of implementation of the present invention and are not intended to limit the scope of the present invention.

[0027] Example 1

[0028] The steps for preparing phenanthrene-quaternized fluorescent polymers (containing 20% ​​phenanthrene after quaternization) are as follows: (1) Under ice bath conditions, using dichloromethane (8.0 mL) as solvent, phenanthrene (2 mmol) and diphenyl ether (8 mmol) were added to a round-bottom flask (100 mL) containing a magnetic stir bar. After stirring evenly, N-methyl-4-piperidinone (10.50 mmol) was added, and trifluoromethanesulfonic acid (94.50 mmol) was slowly added dropwise. The reaction was stirred for 3 h under ice bath conditions. When the solution gradually turned red and viscous, the polymer solution was poured into deionized water and stirred slowly to produce a white solid. The white solid was collected by filtration and washed repeatedly with deionized water until the pH of the washing solution was neutral. The collected white solid product was then dried at 65 °C for 24 h to obtain a polyaryl ketone polymer with a weight-average molecular weight of 2.16 × 10⁻⁶. 4 This is recorded as containing 20% ​​Fiber. (2) The synthetic route of step (1) is as follows: , The structural formulas of polyaryl ketone polymers are as follows: , In the above structural formula, x = 20%; (2) Add polyaryl ketone polymer (1.26 g), excess potassium carbonate (0.72 g) and iodomethane (0.88 mL) to a round-bottom flask (100 mL) containing a magnetic flask. Use N-methylpyrrolidone (20 mL) as solvent and stir in an oil bath at 40 °C for 24 h in the dark to obtain a yellow solution. Pour the yellow solution into ethyl acetate (100 mL) to precipitate a suspension. Filter under reduced pressure to obtain an orange-yellow solid. Wash the obtained orange-yellow solid with ethyl acetate 5 times, then dry it in an oven at 65 °C for 24 h, and then dry it in a vacuum oven at 150 °C for 24 h to obtain a phenanthrene-quaternized fluorescent polymer, which is denoted as quaternized phenanthrene 20%.

[0029] The infrared absorption spectra of 20% phenanthrene and 20% phenanthrene after quaternization are shown in the attached instruction manual. Figure 1 (a) and Figure 1 As shown in (b).

[0030] The 1H NMR spectra of 20% phenanthrene and 20% phenanthrene after quaternization are shown in the attached instructions. Figure 2 (a) and Figure 2 As shown in (b).

[0031] The images show that the integral values ​​of each absorption peak of the synthesized polymer are consistent with the number of protons in the designed structural formula, and the functional group regions can also correspond one-to-one with its structural formula.

[0032] Differential thermal scanning and thermogravimetric analysis (TLC) curves for 20% phenanthrene and quaternized 20% phenanthrene are shown in the attached instruction manual. Figure 4(a) (b) and Figure 4 As shown in (c) and (d), the images show that the thermal decomposition stability of both the 20% phenanthrene-containing and quaternized 20% phenanthrene-containing polymers is above 200 °C, indicating that the polymers have good thermal stability.

[0033] The fluorescence intensity test results of quaternized aqueous solutions containing 20% ​​phenanthrene at different mass concentrations under an excitation wavelength of 430 nm are shown in the instruction manual. Figure 5 As shown in the image, the fluorescence intensity of aqueous solutions containing 20% ​​phenanthrene after quaternization is stronger than that of aqueous solutions containing 10% or 30% phenanthrene after quaternization, respectively.

[0034] Comparative Example 1-1

[0035] The steps for preparing phenanthrene-quaternized fluorescent polymers (containing 10% phenanthrene after quaternization) are as follows: (1) Under ice bath conditions, using dichloromethane (8.0 mL) as solvent, phenanthrene (1 mmol) and diphenyl ether (9 mmol) were added to a round-bottom flask (100 mL) containing a magnetic stir bar. After stirring evenly, N-methyl-4-piperidinone (10.50 mmol) was added, and trifluoromethanesulfonic acid (94.50 mmol) was slowly added dropwise. The reaction was stirred for 3 h under ice bath conditions. When the solution gradually turned red and viscous, the polymer solution was poured into deionized water and stirred slowly to produce a white solid. The white solid was collected by filtration and washed repeatedly with deionized water until the pH of the washing solution was neutral. The collected white solid product was then dried at 65 °C for 24 h to obtain a polyaryl ketone polymer with a weight-average molecular weight of 2.56 × 10⁻⁶. 4 This is recorded as containing 10% Fiber. (2) The synthetic route of step (1) is as follows: , The structural formulas of polyaryl ketone polymers are as follows: , In the above structural formula, x = 10%; (2) Add polyaryl ketone polymer (1.76 g), excess potassium carbonate (1.00 g) and iodomethane (1.23 mL) to a round-bottom flask (100 mL) containing a magnetic ball. Use N-methylpyrrolidone (20 mL) as solvent and stir in an oil bath at 40 °C for 24 h in the dark to obtain a yellow solution. Pour the yellow solution into ethyl acetate (100 mL) to precipitate a suspension. Filter under reduced pressure to obtain an orange-yellow solid. Wash the obtained orange-yellow solid with ethyl acetate 5 times, then dry it in an oven at 65 °C for 24 h, and then dry it in a vacuum oven at 150 °C for 24 h to obtain a phenanthrene-quaternized fluorescent polymer, which is denoted as quaternized 10% phenanthrene.

[0036] The infrared absorption spectra of 10% phenanthrene and 10% phenanthrene after quaternization are shown in the attached instructions. Figure 1 (a) and Figure 1 As shown in (b).

[0037] The 1H NMR spectra of 10% phenanthrene and 10% phenanthrene after quaternization are shown in the attached instructions. Figure 2 (a) and Figure 2 As shown in (b).

[0038] Differential thermal scanning and thermogravimetric analysis (TLC) curves for 10% phenanthrene and quaternized 10% phenanthrene are shown in the attached instructions. Figure 4 (a) (b) and Figure 4 As shown in (c) and (d), the images show that the thermal decomposition stability of the polymer containing 10% phenanthrene and the quaternized 10% phenanthrene is above 200 °C, indicating that the polymer has good thermal stability.

[0039] The fluorescence intensity test results of quaternized aqueous solutions containing 10% phenanthrene at different mass concentrations under an excitation wavelength of 430 nm are shown in the instruction manual. Figure 5 As shown.

[0040] Comparative Examples 1-2

[0041] The steps for preparing phenanthrene-quaternized fluorescent polymers (containing 30% phenanthrene after quaternization) are as follows: (1) Under ice bath conditions, using dichloromethane (8.0 mL) as solvent, phenanthrene (3 mmol) and diphenyl ether (7 mmol) were added to a round-bottom flask (100 mL) containing a magnetic stir bar. After stirring evenly, N-methyl-4-piperidinone (10.50 mmol) was added, and trifluoromethanesulfonic acid (94.50 mmol) was slowly added dropwise. The reaction was stirred for 3 h under ice bath conditions. When the solution gradually turned red and viscous, the polymer solution was poured into deionized water and stirred slowly to produce a white solid. The white solid was collected by filtration and washed repeatedly with deionized water until the pH of the washing solution was neutral. The collected white solid product was then dried at 65 °C for 24 h to obtain a polyaryl ketone polymer with a weight-average molecular weight of 2.47 × 10⁻⁶. 4 This is recorded as containing 30% Fiber. (2) The synthetic route of step (1) is as follows: , The structural formulas of polyaryl ketone polymers are as follows: , In the above structural formula, x = 30%; (2) Add polyaryl ketone polymer (1.21 g), excess potassium carbonate (0.69 g) and iodomethane (0.85 mL) to a round-bottom flask (100 mL) containing a magnetic flask. Use N-methylpyrrolidone (20 mL) as solvent and stir in an oil bath at 40 °C for 24 h in the dark to obtain a yellow solution. Pour the yellow solution into ethyl acetate (100 mL) to precipitate a suspension. Filter under reduced pressure to obtain an orange-yellow solid. Wash the obtained orange-yellow solid with ethyl acetate 5 times, then dry it in an oven at 65 °C for 24 h, and then dry it in a vacuum oven at 150 °C for 24 h to obtain a phenanthrene-quaternized fluorescent polymer, which is denoted as quaternized phenanthrene-containing 30%.

[0042] The infrared absorption spectra of 30% phenanthrene and 30% phenanthrene after quaternization are shown in the attached instruction manual. Figure 1 (a) and Figure 1 As shown in (b).

[0043] The 1H NMR spectra of 30% phenanthrene and 30% phenanthrene after quaternization are shown in the attached instructions. Figure 2 (a) and Figure 2 As shown in (b).

[0044] Differential thermal scanning and thermogravimetric analysis (TLC) curves for 30% phenanthrene and 30% phenanthrene after quaternization are shown in the attached instruction manual. Figure 4 (a) (b) and Figure 4 As shown in (c) and (d), the images show that the thermal decomposition stability of both the 30% phenanthrene-containing and quaternized 30% phenanthrene-containing polymers is above 200 °C, indicating that the polymers have good thermal stability.

[0045] The fluorescence intensity test results of quaternized aqueous solutions containing 30% phenanthrene at different mass concentrations under an excitation wavelength of 430 nm are shown in the instruction manual. Figure 5 As shown.

[0046] Example 2

[0047] The steps for preparing a thermosensitive dry gel are as follows: N-Isopropylacrylamide (11.50 mmol) and acrylamide (11.50 mmol) were dissolved in 10 mL of deionized water at a molar ratio of 1:1 to form a homogeneous precursor solution. Then, the crosslinking agent N-N'-methylenebisacrylamide (BIS) (0.07 mmol), the initiator ammonium persulfate (APS) (0.04 mmol), and the accelerator tetramethylethylenediamine (TEMED) (0.05 mmol) were dissolved in the above precursor solution. The mixture was magnetically stirred at room temperature to obtain a homogeneous solution. The resulting mixture was injected into a 5 cm × 5 cm × 5 cm cubic mold and allowed to stand at room temperature for 10 min for prepolymerization. The sample was then placed in a refrigerator at -10 °C and allowed to stand for 3 h to react, finally obtaining the thermosensitive hydrogel P (NIPAm-co-AAm). The obtained P (NIPAm-co-AAm) was dried in an oven at 65 °C for 24 h to obtain a thermosensitive dry gel, which was sealed and stored for later use.

[0048] The synthetic route for thermosensitive dry gel is as follows: .

[0049] The infrared spectrum and SEM image of the thermosensitive dry gel are shown in the attached instructions. Figure 3 a and Figure 3 As shown in image b, the functional groups of the thermosensitive xerogel correspond one-to-one with its structural formula, indicating successful synthesis. The thermosensitive xerogel exhibits a porous structure, giving it excellent water absorption capacity.

[0050] The stress-strain curve of the thermosensitive hydrogel P (NIPAm-co-AAm) obtained in Example 2 (sample size 3.0cm × 3.0cm × 1cm, tested at room temperature (25℃), compression rate set to 0.1 mm / s) is shown in the attached instruction manual. Figure 8 As shown.

[0051] Example 3

[0052] The following steps were taken to prepare a 20% thermosensitive fluorescent composite hydrogel: 0.0472g of the thermosensitive dry gel obtained in Example 2 was added to 10 mL of an aqueous solution of 20 mg / mL phenanthrene-quaternized fluorescent polymer (containing 20% ​​phenanthrene after quaternization) for complete immersion and allowed to stand for adsorption for different times. After the adsorption was completed, the solid product was collected by solid-liquid separation to obtain 20% thermosensitive fluorescent composite hydrogel.

[0053] The stress-strain curve of the 20% thermosensitive fluorescent composite hydrogel obtained in Example 3 (sample size 3.0cm × 3.0cm × 1cm, tested at room temperature, compression rate set to 0.1 mm / s) is shown in the attached instruction manual. Figure 8 As shown in the image, the mechanical properties of the thermosensitive fluorescent composite hydrogel are significantly improved compared to those of the pure hydrogel.

[0054] Comparative Example 3-1

[0055] The steps for preparing a 10% thermosensitive fluorescent composite hydrogel are as follows: 0.0472g of the thermosensitive dry gel obtained in Example 2 was added to 10mL of an aqueous solution of 20mg / mL phenanthrene-quaternized fluorescent polymer (containing 10% phenanthrene after quaternization) for complete immersion and allowed to stand for adsorption for different times. After the adsorption was completed, the solid product was collected by solid-liquid separation to obtain 10% thermosensitive fluorescent composite hydrogel.

[0056] Comparative Example 3-2

[0057] The steps for preparing a 30% thermosensitive fluorescent composite hydrogel are as follows: 0.0472g of the thermosensitive dry gel obtained in Example 2 was added to 10mL of an aqueous solution of 20mg / mL phenanthrene-quaternized fluorescent polymer (containing 30% phenanthrene after quaternization) for complete immersion and allowed to stand for adsorption for different times. After the adsorption was completed, the solid product was collected by solid-liquid separation to obtain 30% thermosensitive fluorescent composite hydrogel.

[0058] The fluorescence intensity measurements of the thermosensitive fluorescent composite hydrogels obtained in Comparative Examples 3-1 and 3-2 at an excitation wavelength of 430 nm are shown in the appendix to the instruction manual. Figure 6 (Figures a and b represent the contents of 10% and 30% phenanthrene after quaternization, respectively) and Figure 7 (The thermosensitive fluorescent composite hydrogel obtained in Example 3) is shown in the image. The image shows that the 20% thermosensitive fluorescent composite hydrogel obtained had the highest maximum fluorescence intensity when the static adsorption time was 18 hours. The maximum fluorescence intensity of the thermosensitive fluorescent composite hydrogel obtained in Example 3 is significantly stronger than that of the thermosensitive fluorescent composite hydrogels obtained in Comparative Examples 3-1 and 3-2, respectively.

[0059] Amino acid fluorescence detection: Application 1 Take 0.06 g of the thermosensitive fluorescent composite hydrogel obtained in Example 3 (allowed to stand for 18 h for adsorption) and add it to 10 mL of aqueous solutions of different amino acids, each with a concentration of 0.02 µg / mL. Allow it to stand for 5 min for adsorption, then remove it, filter it with filter paper to remove moisture, and test its maximum fluorescence intensity at an excitation wavelength of 430 nm. The test results are shown in the appendix to the instruction manual. Figure 9 As shown in the image, the thermosensitive fluorescent composite hydrogel obtained in Example 3 has a better fluorescence response to glutamate.

[0060] Application 2

[0061] In Example 3, 5 mL of an aqueous solution of phenanthrene-quaternized fluorescent polymer (containing 20% ​​phenanthrene after quaternization) with a mass concentration of 20 mg / mL was added to 3 mL of an aqueous solution of glutamic acid with a concentration of 0.02 µg / mL. The mixture was allowed to stand for adsorption for 5 min, then removed and filtered through filter paper to remove moisture. The maximum fluorescence intensity was measured at an excitation wavelength of 430 nm. The test results are shown in the appendix to the instruction manual. Figure 10 As shown.

[0062] The test results show that the maximum fluorescence intensity of the pure phenanthrene quaternized fluorescent polymer is significantly lower than that of the thermosensitive fluorescent composite hydrogel obtained in Example 3 (after static adsorption for 18 hours). This indicates that the cross-entangled double network structure formed between the phenanthrene quaternized fluorescent polymer and the thermosensitive hydrogel helps to improve the fluorescence detection sensitivity of the thermosensitive fluorescent composite hydrogel for glutamic acid in solution. The thermosensitive hydrogel does not only play a supporting role as a carrier.

[0063] Application 3

[0064] Quantitative analysis of glutamate using fluorescence detection: I. The fluorescence detection method for glutamate is as follows: (1) The gradient concentrations of the glutamic acid aqueous solution were 0.02 µg / mL, 0.04 µg / mL, 0.06 µg / mL, 0.08 µg / mL and 0.10 µg / mL, respectively; (2) 0.5 g of the thermosensitive fluorescent composite hydrogel obtained in Example 3 was added to 10 mL of glutamic acid aqueous solutions of varying concentrations to obtain five sample solutions. After standing for 5 min for adsorption, the thermosensitive fluorescent composite hydrogel was collected by filtration with filter paper. The maximum fluorescence intensity of the five sample solutions was then tested at an excitation wavelength of 430 nm. The test results are shown in the appendix to the instruction manual. Figure 11 As shown in the image, the fluorescence intensity gradually increases with increasing glutamate concentration; (3) Using the maximum fluorescence intensity of the thermosensitive fluorescent composite hydrogels collected separately as the Y value and the amino acid aqueous solutions of the corresponding gradient concentrations as the X value, a standard curve was plotted as shown in the appendix to the instruction manual. Figure 11 As shown, the linear relationship y = 1434629X + 351122 is obtained, and the detection limit (LOD) value is 9 × 10⁻⁶. -3 µg / mL; (4) Add 0.5g of the thermosensitive fluorescent composite hydrogel obtained in Example 3 to a 10mL solution of the glutamic acid to be tested, let it stand for 5min for adsorption, collect the solid product by solid-liquid separation, and test the fluorescence intensity of the obtained solid product. When the fluorescence intensity falls within the fluorescence intensity range defined by the linear relationship obtained in step (3), substitute the measured fluorescence intensity into the linear relationship obtained in step (3) to calculate the concentration of glutamic acid in the corresponding glutamic acid solution. When the fluorescence intensity does not fall within the fluorescence intensity range defined by the linear relationship obtained in step (3), remove the glutamic acid solution from the solution. The solution is continuously diluted with water until 0.5g of the thermosensitive fluorescent composite hydrogel obtained in Example 3 is added to a 10mL diluted glutamic acid solution to be tested. After standing for 5min for adsorption, the solid product is collected by solid-liquid separation. When the fluorescence intensity of the obtained solid product falls within the fluorescence intensity range defined by the linear relationship obtained in step (3), the fluorescence intensity obtained is substituted into the linear relationship obtained in step (3) to calculate the concentration of glutamic acid in the diluted glutamic acid solution to be tested. The concentration of glutamic acid in the diluted glutamic acid solution to be tested is multiplied by the dilution factor to obtain the concentration of glutamic acid in the solution to be tested.

[0065] Application 4

[0066] The desorption and reuse method is as follows: The composite hydrogel, after fluorescence detection using Application 3, was immersed in a 20 mL hot water bath at 75°C for glutamic acid desorption for 5 min. After cooling to room temperature, the composite hydrogel was collected by filtration through filter paper. The adsorption-desorption-resorption test was then performed on the glutamic acid aqueous solution using the method described in Application 3. The test results are shown in the appendix to the instruction manual. Figure 12 As shown in the image, the difference between the maximum fluorescence intensity of the composite hydrogel after adsorbing glutamic acid and after desorption and re-adsorption gradually decreases with the increase of the number of adsorption-desorption-resorption cycles, indicating that the reusability of the composite hydrogel gradually decreases with the increase of the number of adsorption-desorption cycles.

[0067] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A temperature-sensitive fluorescent composite hydrogel for the fluorescent detection of amino acids, characterized in that, This product is obtained by adsorbing and encapsulating a phenanthrene-quaternized fluorescent polymer into the three-dimensional polymer network structure of a hydrogel through hydrophilic interaction during the crosslinking of a thermosensitive dry gel in an aqueous solution. The general structural formula of the phenanthrene-quaternized fluorescent polymer is as follows: ， In the above structural formula, x, 1-x represents the proportion of the repeating structural unit in the entire polymer, and the value range of x is 20%, the weight average molecular weight of the phenanthryl quaternary ammonium fluorescent polymer is 2.1×10 4 2.2×10 4 .

2. The thermosensitive fluorescent composite hydrogel for amino acid fluorescence detection according to claim 1, characterized in that, The thermosensitive dry gel is a product obtained by cross-linking N-isopropylacrylamide with acrylamide and N-N'-methylenebisacrylamide in deionized water to form a hydrogel, followed by drying. The lower critical dissolution temperature of the thermosensitive dry gel after saturation with water is 70°C.

3. The thermosensitive fluorescent composite hydrogel for amino acid fluorescence detection according to claim 2, characterized in that, The preparation method of the thermosensitive dry gel includes the following steps: (1) Dissolve 11.50 mmol N-isopropylacrylamide and 11.50 mmol acrylamide in 10 mL of deionized water to form a homogeneous precursor solution; (2) Then, 0.07 mmol BIS, 0.04 mmol APS and 0.05 mmol TEMED were dissolved in the precursor solution and magnetically stirred at room temperature to form a homogeneous mixture; (3) Inject the mixture obtained in step (2) into the mold and let it stand at room temperature for at least 10 min for prepolymerization. Then place it in a refrigerator at 0℃~-10℃ and let it stand for at least 3 h to finally obtain P(NIPAm-co-AAm). After drying, a thermosensitive dry gel is obtained and sealed for storage.

4. The thermosensitive fluorescent composite hydrogel for amino acid fluorescence detection according to claim 1, characterized in that, The preparation method of the phenanthrene quaternized fluorescent polymer includes the following steps: (1) Phenanthrene, diphenyl ether, and N-methyl-4-piperidinone undergo Friedel-Crafts acylation polymerization under superacid catalysis to obtain polyaryl ketone polymers, wherein the molar ratio of phenanthrene to diphenyl ether and N-methyl-4-piperidinone is 2:8:10.5; the general structural formula of the polyaryl ketone polymers is as follows: ； In the above general formula, x, 1-x represents the proportion of repeating structural units in the entire polymer, and the value of x ranges from 20%. (2) By Menshutkin quaternization reaction, the side chain tertiary amine of polyarylene ketone polymers is converted into quaternary ammonium salt groups to obtain phenanthrene quaternized fluorescent polymers.

5. The thermosensitive fluorescent composite hydrogel for amino acid fluorescence detection according to claim 1, characterized in that, The preparation method of the thermosensitive fluorescent composite hydrogel includes the following steps: The thermosensitive dry gel was completely impregnated in an aqueous solution of phenanthrene-quaternized fluorescent polymer and allowed to stand for 18 hours for adsorption. After the adsorption was completed, the solid product was collected by solid-liquid separation to obtain the thermosensitive fluorescent composite hydrogel.

6. The thermosensitive fluorescent composite hydrogel for amino acid fluorescence detection according to claim 5, characterized in that, The mass of the thermosensitive dry gel is 0.0472 g, the mass concentration of the phenanthrene quaternized fluorescent polymer aqueous solution is 20 mg / mL, and the volume of the phenanthrene quaternized fluorescent polymer aqueous solution is 10 mL.

7. The thermosensitive fluorescent composite hydrogel for amino acid fluorescence detection according to claim 1, characterized in that, The amino acid in question is glutamic acid.

8. A method for quantitative detection of amino acids using fluorescence, characterized in that, Includes the following steps: (1) Add a fixed amount of the thermosensitive fluorescent composite hydrogel obtained by any one of claims 1-7 to an aqueous solution of at least 5 different concentrations of amino acids of the same volume, allow it to stand for the same amount of time to adsorb, collect the thermosensitive fluorescent composite hydrogel by solid-liquid separation, and measure the maximum fluorescence intensity of different thermosensitive fluorescent composite hydrogels at a specific excitation wavelength. (2) Using the maximum fluorescence intensity of the thermosensitive fluorescent composite hydrogels collected separately as the Y value and the amino acid aqueous solutions of the corresponding gradient concentrations as the X value, a standard curve was plotted to obtain the linear relationship. (3) Add the thermosensitive fluorescent composite hydrogel to the amino acid solution to be tested, with the same volume as the amino acid aqueous solution in step (1), according to the amount added in step (1). Allow the hydrogel to stand for the same amount of time as in step (1). Collect the solid product through solid-liquid separation and test the fluorescence intensity of the obtained solid product. When the fluorescence intensity falls within the fluorescence intensity range defined by the linear relationship obtained in step (2), substitute the measured fluorescence intensity into the linear relationship obtained in step (2) to calculate the concentration of amino acids in the corresponding amino acid solution to be tested. When the fluorescence intensity does not fall within the fluorescence intensity range defined by the linear relationship obtained in step (2), add the amino acid solution to be tested... The amino acid solution is continuously diluted with deionized water until the temperature-sensitive fluorescent composite hydrogel is added to the diluted amino acid solution to be tested, which has the same volume as the amino acid aqueous solution in step (1). The static adsorption time is the same as in step (1). Solid products are collected by solid-liquid separation. When the fluorescence intensity of the obtained solid product falls within the fluorescence intensity range defined by the linear relationship obtained in step (3), the fluorescence intensity obtained by the test is substituted into the linear relationship obtained in step (3) to calculate the concentration of amino acids in the diluted amino acid solution to be tested. The concentration of amino acids in the diluted amino acid solution to be tested is multiplied by the dilution factor to obtain the concentration of glutamic acid in the amino acid solution to be tested.

9. The method for quantitative detection of amino acids by fluorescence according to claim 8, characterized in that, The static adsorption time in step (1) is 5 min.

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