A method for preparing an enzyme-metal organic framework composite material by amino acid assisted biomineralization

Abstract

The application discloses a method for preparing enzyme-metal organic framework composite materials by amino acid assisted biomimetic mineralization, and relates to the fields of metal organic framework composite materials and enzyme engineering; the method comprises the following steps: 1) mixing serine and metal ions in a solvent to form secondary structure units carrying negative charges; 2) sequentially adding enzymes and ligands to perform a mineralization reaction, so that the enzyme-metal organic framework composite material is obtained; the preparation method has universal applicability, is suitable for the construction of various enzymes, and has green and mild synthesis conditions, so that the industrialized scale production is facilitated; meanwhile, the enzyme in the material has high activity and high loading capacity, has high efficiency in ECL detection, and is suitable for popularization and application.

Description

Technical Field

[0001] This application relates to the fields of metal-organic framework composites and enzyme engineering, and in particular to a method for preparing enzyme-metal-organic framework composites using amino acid-assisted biomineralization. Background Technology

[0002] Enzyme-metal-organic framework (MOF) composites are a class of enzymes immobilized using MOF materials, including composites that anchor enzymes to the outer surface of MOF particles or encapsulate them in MOF cavities, thereby improving enzyme stability and preventing enzyme denaturation under harsh environmental conditions (“Armor-Plating” Enzymes with Metal–Organic Frameworks, MOFs, Angewandte Chemie International Edition, 2020, 59, 8786-8798).

[0003] Enzyme-MOFs can be used to prepare biosensors for biological detection (such as H1N1 influenza viruses), especially for preparing signal probes based on electrochemiluminescence (ECL) sensors. In this type of sensor, the enzyme catalyzes co-reaction reagents to generate highly active free radicals, which then collide with the luminescent material loaded in the MOF to emit light, enabling signal amplification and accurate detection. In biosensor detection, enzyme-MOF materials generally require: 1) High enzyme activity: Higher enzyme activity catalyzes the generation of more free radicals, and MOF encapsulation protects the enzyme from environmental damage; 2) Numerous enzyme binding sites: MOFs provide sufficient binding sites to achieve high loading while avoiding damage to the enzyme's active conformation due to excessive interaction; 3) For ECL probes, the enzyme and the luminescent material loaded in the MOF (free radicals have short lifetimes) must be close to each other to improve the collision efficiency of free radicals and luminescent materials, achieving sufficient effective collisions.

[0004] Currently reported methods for preparing enzyme-MOFs include post-synthetic modification, co-precipitation, and biomimetic mineralization. In post-synthetic modification, the pore size of the MOF material must be comparable to or larger than the three-dimensional size of the enzyme to trap the enzyme within the MOF cavity. However, most current MOF materials are microporous or small-pore mesoporous, which cannot effectively trap enzymes. In co-precipitation, the enzyme undergoes a solid-state transformation on the surface of spontaneously growing MOFs through heterocrystalline deposition, precipitating inside the forming MOF crystal or in the interstitial lattice. During this process, the enzyme may lose activity due to ligand-induced denaturation, or the diffusion of the catalytic substrate may be restricted due to insufficient pore size or porosity of the MOFs, leading to a decrease in enzyme catalytic activity. Biomimetic mineralization utilizes the surface charge of the enzyme to attract metal ions, thereby directly inducing local supersaturation and rapid nucleation of MOFs on the enzyme surface, achieving efficient encapsulation of enzymes and other biomolecules in MOFs. However, biomimetic mineralization still faces inherent limitations: for example, encapsulation efficiency and biological activity largely depend on the surface charge of the enzyme. Traditional biomimetic mineralization methods usually require the enzyme surface to be negatively charged to attract metal ions. This method fails for enzymes with high isoelectric points that are positively charged under physiological conditions (such as HRP with an isoelectric point pI≈8.8), and can only degenerate into an inefficient co-precipitation method, thus limiting its universality. In addition, direct coordination between metal ions and enzyme surface residues may induce irreversible conformational changes, leading to the loss of catalytic function.

[0005] The preparation of enzyme-MOF composites using amino acids is an emerging method. For example, the paper "A Convenient and Versatile Amino-Acid-Boosted Biomimetic Strategy for the Nondestructive Encapsulation of Biomacromolecules within Metal–Organic Frameworks" (Angewandte Chemie International Edition) proposes an "amino acid-enhanced one-pot encapsulation (AAOPE)" strategy for encapsulating various biomacromolecules (including positively charged enzymes) within metal-organic frameworks (MOFs). The key mechanism utilizes cysteine ​​(Cys) as an amino acid modifier, while simultaneously introducing polyvinylpyrrolidone (PVP) as a polymeric auxiliary. PVP forms a complex with the protein surface, enriching Cys around the protein through hydrogen bonding and other interactions. Cys then interacts with metal ions (such as Zn²⁺). +The strong coordination of metallothionein (similar to the mechanism of metallothionein) accelerates the formation of pre-nucleated clusters on the protein surface, thereby inducing the rapid nucleation and growth of MOFs (such as ZIF-8) around the protein, achieving efficient encapsulation. This method has successfully encapsulated various positively charged proteins (such as HRP, cytochrome c, etc.); however, it has the following shortcomings: 1) The reaction system is complex and requires the introduction of additional polymers: This strategy must use both Cys and PVP as additives. PVP is a polymer that acts as an "anchor" to enrich Cys on the enzyme surface, increasing the complexity of the synthesis system. Moreover, as an exogenous polymer, PVP may cause non-specific adsorption or biocompatibility problems in some biological applications (such as in vivo drug delivery, biosensing). The addition of additional components increases the complexity of the synthesis system. The difficulty of purification and characterization; in particular, the introduction of exogenous polymeric auxiliaries increases the complexity and cost of the system, making it more difficult to precisely control the reaction conditions, which is not conducive to the industrial scale-up and application of the method; 2) The encapsulation mechanism relies on PVP-Cys synergy rather than the direct regulation of enzyme surface charge. The essence of its strategy is to artificially construct a PVP "enrichment layer" on the enzyme surface through the physicochemical interaction of PVP and Cys, thereby bypassing the limitation of the enzyme's own surface charge and increasing the difficulty for the free radicals generated by enzyme catalysis to reach the luminescent body in MOFs (increased diffusion distance, reduced collision efficiency, leading to a decrease in ECL intensity); 3) As a polymeric compound, PVP has electrochemical inertness, which affects the electron transfer in the ECL process, thereby reducing the ECL signal intensity.

[0006] The paper "Biomimetic Mineralization of Large Enzymes Utilizing a Stable Zirconium-Based Metal-Organic Frameworks" (Journal of the American Chemical Society, J. Am. Chem. Soc. 2024, 146, 5108-5117) discloses a method for synthesizing Zr-based MOFs (UiO-66-F4) using amino acids as regulators under aqueous phase and mild temperature (40-80℃) conditions. This method utilizes an in-situ encapsulation strategy to embed multiple enzymes (HRP, lysozyme, lipase) within UiO-66-F4. This method replaces traditional regulators with amino acids: the synthesis of traditional Zr-MOFs (such as UiO-66) typically requires high temperatures (>100°C) and organic solvents (DMF, DEF) using regulators such as benzoic acid and acetic acid. This method utilizes amino acids such as serine and glycine, which can replace traditional regulators under aqueous phase and mild conditions, resulting in milder reaction conditions. Furthermore, the enzymes are directly mixed with MOF precursors (Zr(IV) salt, fluoroterephthalic acid, serine) during the MOF crystallization process. The enzyme is embedded within the framework; however, it also has shortcomings: 1) The encapsulation mechanism is passive and does not achieve active charge adaptation. It uses "passive" in-situ coprecipitation as an encapsulation strategy, simply mixing the enzyme, metal ions, ligands, and amino acid regulators, and physically embedding the enzyme during MOF crystallization. The amino acids are only used as regulators ("pH buffer" / "crystallization regulator") to promote MOF crystallization, without actively regulating the charge state of secondary structural units (SBUs) to adapt to the surface charge of the enzyme; 2) The encapsulation efficiency is low. For positively charged HRP, this strategy fails to actively enrich enzyme molecules through electrostatic attraction, resulting in low encapsulation efficiency (HRP loading is only about 1.6 wt%), leading to fewer free radicals generated by enzyme catalysis, thus affecting the ECL signal; 3) During passive coprecipitation, MOF crystals grow randomly around the enzyme molecules, lacking regulation of the interaction between the MOF precursor and the active region on the enzyme surface, which may affect enzyme activity. Its Michaelis constant Km (the substrate concentration when the enzyme-catalyzed reaction reaches half of the maximum rate Vmax) is only 2.25 mM. Therefore, providing an enzyme-MOF composite material with mild reaction conditions, high enzyme loading, and enzyme that retains its original secondary structure and biological activity after encapsulation has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] To address the aforementioned issues, this application provides a method for preparing enzyme-metal-organic framework (MOF) composite materials via amino acid-assisted biomineralization. This method is used to efficiently encapsulate positively charged enzymes to prepare high-performance enzyme-MOF composite materials. The method uses positively charged horseradish peroxidase (HRP) as a model enzyme, PCN-224 (a Zr-based MOF composed of Zr(IV) clusters and tetrakis(4-carboxyphenyl)porphyrin (ZnTCPP) ligands) as a framework, and serine as an auxiliary agent to prepare enzyme-metal-organic framework (MOF) composite materials.

[0008] Specifically, this application is implemented through the following technical solution: First, this application provides a method for preparing an enzyme-metal-organic framework composite material, comprising the following steps: a) Serine is mixed with metal ions in a solvent to form secondary structural units (SBUs) with specific charges. Furthermore, the aforementioned metal ions include Zr 4+ Zn² + Co² + Ni² + It possesses appropriate solubility and coordination ability in the aqueous phase, while also meeting biocompatibility requirements.

[0009] The solvents mentioned above can be ultrapure water or buffer solutions (such as Tris-HCl buffer, HEPES buffer, etc.).

[0010] After mixing metal ions with a solvent as described above, the metal ions in the solvent (such as Zr) 4+ The preferred concentration of the metal ion is 0.03-0.15 mmol / mL (corresponding to a ZrOCl2 concentration of approximately 10-48 mg / mL). If the concentration of metal ions in the solvent is too low, the nucleation rate of MOFs will be slow and the crystallinity will be low; if the concentration is too high, it may cause MOFs to precipitate too quickly and have an inhomogeneous structure. Furthermore, excessive metal ions may non-specifically coordinate with amino acid residues on the enzyme surface, inducing conformational changes or even inactivation of the enzyme. In one embodiment of this application, the mass ratio of metal ions Zr(IV) to serine is ZrOCl2·8H2O:serine = 41:63-220 (corresponding to a molar ratio of 1:4.7-16.5).

[0011] The aforementioned serine provides a negative charge, acting as a structure-directing agent and a protective "buffer layer." Together with Zr(IV), it regulates the charge of SBUs, guides MOF crystallization, and protects enzyme activity. SBUs are negatively charged (and subsequently require positively charged HRP loading). In this application, the charge of SBUs is adjusted by regulating the ratio of Zr(IV) to serine, ranging from -10 mV to -35 mV. In one embodiment, the zeta potential of the SBUs is -18 mV.

[0012] b) The enzyme to be encapsulated is added to the mixture in step a, and the SBUs are induced to accumulate on the enzyme surface by utilizing the electrostatic interaction between the enzyme and SBUs; in this application, the enzyme carries a positive charge.

[0013] In practice, a relatively excessive amount of SBUs is generally added to ensure the formation of a complete SBUs enrichment layer on the enzyme surface; preferably, the added SBUs (in Zr) 4+ The molar ratio of the enzyme to the total molecular weight is 2700-8000:1; in one embodiment of this application, the molar ratio is 5600:1; wherein the molecular weight of the enzyme is 44 kDa.

[0014] c) An organic ligand is added, and a mineralization reaction is carried out under mild conditions for about 5 minutes, allowing MOFs to nucleate and grow on the enzyme surface, forming an enzyme-MOF composite material. In this application, the term "mild conditions" refers to an aqueous solution reaction at room temperature (not exceeding 40°C).

[0015] The aforementioned organic ligands include one of ZnTCPP, tetra(4-carboxyphenyl)porphyrin (TCPP), and 1,3,6,8-tetra(4-carboxyphenyl)pyrene (TBAPy). These ligands are both luminescent and have carboxyl functional groups, and can form stable MOF structures with Zr.

[0016] The molar ratio of the added organic ligand to the enzyme is 400-600:1, preferably 450-550:1, more preferably 500-550:1, and in one embodiment of this application, the molar ratio is 515:1.

[0017] The molar ratio of the added organic ligand to Zr(IV) is approximately 1:7 to 14, preferably 1:8 to 12, more preferably 1:10 to 12, and in one embodiment of this application, the molar ratio is 1:10.9.

[0018] Secondly, this application provides an enzyme-metal-organic framework composite material obtained by the above method; this material has high crystallinity, high enzyme loading, and the encapsulated enzyme retains its original secondary structure and biological activity.

[0019] Third, this application provides the application of the enzyme-metal-organic framework composite material prepared by the above method as a detection signal probe. For example, the strong coordination between the metal clusters (such as Zr-O clusters) and phosphate groups of the prepared enzyme-MOF composite material can be used to stably immobilize biorecognition molecules (such as aptamers and antibodies). The above-mentioned method for preparing detection signal probes using enzyme-MOF materials is a conventional technique in this field, as seen in literature such as "A zirconium–organic framework nanosheet-based aptasensor with outstanding electrochemical sensing performance" (Inorganic Chemistry Communications, 2022, 145, 109970) and "Impedimetric aptasensor based on zirconium–cobalt metal–organic framework for detection of carcinoembryonic antigen" (Microchimica Acta, 2022, 189, 338).

[0020] Furthermore, the above applications include using the prepared enzyme-MOF composite material as a detection probe for an electrochemiluminescence biosensor to detect biomarkers; in one embodiment of this application, the biomarker includes H1N1 viral hemagglutinin (HA). In detection, the enzyme-MOF material achieves energy transfer and signal amplification by co-localizing short-lived active free radicals generated by enzyme catalysis with the MOF luminescent material at the nanoscale, thereby achieving the detection purpose.

[0021] In the above synthetic scheme, serine plays a dual key role: (1) Pre-assembled negatively charged secondary structural units (SBUs): The carboxyl and hydroxyl groups of serine coordinate with Zr(IV) to form negatively charged SBUs in the aqueous phase. By adjusting the ratio of serine to Zr(IV), the charge state of the SBUs can be precisely controlled to adapt to the surface charge of enzymes with different isoelectric points, achieving either an "opposite charge state" or a "charge-switching state required for electrostatic attraction." This expands the universality of the method and avoids the passive dependence of traditional methods on the enzyme's own charge.

[0022] (2) Electrostatically induced enzyme enrichment and protective "buffer layer" formation: Under the reaction conditions, the HRP surface is positively charged (pI≈8.8), which electrostatically attracts the negatively charged SBUs, causing the SBUs to accumulate on the HRP surface, forming local supersaturation. This induces the nucleation and growth of MOFs on the enzyme surface, achieving efficient encapsulation. At the same time, serine forms a "buffer layer" between the enzyme and MOFs through weak interactions such as hydrogen bonding and electrostatics, avoiding direct rigid coordination of metal ions with the enzyme surface, thereby protecting the enzyme's native conformation and catalytic activity.

[0023] Compared to the literature "Angew. Chem. Int. Ed. 2019, 58, 1463-1467", the method of this application forms negatively charged secondary structural units (SBUs) by pre-assembling serine with Zr(IV), and directly enriches positively charged HRP by electrostatic attraction. The mechanism is more direct and simpler, and no polymeric additives are required. The method of this application is more conducive to clarifying the enzyme encapsulation mechanism and avoiding the influence of PVP on the biosensing process: electrochemically inert PVP forms a physical barrier at the electrode interface, which hinders electron transfer and thus affects the ECL performance of the material and the sensitivity of subsequent ECL sensing detection.

[0024] Compared to the literature "J. Am. Chem. Soc. 2024, 146, 5108-5117" which lacks ECL luminescence properties, this application utilizes serine to form SBUs with a specific charge with Zr(IV), achieving "active" encapsulation to form negatively charged SBUs, thus realizing electrostatic attraction and efficient encapsulation of positively charged HRPs (loading 6.5 wt%). The MOFs (PCN-224) selected in this invention possess electrochemiluminescence (ECL) activity. PCN-224 can not only serve as an enzyme carrier, but its own ZnTCPP ligand also exhibits excellent ECL luminescence performance. Through nanoscale "catalytic-luminescence co-localization," the diffusion distance of the active intermediate is significantly shortened, achieving efficient energy transfer and signal amplification. Furthermore, by utilizing the Zr-O cluster in PCN-224 to form a stable Zr-OPO3 bond with an aptamer containing a phosphate group, an ultrasensitive H1N1 biosensor was constructed.

[0025] Compared with existing methods for preparing enzyme-MOF composite materials, the preparation strategy provided in this application has the following advantages: 1. Wide applicability, expanding the scope of enzyme application: The amino acid-assisted biomineralization strategy proposed in this invention introduces amino acids to coordinate with metal ions during the pre-assembly stage before MOF synthesis. By adjusting the amount of serine added, the charge of SBUs is regulated (in the example, without serine, the zeta potential of ZrOCl2 is +40 mV. As the amount of serine added increases, the zeta potential gradually decreases and reverses to a negative charge). This successfully achieves efficient encapsulation of positively charged enzymes (HRPs), breaking through the dependence of traditional biomimetic mineralization methods on enzyme surface charge, and has broad applicability.

[0026] 2. The synthesis conditions are green and mild, facilitating industrial-scale production: The entire synthesis process is completed under mild conditions of aqueous phase and 37°C, avoiding the use of high temperatures, toxic organic solvents, and harmful regulators, making it more enzyme-friendly; at the same time, the obtained MOF materials have high crystallinity (e.g., XRD). Figure 2 As shown in the figure, it still maintains its original structure and is not affected by enzymes or serine, thus ensuring the stability and performance of the material.

[0027] 3. The enzyme exhibits high activity and high loading capacity: In the enzyme-MOF materials prepared by this method, serine forms a "buffer layer" between the enzyme and MOFs, protecting the enzyme conformation through non-covalent interactions rather than direct rigid coordination, effectively avoiding the destruction of the enzyme conformation by metal ions; at the same time, through the charge matching between SBUs and the enzyme, SBUs are enriched around the enzyme through electrostatic interactions before nucleation and growth, achieving a high loading capacity (6.5 wt%) while maximizing the preservation of the enzyme's catalytic activity (the α-helix content remains almost unchanged).

[0028] 4. High ECL efficiency: By co-encapsulating the enzyme catalytic center and the ECL luminescent center within a nanoscale MOF framework, the diffusion distance of short-lived active free radicals (•OH) is greatly shortened, energy dissipation is significantly reduced, and efficient signal amplification is achieved. In the embodiments of this application, the ECL intensity is increased by 9 times compared to the comparative example.

[0029] 5. Excellent sensor performance: The ECL biosensor / detection probe constructed based on this composite material has better detection sensitivity (LOD of 0.03 ng / mL) and selectivity for H1N1 HA than most existing reports, providing a new technical platform for rapid and accurate diagnosis of influenza virus. Attached Figure Description

[0030] Figure 1 The zeta potential of ZrOCl2, SBUs synthesized from ZrOCl2 and serine, and HRP enzyme solution was detected during the preparation of HRP@PCN-224(Ser)-2.

[0031] Figure 2 This is a schematic diagram of the preparation process of HRP@PCN-224(Ser) in Example 1.

[0032] Figure 3 The reaction kinetics (enzyme activity) of HRP@PCN-224(Ser) oxidizing TMB with different serine contents are as follows; Where a is the control group HRP@PCN-224 (Ser: 0 mg), and be are HRP@PCN-224(Ser)-1 (serine: 63 mg), HRP@PCN-224(Ser)-2 (Ser: 115 mg), HRP@PCN-224(Ser)-3 (Ser: 168 mg), and HRP@PCN-224(Ser)-4 (Ser: 220 mg) in sequence.

[0033] Figure 4 ECL potential curves of HRP@PCN-224(Ser) with different serine contents in an aqueous solution containing 25 mM H2O2; In this context, a represents the control group HRP@PCN-224 (serine: 0 mg), and be represents HRP@PCN-224(Ser)-1 (Ser: 63 mg), HRP@PCN-224(Ser)-2 (Ser: 115 mg), HRP@PCN-224(Ser)-3 (Ser: 168 mg), and HRP@PCN-224(Ser)-4 (Ser: 220 mg) in sequence.

[0034] Figure 5 shows the load detection results of HRP; Wherein, (A) is the Bradford method calibration curve for determining protein concentration using bovine serum albumin (BSA) as a standard; (B) is the HRP loading in HRP@PCN-224(Ser) composites synthesized with different serine amounts. a: HRP@PCN-224, b: HRP@PCN-224(Ser)-1, c: HRP@PCN-224(Ser)-2, d: HRP@PCN-224(Ser)-3, e: HRP@PCN-224(Ser)-4.

[0035] Figure 6 XRD patterns of HRP@PCN-224 and HRP@PCN-224(Ser).

[0036] Figure 7 shows the secondary structure detection results of free HRP and HRP@PCN-224(Ser)-2.

[0037] Figure 8 The results show the catalytic activity of HRP@PCN-224 and HRP@PCN-224(Ser)-2.

[0038] Figure 9 The biological activity retained by free HRP and HRP@PCN-224(Ser) after treatment with DMF and MeOH.

[0039] Figure 10 The results are from the ECL amplification experiment. Among them, (A) is the cyclic voltammogram of HRP@PCN-224 and HRP@PCN-224(Ser)-2 in an aqueous solution containing 25 mM H2O2; (B) is the ECL-potential curve of HRP@PCN-224 and HRP@PCN-224(Ser)-2 in an aqueous solution containing 25 mM H2O2; (C) is the statistical graph of ECL intensity after adding 10 mM of different quenchers (IPA: •OH quencher, BQ: O2•⁻ quencher); (D) is the schematic diagram of the ECL signal amplification mechanism of HRP@PCN-224(Ser)-2.

[0040] Figure 11 This is a schematic diagram of the ECL detection principle.

[0041] Figure 12 The results of H1N1 virus detection using the ECL biosensor; Among them, (A) ECL response of the biosensor after assembly: a: GCE / HRP@PCN-224(Ser)-2, b: GCE / HRP@PCN-224(Ser)-2 / apt 1, c: GCE / HRP@PCN-224(Ser)-2 / apt 1 / BSA / HA, d: GCE / HRP@PCN-224(Ser)-2 / apt 1 / BSA / H1N1 / apt 2-Fc; (B) ECL response results of the biosensor to different concentrations of H1N1 hemagglutinin; (C) Calibration curve of H1N1 hemagglutinin detection; (D) Specificity of the biosensor to different influenza subtypes. Detailed Implementation

[0042] Sources of raw materials and reagents involved in the examples: Zirconium oxychloride octahydrate (ZrOCl2·8H2O), tetraoctylammonium bromide (TOAB), potassium chloride (KCl), sodium hydroxide (NaOH), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), hydrogen peroxide solution, N,N-dimethylformamide (DMF), ethanol and methanol were purchased from Aladdin (Shanghai, China).

[0043] Zinc(II) tetra(4-carboxyphenyl)porphyrin (ZnTCPP) was purchased from Maclean's Reagent Co., Ltd. (Shanghai, China).

[0044] The H1N1 HA antigen was purchased from Nanjing Fuxiao Biotechnology Co., Ltd.

[0045] HRP enzyme solution (10 mg / mL), 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric kit, Bradford protein assay kit, and serine (L-serine) were all purchased from Shanghai Sangon Biotech Co., Ltd. (Shanghai, China).

[0046] Example 1: Preparation of enzyme-MOF composite materials The following examples illustrate the principle / process diagram of composite materials. Figure 1 As shown, the specific steps are as follows:

[0047] 1) Pre-assembled negatively charged secondary structural units (SBUs): ZrOCl2·8H2O (41 mg, 0.127 mmol) was dissolved in 1.9 mL of ultrapure water with different amounts of serine (63 mg, 115 mg, 168 mg, and 220 mg, corresponding to samples 1-4). The pH was adjusted to 7.0 with 1 M NaOH solution (to protect the activity of subsequently added enzymes) to obtain mixed solutions, which were then numbered 1-4. A control group without serine was also set up. 2) Enzyme-induced nucleation and encapsulation: Add 0.1 mL of HRP enzyme solution (10 mg / mL) to each of the mixtures 1-4 obtained in step 1, and mix well; Since HRP is positively charged under reaction conditions, the charge of the added HRP enzyme solution in this embodiment, as detected by zeta potential, is approximately +12 mV. The pre-assembled SBUs in step 1 are negatively charged, and through electrostatic attraction, the SBUs accumulate on the HRP surface, forming localized supersaturation. The zeta potential detection results of the reagents during the preparation of HRP@PCN-224(Ser)-2 are as follows: Figure 2 As shown.

[0048] 3) Preparation of ligand solution: Separately dissolve 10 mg of ZnTCPP in 2 mL of deionized water, adjust the pH to 7.0 with 1 M NaOH solution to obtain the ligand solution; prepare multiple sets of ligand solutions using the same method for later use.

[0049] 4) Growth of MOFs: Under stirring at room temperature, the ligand solution was added dropwise to the well-mixed solution from step 2, and stirring was continued for 10 min. The mixture was then transferred to a reaction vessel and reacted at 37 °C for 12 h. This induced rapid and uniform nucleation and growth of MOFs on the HRP surface, ultimately encapsulating the HRP within a PCN-224 framework. The concentration of Zr(IV) was approximately 0.064 mmol / mL, the molar ratio of added organic ligand to enzyme was 515:1, and the molar ratio of organic ligand to Zr(IV) was 1:10.9.

[0050] After the reaction was completed, the precipitate was collected by centrifugation (12000 rpm, 5 min), washed thoroughly with deionized water, and finally freeze-dried under vacuum at -50℃ for 24 h to obtain a powdered product. Depending on the amount of serine used, the products were named HRP@PCN-224(Ser)-1 to HRP@PCN-224(Ser)-4, and a control group HRP@PCN-224(Ser)-0 (without serine) was included. The products will be collectively referred to as HRP@PCN-224(Ser) below.

[0051] The performance of the obtained HRP@PCN-224(Ser) was tested, and the results are as follows: 1. Enzyme activity and ECL potential detection experiments Depend on Figure 3 Enzyme loading data showed that when the serine dosage exceeded 115 mg, the enzyme loading almost ceased to increase; similar data on TMB enzyme activity showed that when the serine dosage exceeded 115 mg, the enzyme's ability to catalyze TMB oxidation only increased slightly; and Figure 4 ECL detection data showed that the ECL intensity was highest when the serine dosage was 115 mg. This may be because when serine is in excess, it may introduce an interfacial barrier, thereby slowing down electron transfer or limiting the mass transport of hydrogen peroxide and active intermediates.

[0052] 2. Loading and Crystallinity Testing Experiment Figure 5To determine the HRP loading in MOFs using the Bradford protein assay kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) via the Bradford protein assay method (specific detection steps refer to the kit instructions): (A) shows the Bradford method calibration curve using bovine serum albumin (BSA) as a standard to determine protein concentration. For actual sample measurements, 20 μL of 1 mg / mL sample was added to a 96-well plate, followed by 200 μL of Coomassie Brilliant Blue G-250 reagent. After incubation for 5 minutes, the solution was collected and detected using a UV-Vis spectrophotometer. (B) shows the HRP loading in HRP@PCN-224(Ser) composites synthesized with different serine amounts. It is evident that the HRP concentration is directly proportional to the absorbance at 595 nm. The HRP loading with serine assistance reached 6.5 wt%, significantly higher than the 2.0 wt% in the serine-free control group.

[0053] PXRD analysis showed that HRP@PCN-224(Ser) had the same high crystallinity as simulated PCN-224. Figure 6 The crystal structure of PCN-224 is disclosed in the literature “Construction of Ultrastable Porphyrin Zr Metal−Organic Frameworks through Linker Elimination” (Journal of the American Chemical Society, 2013, 135, 17105−17110).

[0054] above Figure 5 , Figure 6 The test data demonstrate the high enzyme loading and high crystallinity prepared by the above method. Final performance optimization was performed using: 1) the Bradford method to determine the enzyme loading; 2) the TMB method to determine enzyme activity; and 3) the ECL performance test results. It was found that when the serine dosage was 115 mg (i.e., HRP@PCN-224(Ser)-2), the resulting composite material achieved optimal performance in terms of enzyme loading, catalytic activity, and ECL performance. Therefore, this was selected as the preferred embodiment and used for subsequent characterization and application studies.

[0055] 3. Enzyme conformation retention experiment FT-IR amide I band Gaussian fitting quantitative analysis showed that the α-helix content of encapsulated HRP (18.9%) was almost identical to that of free HRP (19.5%), indicating that the active site structure was well protected. Figure 7 ).

[0056] Figure 7 This was achieved by testing the amide I band (wavenumber range 1610 to 1700 cm⁻¹) in Fourier transform infrared (FT-IR) spectra. −1 This range is mainly attributed to the CO stretching vibration of the peptide chain (approximately 80%), and is highly sensitive to conformational changes in the enzyme. (A) and (B) show the secondary structure detection results for free HRP and HRP@PCN-224(Ser)-2, respectively. Due to differences in hydrogen bonding patterns and molecular geometry, different secondary structures of HRP exhibit slightly different wavenumbers in the amide I band region. The native β-sheet structure is typically located at 1623-1641 cm⁻¹. −1 The peak at 1642-1650 cm⁻¹ is correlated with the peak at 1642-1650 cm⁻¹. −1 The nearby bands are attributed to a disordered coiled conformation. The α-helical structure is located at 1648–1657 cm⁻¹. −1 The surrounding area exhibits a characteristic zone, while 1662-1686 cm −1 The bands within the range may be attributed to the β-turn. To identify these peaks, the second derivative of the amide I band was determined in this embodiment. The peak positions were determined by the positions of the characteristic peaks appearing in the second derivative and the wavenumber range corresponding to each conformation theory. Then, peak separation was performed on PeakFit software, and the proportion of each structure was determined by the ratio between the area of ​​each peak and the total area.

[0057] 4. Catalytic activity detection experiment Figure 8 To determine the catalytic activity of HRP, the following procedure was used: The catalytic reaction between HRP and H₂O₂ (which oxidizes TMB) was employed as a method to calibrate HRP activity. In a 96-well microplate, 100 μL of acetate buffer (0.2 M, pH = 4), 5 μL of TMB solution, and 20 μL of HRP@PCN-224(Ser)-2 solution (1 mg / mL) were added. After shaking and mixing for 2 minutes, 2 μL of 100 mM H₂O₂ solution was added, and incubation was performed for different time periods. The reaction was then terminated by adding 5 μL of 2 M H₂SO₄ solution. The absorbance at 450 nm was measured, an absorbance-time curve was plotted, and the activity of HRP was evaluated.

[0058] HRP is a peroxidase, which catalyzes the generation of hydroxyl radicals (•OH) from H₂O₂. These •OH radicals then catalyze the oxidation of TMB. Figure 8 The Abs 450 nm shown refers to the UV characteristic absorption peak at 450 nm after TMB is oxidized to oxTMB and sulfuric acid is added as a stop solution. It can be seen that compared with the serine-free complex, HRP@PCN-224(Ser)-2 has 8 times more catalytic activity for TMB, and has stronger substrate affinity and catalytic efficiency.

[0059] 5. Stability testing After treatment in 25% DMF or MeOH for 10 min, the enzyme activity assay results are as follows: Figure 9 As shown, HRP@PCN-224(Ser)-2 can still maintain 74%-85% of its initial activity, while the activity of free HRP is almost completely lost, proving the effective protective effect of the MOF shell.

[0060] 6. ECL Scale-up Experiment The ECL device used in the examples is an MPI-EIII full-spectrum electrochemiluminescence detector (purchased from Xi'an Ruimai Analytical Instrument Co., Ltd.).

[0061] In all examples, electrochemical studies were conducted using a conventional three-electrode system: a platinum wire electrode and an Ag / AgCl electrode were used as the counter and reference electrodes, respectively. A modified glassy carbon electrode (GCE, 5 mm in diameter) was used as the working electrode. HRP@PCN-224 and HRP@PCN-224(Ser)-2 (1 mg / mL, 10 μL) were coated onto the surface of the glassy carbon electrode. To improve electrode stability, after drying at room temperature, 10 μL of TOAB solution (prepared by ultrasonically dispersing 10 mM TOAB in ethanol) was coated onto the GCE surface. The ECL experiments used a 10 mM pH 7.0 HEPES solution containing 25 Mm H2O2 and 0.1 M KCl as the electrolyte. The scan range was set from -1.7 V to 0 V (start potential: 0 V, scan direction: negative, scan rate: 50 mV s⁻¹).

[0062] Standard CV test results, such as Figure 10 As shown in (A), when a negative potential is applied, the HRP in HRP@PCN-224(Ser)-2 can efficiently catalyze the reduction of H2O2, generating a large number of hydroxyl radicals (•OH) near the electrode surface. At the same time, the ZnTCPP ligand in the PCN-224 framework undergoes a two-step reduction to generate the strongly reducing anionic radical ZnTCPP²⁻.

[0063] ECL test results are as follows Figure 10 As shown in (B), the ECL intensity of HRP@PCN-224(Ser)-2 is increased by approximately 9 times compared to the complex without serine assistance. Because HRP and ZnTCPP are co-encapsulated within a nanoscale MOF framework, the diffusion distance between •OH and ZnTCPP²⁻ is extremely short, resulting in efficient collisions and electron transfer, exciting ZnTCPP to an excited state (ZnTCPP²⁻*). The excited-state ZnTCPP²⁻* returns to the ground state and releases photons, generating the ECL signal.

[0064] Figure 10 In section (C), a free radical quenching experiment was conducted. Specifically, 10 mM benzoquinone (BQ) and IPA (isopropanol) were added to the electrolyte used in section (B) for ECL testing to quench superoxide radicals and hydroxyl radicals, respectively. The experimental results showed that the ECL signal significantly decreased only when IPA was added, suggesting that hydroxyl radicals (•OH) are the main free radicals involved in the ECL process.

[0065] Based on the reduction process corresponding to the two reduction peaks in the CV test, the elution position of ECL, and the results after adding the quencher, the simulated ECL mechanism diagram is as follows: Figure 10 As shown in (D).

[0066] Example 2: Construction and Experiment of an ECL Biosensor for Detecting H1N1 Virus The principle / process for detecting the H1N1 virus in this embodiment is as follows: Figure 11 As shown, the HRP@PCN-224(Ser)-2 material prepared in Example 1 was used to construct an ECL biosensor for detecting the H1N1 virus: The fabrication steps of the H1N1 ECL biosensor are as follows: 1) A conventional three-electrode system was used: a platinum wire electrode and an Ag / AgCl electrode were used as the counter electrode and reference electrode, respectively, and a modified glassy carbon electrode (GCE, 5 mm in diameter) was used as the working electrode; the gold foil electrode (GCE) was ground with Al2O3 powder of 0.3 and 0.05 μm, then ultrasonically treated in ethanol and rinsed with deionized water for later use.

[0067] 2) 10 μL of HRP@PCN-224(Ser)-2 (1 mg / mL aqueous solution) was dropped onto GCE and dried at room temperature to obtain GCE / HRP@PCN-224(Ser); then 20 μL of aptamer 1 solution (apt1, 1 μM) for capturing H1N1 HA protein was dropped onto the modified electrode and incubated at 37 °C for 2 hours to immobilize the capture probe.

[0068] After incubation, the electrode was thoroughly rinsed with ultrapure water to remove unbound apt1, yielding GCE / HRP@PCN-224(Ser)-2 / apt1. To block non-specific binding sites, 20 μL of bovine serum albumin (BSA, 1% aqueous solution) was then added to the electrode surface and incubated at 37°C for 1 hour. The electrode was then washed with ultrapure water to obtain GCE / HRP@PCN-224(Ser)-2 / apt1 / BSA / HA.

[0069] For target capture, 20 μL of H1N1 antigen solution at different concentrations was added dropwise to the electrode and incubated at 37°C for 2 hours. Subsequently, the electrode was rinsed with ultrapure water to remove uncaptured antigen. Different concentrations of H1N1 antigen were added to obtain a linear curve, such as... Figure 12 As shown in (C); in practical applications, the solution to be tested is dropped onto the electrode surface, and then the actual concentration of H1N1 can be obtained by mapping the measured ECL intensity to the linear curve.

[0070] Next, 20 μL of ferrocene-labeled aptamer 2 solution (apt2-Fc, 0.5 μM) was dropped onto the electrode and incubated at 37 °C for 2 hours to form a sandwich complex. After washing with ultrapure water and drying with nitrogen, 10 μL of TOAB (10 mM ethanol solution) was applied to the electrode surface to obtain GCE / HRP@PCN-224(Ser)-2 / apt 1 / BSA / H1N1 / apt 2-Fc for subsequent ECL measurements.

[0071] Apt1 (SEQ ID NO: 1): (5′-GGG GGG TCC CTA CGG CGC TAA CCG ATA GGT GTAGCG GGC GGC ACA TGT TCG CGC CAC CGT GCT GCT ACA AC-3′); apt2-Fc (SEQ ID NO: 2): 5′-Fc-C6-TAC TGC ACA CGA CAC CGA CGA CTG TCA CCATCA TCA CCT CGG CGC A-3′); all were synthesized by Shanghai Sangon Biotech.

[0072] The aforementioned aptamer sequence was prepared in reference “Fluorescence / Colorimetric Lateral FlowImmunoassay Based on Dual-Aptamer Domain Recognition and Upconversion SignalAmplification for Sensitive H1N1 Hemagglutinin Detection” (Advanced Functional Materials, 2025, e19871).

[0073] This assay modifies the surface of a glassy carbon electrode (GCE) with HRP@PCN-224(Ser)-2. Utilizing the strong affinity between the Zr-O clusters in PCN-224 and phosphate groups, the 5'-phosphorylated aptamer 1 (apt1) is stably immobilized via Zr-OPO3 bonds. When the target analyte H1N1 HA is present, it is captured by apt1. Subsequently, a ferrocene-labeled aptamer 2 (apt2-Fc) is added, forming an apt1-HA-apt2-Fc "sandwich" structure. Ferrocene, as a highly efficient electron transfer quencher, can quench the ECL signal of HRP@PCN-224(Ser)-2, and the degree of signal reduction is correlated with the concentration of H1N1 HA.

[0074] ECL test conditions: Electrolyte solution 10 mM pH 7.0 HEPES buffer (containing 25 mM H2O2, 0.1 MKCl); Scan range set to -1.7 V to 0 V (start potential: 0 V, scan direction: negative, scan rate: 50 mV s⁻¹); Detection results are as follows: Figure 12 As shown, the sensor constructed based on HRP@PCN-224(Ser)-2 material exhibits a wide linear range (0.1-1000 ng / mL) and an extremely low detection limit (0.03 ng / mL) for the detection of H1N1 HA, which is below the clinical diagnostic threshold. Figure 12 As shown in (D), specificity tests indicate that it has no significant cross-reactivity with other influenza subtypes (H9N2, H7N9, etc.), demonstrating excellent selectivity.

[0075] Table 1 compares the sensitivity of the ECL constructed in this embodiment with that of existing reported H1N1 detection methods: Table 1 Comparison of H1N1 detection sensitivity of different methods

[0076] Methods 1-5 mentioned in Table 1 are disclosed in the following documents in sequence: (1) Xu, M.; Zhao, S.; Lin, C.; Li, Y.; Zhang, W.; Peng, Y.; Xiao, R.;Huang, Z.; Yang, Y. Dual-Mode Lateral Flow Immunoassay Based on "Pompon Mum"-Like Fe3O4@MoS2@Pt Nanotags for Sensitive Detection of Viral Pathogens. ACS Applied Materials Interfaces 2024, 16(9), 11172-11184. (2) Pan, X.; Wang, J.; Zhang, K.; Sun, P.; Shi, J.; Zhi, J.; Cai, Z.;Li, Z.; Wang, D.; Tong, B.; et al. Differential detection of H1N1 virusspiker proteins by two hexaphenylbutadiene isomers based on size-matchingprinciple. Anal. Chim. Acta 2024, 1299 , 342452. (3) Wan, Z.; Gong, H.; Liu, Q.; Wang, Q.; Ma, P. a.; He, F.; Wang,Q.; Ding, H.; Gai, S.; Yang, P. Fluorescence / Colorimetric Lateral FlowImmunoassay Based on Dual‐Aptamer Domain Recognition and Upconversion SignalAmplification for Sensitive H1N1 Hemagglutinin Detection. Adv. Funct. Mater. 2025, 36 (20), e19871. (4) Yadav M.; Arora R.; Dhanda M.; Singh G.; Mohan H.; Lata S. TiO2-guanine as a new amalgamation compound for fabrication of a disposablebiosensor with high sensitivity and rapid detection of H1N1 swine fu virus. Microchim. Acta 2023, 190 , 412. (5) Lin, J.; Liu, J.; Xu, J. Ultrasensitive electrochemical immunoassay for screening the influenza A (H1N1) virus based on atomically Ru-dispersed nitrogen-doped carbon. New Journal of Chem. 2023, 47 (4), 1685-1690. As shown in Table 1, the linear range and limit of detection (LOD) of the ECL sensor prepared in this application are superior to those reported in the prior art, and it can effectively meet the requirements for ultra-trace target detection. This demonstrates that the amino acid-assisted biomineralization strategy proposed in this application can successfully prepare high-performance enzyme-MOF signal probes, providing a reliable technical platform for the rapid and accurate diagnosis of influenza viruses.

Claims

1. A method for preparing enzyme-metal-organic framework composite materials by amino acid-assisted biomineralization, characterized in that: The specific steps are as follows: 1) Serine is mixed with metal ions in a solvent to form negatively charged secondary structural units; the metal ions include Zr. 4+ Zn² + Co² + Ni² + At least one of them; 2) Add an enzyme to the secondary structural unit obtained in step 1), and then add a ligand to carry out a mineralization reaction to obtain the enzyme-metal-organic framework composite material.

2. The method according to claim 1, characterized in that, Step 1) The solvent is either deionized water or a buffer solution; the buffer solution includes either Tris-HCl buffer or HEPES buffer.

3. The method according to claim 1, characterized in that, Step 2) The ligand includes at least one of tetrakis(4-carboxyphenyl)porphyrin zinc(II), tetrakis(4-carboxyphenyl)porphyrin, and 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene.

4. The method according to claim 1, characterized in that, In step 1), after the serine and metal ions are mixed in the solvent, the concentration of the metal ions in the solvent is 0.03-0.15 mmol / mL.

5. The method according to claim 1, characterized in that, Step 2) The molar ratio of the added ligand to the metal ion is 1:7 to 14; the metal ion is Zr. 4+ .

6. The method according to claim 5, characterized in that, Step 1) The Zr 4+ Provided by ZrOCl2·8H2O, the molar ratio of ZrOCl2·8H2O to serine is 1:4.7-16.

5.

7. The method according to claim 6, characterized in that, Step 2) The added ligand and Zr 4+ The molar ratio is 1:8-12, and the molar ratio of the added ligand to the enzyme is 400-600:

1.

8. The enzyme-metal-organic framework composite material prepared by any one of claims 1-7.

9. The application of the enzyme-metal-organic framework composite material prepared by any one of claims 1-7 as a detection signal probe.

10. The application as described in claim 9, characterized in that, The application refers to the use of the enzyme-metal-organic framework composite material as a detection probe in an electrochemiluminescence biosensor.

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