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CN-121975786-A - Multilayer structure's enzyme immobilization and fluorescence bimodulus detects and uses Fe3O4@SiO2ZIF-8 compound and preparation method thereof

CN121975786ACN 121975786 ACN121975786 ACN 121975786ACN-121975786-A

Abstract

The invention relates to the technical field of nano materials and biochemistry, in particular to an enzyme immobilization and fluorescence dual-mode detection Fe 3 O 4 @SiO 2 @ZIF-8 compound based on a space isolation structure and a preparation method thereof. The composite compound is of a multilayer structure which sequentially comprises a Fe 3 O 4 core, a SiO 2 isolation layer, a ZIF-8 shell layer and an outer carbon quantum dot from inside to outside, enzyme molecules are loaded in a pore canal of the ZIF-8 shell layer, wherein Fe 3 O 4 is magnetic nanoparticles, siO 2 is a continuous compact silicon dioxide layer coated on the surface of Fe 3 O 4 , ZIF-8 is a metal organic framework material formed by in-situ growth on the surface of SiO 2 , the carbon quantum dot is fixed on the outer surface of ZIF-8/SiO 2 through chemical bonding, and the enzyme is loaded in the ZIF-8 in a pore canal diffusion mode and is fixed through light crosslinking.

Inventors

  • WANG MIN
  • LUO HONGLI
  • CHEN PANPAN
  • ZHANG MANYAO
  • CHENG YANJIE
  • LIU SHUANG
  • WU HAOJIE

Assignees

  • 西南医科大学附属医院

Dates

Publication Date
20260505
Application Date
20251231

Claims (10)

  1. 1. The Fe 3 O 4 @SiO 2 @ZIF-8 compound for enzyme immobilization and fluorescence dual-mode detection with a multilayer structure is characterized by comprising a multilayer structure of a Fe 3 O 4 core, a SiO 2 isolation layer, a ZIF-8 shell layer and an outer carbon quantum dot from inside to outside in sequence, wherein enzyme molecules are loaded in a pore canal of the ZIF-8 shell layer, and the Fe 3 O 4 is a magnetic nanoparticle; the SiO 2 is a continuous compact silicon dioxide layer coated on the surface of the Fe 3 O 4 ; The ZIF-8 is a metal organic framework material formed by in-situ growth on the surface of the SiO 2 ; The carbon quantum dots are fixed on the outer surface of the ZIF-8/SiO 2 through chemical bonding; The enzyme is loaded in ZIF-8 through a pore canal diffusion mode and is immobilized through light crosslinking.
  2. 2. The composite compound of claim 1, wherein the SiO 2 isolation layer has a thickness of 5-20 nm.
  3. 3. The composite compound of claim 1, wherein the ZIF-8 shell has a thickness of 20-60 nm.
  4. 4. The composite compound of claim 1, wherein the carbon quantum dots are immobilized to the outer surface of the composite material by amide bonds, silane bonds, or quaternary ammonium salt bonds.
  5. 5. The composite compound of claim 1 wherein the ZIF-8 outer surface further has an alkylsilane layer.
  6. 6. The composite compound of claim 5, wherein the alkylsilane layer is Methyltriethoxysilane (MTES) or ethyltriethoxysilane (ETES).
  7. 7. The compound of claim 1, wherein the carbon quantum dots are nitrogen-doped carbon dots, sulfur-doped carbon dots, or sulfur-nitrogen co-doped carbon dots.
  8. 8. The complex of claim 1, wherein the enzyme is at least one of thioredoxin reductase (THR), oxidoreductase, peroxidase, glucose oxidase, lactate oxidase, or lipase.
  9. 9. A method for preparing the compound according to any one of claims 1 to 8, comprising the steps of: (1) Preparing Fe 3 O 4 cores by a coprecipitation method; (2) The hydrolysis of TEOS forms a SiO 2 coating layer on the surface of Fe 3 O 4 ; (3) Silanization treatment is carried out on the surface of SiO 2 ; (4) A ZIF-8 shell layer grows in situ on the outer layer of SiO 2 ; (5) Coupling the carbon quantum dots to the outer surface of ZIF-8 or SiO 2 through an EDC/NHS system; (6) Enzyme molecules enter ZIF-8 pore channels through diffusion; (7) And (3) carrying out light cross-linking fixation on the enzyme-carrying structure by using low-concentration genipin or glutaraldehyde.
  10. 10. The application of the compound of any one of claims 1-8 in fluorescence detection, which is characterized in that a fluorescence signal channel is constructed through outer carbon quantum dots and is used for detecting thioredoxin substrates, redox compounds or free active small molecules.

Description

Fe 3O4@SiO2 @ZIF-8 compound for enzyme immobilization and fluorescence dual-mode detection with multilayer structure and preparation method thereof Technical Field The invention relates to the technical field of nano materials and biochemistry, in particular to an enzyme immobilization and fluorescence dual-mode detection Fe 3O4@SiO2 @ZIF-8 compound based on a space isolation structure and a preparation method thereof. Background In recent years, metal-organic framework materials (Metal-OrganicFrameworks, MOFs) have received wide attention in the fields of enzyme immobilization, biocatalysis, fluorescence sensing, drug delivery and the like due to their high specific surface area, controllable pore structure and good designability. The ZIF-8 is used as a typical MOF material, has the characteristics of stable structure, proper pore diameter, mild synthesis conditions and the like, and has been widely applied to construction of a composite system of enzyme load and active molecule encapsulation. On the other hand, the enzyme is used as a high-efficiency and specific biocatalyst, has softer natural conformation, has higher sensitivity to temperature, solvent environment, metal ions and surface interaction, and is easy to be inactivated or change in conformation. By immobilizing the enzyme on the pore canal or the surface of the nano-carrier, the enzyme can be recycled, the stability of the enzyme is improved, and the operation performance of the enzyme in a complex system is improved. However, the existing enzyme immobilization methods still have the following general problems: (1) Conformational disruption of the enzyme during immobilization leads to reduced activity; (2) Non-specific adsorption or strong interactions between the carrier and the enzyme cause inactivation of the enzyme molecule; (3) When enzymes are encapsulated inside MOFs, fluorescent probes, metal ions or organic ligands may affect the enzymatic reaction; (4) Exogenous organic molecules (such as carbon dots, dyes and the like) can easily compete with metal ions for coordination in the nucleation process of the carrier, so that MOF structural defects or stability reduction is caused. Magnetic nanomaterials (such as Fe 3O4) are commonly used to construct reusable enzyme load platforms due to their convenient magnetic response separation capability. However, the bare Fe 3O4 has poor stability in aqueous phase, and the surface is prone to deposit impurities and affect enzyme binding, so the surface modification is often performed by materials such as silica (SiO 2). The SiO 2 shell layer not only can improve the stability of particles, but also provides reaction sites for further functionalization of the surface, and is an important intermediate layer material for constructing a composite multilayer structure. Carbon Dots (CDs) are used as a novel fluorescent nanomaterial with excellent light stability, low biotoxicity and good water solubility, and are often used as fluorescent signal reading units of a MOF-enzyme composite system. However, direct coupling of CDs with enzymes or co-participation in the MOF nucleation process can easily lead to problems of reduced enzyme activity, unstable fluorescent signals or uncontrolled MOF nucleation structure. Therefore, how to ensure that the enzyme activity is maintained, the MOF structure is complete and the fluorescence signal is stable in the same compound system becomes one of the research difficulties in the current field. In the prior art, a novel composite structure which can realize enzyme immobilization and fluorescence detection function integration while keeping enzyme conformation intact, avoiding nucleation interference and improving structural stability is not yet available. Therefore, developing a compound of Fe 3O4@SiO2 @ ZIF-8 with a spatial hierarchy structure, and reducing interference between components by layering and assembling enzymes and fluorescent units has become a technical problem to be solved in the art. Disclosure of Invention Aiming at the technical problems that the enzyme activity is easy to be damaged, the metal-organic framework structure is easy to be interfered, the fluorescent signal is unstable, the repeated use is difficult to realize and the like in the existing enzyme immobilization and fluorescent detection integrated system, the invention provides the compound with enzyme immobilization and fluorescent response and the preparation method thereof. The multi-layer structure of the Fe 3O4 core, the SiO 2 isolation layer, the ZIF-8 shell layer and the outer carbon quantum dots is prepared from inside to outside, so that the high activity retention of enzyme, the stability of MOF nucleation and the stable reading of fluorescent signals are realized, and the problems of mutual interference in the prior art are solved. In order to solve the technical problems, the invention provides a Fe 3O4@SiO2 @ZIF-8 compound for enzyme immobilization and fluorescence dual-mode