Multilayer-structure Fe3O4 (at) SiO2 (at) ZIF-8 composite compound for enzyme immobilization and fluorescence dual-mode detection and preparation method of multilayer-structure Fe3O4 (at) SiO2 (at) ZIF-8 composite compound

Abstract

The invention relates to the technical field of nano materials and biochemistry, in particular to a Fe3O4 (at) SiO2 (at) ZIF-8 composite compound for enzyme immobilization and fluorescence dual-mode detection based on a spatial isolation structure and a preparation method of the Fe3O4 (at) SiO2 (at) ZIF-8 composite compound. The composite compound has a multi-layer structure which sequentially comprises a Fe3O4 core, a SiO2 isolation layer, a ZIF-8 shell layer and an outer layer carbon quantum dot from inside to outside, enzyme molecules are loaded in pore channels of the ZIF-8 shell layer, and Fe3O4 is magnetic nanoparticles; the SiO2 is a continuous compact silicon dioxide layer coated on the surface of the Fe3O4; the ZIF-8 is a metal organic framework material formed by in-situ growth on the surface of SiO2; the carbon quantum dots are fixed on the outer surface of the ZIF-8 / SiO2 through chemical bonding; the enzyme is loaded in the ZIF-8 in a pore diffusion manner and is fixed by light cross-linking.

Description

Technical Field

[0001] This invention relates to the fields of nanomaterials and biochemistry, and in particular to a Fe3O4@SiO2@ZIF-8 composite compound for enzyme immobilization and fluorescence dual-mode detection based on a spatially isolated structure and its preparation method. Background Technology

[0002] In recent years, metal-organic frameworks (MOFs) have attracted widespread attention in fields such as enzyme immobilization, biocatalysis, fluorescence sensing, and drug delivery due to their high specific surface area, tunable pore structure, and good designability. Among them, ZIF-8, as a typical MOF material, has the characteristics of structural stability, moderate pore size, and mild synthesis conditions, and has been widely used to construct composite systems for enzyme loading and active molecule encapsulation.

[0003] On the other hand, enzymes, as highly efficient and specific biocatalysts, have relatively soft natural conformations and are highly sensitive to temperature, solvent environment, metal ions, and surface interactions, making them prone to inactivation or conformational changes. Immobilizing enzymes on the pores or surface of nanocarriers can enable enzyme reuse, improve enzyme stability, and enhance their operational performance in complex systems. However, existing enzyme immobilization methods still suffer from the following common problems:

[0004] (1) The conformational disruption of the enzyme during the immobilization process leads to a decrease in activity;

[0005] (2) Non-specific adsorption or strong interaction between the carrier and the enzyme causes enzyme molecule inactivation;

[0006] (3) When the enzyme is encapsulated inside the MOF, fluorescent probes, metal ions or organic ligands may affect the enzyme reaction;

[0007] (4) During the nucleation process of the support, exogenous organic molecules (such as carbon dots, dyes, etc.) are prone to compete with metal ions for coordination, which can cause structural defects or decreased stability of MOF.

[0008] Magnetic nanomaterials (such as Fe3O4) are often used to construct reusable enzyme loading platforms due to their convenient magnetic response separation capabilities. However, bare Fe3O4 exhibits poor stability in aqueous solutions, and impurities easily deposit on its surface, affecting enzyme binding. Therefore, surface modification with materials such as silica (SiO2) is frequently employed. The SiO2 shell not only improves particle stability but also provides reaction sites for further surface functionalization, making it an important intermediate layer material for constructing composite multilayer structures.

[0009] Carbon quantum dots (CDs), as a novel fluorescent nanomaterial, possess excellent photostability, low biotoxicity, and good water solubility, and are often used as fluorescence signal readout units in MOF-enzyme complex systems. However, directly coupling CDs with enzymes or having them participate in the MOF nucleation process can easily lead to reduced enzyme activity, unstable fluorescence signals, or uncontrolled MOF nucleation structures. Therefore, ensuring "maintained enzyme activity, intact MOF structure, and stable fluorescence signal" within the same complex system has become one of the current research challenges in this field.

[0010] Currently, there is a lack of novel composite structures that can integrate enzyme immobilization and fluorescence detection functions while maintaining the integrity of the enzyme conformation, avoiding nucleation interference, and improving structural stability. Therefore, developing a Fe3O4@SiO2@ZIF-8 composite compound with a spatial hierarchical structure and reducing interference between components by assembling the enzyme and fluorescence unit in layers has become an urgent technical problem to be solved in this field. Summary of the Invention

[0011] This invention addresses the technical problems of existing integrated enzyme immobilization and fluorescence detection systems, such as easily impaired enzyme activity, susceptibility to interference with the metal-organic framework structure, unstable fluorescence signals, and difficulty in reusability. It provides a composite compound with both enzyme immobilization and fluorescence response, along with its preparation method. By preparing a multilayer structure consisting of an Fe3O4 core, a SiO2 insulating layer, a ZIF-8 shell, and an outer layer of carbon quantum dots from the inside out, high enzyme activity is maintained, MOF nucleation stability is achieved, and fluorescence signal readout is stable, thus resolving some of the mutual interference problems in existing technologies.

[0012] To address the aforementioned technical problems, this invention provides a multilayered Fe3O4@SiO2@ZIF-8 composite compound for enzyme immobilization and fluorescence dual-mode detection. The composite compound has a multilayered structure consisting of an Fe3O4 core, a SiO2 isolation layer, a ZIF-8 shell, and an outer layer of carbon quantum dots, arranged sequentially from the inside out. The enzyme molecules are loaded inside the pores of the ZIF-8 shell. The Fe3O4 is a magnetic nanoparticle.

[0013] The SiO2 is a continuous and dense silicon dioxide layer coated on the surface of Fe3O4;

[0014] The ZIF-8 is a metal-organic framework material grown in situ on the surface of SiO2;

[0015] The carbon quantum dots are chemically bonded to the outer surface of ZIF-8 / SiO2;

[0016] The enzyme is loaded inside ZIF-8 via pore diffusion and immobilized by mild cross-linking.

[0017] Preferably, the thickness of the SiO2 isolation layer is 5–20 nm.

[0018] Preferably, the thickness of the ZIF-8 shell is 20–60 nm.

[0019] Preferably, carbon quantum dots are fixed to the outer surface of the composite material through amide bonds, silane bonds, or quaternary ammonium salt bonds.

[0020] Preferably, the outer surface of the ZIF-8 further has an alkylsilane layer.

[0021] Preferably, the alkylsilane layer is methyltriethoxysilane (MTES) or ethyltriethoxysilane (ETES).

[0022] Preferably, the carbon quantum dots are nitrogen-doped carbon dots, sulfur-doped carbon dots, or sulfur-nitrogen co-doped carbon dots.

[0023] Preferably, the enzyme is at least one of thioredoxin reductase (THR), oxidoreductase, peroxidase, glucose oxidase, lactate oxidase, or lipase.

[0024] A second aspect of the present invention provides a method for preparing the composite compound, comprising the following steps:

[0025] (1) Fe3O4 cores were prepared by coprecipitation method;

[0026] (2) TEOS hydrolysis forms a SiO2 coating layer on the Fe3O4 surface;

[0027] (3) Silanization treatment of SiO2 surface;

[0028] (4) In-situ growth of ZIF-8 shell on the outer layer of SiO2;

[0029] (5) Carbon quantum dots are coupled to the outer surface of ZIF-8 or SiO2 through the EDC / NHS system;

[0030] (6) Enzyme molecules diffuse into the ZIF-8 channels;

[0031] (7) The enzyme carrier structure was lightly cross-linked and fixed using low concentrations of genipin (geniposide) or glutaraldehyde.

[0032] The third aspect of this invention provides the application of the aforementioned composite compound in fluorescence detection, wherein a fluorescence signal channel is constructed through an outer carbon quantum dot for detecting thioredoxin substrates, redox compounds, or free active small molecules.

[0033] By adopting the above technical solution, the present invention has the following beneficial effects:

[0034] In this invention, because the enzyme does not directly contact the carbon quantum dots or participate in the metal ion nucleation process, conformational damage is significantly reduced; post-enzyme loading and mild cross-linking methods enable an activity retention rate of over 90%. Simultaneously, the carbon dots are confined to the outer layer and do not participate in Zn... 2+ Coordination significantly reduces skeletal defects, enabling ZIF-8 to maintain a high specific surface area and intact crystal form, thus improving structural stability.

[0035] In addition, carbon quantum dots are located on the outer layer of MOF, avoiding interference from the pore environment on fluorescence behavior. The signal is not easily quenched, drifted or blue-shifted, making it suitable for ratiometric or single-channel fluorescence detection.

[0036] Meanwhile, the Fe3O4 core introduces magnetic response capability, the enzyme is firmly immobilized, and the enzyme activity retention rate can still reach 80-90% after 10 cycles of use of the composite material. Attached Figure Description

[0037] 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.

[0038] Figure 1 A flowchart illustrating the preparation process of the Fe3O4@SiO2@ZIF-8 composite compound provided by this invention;

[0039] Figure 2 This is a transmission electron microscope image of CDs.

[0040] Figure 3 This is a scanning electron microscope image of Fe3O4.

[0041] Figure 4 This is a transmission electron microscope (TEM) image of Fe3O4.

[0042] Figure 5 This is a scanning electron microscope image of the THR@Fe3O4@SiO2@ZIF-8-CDs composite. Detailed Implementation

[0043] Existing research on MOF complexes for synthesizing CDs-modified enzymes indicates that the enzyme / carbon quantum dot complex system still has several shortcomings: First, direct coupling of carbon quantum dots to enzyme molecules on the surface or their participation in the nucleation process of ZIF-8 can disturb the native conformation of the enzyme, leading to a significant reduction in enzyme activity; second, the carboxyl and thiol groups on the surface of carbon quantum dots readily interact with Zn. 2+Competition for coordination interferes with the lattice growth of ZIF-8, leading to increased defects in the resulting framework structure, a significant decrease in specific surface area, and even reduced material stability in solvent environments. Third, placing fluorescent probes inside MOF channels is affected by the electronic environment within the pores, causing blue shift of the emission peak, fluorescence quenching, and signal drift, thus affecting detection reliability. Fourth, the enzyme immobilization method in this type of structure is relatively simple, making it prone to enzyme leakage and poor recyclability.

[0044] Based on the problems encountered in actual research and development, this invention provides a Fe3O4@SiO2@ZIF-8 composite compound for enzyme immobilization and fluorescence dual-mode detection with a multilayer structure. The specific preparation method and structure are adjusted to maintain high enzyme activity and the complete MOF structure while obtaining stable fluorescence output and significantly improving the structural stability under cyclic use and solvent environment.

[0045] The present invention will be further explained below with reference to specific embodiments.

[0046] Example 1

[0047] This embodiment provides a THR@Fe3O4@SiO2@ZIF-8-CDs composite, the specific preparation method of which is as follows:

[0048] (1) Fe3O4 nanoparticles: commercially available.

[0049] (2) Preparation of Fe3O4@SiO2

[0050] 200 mg of Fe3O4 nanoparticles were added to a mixed solution of 100 mL ethanol and 20 mL deionized water and ultrasonically dispersed for 10 min. 2 mL of ammonia was added to the system, and the mixture was stirred for 10 min. Then, 0.5 mL of tetraethoxysilane (TEOS) was added dropwise in five portions (2 min intervals) while stirring. Stirring was continued at room temperature for 6 h to allow TEOS to hydrolyze and form a silica coating layer on the Fe3O4 surface. After the reaction was complete, the mixture was magnetically separated, washed three times with ethanol, and vacuum dried at 50 °C for 6 h to obtain Fe3O4@SiO2 composite particles.

[0051] (3) Amination treatment of Fe3O4@SiO2

[0052] The above-mentioned Fe3O4@SiO2 (200 mg) was added to 50 mL of anhydrous ethanol and sonicated for 5 min to ensure uniform dispersion. Then, 0.2 mL of 3-aminopropyltriethoxysilane (APTES) was added under stirring, and the mixture was refluxed at 60 °C for 4 h to allow the silane coupling agent to form a covalently bonded amino layer on the silica surface. After the reaction was complete, the mixture was magnetically separated, washed three times with ethanol, and vacuum dried at 50 °C for 2 h to obtain Fe3O4@SiO2-NH2 particles.

[0053] (4) In-situ growth of ZIF-8 on Fe3O4@SiO2 surface

[0054] Prepare the following two methanol solutions respectively:

[0055] Solution A: Dissolve 2.97 g of zinc nitrate hexahydrate (Zn(NO3)2·6H2O) in 50 mL of methanol;

[0056] Solution B: Dissolve 8.0 g of 2-methylimidazole (HmIm) in 50 mL of methanol.

[0057] 200 mg of Fe3O4@SiO2-NH2 was added to solution B and dispersed under magnetic stirring for 10 min. Then, solution A was quickly poured into solution B containing Fe3O4@SiO2-NH2, and the mixture was stirred at room temperature for 3 h, followed by standing for 12 h to allow ZIF-8 to grow uniformly on the outer surface of SiO2. After the reaction, the solid was magnetically separated and washed successively with methanol (20 mL × 3) and deionized water once. The obtained solid was vacuum dried at 40 °C for 6 h to obtain Fe3O4@SiO2@ZIF-8 composite particles.

[0058] (5) Preparation of S / N doped carbon dots (CDs)

[0059] Dissolve 0.6 g of citric acid and 0.3 g of thiourea in 20 mL of deionized water, transfer to a 50 mL high-pressure reactor lined with polytetrafluoroethylene, and hydrothermally react at 180 °C for 6 h. After natural cooling, collect the supernatant, filter through a 0.22 μm filter membrane, and centrifuge at 10000 rpm for 10 min to remove large particles. The resulting light yellow solution is the mother liquor of S / N doped carbon dots (CDs).

[0060] (6) Surface coupling fixation of CDs (located on the outer layer of ZIF-8)

[0061] Fe3O4@SiO2-NH2 (100 mg) was dispersed in 20 mL of MES buffer (50 mM, pH 6.0).

[0062] Take 10 mL of CDs solution, add 15 mg EDC and 9 mg NHS, activate at room temperature for 15 min, then add to the above Fe3O4@SiO2-NH2 suspension, and gently shake at room temperature for 4 h to allow the carboxyl groups of CDs to couple with the amino groups on the SiO2 surface via amide bonds. After the reaction is complete, magnetic separation is performed, and the sample is washed three times with PBS buffer (pH 7.4) to obtain Fe3O4@SiO2-NH2@ZIF-8-CDs.

[0063] (7) Post-loading of THR enzyme and mild in-well cross-linking fixation

[0064] Fe3O4@SiO2-NH2@ZIF-8-CDs (50 mg) were dispersed in 10 mL PBS (50 mM, pH 7.4) and gently stirred at 4 °C.

[0065] Separately, 10 mg of THR enzyme was dissolved in 2 mL of PBS (pH 7.4). The enzyme solution was slowly added dropwise to a suspension containing Fe3O4@SiO2-NH2@ZIF-8-CDs and incubated at 4 °C for 12 h to allow the enzyme to diffuse into the ZIF-8 pores. After incubation, the enzyme was magnetically separated and washed twice with PBS to remove unloaded free enzyme.

[0066] Subsequently, the enzyme-carrying complex was redispersed in 5 mL of PBS (pH 7.4), and genipin (geniposide) was added to a final concentration of 0.2% (w / v). Crosslinking was then carried out by gentle shaking at 4 °C for 1.5 h. After crosslinking, the particles were washed three times with PBS to obtain THR@Fe3O4@SiO2-NH2@ZIF-8-CDs particles.

[0067] (8) Hydrophobic treatment of the outer layer

[0068] The above enzyme-carrying complex was dispersed in 20 mL of anhydrous ethanol, and 0.2 mL of methyltriethoxysilane (MTES) was added. The mixture was gently shaken at room temperature for 2 h to form a low-density short-chain hydrophobic layer on the outer layer of ZIF-8. After the reaction was completed, the solid was collected by magnetic separation, washed three times with ethanol, and dried under vacuum at 40 °C for 2 h to obtain the final product Fe3O4@SiO2-NH2@ZIF-8-CDs.

[0069] Figure 2 The transmission electron microscope (TEM) image of CDs shows that the synthesized carbon dots are uniform and clear, with the carbon dot particle size ranging from 1 to 4 nm and the median average diameter being about 2 nm.

[0070] Figure 3 The image shows a scanning electron microscope (SEM) image of Fe3O4. It can be seen that Fe3O4 NPs are spherical with relatively uniform particle size, mostly around 20 nm.

[0071] Figure 4 The image shows a transmission electron microscope (TEM) image of Fe3O4. It can be seen that Fe3O4 NPs have a distinct lattice with a lattice spacing of about 0.16 nm.

[0072] Figure 5 The image shows a scanning electron microscope (SEM) image of the THR@Fe3O4@SiO2@ZIF-8-CDs composite. The composite exhibits a smooth and well-defined surface under the microscope. While the sample appears to be approximately hexagonal, it is actually a rhombic dodecahedron.

[0073] Comparative Example 1

[0074] The steps are as follows:

[0075] (1) Preparation of S / N-doped carbon dots (CDs)

[0076] The procedure is exactly the same as step (5) in Example 1: Dissolve 0.6g citric acid and 0.3g thiourea in 20mL deionized water, transfer to a 50mL polytetrafluoroethylene-lined autoclave, hydrothermally heat at 180℃ for 6h, cool, filter at 0.22μm and centrifuge at 10000rpm for 10min, and take the supernatant as CDs mother liquor for later use.

[0077] (2)THR@CDs conjugate

[0078] 1. Dissolve 10 mg THR in 2 mL PBS (pH 7.4) and incubate at 4 °C.

[0079] 2. Take 10 mL of CDs stock solution, add 15 mg of EDC and 9 mg of NHS, and activate by shaking at room temperature for 15 min.

[0080] 3. Mix the activated CDs solution with the THR solution and gently shake at room temperature for 2 hours to allow the carboxyl groups of CDs to form amide bonds with the amine groups on the enzyme surface, thus obtaining THR@CDs.

[0081] 4. Remove residual EDC / NHS and free small molecules from THR@CDs by dialysis (MWCO 10kDa), and collect the THR@CDs suspension for later use.

[0082] (3) In-situ growth of Fe3O4@ZIF-8 and encapsulation of THR@CDs

[0083] 1. Disperse commercially available Fe3O4 (200mg) in 50mL HmIm (8.0g dissolved in 50mL methanol) and sonicate for 5-10 minutes to fully disperse.

[0084] 2. Add the THR@CDs suspension (containing an equal amount of CD / protein as in Example 1) obtained in step (2) to the Fe3O4 / HmIm suspension and stir gently to mix.

[0085] 3. Quickly add Zn(NO3)2·6H2O (2.97g dissolved in 50mL methanol, solution A) to the above mixture, stir at room temperature for 3h, and let stand for 12h to complete crystallization, so that THR@CDs are embedded or adsorbed during the nucleation / growth of ZIF-8 to form THR@CDs@Fe3O4@ZIF-8.

[0086] 4. After the reaction was completed, the solid was collected by magnetic separation, washed with methanol (20 mL × 3) and deionized water once, and dried under vacuum at 40 °C for 6 h to obtain THR@CDs@Fe3O4@ZIF-8 dry powder.

[0087] Comparative Example 2

[0088] The steps are as follows:

[0089] (1) Fe3O4 dispersion

[0090] Take 200 mg of commercially available Fe3O4 and disperse it according to the method described in Example 1 before coating Fe3O4@SiO2. Use 100 mL of ethanol and 20 mL of deionized water to sonicate for 10 min. Set aside for use.

[0091] (2) In-situ growth of ZIF-8 on Fe3O4 surface

[0092] 1. Prepare solution A (2.97 g Zn(NO3)2·6H2O dissolved in 50 mL methanol) and solution B (8.0 g HmIm dissolved in 50 mL methanol).

[0093] 2. Disperse 200 mg Fe3O4 in solution B and stir for 10 min.

[0094] 3. Quickly add solution A, stir at room temperature for 3 hours, and let stand for 12 hours to allow ZIF-8 to form a shell on the Fe3O4 surface.

[0095] 4. The sample was magnetically separated and washed three times with methanol and once with deionized water. It was then vacuum dried at 40°C for 6 hours to obtain Fe3O4@ZIF-8.

[0096] (3) CD synthesis and coupling

[0097] CDs were synthesized according to step (5) in Example 1; then the CDs (10 mL) were activated with 15 mg EDC / 9 mg NHS for 15 min and mixed with Fe3O4@ZIF-8 and gently shaken at room temperature for 4 h to allow the CDs to form amide bonds with the surface amine groups; magnetic separation and washing with PBS 3 times were performed to obtain Fe3O4@ZIF-8-CDs.

[0098] (4) THR post-loading and cross-linking with genipin (geniposide) (same as in Example 1)

[0099] 1. Disperse Fe3O4@ZIF-8-CDs (50mg) in 10mL PBS (pH 7.4) and shake gently at 4℃.

[0100] 2. Dissolve 10 mg THR in 2 mL PBS and slowly add it dropwise into the particle suspension. Incubate at 4 °C for 12 h to allow the enzyme to diffuse into the ZIF-8 channels.

[0101] 3. After magnetic separation and washing twice with PBS, the mixture was redispersed in 5 mL of PBS, and genipin (geniposide) (final concentration 0.2% w / v) was added. The mixture was crosslinked at 4 °C for 1.5 h, followed by washing three times to obtain THR@Fe3O4@ZIF-8-CDs.

[0102] (5) Disperse THR@Fe3O4@ZIF-8-CDs in 20mL of anhydrous ethanol, add 0.2mL of MTES, shake gently at room temperature for 2h, magnetically separate and wash 3 times with ethanol, and vacuum dry at 40℃ for 2h to obtain the final product.

[0103] Comparative Example 3

[0104] The steps are as follows:

[0105] (1) Preparation and amination of Fe3O4@SiO2

[0106] Steps (1), (2), and (3) of Example 1 were exactly the same: Fe3O4@SiO2-NH2 (200 mg) was obtained.

[0107] (2) Introducing CDs into the ZIF-8 nucleation system

[0108] 1. Prepare solution A (Zn(NO3)2 2.97g / 50mL methanol) and solution B (HmIm 2-methylimidazole 8.0g / 50mL methanol).

[0109] 2. Disperse Fe3O4@SiO2-NH2 (200mg) in solution B and stir for 10min.

[0110] 3. Add 3 mL of CDs stock solution to solution B and shake gently for 5 min to disperse CDs in the nucleation system.

[0111] 4. Quickly pour solution A into solution B containing Fe3O4@SiO2-NH2 and CDs, stir at room temperature for 3 hours, and let stand for 12 hours to complete crystallization, forming Fe3O4@SiO2@ZIF-8 (containing CDs).

[0112] 5. Magnetic separation followed by washing with methanol three times and deionized water once, then vacuum drying at 40℃ for 6 hours.

[0113] (4) Post-enzyme loading and cross-linking with genipin (geniposide) (same as Example 1)

[0114] Fe3O4@SiO2@ZIF-8 (containing CD) (50 mg) was dispersed in 10 mL PBS (pH 7.4) and incubated at 4 °C; 10 mg THR was dissolved in 2 mL PBS, slowly added dropwise and incubated for 12 h; after magnetic separation and washing, it was lightly crosslinked with genipin (0.2%) at 4 °C for 1.5 h, washed and dried to obtain the final product.

[0115] (5) Hydrophobication of the outer layer (same as in Example 1)

[0116] MTES (0.2 mL) was grafted into ethanol for 2 h.

[0117] Comparative Example 4

[0118] The steps are as follows:

[0119] 1. The steps (1) to (6) of Example 1 are exactly the same;

[0120] 2. Post-enzyme loading: Fe3O4@SiO2@ZIF-8-CDs (50mg) were dispersed in 10mL PBS, and 10mg THR (2mL PBS solution) was slowly added at 4℃ and incubated for 12h; magnetic separation and washing twice with PBS were performed; genipin (geniposide) was not added.

[0121] 3. Hydrophobication of the outer layer: Grafting with 0.2 mL of MTES in ethanol for 2 h as in step (8) of Example 1.

[0122] Comparative Example 5: No outer layer hydrophobicity (other aspects are the same as Example 1)

[0123] The steps are as follows:

[0124] 1. Complete steps (1) to (7) of Example 1 (including genipin (geniposide) crosslinking);

[0125] 2. Omit step (8) of Example 1, outer layer hydrophobication (MTES grafting) treatment; direct magnetic separation, PBS washing and preservation of the product for testing.

[0126] Performance testing

[0127] Test 1: The morphology, size, and surface functional groups of the CDs prepared in Example 1 were first examined. TEM images showed that the prepared CDs had good dispersibility, exhibited a quasi-spherical morphology, and had an average particle size of 2.6 nm.

[0128] Test 2:

[0129] This assay used a reaction system where substrate S-2238 (HD-Phe-Pip-Arg-pNA) was hydrolyzed under THR catalysis to generate p-nitrophenol (pNP) to determine enzyme activity. The absorption peak area of ​​pNP at 405 nm was detected by Kappa electrophoresis (CE) to quantify the enzyme reaction rate.

[0130] 1. Determination of the activity of free THR

[0131] (1) Preparation of the reaction system:

[0132] Take 100 μL of THR solution; add 100 μL of S-2238 solution (1 mM, pH 7.4).

[0133] (2) Incubate at 25℃ for 15 min.

[0134] (3) Immediately after the reaction is complete, freeze at -20℃ for 5 minutes to terminate the reaction.

[0135] (4) After thawing, the reaction mixture is directly used for CE analysis to measure the peak area or peak height of pNP (405nm).

[0136] 2. Activity determination of immobilized THR

[0137] (1) The composite materials obtained in the examples and comparative examples were ultrasonically dispersed for 2 min to obtain 10 mg·mL⁻¹ -1 Uniform suspension.

[0138] (2) Take 100 μL of enzyme suspension and add it to 100 μL of 1 mM S-2238 solution, and incubate at 25℃ for 15 min.

[0139] (3) After the reaction is complete, use an external magnet to quickly separate the enzyme-carrying particles, and the reaction will be terminated immediately.

[0140] (4) Collect the supernatant and inject it into CE to analyze the pNP (405nm) absorption peak.

[0141] 3. CE analysis conditions

[0142] Capillary: Uncoated fused silica capillary, 75 μm.d. × 28.5 cm (effective length 20 cm)

[0143] Sample injection: 50 mbar × 5 s pressure injection

[0144] Separation voltage: 20kV

[0145] Detection wavelength: 405nm

[0146] Temperature: 25℃

[0147] Electrophoresis buffer: 30mM Tris-HCl (pH 7.4)

[0148] 4. CE capillary pretreatment

[0149] The new capillary tube was rinsed sequentially with 1M NaOH (30 min) - water (20 min) - 0.1M HCl (30 min) - water (20 min) and then purged with nitrogen for 15 min.

[0150] Before each experiment, rinse with 1M NaOH, water, and 30mM Tris-HCl for 5 minutes each.

[0151] Between each run, rinse with 1M NaOH, water, and Tris-HCl for 3 minutes each.

[0152] After each day's experiment, rinse with 1M NaOH for 5 minutes, then rinse with water for 10 minutes.

[0153] Data processing

[0154] The peak area of ​​pNP at 405 nm in the CE spectrum is used as the reaction rate = Aimmobilized / Afree enzyme; where: Aimmobilized is the peak area of ​​pNP generated by the immobilized enzyme reaction; Afree enzyme is the peak area of ​​pNP generated by an equal amount of free enzyme.

[0155] (II) THR Cyclic Performance Test

[0156] (1) After each reaction, the enzyme-carrying particles were separated using an external magnet and washed three times with PBS (pH 7.4).

[0157] (2) Add the particles back into a new substrate solution (1 mM S-2238) and carry out the next round of reaction.

[0158] (3) Repeat the process 10 times consecutively, recording the pNP peak area each time. Relative activity calculation formula: n = An / A1.

[0159] (III) Solvent stability test of THR

[0160] (1) The enzyme-carrying complex (10 mg·mL) -1 Dispersed in PBS containing 10% (v / v) DMSO and incubated at 25°C for 1 hour.

[0161] (2) After incubation, separate the particles and add 1 mM S-2238 for routine enzyme activity testing.

[0162] (3) Calculate the relative activity after solvent resistance: Residual activity = A DMSO / A PBS .

[0163] (iv) THR Leakage Rate Test

[0164] (1) Disperse the enzyme-carrying complex (10 mg) in 2 mL PBS (pH 7.4) and shake at 25 °C and 100 rpm for 24 h.

[0165] (2) The supernatant was separated magnetically, and the protein content was determined by BCA protein quantification method.

[0166] (3) Leakage rate calculation: Leakage rate (%) = m supernatant protein / m initially loaded enzyme.

[0167] (v) Fluorescence stability test (for the outer fixation structure of CDs)

[0168] (1) The composite particles of equal mass were dispersed in PBS and the excitation wavelength was set as the main excitation wavelength of CDs, λex.

[0169] (2) Record the decay of the emission peak (λem≈420-480nm) over time.

[0170] (3) Compare the fluorescence intensity retention rate of the sample of the present invention with that of the comparative example: fluorescence retention rate = It / I0; where I0 is the initial fluorescence intensity and It is the intensity after storage for t days.

[0171] Table 1 Test Results

[0172]

[0173]

[0174] The THR@Fe3O4@SiO2@ZIF-8-CDs composite compound provided by this invention significantly outperforms the comparative example in various properties, including enzyme activity retention, cyclic stability, resistance to solvent disturbance, and fluorescence output stability. In particular, Example 1 adjusted the order of the components.

[0175] The initial relative activity of Example 1 was 93.5%, which was significantly higher than that of Comparative Example 1 (54.8%), and also higher than that of Comparative Example 2 (81.4%) and Comparative Example 3 (78.2%), which had incomplete structures or missing operation steps. This indicates that the enzyme post-loading method used in this invention can maintain the enzyme conformation to the maximum extent.

[0176] Example 1 maintained 92.7% activity after 10 cycles, the highest value; in contrast, the activity of uncrosslinked Comparative Example 4 (49.3%) and Comparative Example 1 (62.5%) decreased significantly, demonstrating that genipin crosslinking is a key step in improving cycle stability.

[0177] Under conditions containing 10% DMSO, the residual activity of Example 1 reached 88.3%, which was significantly higher than that of Comparative Example 1 (48.2%), Comparative Example 3 (64.8%) and Comparative Example 5 (59.3%) without hydrophobication treatment, further verifying that the hydrophobication of the outer MTES layer plays a key role in improving the stability of the enzyme in polar perturbation environments.

[0178] This is because ZIF-8(Zn 2+ -2-mIm coordination) has a typical sodalite-type framework with a pore size of approximately Hole window approx. They exhibit weak hydrophobicity. The pores of these MOFs provide an environment that prevents proteins from being damaged by external polar molecules. Carbon dots (CDs) typically contain carboxyl, hydroxyl, or amine groups; if they directly react with Zn... 2+ Upon contact, carboxyl-Zn groups will form. 2+ Coordination competition; the example uses external fixation via amide bonds (EDC / NHS), with CDs located on the outer surface of the ZIF-8 shell, not entering the pores, thus maintaining the integrity of the framework crystal and stabilizing the fluorescence peak position. After hydrolysis of MTES (methyltriethoxysilane), a short-chain Si-O-Si-CH3 hydrophobic layer is formed, which can stabilize the surface states of CDs and ensure that the fluorescence does not drift.

[0179] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A multilayered enzyme immobilization and fluorescence dual-mode detection Fe3O4@SiO2@ZIF-8 composite compound, characterized in that, The composite compound has a multilayer structure consisting of an Fe3O4 core, a SiO2 isolation layer, a ZIF-8 shell, and an outer carbon quantum dot layer, from the inside out. The enzyme molecules are loaded inside the pores of the ZIF-8 shell. The Fe3O4 is a magnetic nanoparticle. The SiO2 is a continuous and dense silicon dioxide layer coated on the surface of Fe3O4; The ZIF-8 is a metal-organic framework material grown in situ on the surface of SiO2; The carbon quantum dots are chemically bonded to the outer surface of ZIF-8 / SiO2; The enzyme is loaded inside ZIF-8 via pore diffusion and immobilized by mild cross-linking.

2. The composite compound according to claim 1, characterized in that, The thickness of the SiO2 isolation layer is 5–20 nm.

3. The composite compound according to claim 1, characterized in that, The thickness of the ZIF-8 shell is 20–60 nm.

4. The composite compound according to claim 1, characterized in that, Carbon quantum dots are fixed to the outer surface of composite materials through amide bonds, silane bonds, or quaternary ammonium salt bonds.

5. The composite compound according to claim 1, characterized in that, The outer surface of the ZIF-8 further has an alkylsilane layer.

6. The composite compound according to claim 5, characterized in that, The alkylsilane layer is methyltriethoxysilane (MTES) or ethyltriethoxysilane (ETES).

7. The composite compound according to claim 1, characterized in that, The carbon quantum dots are nitrogen-doped carbon dots, sulfur-doped carbon dots, or sulfur-nitrogen co-doped carbon dots.

8. The composite compound according to claim 1, characterized in that, The enzyme is at least one of thioredoxin reductase (THR), oxidoreductase, peroxidase, glucose oxidase, lactate oxidase, or lipase.

9. A method for preparing the composite compound according to any one of claims 1 to 8, characterized in that, Includes the following steps: (1) Fe3O4 cores were prepared by coprecipitation method; (2) TEOS hydrolysis forms a SiO2 coating layer on the Fe3O4 surface; (3) Silanization treatment of SiO2 surface; (4) In-situ growth of ZIF-8 shell on the outer layer of SiO2; (5) Carbon quantum dots are coupled to the outer surface of ZIF-8 or SiO2 through the EDC / NHS system; (6) Enzyme molecules diffuse into the ZIF-8 channels; (7) The enzyme carrier structure was lightly cross-linked and fixed using low concentrations of genipin or glutaraldehyde.

10. The application of the composite compound according to any one of claims 1 to 8 in fluorescence detection, characterized in that, A fluorescent signal channel is constructed using outer carbon quantum dots to detect thioredoxin substrates, redox compounds, or free active small molecules.

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