Preparation method of bio-based aggregate microspheres

Abstract

The preparation method comprises the following steps: uniformly mixing a polyelectrolyte solution with positive charges and an adenosine triphosphate solution with negative charges to obtain condensed fluid drops; adding dye, biomacromolecules or nano-particles into the condensed body liquid drops to obtain condensed body liquid drops loaded with guest molecules; and incubating the condensate droplets loaded with the guest molecules, adding a sodium silicate solution, and centrifugally cleaning to obtain the condensate microspheres. According to the invention, a biomimetic mineralization method is proposed for the first time, SiO2 is used as a membrane component, condensed liquid drops are converted into solid microspheres with stable mechanical strength, and a new idea is provided for overcoming the stability bottleneck of the condensed liquid drops.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for preparing bio-based condensed microspheres. The prepared microspheres have adjustable sizes and can efficiently encapsulate and maintain the activity of various functional substances (such as enzymes, proteins, and nanoparticles). Background Technology

[0002] Functionalized microspheres, especially those based on biomolecules, have broad application prospects in drug delivery, biosensing, and enzyme immobilization. Ideal bio-based microspheres should possess high biocompatibility, excellent loading capacity for active substances (such as enzymes, proteins, and nucleic acids), and a mild preparation environment that maintains their activity. However, current mainstream preparation methods (such as polymerization, emulsion-solvent evaporation, and spray drying) often face significant challenges: their processes are often complex and demanding, involving high temperatures, stirring, organic solvents, or extreme pH values, which can easily damage active substances, leading to enzyme inactivation, protein denaturation, or nucleic acid degradation; simultaneously, the use of organic solvents may introduce residual risks, threatening the activity of the loaded material and product safety; furthermore, these methods also have limitations in achieving high-concentration and high-efficiency loading of active substances.

[0003] To address the aforementioned challenges, condensate droplets formed by liquid-liquid phase separation (LLPS) exhibit unique advantages: their formation process typically occurs spontaneously under near-physiological conditions (room temperature, neutral pH, aqueous phase), providing a mild platform for encapsulating highly active and sensitive biomolecules; simultaneously, condensate droplets possess selective adsorption capabilities for guest molecules (such as enzymes, dyes, drugs, and nanoparticles) and can form a high-concentration biomolecular microenvironment. Despite these advantageous properties, the lack of a shell severely limits their stability, making them prone to aggregation and pH / salt-induced dissociation, leading to leakage and loss of function, thus failing to meet the application requirements for long-term storage, recyclability, or complex physiological / industrial environments.

[0004] Physicochemical strategies for improving stability (membrane formation) often rely on polymer materials, resulting in flexible protective layers with insufficient mechanical strength and easy degradation. Nature provides an excellent example: organisms such as diatoms can construct inorganic protective shells with high mechanical strength and excellent chemical stability through biomineralization processes under mild conditions. Inspired by this, biomimetic mineralization technology has been used to enhance the performance of microspheres or microcapsules.

[0005] However, how to efficiently and controllably apply biomimetic mineralization strategies to aggregate droplets, and construct solid bio-based aggregate microspheres with a robust outer shell while retaining the high bioactivity loading capacity of the core and endowing it with excellent stability under mild aqueous conditions, remains a key technical challenge that urgently needs to be overcome in this field. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0007] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0008] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing bio-based condensate microspheres.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for preparing bio-based condensate microspheres, comprising: mixing a positively charged polyelectrolyte solution and a negatively charged adenosine triphosphate (ATP) solution uniformly to obtain condensate droplets; adding dyes, biomacromolecules or nanoparticles to the condensate droplets to obtain condensate droplets loaded with guest molecules; incubating the condensate droplets loaded with guest molecules for a period of time, adding sodium silicate solution, and then centrifuging and washing to obtain condensate microspheres.

[0010] In a preferred embodiment of the method for preparing the bio-based condensed microspheres of the present invention, the positively charged polyelectrolyte comprises quaternized chitosan (QCS), polydiallyldimethylammonium chloride (PDDA), protamine sulfate (Prot), and polylysine (Plys), preferably QCS.

[0011] In a preferred embodiment of the method for preparing bio-based condensed microspheres according to the present invention, the positively charged polyelectrolyte solution and the negatively charged adenosine triphosphate (ATP) solution are mixed evenly, wherein the mass ratio of QCS to ATP is 0.1 to 1.25, preferably 1.0.

[0012] In a preferred embodiment of the method for preparing bio-based aggregate microspheres according to the present invention, the dyes include methylene blue, crystal violet, malachite green, rhodamine B, sodium fluorescein, nilored red, fluorescein isothiocyanate (FITC), and rhodamine B isothiocyanate (RITC).

[0013] In a preferred embodiment of the method for preparing bio-based condensed microspheres according to the present invention, the biomacromolecules include horseradish peroxidase (HRP), glucose oxidase (GOx), lipase (CALB), laccase, urease, dextran, and phycocyanin.

[0014] In a preferred embodiment of the preparation method of the bio-based condensed microspheres of the present invention, the nanoparticles include gold nanoparticles, iron oxide nanoparticles, and silver nanoparticles.

[0015] In a preferred embodiment of the method for preparing the bio-based aggregate microspheres of the present invention, the volume ratio of the dye to the aggregate droplets is 0.025 to 0.25:1.

[0016] The volume ratio of the biomacromolecules to the aggregated droplets is 0.025–0.25:1;

[0017] The volume ratio of the nanoparticles to the aggregated droplets is 0.25 to 2.5:1.

[0018] In a preferred embodiment of the method for preparing the bio-based aggregate microspheres of the present invention, the static incubation period is 0.1 to 30 minutes.

[0019] In a preferred embodiment of the method for preparing the bio-based condensate microspheres of the present invention, the sodium silicate solution has a concentration of 0.5 wt% to 5 wt%, preferably 5 wt%; and the volume ratio of the sodium silicate solution to the condensate droplets loaded with guest molecules is 0.025 to 0.25:1.

[0020] Another objective of this invention is to overcome the shortcomings of the prior art and provide a bio-based aggregate microsphere, wherein the bio-based aggregate microsphere is a SiO2 aggregate microsphere with good encapsulation ability and environmental tolerance.

[0021] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of bio-based condensate microspheres in the preparation of immobilized enzyme carriers.

[0022] The beneficial effects of this invention are:

[0023] (1) This invention proposes for the first time to transform aggregated droplets into mechanically stable solid microspheres by using SiO2 as a membrane component through a biomimetic mineralization method, which also provides a new approach to overcome the bottleneck of aggregated droplet stability.

[0024] (2) The structure of the aggregated microspheres prepared by this invention is controllable. The particle size of the microspheres can be controlled by changing the mass ratio of QCS and ATP and the incubation time of the aggregated droplets for different application scenarios. In addition, the SiO2 surface can be functionalized to meet diverse research and application needs.

[0025] (3) The aggregated microspheres prepared by this invention have good enrichment capabilities, including dyes, bioactive molecules and nanoparticles, and the preparation conditions are simple and mild, without involving organic reagents. They can effectively retain the activity of the encapsulated substances and reduce potential harm to organisms. At the same time, they have certain potential for large-scale production and have broad application prospects in the biomedical field. Attached Figure Description

[0026] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0027] Figure 1 This is a scanning electron microscope image of the morphology of the aggregated microspheres in Embodiment 1 of the present invention.

[0028] Figure 2 This is a scanning electron microscope image of the morphology of the aggregated microspheres in Embodiment 2 of the present invention.

[0029] Figure 3 This is a scanning electron microscope image of the morphology of the aggregated microspheres in Embodiment 5 of the present invention.

[0030] Figure 4 This is a morphological image of the aggregated microspheres loaded with FITC-GOx in Embodiment 8 of the present invention under a laser confocal microscope.

[0031] Figure 5 This is a morphological image of the RITC-HRP-loaded aggregated microspheres in Embodiment 9 of the present invention under a laser confocal microscope.

[0032] Figure 6 This is a transmission electron microscope image of the aggregated microspheres loaded with gold nanoparticles in Example 11 of the present invention.

[0033] Figure 7 This is a morphological image of the PDDA / ATP aggregate microspheres in Example 12 of the present invention under an optical microscope.

[0034] Figure 8 This is a morphological image of the Prot / ATP condensate microspheres in Example 13 of the present invention under an optical microscope.

[0035] Figure 9 This is an appearance diagram of the red fluorescent product, halogenated ...

[0036] Figure 10 This is a diagram of the morphology of the QCS / ATP condensate microspheres in Comparative Example 1 of this invention under an optical microscope.

[0037] Figure 11 This is a diagram of the morphology of the QCS / ATP condensate microspheres in Comparative Example 2 of this invention under an optical microscope. Detailed Implementation

[0038] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0039] The experimental materials used in this invention embodiment are as follows: QCS (90% substitution degree) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; ATP, FITC, and phycocyanin were purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd.; Rhodamine B, crystal violet, RITC, HRP, and Prot were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; methylene blue, chloroauric acid, trisodium citrate, glucose, silver nitrate, and iron tetroxide nanoparticles were purchased from Sinopharm Chemical Reagent Co., Ltd.; sodium silicate, FITC-glucan, laccase, urease, and PDDA were purchased from Shanghai Titan Technology Co., Ltd.; Amplex Red was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; GOx was purchased from Beijing Bailingwei Technology Co., Ltd.; malachite green and Plys were purchased from Beijing Wokai Biotechnology Co., Ltd.; sodium fluorescein was purchased from Beijing Innocare Technology Co., Ltd.; Nile red and lipase were purchased from Sigma-Aldrich (USA), all of which are commercially available products.

[0040] Example 1:

[0041] (1) Dissolve QCS powder in water to prepare a QCS solution with a mass concentration of 4 mg / mL;

[0042] (2) Dissolve ATP powder in water to prepare an ATP solution with a mass concentration of 4 mg / mL;

[0043] (3) Add 200 μL of the pre-prepared ATP solution to 200 μL of QCS solution, invert the solution 3 times, and let it stand for 30 min to obtain aggregate droplets;

[0044] (4) Add 50 μL of 5 wt% sodium silicate solution to the above aggregate droplets, incubate for 10 min, then centrifuge and wash to obtain aggregate microspheres. See [link to product details]. Figure 1 .

[0045] Example 2:

[0046] (1) Dissolve QCS powder in water to prepare a QCS solution with a mass concentration of 4 mg / mL;

[0047] (2) Dissolve ATP powder in water to prepare an ATP solution with a mass concentration of 4 mg / mL;

[0048] (3) Add 200 μL of pre-prepared ATP solution to 200 μL of QCS solution, invert 3 times to obtain aggregate droplets;

[0049] (4) Add 50 μL of 5 wt% sodium silicate solution to the freshly prepared aggregate droplets, incubate for 10 min, then centrifuge and wash to obtain aggregate microspheres. See [link to product details]. Figure 2 .

[0050] Example 3:

[0051] (1) Dissolve QCS powder in water to prepare a QCS solution with a mass concentration of 3 mg / mL;

[0052] (2) Dissolve ATP powder in water to prepare an ATP solution with a mass concentration of 4 mg / mL;

[0053] (3) Add 200 μL of the pre-prepared ATP solution to 200 μL of QCS solution, invert the solution 3 times, and let it stand for 30 min to obtain aggregate droplets;

[0054] (4) Add 50 μL of 5 wt% sodium silicate solution to the above aggregate droplets, incubate for 10 min, and then centrifuge and wash to obtain aggregate microspheres.

[0055] Example 4:

[0056] (1) Dissolve QCS powder in water to prepare a QCS solution with a mass concentration of 2 mg / mL;

[0057] (2) Dissolve ATP powder in water to prepare an ATP solution with a mass concentration of 4 mg / mL;

[0058] (3) Add 200 μL of the pre-prepared ATP solution to 200 μL of QCS solution, invert the solution 3 times, and let it stand for 30 min to obtain aggregate droplets;

[0059] (4) Add 50 μL of 5 wt% sodium silicate solution to the above aggregate droplets, incubate for 10 min, and then centrifuge and wash to obtain aggregate microspheres.

[0060] Example 5:

[0061] (1) Dissolve QCS powder in water to prepare a QCS solution with a mass concentration of 1 mg / mL;

[0062] (2) Dissolve ATP powder in water to prepare an ATP solution with a mass concentration of 4 mg / mL;

[0063] (3) Add 200 μL of the pre-prepared ATP solution to 200 μL of QCS solution, invert the solution 3 times, and let it stand for 30 min to obtain aggregate droplets;

[0064] (4) Add 50 μL of 5 wt% sodium silicate solution to the above aggregate droplets, incubate for 10 min, then centrifuge and wash to obtain aggregate microspheres. See [link to product details]. Figure 3 .

[0065] Example 6:

[0066] (1) Dissolve QCS powder in water to prepare a QCS solution with a mass concentration of 4 mg / mL;

[0067] (2) Dissolve ATP powder in water to prepare an ATP solution with a mass concentration of 4 mg / mL;

[0068] (3) Add 200 μL of pre-prepared ATP solution to 200 μL of QCS solution, then add 50 μL of 0.5 wt% sodium fluorescein, invert 3 times and let stand for 30 min to obtain aggregate droplets;

[0069] (4) Add 50 μL of 5 wt% sodium silicate solution to the above aggregate droplets, incubate for 10 min, and then centrifuge and wash to obtain aggregate microspheres loaded with sodium fluorescein.

[0070] Example 7:

[0071] (1) Dissolve QCS powder in water to prepare a QCS solution with a mass concentration of 4 mg / mL;

[0072] (2) Dissolve ATP powder in water to prepare an ATP solution with a mass concentration of 4 mg / mL;

[0073] (3) Add 200 μL of pre-prepared ATP solution to 200 μL of QCS solution, then add 50 μL of 0.5 wt% FITC fluorescently labeled dextran, invert 3 times and let stand for 30 min to obtain aggregate droplets.

[0074] (4) Add 50 μL of 5 wt% sodium silicate solution to the above aggregate droplets, incubate for 10 min, and then centrifuge and wash to obtain aggregate microspheres loaded with FITC-glucan.

[0075] Example 8:

[0076] (1) Dissolve QCS powder in water to prepare a QCS solution with a mass concentration of 4 mg / mL;

[0077] (2) Dissolve ATP powder in water to prepare an ATP solution with a mass concentration of 4 mg / mL;

[0078] (3) Add 200 μL of pre-prepared ATP solution to 200 μL of QCS solution, then add 50 μL of 0.5 wt% FITC fluorescently labeled GOx, invert 3 times and let stand for 30 min to obtain aggregate droplets.

[0079] (4) Add 50 μL of 5 wt% sodium silicate solution to the above aggregate droplets, incubate for 10 min, and then centrifuge and wash to obtain FITC-GOx-loaded aggregate microspheres. See Figure 4 .

[0080] Example 9:

[0081] (1) Dissolve QCS powder in water to prepare a QCS solution with a mass concentration of 4 mg / mL;

[0082] (2) Dissolve ATP powder in water to prepare an ATP solution with a mass concentration of 4 mg / mL;

[0083] (3) Add 200 μL of pre-prepared ATP solution to 200 μL of QCS solution, then add 50 μL of 0.5 wt% Rhodamine B isothiocyanate (RITC) fluorescently labeled HRP, invert 3 times and let stand for 30 min to obtain aggregate droplets.

[0084] (4) Add 50 μL of 5 wt% sodium silicate solution to the above aggregate droplets, incubate for 10 min, and then centrifuge and wash to obtain RITC-HRP-loaded aggregate microspheres. See Figure 5 .

[0085] Example 10:

[0086] (1) Dissolve QCS powder in water to prepare a QCS solution with a mass concentration of 4 mg / mL;

[0087] (2) Dissolve ATP powder in water to prepare an ATP solution with a mass concentration of 4 mg / mL;

[0088] (3) Add 200 μL of pre-prepared ATP solution to 200 μL of QCS solution, then add 50 μL of 0.5 wt% phycocyanin, invert 3 times and let stand for 30 min to obtain aggregate droplets;

[0089] (4) Add 50 μL of 5 wt% sodium silicate solution to the above aggregate droplets, incubate for 10 min, and then centrifuge and wash to obtain aggregate microspheres loaded with phycocyanin.

[0090] Example 11:

[0091] (1) 10 mL of trisodium citrate solution (38.8 mM) was injected into 100 mL of chloroauric acid solution (0.9 mM), and the mixture was heated and refluxed for 20 min under vigorous stirring. After cooling to room temperature, gold nanoparticles with a particle size of 12 nm were obtained.

[0092] (2) Dissolve QCS powder in water to prepare a QCS solution with a mass concentration of 4 mg / mL;

[0093] (3) Dissolve ATP powder in gold nanoparticle solution to prepare ATP mixed solution with a mass concentration of 4 mg / mL;

[0094] (4) Add 200 μL of the pre-prepared ATP mixed solution to 200 μL of QCS solution, invert 3 times and let stand for 30 min to obtain aggregate droplets loaded with gold nanoparticles.

[0095] (5) Add 50 μL of 5wt% sodium silicate solution to the above aggregated droplets, incubate for 10 min, and then centrifuge and wash to obtain aggregated microspheres loaded with gold nanoparticles. See Figure 6 .

[0096] Example 12:

[0097] (1) Dissolve PDDA powder in water to prepare a PDDA solution with a mass concentration of 10 mg / mL;

[0098] (2) Dissolve ATP powder in water to prepare an ATP solution with a mass concentration of 10 mg / mL;

[0099] (3) Add 200 μL of pre-prepared ATP solution to 200 μL of PDDA solution, and invert the solution 3 times to obtain aggregate droplets;

[0100] (4) Add 50 μL of 5 wt% sodium silicate solution to the freshly prepared aggregate droplets, incubate for 10 min, then centrifuge and wash to obtain PDDA / ATP aggregate microspheres. See [link to product details]. Figure 7 .

[0101] Example 13:

[0102] (1) Dissolve the Prot powder in water to prepare a Prot solution with a mass concentration of 10 mg / mL;

[0103] (2) Dissolve ATP powder in water to prepare an ATP solution with a mass concentration of 10 mg / mL;

[0104] (3) Add 200 μL of pre-prepared ATP solution to 200 μL of Prot solution, and invert the solution 3 times to obtain aggregate droplets;

[0105] (4) Add 50 μL of 5 wt% sodium silicate solution to the above-mentioned aggregate droplets, incubate for 10 min, then centrifuge and wash to obtain Prot / ATP aggregate microspheres. See [link to product description]. Figure 8 .

[0106] Example 14:

[0107] (1) Dissolve Plys powder in water to prepare a Plys solution with a mass concentration of 10 mg / mL;

[0108] (2) Dissolve ATP powder in water to prepare an ATP solution with a mass concentration of 10 mg / mL;

[0109] (3) Add 200 μL of the pre-prepared ATP solution to 200 μL of Plys solution, and invert the solution 3 times to obtain aggregate droplets;

[0110] (4) Add 50 μL of 5 wt% sodium silicate solution to the above aggregate droplets, incubate for 10 min, and then centrifuge and wash to obtain Plys / ATP aggregate microspheres.

[0111] Example 15:

[0112] (1) Condensed microspheres loaded with GOx and HRP were prepared according to the methods in Examples 8 and 9, respectively;

[0113] (2) Add 5 μL of the fluorescent substrate Amplex Red to 300 μL of the enzyme-carrying microsphere mixture prepared above. After incubation for 5 min, add 20 μL of glucose (1 mM) to trigger the GOx-mediated catalytic reaction. The generated hydrogen peroxide can react with Amplex Red under the catalysis of HRP to produce the red fluorescent substance halogen. See Figure 9 The significant fluorescence signal demonstrated the effective execution of the enzyme cascade catalysis and verified the excellent preservation of enzyme activity by the preparation method of this invention.

[0114] Comparative Example 1

[0115] (1) Dissolve QCS powder in water to prepare a QCS solution with a mass concentration of 4 mg / mL;

[0116] (2) Dissolve ATP powder in water to prepare an ATP solution with a mass concentration of 4 mg / mL;

[0117] (3) Add 200 μL of pre-prepared ATP solution to 200 μL of QCS solution, invert 3 times to obtain aggregate droplets;

[0118] (4) Add 50 μL of 0.5 wt% sodium silicate solution to the freshly prepared aggregate droplets, incubate for 10 min, then centrifuge and wash to obtain aggregate microspheres. See [link to product details]. Figure 10 The results showed that when the sodium silicate concentration was insufficient, it could not effectively assemble on the surface of the droplets and it was difficult to form microspheres.

[0119] Comparative Example 2

[0120] (1) Dissolve QCS powder in water to prepare a QCS solution with a mass concentration of 5 mg / mL;

[0121] (2) Dissolve ATP powder in water to prepare an ATP solution with a mass concentration of 4 mg / mL;

[0122] (3) Add 200 μL of the pre-prepared ATP solution to 200 μL of QCS solution, invert the solution 3 times, and let it stand for 30 min to obtain aggregate droplets;

[0123] (4) Add 50 μL of 5 wt% sodium silicate solution to the above aggregate droplets, incubate for 10 min, then centrifuge and wash to obtain aggregate microspheres. See [link to product details]. Figure 11 The results showed that when the QCS concentration exceeded a certain proportion, it could only form large aggregates that were difficult to disperse by ultrasound.

[0124] This invention provides a novel method for preparing bio-based aggregated microspheres based on biomimetic mineralization. The core of this method lies in directly constructing bio-based aggregated microspheres with a robust silica shell through a biomimetic mineralization process under mild aqueous conditions. Compared to existing microsphere preparation technologies (such as polymerization and emulsification methods), which often involve complex processes, harsh conditions (high temperature, high pressure, organic solvents), and the resulting damage to bioactive substances, this method requires only simple mixing and can be completed at room temperature and pressure without organic solvents. This maximizes the preservation of the activity of the encapsulated substances (especially sensitive biomolecules such as enzymes) and eliminates the risk of residual organic solvents, significantly improving the biosafety of the product. The particle size of the SiO2 mineralized microspheres prepared by this method can be precisely controlled by the concentration and ratio of key biomolecules (such as QCS and ATP) and the reaction time. Simultaneously, the SiO2 shell is easily functionalized, endowing the microspheres with diverse surface properties. The resulting microspheres possess excellent mechanical strength, chemical stability, and environmental tolerance, and can efficiently enrich and encapsulate various functional substances (such as dyes, bioactive molecules, enzymes, and nanoparticles), making them particularly suitable as immobilized enzyme carriers for biocatalysis. This preparation method is simple, environmentally friendly, low-cost, and has scalability potential, opening new avenues for developing high-performance bio-based microspheres for applications in bioreactors, drug delivery, and cosmetics.

[0125] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing bio-based condensate microspheres, characterized in that: include, A positively charged polyelectrolyte solution and a negatively charged adenosine triphosphate solution are mixed evenly to obtain aggregated droplets. Adding dyes, biomacromolecules, or nanoparticles to aggregate droplets yields aggregate droplets loaded with guest molecules. After incubating the aggregated droplets loaded with guest molecules, sodium silicate solution was added, and the mixture was centrifuged and washed to obtain aggregated microspheres.

2. The preparation method according to claim 1, characterized in that: The positively charged polyelectrolytes include quaternized chitosan, polydiallyldimethylammonium chloride, protamine sulfate, and polylysine.

3. The preparation method according to claim 1 or 2, characterized in that: The positively charged polyelectrolyte solution and the negatively charged adenosine triphosphate solution are mixed evenly, wherein the mass ratio of the positively charged polyelectrolyte to the negatively charged adenosine triphosphate is 0.1 to 1.25:

1.

4. The preparation method according to claim 1, characterized in that: The dyes include methylene blue, crystal violet, malachite green, rhodamine B, sodium fluorescein, nilored, fluorescein isothiocyanate, and rhodamine B isothiocyanate. The biomacromolecules include horseradish peroxidase, glucose oxidase, lipase, laccase, urease, dextran, and phycocyanin.

5. The preparation method according to claim 1 or 4, characterized in that: The nanoparticles include gold nanoparticles, iron oxide nanoparticles, and silver nanoparticles.

6. The preparation method according to claim 5, characterized in that: The volume ratio of the dye to the aggregate droplets is 0.025–0.25:1; The volume ratio of the biomacromolecules to the aggregated droplets is 0.025–0.25:1; The volume ratio of the nanoparticles to the aggregated droplets is 0.25 to 2.5:

1.

7. The preparation method according to claim 1, characterized in that: The incubation process, wherein the incubation time is 0.1 to 30 minutes.

8. The preparation method according to claim 1, characterized in that: The sodium silicate solution has a concentration of 0.5 wt% to 5 wt%; the volume ratio of the sodium silicate solution to the aggregate droplets loaded with guest molecules is 0.025 to 0.25:

1.

9. The bio-based condensate microspheres prepared by any one of claims 1 to 8, characterized in that: The bio-based condensate microspheres are SiO2 condensate microspheres, which have good encapsulation ability and environmental tolerance.

10. The application of the bio-based condensate microspheres of claim 9 in the preparation of immobilized enzyme carriers.

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