Sodium alginate AIE fluorescent microspheres and a preparation method thereof

Abstract

The application belongs to the field of chemical synthesis and fluorescent sensing, and particularly relates to a sodium alginate AIE fluorescent microsphere and a preparation method thereof. The method comprises the following steps: dissolving AIE dye and sodium alginate in water, dispersing the mixture in a non-polar organic solvent to obtain a mixed solution, preparing a micro-nano emulsion by ultrasonic under ice bath, adding a calcium chloride solution for solidification, centrifugal washing, and re-dissolving, so as to obtain the sodium alginate AIE fluorescent microsphere. The preparation method has the advantages of simplicity, high efficiency, mildness, stability and superior performance. The AIE fluorescent microsphere prepared by the method has controllable size, high dye loading capacity, good fluorescent performance, super-hydrophilic surface, good biocompatibility, and is easy to be directly applied in fluorescent sensing.

Description

Technical Field

[0001] This invention belongs to the fields of chemical synthesis and fluorescence sensing, and specifically relates to a sodium alginate AIE fluorescent microsphere and its preparation method. Background Technology

[0002] Fluorescent sensors are widely used in medical diagnostics, environmental monitoring, and food safety. Compared to traditional colorimetric sensors, they have stronger resistance to background interference and higher signal output intensity. They can increase the intensity of incident light to increase the intensity of emitted light, thereby improving sensitivity. Commonly used fluorescent sensors generally include organic fluorescent dyes, quantum dots, nanoclusters, and fluorescent microspheres. Among these, fluorescent microspheres, compared to the other three, have a higher signal intensity per unit particle because each particle carries a large amount of luminescent material. Furthermore, the surface of the microsphere protects the loaded fluorescent material, making it more advantageous for applications in the sensing field.

[0003] However, traditional fluorescent materials, such as conventional organic fluorescent dye molecules and quantum dots, tend to spontaneously aggregate in the aqueous phase, leading to a significant reduction or even complete disappearance of the emitted fluorescence signal. This is the aggregation-induced quenching (ACQ) phenomenon commonly found in conventional fluorescence. This phenomenon is even more unavoidable inside fluorescent microspheres. Therefore, conventional fluorescent microspheres are primarily prepared using a swelling method that artificially controls the spacing between loaded fluorescent materials. The advantage of this method is uniform particle size and even distribution of the fluorescent material within the microspheres, thus minimizing the occurrence of ACQ. However, this significantly reduces the loading capacity, thus limiting the fluorescence intensity of conventional fluorescent microspheres.

[0004] Sensor development involves more than just pursuing signal output strength; it also requires consideration of sensor stability within the system. Key factors influencing sensor stability include the coupling efficiency between the microspheres and the recognition unit, coupling activity, and the monodispersity after coupling. All of these factors are closely related to the surface hydrophilicity and biocompatibility of the fluorescent microspheres.

[0005] Therefore, based on the above, it is essential to prepare a new substrate for fluorescent microspheres and apply it to fluorescent sensors. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide sodium alginate AIE fluorescent microspheres and their preparation method, specifically adopting the following technical solution:

[0007] According to a first aspect of the present invention, a method for preparing sodium alginate AIE fluorescent microspheres is provided, comprising the following steps:

[0008] AIE dye and sodium alginate were dissolved in water and dispersed in a non-polar organic solvent to obtain a mixture. Then, under ice bath conditions, the mixture was ultrasonically prepared into a micro / nano emulsion. Calcium chloride solution was added for solidification, followed by centrifugation, washing, and resolubilization to finally obtain sodium alginate AIE fluorescent microspheres.

[0009] This invention uses sodium alginate as a raw material. Natural sodium alginate hydrogel possesses superhydrophilic properties and excellent biocompatibility due to its multi-hydroxyl structure, ensuring the overall superhydrophilicity of the microspheres within the system. Sodium alginate hydrogel exhibits good film-forming properties, preventing leakage of the loaded material. Furthermore, the carboxyl groups in sodium alginate make it easily modifiable, facilitating coupling with recognition units. Based on this, this invention employs a reverse microemulsion method, i.e., a water-in-oil strategy. On one hand, the microspheres are bound in the aqueous phase, thus forming superhydrophilic properties. On the other hand, a large amount of fluorescent material to be loaded is compressed within the water-in-oil micro / nano droplets. By solidifying the microsphere substrate, high-load coating of the fluorescent material is achieved, generating the AIE effect, ultimately synthesizing superhydrophilic, highly biocompatible, and strongly fluorescent AIE microspheres.

[0010] As a further preferred embodiment, the above-mentioned AIE dye is a hydrophilic AIE dye, including at least one of tetraphenylphenyl carboxylic acids and their derivatives, tetraphenylphenyl phenolic alcohols and their derivatives, and tetraphenylphenyl nitriles and their derivatives. These materials all have good hydrophilicity and strong AIE fluorescence properties.

[0011] As a further preferred embodiment, the aforementioned nonpolar organic solvent includes at least one of aliphatic hydrocarbons and their derivatives and aromatic hydrocarbons and their derivatives. These solvents are insoluble in water, resulting in a higher quality emulsion.

[0012] As a further preferred embodiment, the ratio of AIE dye, sodium alginate, and water is 1 mg-100 mg: 1 mg-50 mg: 1 mL. The high concentration of AIE dye is used to achieve the AIE effect and significantly increase the loading capacity, resulting in highly fluorescent microspheres.

[0013] As a further preferred embodiment, the steps for forming the micro / nano emulsion described above are as follows:

[0014] The mixture was prepared by ultrasonic emulsification using a cell disruptor under ice bath conditions. The emulsification time was 3-30 minutes, and the emulsification power was 120 W-600 W. If the degree of emulsification was insufficient (emulsification time was too short), the final microsphere yield would be low and the quality would be poor. If the emulsification was excessive (emulsification time was too long), the system temperature would rise, organic solvent would be lost, the system would become unbalanced, and the quality of the microspheres would be uncontrollable.

[0015] As a further preferred embodiment, the specific process of the above-mentioned curing is as follows:

[0016] Add a calcium chloride solution of 1 mg / mL to 100 mg / mL while stirring at 20 r / min to 200 r / min. After the addition is complete, continue stirring for 1 h to 12 h until solidification is complete. Calcium chloride is the most common calcium salt and has excellent solubility, which is more conducive to the reaction.

[0017] As a further preferred embodiment, the specific steps of the above-mentioned centrifugal washing are as follows:

[0018] First, centrifuge at 3000 r / min-10000 r / min for 10 min-30 min, then wash three times with a 5%-50% ethanol aqueous solution as the washing agent, and finally wash with ultrapure water until the supernatant is clear after centrifugation. Washing with ethanol aqueous solution can completely remove residual organic solvents and AIE dyes.

[0019] As a further preferred embodiment, the reconstituted solution is ultrapure water or a phosphate buffer solution. This facilitates the subsequent direct connection of the microspheres to the recognition unit.

[0020] In another aspect, the present invention provides sodium alginate AIE fluorescent microspheres prepared by the above-described method. The particle size of these AIE fluorescent microspheres is 0.05 μm to 5 μm. This particle size range essentially covers the potential applications in biosensing.

[0021] The beneficial effects of this invention are as follows: This invention innovatively utilizes a reverse microemulsion method, which can form water-in-oil droplets with controllable size. Simultaneously, using sodium alginate as the matrix, the microspheres exhibit superhydrophilic properties and excellent biocompatibility, particularly beneficial for applications in biosensing. Furthermore, sodium alginate, as the microsphere matrix, loads a large amount of AIE dye, overcoming the fluorescence self-quenching problem caused by excessively high dye concentrations in traditional fluorescent microspheres, thus ensuring superior fluorescence performance. In addition, AIE is completely opposite to the traditional ACQ effect, truly overcoming the adverse effects of the traditional ACQ effect and making high-sensitivity online sensing monitoring easier. The preparation method provided by this invention has the advantages of simplicity, efficiency, mildness, stability, and superior performance. The prepared AIE fluorescent microspheres have controllable size, large dye loading, good fluorescence performance, and a superhydrophilic surface with good biocompatibility, making them easy to apply directly in fluorescence sensing. Attached Figure Description

[0022] Figure 1 The diagram illustrates the principle of preparing sodium alginate AIE fluorescent microspheres according to the present invention; wherein 1 is a nonpolar organic solvent, 2 is ultrapure water, 3 is sodium alginate, 4 is AIE dye, and 5 is calcium ions;

[0023] Figure 2The diagram shows the process for preparing sodium alginate AIE fluorescent microspheres according to the present invention;

[0024] Figure 3 The image shown is a microscopic characterization diagram of sodium alginate AIE (tetraphenyltetracarboxylic acid, TCBPE) fluorescent microspheres prepared by the method of the present invention.

[0025] Figure 4 The figure shows the excitation and emission spectra of sodium alginate AIE (TCBPE) fluorescent microspheres prepared by the method of the present invention.

[0026] Figure 5 The image shows a comparison of sodium alginate AIE (TCBPE) fluorescent microspheres prepared by the method of this invention under natural light and ultraviolet light.

[0027] Figure 6 The image shows sodium alginate AIE (TCPE) fluorescent microspheres prepared according to Example 2 of the method of the present invention, applied to the immunochromatographic detection of Escherichia coli O157:H7 (…). E. coli Physical image and standard curve of O157:H7. Detailed Implementation

[0028] The following will provide a clear and complete description of the concept, specific structure, and technical effects of the present invention in conjunction with the embodiments and accompanying drawings, so as to fully understand the purpose, solution, and effects of the present invention. It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other.

[0029] Example 1

[0030] A method for preparing sodium alginate AIE fluorescent microspheres includes the following steps:

[0031] (1) Preparation of aqueous solution: Weigh 10 mg of AIE dye TCBPE and 50 mg of sodium alginate, and dissolve them in 5 mL of ultrapure water at 50 °C with stirring.

[0032] (2) Preparation of water-in-oil microemulsion: Add the above mixture to 20 mL of n-octane, place in an ice bath at 0°C, and ultrasonically emulsify for 10 min using an ultrasonic disruptor at 360W power;

[0033] (3) Calcium ion solidified microspheres: Transfer the above microemulsion to a beaker, add 1 mL of 20 mg / mL calcium chloride solution dropwise while stirring slowly at 50 r / min, and keep stirring at 10 r / min for 6 h to solidify completely;

[0034] (4) Washing and resuspension: The above reaction end product was centrifuged at 8500 r / min for 20 min, the supernatant was discarded, the precipitate was washed three times with 10% ethanol aqueous solution, and then washed several times with ultrapure water until the supernatant after centrifugation was clear. The washed precipitate was redissolved in 5 mL of phosphate buffer solution (0.01 M, pH=7.4) to obtain 200 nm sodium alginate AIE fluorescent microspheres, which were stored in the dark at 4℃.

[0035] The molecular structure of TCBPE prepared in the above method is shown below:

[0036] .

[0037] Example 2

[0038] A method for preparing sodium alginate AIE fluorescent microspheres includes the following steps:

[0039] (1) Preparation of aqueous solution: Weigh 10 mg of AIE dye TCPE and 80 mg of sodium alginate, and dissolve them in 10 mL of ultrapure water at 40°C with stirring.

[0040] (2) Preparation of water-in-oil microemulsion: Add the above mixture to 25 mL of n-hexane, place in an ice bath at 0°C, and ultrasonically emulsify for 15 min using an ultrasonic disruptor at 120W power.

[0041] (3) Calcium ion solidified microspheres: Transfer the above microemulsion to a beaker, add 1 mL of 50 mg / mL calcium chloride solution dropwise while stirring slowly at 30 r / min, and keep stirring at 10 r / min for 8 h to solidify completely;

[0042] (4) Washing and resuspension: The final product of the above reaction was centrifuged at 6000 r / min for 15 min, the supernatant was discarded, the precipitate was washed three times with 20% ethanol aqueous solution, and then washed several times with ultrapure water until the supernatant after centrifugation was clear. The washed precipitate was redissolved in 8 mL of phosphate buffer solution (0.01 M, pH=7.4) to obtain 1000 nm sodium alginate AIE fluorescent microspheres, which were stored in the dark at 4℃.

[0043] The molecular structure of TCPE in the above preparation method is shown below:

[0044] .

[0045] Example 3

[0046] A method for preparing sodium alginate AIE fluorescent microspheres includes the following steps:

[0047] (1) Preparation of aqueous solution: Weigh 10 mg of AIE dye TCPE and 100 mg of sodium alginate, and dissolve them in 20 mL of ultrapure water at 40°C with stirring.

[0048] (2) Preparation of water-in-oil microemulsion: Add the above mixture to 40 mL of n-hexane, place in an ice bath at 0°C, and ultrasonically emulsify for 10 min using an ultrasonic disruptor at 100W power;

[0049] (3) Calcium ion solidified microspheres: Transfer the above microemulsion to a beaker, add 1 mL of 80 mg / mL calcium chloride solution dropwise while stirring slowly at 30 r / min, and keep stirring at 10 r / min for 10 h to solidify completely;

[0050] (4) Washing and resuspension: The final product of the above reaction was centrifuged at 4000 r / min for 15 min, the supernatant was discarded, the precipitate was washed three times with 20% ethanol aqueous solution, and then washed several times with ultrapure water until the supernatant after centrifugation was clear. The washed precipitate was redissolved in 10 mL of phosphate buffer solution (0.01 M, pH=7.4) to obtain 3 μm sodium alginate AIE fluorescent microspheres, which were stored in the dark at 4℃.

[0051] The molecular structure of TCPE in the above preparation method is shown below:

[0052] .

[0053] Figure 3 The image shown is a microscopic characterization diagram of sodium alginate AIE (tetrabutylstyrene tetracarboxylic acid, TCBPE) fluorescent microspheres prepared by the method of this invention; Figure 3 It can be seen that the microspheres of the present invention have a distinct core-shell structure, with the core being an aggregate of AIE dye and the shell being sodium alginate gel with a thickness of 261.7 nm. The overall true average particle size of the microspheres is 1309.3 nm, the hydrated particle size is 1429.0 nm, and the particle monodispersity index (PDI) is 0.158.

[0054] Figure 4 The image shows the excitation and emission spectra of sodium alginate AIE (TCBPE) fluorescent microspheres prepared by the method of this invention; Figure 4 It is known that the maximum excitation wavelength of the microspheres of the present invention is 376 nm and the maximum emission wavelength is 465 nm.

[0055] Figure 5 The image shows a comparison of sodium alginate AIE (TCBPE) fluorescent microspheres prepared by the method of this invention under natural light and ultraviolet light; Figure 5It is known that the AIE dye TCBPE is clear and transparent when dissolved in aqueous solution (0.01 mg / mL), and exhibits very weak fluorescence under dark ultraviolet light. The two batches of microspheres prepared by loading TCBPE appear pale yellow-white under natural light and exhibit strong fluorescence under dark ultraviolet light.

[0056] Example 4

[0057] A practical application of sodium alginate AIE fluorescent microspheres in immunochromatography includes the following steps:

[0058] (1) Probe preparation: AIE fluorescent microspheres were dispersed in borate buffer solution (0.02 M, pH=8.0) to a final concentration of 0.05 mg / mL, and sonicated for 2 min to ensure complete dispersion. 1 / 10 volume of antibody was added dropwise. E. coli O157:H7 monoclonal antibody solution (100 μg / mL) was labeled for 2 h, and then 1 / 10 volume of bovine serum albumin solution (10%, w / v) was added for blocking for 2 h. After the reaction was completed, the mixture was centrifuged at 4℃ and 8000 r / min for 15 min, the supernatant was discarded, and the mixture was reconstituted with 1 / 10 volume of PBS and stored at 4℃ for later use.

[0059] (2) Preparation of immunochromatographic test strips: Anti-... E. coli O157:H7 polyclonal antibody (1.0 mg / mL) and donkey anti-mouse secondary antibody (0.5 mg / mL) were sprayed onto a nitrocellulose membrane as the detection line (T) and test line (C). After drying, the membrane was assembled with the sample pad, absorbent paper, and test strip base plate and then cut into strips for later use.

[0060] (3) Establishing a standard curve: E. coli O157:H7 was spiked into PBS to achieve final concentrations of 0 and 1 × 10⁻⁶, respectively. 2 2×10 2 1×10 3 2×10 3 1×10 4 2×10 4 1×10 5 2×10 5 1×10 6 2×10 6 1×10 7 Transfer the spiked solution to the ELISA wells, add 3 μL of probe, incubate for 3 min, insert the test strip, and read the value after 15 min of swirling.

[0061] Figure 6 The image shows sodium alginate AIE (TCPE) fluorescent microspheres prepared by the method of the present invention (Example 2) applied to immunochromatographic detection. E. coli Physical image and standard curve of O157:H7. Figure 6 'a' is a picture of the actual object, and... E. coli As the concentration of O157:H7 increases, the fluorescence of the T line gradually increases until a hook effect appears. Figure 6 b is its standard curve, with the linear equation y = 1038ln(x) − 7014.9, a linear correlation coefficient (R²) of 0.9823, and a calculated limit of detection of 869 CFU / mL.

[0062] Although the description of the invention has been quite detailed and particularly of several described embodiments, it is not intended to limit it to any of these details or embodiments or any particular embodiment, but should be considered as providing a broad possible interpretation of the claims by referring to the appended claims and taking into account the prior art, thereby effectively covering the intended scope of the invention. Furthermore, the invention has been described above with respect to embodiments foreseeable by the inventors in order to provide a useful description, and non-substantial modifications to the invention that have not yet been foreseen may still represent equivalent modifications.

Claims

1. A method for preparing sodium alginate AIE fluorescent microspheres, characterized in that, Includes the following steps: AIE dye and sodium alginate were dissolved in water and dispersed in a non-polar organic solvent to obtain a mixture. Then, under ice bath conditions, the mixture was ultrasonically prepared into a micro / nano emulsion. Calcium chloride solution was added for solidification, followed by centrifugation, washing, and resolubilization to finally obtain sodium alginate AIE fluorescent microspheres. The AIE dye is a hydrophilic AIE dye, including at least one of tetrastyrylcarboxylic acids and their derivatives, tetrastyrylphenols and their derivatives, and tetrastyryl nitriles and their derivatives.

2. The preparation method according to claim 1, characterized in that, The nonpolar organic solvent includes at least one of aliphatic hydrocarbons and their derivatives and aromatic hydrocarbons and their derivatives.

3. The preparation method according to claim 1, characterized in that, The ratio of AIE dye, sodium alginate, and water is 1 mg-100 mg: 1 mg-50 mg: 1 mL.

4. The preparation method according to claim 1, characterized in that, The specific steps for forming the micro / nano emulsion are as follows: The mixture was prepared by ultrasonic emulsification using a cell disruptor under ice bath conditions, with an emulsification time of 3-30 minutes and an emulsification power of 120 W-600 W.

5. The preparation method according to claim 1, characterized in that, The specific curing process is as follows: Add calcium chloride solution at a speed of 20 r / min-200 r / min, with a concentration of 1 mg / mL-100 mg / mL. Continue stirring for 1 h-12 h until complete curing.

6. The preparation method according to claim 1, characterized in that, The specific steps of the centrifugal washing are as follows: First, centrifuge at 3000 r / min-10000 r / min for 10 min-30 min, then wash three times with 5%-50% ethanol aqueous solution as washing agent, and finally wash with ultrapure water until the supernatant after centrifugation is clear.

7. The preparation method according to claim 1, characterized in that, The reconstituted solution is ultrapure water or phosphate buffer solution.

8. A sodium alginate AIE fluorescent microsphere, characterized in that, It is obtained by any one of the preparation methods of claims 1-7.

9. The sodium alginate AIE fluorescent microspheres according to claim 8, characterized in that, The particle size of the AIE fluorescent microspheres is 0.05 μm to 5 μm.

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