Aperture-adjustable graded porous metal organic framework sensing probe as well as preparation method and application thereof

Abstract

The invention discloses a graded porous metal organic framework sensing probe with adjustable aperture and a preparation method and application thereof, and the preparation method comprises the following steps: S1, under the synergistic effect of salt and a hydrophobic oil phase solvent, obtaining graded porous metal organic framework nanoparticles with open large cage-shaped mesoporous channels and microporous crystal walls; s2, loading NAD < + > cofactors into the graded porous metal organic framework nanoparticles; s3, glucose dehydrogenase, beta-hydroxybutyrate dehydrogenase, L-lactic dehydrogenase and D-lactic dehydrogenase are loaded into mesoporous channels of the organic framework nanoparticles respectively, and the graded porous metal organic framework sensing probes fixed with the dehydrogenase and adjustable in multiple pore diameters are obtained. The probe can be used for rapidly, accurately and synchronously detecting glucose, beta-hydroxybutyric acid, L-lactic acid and D-lactic acid biomarkers in a human serum sample, and is particularly suitable for screening and diagnosing different types of diabetic acidosis.

Description

Technical Field

[0001] The present invention relates to enzyme immobilization and analytical chemistry, and more particularly to a hierarchically porous metal-organic framework sensing probe with adjustable pore size, and a preparation method and application thereof. Background Art

[0002] Disorders of metabolic pathways, such as carbohydrates, acids, and lipids, are closely related to diseases such as diabetes, obesity, cardiovascular diseases, and cancer (Nat. Rev. Urol. 2019, 16, 339-362). Diabetic acidosis is an acute complication of diabetes, typically manifested as diabetic ketoacidosis (DKA) or lactic acidosis (Pediatr. Nephrol. 2018, 33, 673-681). DKA results from insufficient insulin, leading to abnormal fat metabolism and excessive accumulation of ketone bodies (such as β-hydroxybutyrate, acetoacetate), thereby increasing blood acidity and disrupting organ function (Anal. Chem. 2020, 92, 2291-2300). Despite improvements in diabetes treatment, in some severe cases, this life-threatening disease remains a major cause of morbidity and mortality in type 1 and type 2 diabetes (Diabetic Med. 2015, 32, 14-23). Elevated levels of L-lactic acid (L-LAc) or D-lactic acid (D-LAc) exacerbate metabolic acidosis, resulting in a more pronounced acidosis state and higher mortality in DKA patients, with a worse recovery effect (J. Crit. Care. 2023, 78, 154377).

[0003] Therefore, rapid, accurate, and synchronous detection of metabolic biomarkers, including glucose (Glu), β-hydroxybutyrate (Hb), L-lactic acid (L-LAc), and D-lactic acid (D-LAc), is crucial for diagnosing and evaluating the severity of diabetic acidosis. Summary of the Invention

[0004] The object of the present invention is to provide a hierarchically porous metal-organic framework sensing probe with adjustable pore size, and a preparation method and application thereof, so as to solve the problem that the existing technology still lacks a method for rapidly, accurately, and synchronously detecting metabolic biomarkers to screen the types of diabetic acidosis.

[0005] To solve the above problems, the present invention adopts the following technical solutions:

[0006] According to the first aspect of the present invention, there is provided a preparation method of a hierarchically porous metal-organic framework sensing probe, which includes the steps: S1, under the synergistic action of a salt and a hydrophobic oil-phase solvent, obtaining hierarchically porous metal-organic framework nanoparticles (HMUiO-66(Zr)) with open large-cage mesoporous channels and microporous crystal walls; S2, adding NAD +Cofactors are loaded into hierarchical porous metal-organic framework nanoparticles to obtain hierarchical porous metal-organic framework nanoparticles with mesoporous channels and immobilized cofactors; S3, glucose dehydrogenase (GDH), β-hydroxybutyrate dehydrogenase (3-HBDH), L-lactate dehydrogenase (L-LDH), and D-lactate dehydrogenase (D-LDH) are respectively loaded into the mesoporous channels of the hierarchical porous metal-organic framework nanoparticles immobilized with cofactors to obtain a variety of hierarchical porous metal-organic framework sensing probes with adjustable pore sizes and immobilized dehydrogenases, NAD + @GDH@HMUiO-66(Zr), NAD + @3-HBDH@HMUiO-66(Zr), NAD + @L-LDH@HMUiO-66(Zr) and NAD + @D-LDH@HMUiO-66(Zr).

[0007] It should be understood that the full English name of HMUiO-66(Zr) is Hierarchical Mesoporous UiO-66(Zr). UiO-66(Zr) is a very classic porous material in metal-organic frameworks (MOFs), with excellent chemical stability and long-term stability.

[0008] According to such a preparation method of the hierarchical porous metal-organic framework sensing probe provided by the present invention, the main inventive point lies in that the synergistic effect of salt and hydrophobic oil-phase solvent is utilized to precisely control the size and structure of mesopores. Among them, the anions of salts (such as Cl - , NO 3 - , Br - , I - and ClO 4 - ) can reduce the harsh reaction conditions required for MOF synthesis and make it compatible with the mild conditions required for self-assembly with soft templates. At the same time, the hydrophobic oil-phase solvent (such as toluene) stabilizes the micelle template by forming strong hydrophobic interactions with the PPO segments of the micelles, preventing surfactant molecules from dissociating from the micelles into the solution due to the interaction between the metal precursor and the PEO segments during the MOF crystallization process.

[0009] According to the present invention, the salt used in step S1 can be NaCl, NaNO 3 , NaBr, NaI, NaClO 4 , KCl, LiCl, MgCl 2 and CaCl 2Any one of them, the hydrophobic oil phase solvent refers to a solvent with low polarity and strong lipophilicity, such as toluene, triethylbenzene, mesitylene, etc. The salt can significantly reduce the reaction energy and promote the crystallization of hierarchical porous metal-organic framework nanoparticles in the aqueous phase. The highly hydrophobic oil phase stabilizes the micelles and emulsions, preventing the premature dissociation of surfactant molecules during the slow growth of hierarchical porous metal-organic frameworks.

[0010] More preferably, the salt is sodium nitrate or sodium chloride, and the hydrophobic oil phase solvent is toluene. The reason is that NO 3 - shows the strongest promoting effect on the crystallization of UiO-66(Zr), enabling the reaction temperature to be reduced to only 40 °C, producing HMUiO-66(Zr) with the highest crystallinity; Cl - The salting-out ion increases the hydrophobicity of the micelles, allowing more toluene to enter the micelle core and enabling the formation of HMUiO-66(Zr) with larger mesopore sizes. Toluene has the highest hydrophobicity among common oil phases (such as triethylbenzene, mesitylene, xylene, toluene, etc.), and the inventors have fully demonstrated that toluene can maximize the expansion of mesopore sizes. Therefore, toluene is most preferably used as the hydrophobic oil phase solvent for pore expansion in the present invention.

[0011] Preferably, the step S1 includes: S11, using a template agent (such as F127, etc.) to direct the formation of metal-organic frameworks, and using the synergistic effect between the salt and the oil phase to hydrothermally generate hierarchical porous metal-organic frameworks (HMUiO-66(Zr)-as) with adjustable mesopore diameters; S12, obtaining hierarchical porous metal-organic framework nanoparticles (HMUiO-66(Zr)) through activation and template removal. More preferably, by regulating the type of salt and the feeding amount of toluene, the mesopore diameter size of the obtained HMUiO-66(Zr) can be continuously adjusted between 6.8 - 18.1 nm. In a preferred embodiment, when using sodium nitrate salt, the toluene feeding amounts are 30 μL, 70 μL, and 100 μL respectively, and the pore diameters of HMUiO-66(Zr) are 6.8 nm, 8.9 nm, and 10.3 nm respectively. Specifically, 100 mg of F127 is dissolved in 6 mL of deionized water. Subsequently, HAc (125 μL, 2.19 mmol), toluene (30 μL, 70 μL, and 100 μL), and NaNO 3 (722 mg, 8.5 mmol) are added to the solution for the synthesis of HMUiO-66(Zr). The mixture is stirred at 30 °C for 1 hour to obtain a homogeneous solution. Then, BDC (166 mg, 1 mmol) is added and stirred for 20 minutes. Subsequently, ZrO(NO 3 ) 2 ·2H 2O (231 mg, 1 mmol). The reaction mixture was stirred in a 60 °C water bath for 24 h. Then, the obtained sample was centrifuged and washed twice with deionized water, followed by two washes with DMF to remove residual unreacted ligands. Then, the synthesized sample was washed with ethanol and soaked in ethanol at 60 °C for two days, with the ethanol being changed daily. Finally, the sample was dried in vacuo at 80 °C for 24 h. In a preferred embodiment, when using sodium chloride salt, an HMUiO-66(Zr) with a pore size of approximately 18.1 nm could be synthesized with a toluene feed amount of 400 μL. Specifically, 100 mg of F127 was dissolved in 6 mL of deionized water. Subsequently, HAc (125 μL, 2.19 mmol), toluene (400 μL, 3.8 mmol), and NaCl (500 mg, 8.5 mmol) were added to the solution for the synthesis of HMUiO-66(Zr). The mixture was stirred at 30 °C for 1 h to obtain a homogeneous solution. Then, BDC (166 mg, 1 mmol) was added and stirred for 20 min. Subsequently, ZrO(NO 3 ) 2 ·2H 2 O (231 mg, 1 mmol). The reaction mixture was stirred in a 60 °C water bath for 24 h. Then, the obtained sample was centrifuged and washed twice with deionized water, followed by two washes with DMF to remove residual unreacted ligands. Then, the synthesized sample was washed with ethanol and soaked in ethanol at 60 °C for two days, with the ethanol being changed daily. Finally, the sample was dried in vacuo at 80 °C for 24 h.

[0012] Preferably, step S2 includes: dissolving glucose dehydrogenase (GDH), β-hydroxybutyrate dehydrogenase (3-HBDH), L-lactate dehydrogenase (L-LDH), and D-lactate dehydrogenase (D-LDH) in Tris-HCl buffer (20 mM, pH 7.4) respectively, adding NAD + @HMUiO-66(Zr), and obtaining a hierarchically porous metal-organic framework sensing probe immobilized with dehydrogenase (NAD + @GDH@HMUiO-66(Zr), NAD + @3-HBDH@HMUiO-66(Zr), NAD + @L-LDH@HMUiO-66(Zr), and NAD + @D-LDH@HMUiO-66(Zr)) by the adsorption method. Specifically, to 0.5 mL of NAD +0.5 mL of GDH, 3-HBDH, L-LDH, and D-LDH solutions (2 mg / mL, dissolved in 20 mM Tris-HCl buffer, pH 7.4) were added to the + @HMUiO-66(Zr) suspension and shaken at 25 °C for 1 hour. The enzyme-loaded NAD was collected by centrifugation, and

[0013] @HMUiO-66(Zr) was washed twice with water and then dispersed in 2 mL of Tris-HCl buffer (20 mM, pH 7.4). As the mesopore size of the hierarchical porous metal-organic framework increased from the traditional microporous structure to 10.6 nm, the loading amounts of GDH, 3-HBDH, L-LDH, and D-LDH increased significantly, from 38, 31, 27, and 28 mg / g to 227, 207, 195, and 169 mg / g, respectively. When the mesopore size further increased to the branched mesopores of 18.1 nm, the loading amounts of GDH, 3-HBDH, L-LDH, and D-LDH decreased to 153, 137, 132, and 114 mg / g to some extent. This decrease in the loading amount is likely due to the excessively large mesopore size, which reduces the number of mesoporous cages available for enzyme immobilization. Therefore, an appropriate mesopore size helps the diffusion and immobilization of enzymes into the material, thereby achieving a higher immobilization efficiency.

[0014] It should be understood that UiO-66(Zr) is a classic MOF structure, which has attracted attention due to its excellent chemical stability and wide applications. However, traditional UiO-66(Zr) mainly has a microporous structure and is difficult to meet the requirements for the immobilization of biological macromolecules (such as enzymes), thus limiting its application in metabolic biomarker sensing probes. However, up to now, it has not been possible to synthesize UiO-66(Zr) with ordered mesopores. The main difficulty lies in that MOF crystallization usually requires high temperature (>90 °C) and organic solvents, while micelle self-assembly requires an aqueous phase and mild temperature (<70 °C). The reaction conditions of the two are fundamentally mismatched, making it extremely difficult to construct a stable mesoporous structure in UiO-66(Zr).

[0015] The key inventive point of the present invention lies in successfully solving this technical problem by discovering the salt-oil synergistic regulation mechanism: 1) Salt reduces the crystallization temperature of UiO-66(Zr), for example NO 3 - promotes the crystallization of UiO-66(Zr), enabling it to form a stable crystal phase in an aqueous low-temperature (40-60 °C) environment; 2) The oil phase can increase the stability of micelles at high temperatures when crystals grow on the surface of micelles; 3) The salt-oil synergistic effect greatly broadens the synthesis window of HMUiO-66(Zr), making its mesopore aperture continuously adjustable (6.8-18.1 nm). This strategy is expected to provide guidance for the synthesis of other HMMOFs.

[0016] According to the second aspect of the present invention, there is provided a hierarchical porous metal-organic framework prepared by the said preparation method for preparing a sensing probe NAD + @GDH@HMUiO-66(Zr), NAD + @3-HBDH@HMUiO-66(Zr), NAD + @L-LDH@HMUiO-66(Zr) and NAD + @D-LDH@HMUiO-66(Zr), where the dehydrogenase and cofactor are respectively immobilized in the mesopores of the hierarchical porous metal-organic framework nanoparticles.

[0017] Another key inventive point of the present invention lies in the innovation at the application level of the probe: 1) The adjustable mesoporous structure optimizes enzyme immobilization and improves sensing sensitivity: The microporous structure of traditional MOFs limits the entry of macromolecules (such as enzymes and cofactors), making it difficult to efficiently immobilize biomolecules. HMUiO-66(Zr) prepared according to the method of the present invention has adjustable mesopores (6.8-18.1 nm), which can match enzymes of different sizes (such as GDH, 3-HBDH, L-LDH, and D-LDH), increase the immobilization amount and activity of the enzyme, and the appropriate mesopore size matches the size of the enzyme, ultimately optimizing the sensitivity performance of the probe; 2) Multi-marker integrated detection improves the ability to monitor diabetic acidosis: Traditional biosensing is mainly based on single-marker detection and cannot achieve comprehensive disease analysis. The present invention integrates four different probes into a sensing array to achieve synchronous detection of Glu, Hb, L-LAc, and D-LAc. Through multi-marker correlation analysis, the progression and type of diabetic acidosis can be monitored more accurately. Compared with single-marker detection, this strategy is more conducive to providing systematic disease diagnosis and improving the ability to monitor acute complications of diabetes.

[0018] According to the third aspect of the present invention, there is provided an application of the above-mentioned hierarchical porous metal-organic framework sensing probe, which has a colorimetric sensing response to Glu, Hb, L-LAc, and D-LAc biomarkers respectively. Since GDH, 3-HBDH, L-LDH, and D-LDH serve as the recognition centers for Glu, Hb, L-LAc, and D-LAc respectively and have high specificity, the quantitative detection of the corresponding metabolic biomarkers in complex biological samples is not interfered. Accordingly, the hierarchical porous metal-organic framework sensing probe through the cascade response system constructed by dehydrogenases and NAD + cofactors is applicable to the specific recognition and detection of Glu, Hb, L-LAc, and D-LAc biomarkers in human serum samples.

[0019] Preferably, the hierarchical porous metal-organic framework sensing probe shows specificity to the biomarker to be detected and has no colorimetric response to other interfering molecules.

[0020] Preferably, the hierarchical porous metal-organic framework sensing probe (NAD + @GDH@HMUiO-66(Zr)) is dispersed in Tris-HCl buffer solution to form a probe solution, and the Glu solution is added to the probe solution for incubation, and its absorbance is measured using a multi-functional microplate reader. Subsequently, using the same method, NAD + @3-HBDH@HMUiO-66(Zr), NAD + @L-LDH@HMUiO-66(Zr), and NAD + @D-LDH@HMUiO-66(Zr) probes are used respectively, and the absorbances of Hb, L-LAc, and D-LAc are measured using a multi-functional microplate reader.

[0021] Preferably, within the concentration range of 0-500 μM of the Glu solution, the hierarchical porous metal-organic framework sensing probe (NAD + @GDH@HMUiO-66(Zr)) is used for colorimetric response to Glu, and the absorbance of the probe and the Glu concentration are linearly fitted and analyzed for the quantitative analysis of Glu. Subsequently, using the same method, NAD + @3-HBDH@HMUiO-66(Zr), NAD + @L-LDH@HMUiO-66(Zr), and NAD + @D-LDH@HMUiO-66(Zr) are used for colorimetric response to Hb, L-LAc, and D-LAc respectively, and the absorbances of the probe and the Hb, L-LAc, and D-LAc concentrations are linearly fitted and analyzed for the quantitative analysis of Hb, L-LAc, and D-LAc.

[0022] Preferably, the lower limits of detection of Glu, Hb, L-LAc and D-LAc solutions are 6.4 μM, 2.2 μM, 35 μM and 24 μM, respectively.

[0023] Accordingly, the hierarchical porous metal-organic framework sensing probes (NAD + @GDH@HMUiO-66(Zr), NAD + @3-HBDH@HMUiO-66(Zr), NAD + @L-LDH@HMUiO-66(Zr) and NAD + @D-LDH@HMUiO-66(Zr)) of the present invention can be used for clinical detection applications, which can directly quantitatively detect the contents of Glu, Hb, L-LAc and D-LAc in human serum to screen diabetes acidosis types. Among them, first, the hierarchical porous metal-organic framework sensing probes (NAD + @GDH@HMUiO-66(Zr), NAD + @3-HBDH@HMUiO-66(Zr), NAD + @L-LDH@HMUiO-66(Zr) and NAD + @D-LDH@HMUiO-66(Zr)) are used to prepare standard curves of Glu, Hb, L-LAc and D-LAc metabolic biomarkers. Subsequently, human sera of different diabetic patients or healthy people are added to the probe solution for incubation, and the absorbance intensity is measured by a multifunctional microplate reader. Through the standard curve, the absorbance intensity is converted into the concentration of the corresponding metabolic biomarker to achieve quantitative detection in serum samples.

[0024] The present invention demonstrates the synergistic effect of the anion of the salt and the oil phase on the soft-template self-assembly of HMUiO-66(Zr) under mild conditions. The anion of the salt reduces the crystallization reaction temperature of HMUiO-66(Zr) to as low as 40 °C, while the oil phase stabilizes the micelle template by strengthening the micelle template structure and preventing surfactant dissociation. The synergistic effect of the salt phase and the oil phase can precisely control the size and structure of the mesopores, and the adjustable range of the pore diameter is 6.8 - 18.1 nm. The tunable mesoporous structure of HMUiO-66(Zr) provides an efficient and versatile platform for applications, especially in biosensing and medical diagnosis. Taking HMUiO-66(Zr) as a representative platform, its open and adjustable mesoporous channels can effectively accommodate dehydrogenases and NAD +Cofactors significantly enhance their enzyme immobilization ability. These probes have high loading efficiency and sensitive biosensing ability, with detection limits for Glu, Hb, L-LAc, and D-LAc as low as 6.4 μM, 2.2 μM, 35 μM, and 24 μM, respectively. By integrating these probes into an array, a 4-input AND-NOR logic gate combination is used to simultaneously detect and analyze changes in these metabolic biomarkers, enabling rapid and accurate screening of diabetes acidosis types.

[0025] A hierarchical porous metal-organic framework sensing probe with adjustable pore size and its preparation method and application provided by the present invention have the following beneficial effects compared with the prior art:

[0026] 1) The key role of the salt-oil synergistic effect not only contributes to the construction of different pore size structures in HMUiO-66(Zr), but also provides valuable insights for the synthesis of other hierarchical mesoporous MOFs;

[0027] 2) By quantitatively detecting the contents of Glu, Hb, L-Lac, and D-LAc in human serum by colorimetry, a simple, reliable, and specific detection method is provided for screening diabetes acidosis types;

[0028] 3) The HMUiO-66(Zr) prepared according to the present invention provides a multifunctional platform, highlighting its potential in biomolecule immobilization and disease diagnosis, and at the same time paving the way for broader applications in biosensing and catalytic processes.

[0029] In summary, the present invention relates to a hierarchical porous metal-organic framework sensing probe with adjustable pore size and its preparation method and application. Under the synergistic effect between anions and the oil phase, the size and structure of mesopores can be precisely controlled, enabling the preparation of a variety of hierarchical porous metal-organic framework sensing probes with adjustable pore sizes immobilized with dehydrogenases under mild conditions. This hierarchical porous metal-organic framework sensing probe is also particularly suitable for the specific detection of Glu, Hb, L-Lac, and D-LAc biomarkers in human serum samples, providing a convenient, rapid, and specific detection technology that can be used for the screening and diagnosis of different types of diabetes acidosis, improving the monitoring ability of acute diabetes complications. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1A is a flow chart of the preparation method of the hierarchical porous metal-organic framework sensing probe provided by the present invention, Figure 1B is a schematic diagram of the hierarchical porous metal-organic framework structure (1) and the sensing probe constructed therefrom and its application (2);

[0031] Figure 2Schematic diagram of (A) HMUiO-66(Zr) according to the present invention, (B) XRD pattern of HMUiO-66(Zr), (C) N 2 adsorption isotherm and corresponding BJH pore size distribution curve (inset), (D, E) SEM images of HMUiO-66(Zr), (F - H) TEM images of HMUiO-66(Zr), (I) STEM image of HMUiO-66(Zr) and corresponding element distribution maps of Zr, O, C, Na, and N;

[0032] Figure 3 XRD patterns of (A) HMUiO-66(Zr) and HMUiO-66(Zr) soaked in aqueous solutions with different pH values for 1 day and (B) HMUiO-66(Zr) and HMUiO-66(Zr) soaked in acidic, neutral, and alkaline aqueous solutions for 14 days according to the present invention;

[0033] Figure 4 FT-IR spectra of (a) F127 and (b) HMUiO-66(Zr) according to the present invention;

[0034] Figure 5 TGA curve of HMUiO-66(Zr) sample according to the present invention;

[0035] Figure 6 Heat map of comparison of diffraction peak intensities of UiO-66(Zr) synthesized in aqueous phase with different anions at different temperatures (A), BJH pore size distribution curves of HMUiO-66(Zr) synthesized at 60 °C (black line), 50 °C (red line), and 40 °C (blue line) with 400 μL toluene (B) and without toluene (C), and TEM images of HMUiO-66(Zr) synthesized at 60 °C (D, G), 50 °C (E, H), and 40 °C (F, I) with 400 μL toluene (D - F) and without toluene (G - I) according to the present invention;

[0036] Figure 7 Schematic of the preparation of HMMOFs by soft template self-assembly under the synergistic effect of (a) toluene and NO 3 - synergistic effect of salt-soluble ions. (b) Toluene and Cl - synergistic effect of salting-out ions;

[0037] Figure 8 Schematic of using NaNO 3SEM images of representative HMUiO-66(Zr) samples synthesized by mediating salts and different amounts of toluene (A, E, I, M) 30, (B, F, J, N) 70, and (C, G, K, O) 100 μL or using NaCl to mediate salts and (D, H, L, P) 400 μL of toluene, (I-L) TEM images, (M-P) BJH pore size distribution curves, and partial corresponding N 2 adsorption isotherms (insets);

[0038] Figure 9 are SEM images of HMUiO-66(Zr) samples synthesized according to the present invention using 400 μL of toluene and 8.5 mmol of salts (A) NaCl, (B) NaNO 3 , (C) NaBr, (D) NaI, and (E) NaClO 4 H 2 O;

[0039] Figure 10 are SEM images of HMUiO-66(Zr) samples synthesized according to the present invention using 400 μL of toluene and 8.5 mmol of salts (A) KCl, (B) LiCl, (C) MgCl 2 and (D) CaCl 2 ;

[0040] Figure 11 are XRD patterns of (A) (a) simulated UiO-66(Zr) and HMUiO-66(Zr) samples synthesized using NaNO 3 to mediate salts and different amounts of toluene (b) 30, (c) 50, (d) 70, (e) 90, and (f) 100 μL; (B) (a) simulated UiO-66(Zr) and XRD patterns of HMUiO-66(Zr) samples synthesized using NaCl to mediate salts and different amounts of toluene (b) 50, (c) 100, (d) 200, (e) 400 μL;

[0041] Figure 12 are SEM images of HMUiO-66(Zr) samples synthesized according to the present invention using NaNO 3 to mediate salts and different amounts of toluene (A, B) 30, (C, D) 50, (E, F) 70, (G, H) 90, and (I, J) 100 μL;

[0042] Figure 13 are (A-E) N of HMUiO-66(Zr) samples synthesized according to the present invention using NaNO 3 to mediate salts and different amounts of toluene (A, F, K) 30, (B, G, L) 50, (C, H, M) 70, (D, I, N) 90, and (E, J, O) 100 μL2 Adsorption curve, (F-J) hysteresis loop part and (K-O) BJH pore size distribution diagram;

[0043] Figure 14 SEM images of HMUiO-66(Zr) samples synthesized using NaCl-mediated salt and different amounts of toluene (A, B) 50, (C, D) 100, (E, F) 200, and (G, H) 400 μL according to the present invention;

[0044] Figure 15 (A-D) N of HMUiO-66(Zr) samples synthesized using NaCl-mediated salt and different amounts of toluene (A, E, I) 50, (B, G, J) 100, (C, G, K) 200, and (D, H, L) 400 μL according to the present invention; 2 Adsorption curve, (E-H) hysteresis loop part and (I-L) BJH pore size distribution diagram;

[0045] Figure 16 (A-D) Study on the effect of the mesopore size of HMUiO-66(Zr) on the loading capacity of (A) GDH, (B) 3-HBDH, (C) L-LDH, and (D) D-LDH according to the present invention, and (E-H) and their colorimetric response study on 25 μM Glu, Hb, L-LAc, and D-LAc;

[0046] Figure 17 Effect of incubation time, Glu concentration, Hb concentration, L-LAc concentration, and D-LAc concentration on the absorbance intensity of (A) NAD + @GDH@HMUiO-66(Zr) probe, (B) NAD + @3-HBDH@HMUiO-66(Zr) probe, (C), NAD + @L-LDH@HMUiO-66(Zr) and (D) NAD + @D-LDH@HMUiO-66(Zr) probe;

[0047] Figure 18 Effect of (A) NAD + @GDH@HMUiO-66(Zr) probe, (B) NAD + @3-HBDH@HMUiO-66(Zr) probe, (C), NAD + @L-LDH@HMUiO-66(Zr) and (D) NAD + Linear fitting curves of the absorbance of @D-LDH@HMUiO-66(Zr) probe varying with the concentrations of Glu, Hb, L-LAc, and D-LAc;

[0048] Figure 19 A 4-input AND-NOR logic gate circuit is constructed by integrating four probes according to the present invention into a sensing array;

[0049] Figure 20 is based on various possible co-existing interferents (A) NAD + @GDH@HMUiO-66(Zr) probe, (B) NAD + @3-HBDH@HMUiO-66(Zr) probe, (C), NAD + @L-LDH@HMUiO-66(Zr) and (D) NAD + Study on the influence of @D-LDH@HMUiO-66(Zr) probe sensing response;

[0050] Figure 21 is based on the study of the influence of (A) simultaneous addition of Glu and other interfering substances on the response of NAD + @GDH@HMUiO-66(Zr) probe, where (1) Glu, (2) Glu + K + , (3) Glu + Na + , (4) Glu + Cl - , (5) Glu + Urea, (6) Glu + Chol, (7) Glu + Cre, (8) Glu + BSA; (B) study on the influence of simultaneous addition of Hb and other interfering substances on the sensing response of NAD + @3-HBDH@HMUiO-66(Zr) probe, where (1) Hb, (2) Hb + K + , (3) Hb + Na + , (4) Hb + Cl - , (5) Hb + Urea, (6) Hb + Chol, (7) Hb + Cre, (8) Hb + BSA; (C) study on the influence of simultaneous addition of L-LAc and other interfering substances on the response of NAD + @L-LDH@HMUiO-66(Zr) probe, where (1) L-LAc, (2) L-LAc + K + , (3) L-LAc + Na + , (4) L-LAc + Cl - , (5) L-LAc + Urea, (6) L-LAc + Chol, (7) L-LAc + Cre, (8) L-LAc + BSA; (D) study on the influence of simultaneous addition of D-LAc and other interfering substances on the response of NAD + @D-LDH@HMUiO-66(Zr) probe, where (1) D-LAc, (2) D-LAc + K+ , (3) D-LAc + Na + , (4) D-LAc + Cl - , (5) D-LAc + Urea, (6) D-LAc + Chol, (7) D-LAc + Cre, (8) D-LAc + BSA;

[0051] Figure 22 is the crosstalk analysis of the array's response to different Glu concentrations according to the present invention (A) in the presence of high concentrations of Hb, L-LAc, and D-LAc; (B) the crosstalk analysis of the array's response to different Hb concentrations in the presence of high concentrations of Glu, L-LAc, and D-LAc; (C) the crosstalk analysis of the array's response to different L-LAc concentrations in the presence of high concentrations of Glu, Hb, and D-LAc; (D) the crosstalk analysis of the array's response to different D-LAc concentrations in the presence of high concentrations of Glu, Hb, and L-LAc;

[0052] Figure 23 is the study of detecting Glu, Hb, L-LAc, and D-LAc in (A) healthy human serum, (B) simulated DKA serum, (C) simulated L-lactic acidosis serum, and (D) simulated D-lactic acidosis serum respectively using an integrated array according to the present invention. Detailed implementation manners

[0053] The following combines with the attached drawings to give the preferred embodiments of the present invention and describes them in detail.

[0054] As Figure 1A shown, it is a flowchart of the preparation method of a hierarchical porous metal-organic framework sensing probe provided by the present invention, which includes the steps: guiding the formation of the metal-organic framework using a template agent (such as F127), and using the synergistic effect between salts and the oil phase to hydrothermally generate a hierarchical porous metal-organic framework with adjustable mesoporous pore size under mild conditions; subsequently, obtaining hierarchical porous metal-organic framework nanoparticles UiO-66(Zr) with adjustable pore size through activation and template removal; loading the NAD + cofactor into the hierarchical porous metal-organic framework nanoparticles to obtain a hierarchical porous metal-organic framework nanoparticle with mesoporous channels and the cofactor immobilized thereon; loading GDH, 3-HBDH, L-LDH, and D-LDH into the mesoporous channels of the hierarchical porous metal-organic framework nanoparticles with the cofactor immobilized thereon respectively to obtain a variety of hierarchical porous metal-organic framework sensing probes with adjustable pore sizes and dehydrogenases immobilized thereon, NAD + @GDH@HMUiO-66(Zr), NAD + @3-HBDH@HMUiO-66(Zr), NAD +@L-LDH@HMUiO-66(Zr) and NAD + @D-LDH@HMUiO-66(Zr).

[0055] As Figure 1B shown, it is the hierarchical porous metal-organic framework structure according to the present invention and the sensing probe constructed therefrom and its application. (1) In the mesopores of the hierarchical porous metal-organic framework sensing probe prepared by the above method, both dehydrogenase and the cofactor of nicotinamide adenine dinucleotide NAD + are accommodated. By utilizing the specific recognition of dehydrogenase for metabolic biomarkers, these enzymes catalyze the oxidation of Glu, Hb, L-LAc, and D-LAc in the mesopores of HMUiO-66(Zr), converting an equal amount of NAD + into NADH. Meanwhile, NADH interacts with the electron transfer agent of PMS, catalyzing the MTT colorimetric reaction and realizing the rapid and intuitive detection of metabolites. (2) Integrating the above four probes into the same sensing array, Glu, Hb, L-LAc, and D-LAc are analyzed simultaneously by color change. When exposed to the corresponding metabolites, each probe will produce a different color response, facilitating the rapid on-site detection of the negativity or positivity of these biomarkers through digital output (0 / 1).

[0056] Example 1

[0057] Synthesis of 1.1 HMUiO-66(Zr)

[0058] At room temperature, 100 mg of F127 was dissolved in 6 mL of deionized water. Subsequently, HAc (125 μL, 2.19 mmol), toluene (30 μL, 70 μL, and 100 μL), and NaNO 3 (722 mg, 8.5 mmol) were added to the solution for the synthesis of HMUiO-66(Zr). The mixture was stirred at 30 °C for 1 hour to obtain a homogeneous solution. Then, BDC (166 mg, 1 mmol) was added and stirred for 20 minutes. Subsequently, ZrO(NO 3 ) 2 ·2H 2 O (231 mg, 1 mmol) was added to the solution. The reaction mixture was stirred in a water bath at 60 °C for 24 hours. After that, the obtained sample was centrifuged and washed twice with clear water, and then washed twice with DMF to remove the residual unreacted ligands. Then, the synthesized sample was washed with ethanol and soaked in ethanol at 60 °C for two days, with the ethanol being replaced every day. Finally, the sample was dried under vacuum at 80 °C for 24 hours.

[0059] Figure 2 where A is in NaNO 3In the presence of toluene as the salt phase and the oil phase, typical HMUiO-66(Zr) with open large cage-like mesopores coupled with a microporous crystal wall was synthesized. Figure 2 In B is the wide-angle X-ray diffraction (XRD) pattern of HMUiO-66(Zr), which is in good agreement with the simulated pattern of UiO-66(Zr), confirming the formation of the crystal framework. Figure 2 In C is N 2 adsorption isotherm and its corresponding Barrett-Joyner-Halenda (BJH) pore size distribution. The sample shows a combination of type I and type IV isotherms, indicating the coexistence of micropores and mesopores in HMUiO-66(Zr). The total BET surface area is approximately 960 m 2 / g, of which the mesoporous specific surface area is 281 m 2 / g. The mesopore size of the adsorption branch was measured by the BJH method to be approximately 10.6 nm. Generally, cage-like mesoporous materials show typical H2-type or H1-type hysteresis loops due to relatively narrow mesopore connections. Interestingly, the adsorption branch of the HMUiO-66(Zr) sample shows a sharp step at P / P 0 of 0.75, corresponding to the presence of mesopores around 10.6 nm. In the range of P / P 0 from 0.6 to 0.85, the adsorption and desorption branches almost coincide, indicating the absence of an obvious hysteresis effect. This phenomenon can be attributed to the unique mesoporous structure of HMUiO-66(Zr), whose small particle size ensures that almost all mesopores are open and accessible, enabling N 2 molecules to enter and exit quickly without an obvious hysteresis effect. Figure 2 In D-E are the scanning electron microscopy (SEM) images of HMUiO-66(Zr). HMUiO-66(Zr) consists of uniform monodisperse spherical NPs with a diameter of 70 - 90 nm ( Figure 2 in D). At higher magnification, uniformly ordered mesopore entrances can be clearly resolved on the surface of HMUiO-66(Zr) NPs ( Figure 2 in E). Figure 2 In F-H are the transmission electron microscopy (TEM) images of HMUiO-66(Zr), which further confirm that HMUiO-66(Zr) is well-dispersed and ordered large mesopores are uniformly distributed throughout the NPs. The small size of HMUiO-66(Zr) NPs makes most mesopores highly open, thus facilitating the effective diffusion of biomacromolecules within HMUiO-66(Zr). This structural feature further explains the absence of a hysteresis loop in the N 2 adsorption isotherm. Figure 2In I, scanning TEM (STEM) and elemental mapping of HMUiO-66(Zr) NPs revealed uniform distribution of Zr, C, and O elements, and the absence of Na and N elements, indicating that the salt of NaNO 3 was completely removed. Figure 3 In A-B is the XRD pattern of HMUiO-66(Zr), which exhibits excellent chemical and thermal stability and can maintain its crystal structure after exposure to solutions with pH values ranging from 1 to 12 ( Figure 3 in A). Additionally, it shows remarkable long-term stability and remains intact even after being placed in acidic, neutral, and alkaline solutions for 14 days ( Figure 3 in B). Figure 4 is the FTIR pattern of HMUiO-66(Zr). The FTIR spectrum indicates that the F127 surfactant template has been completely removed from these HMUiO-66(Zr) structures. Figure 5 is the thermogravimetric pattern of HMUiO-66(Zr). The thermal decomposition temperature of HMUiO-66(Zr) can reach 517 °C, which is similar to the decomposition temperature of HMUiO-66(Zr) reported previously. It is calculated that HMUiO-66(Zr) contains a large number of defects, and each Zr 6 cluster is connected by only 8.98 linkers.

[0060] Figure 6 In A, the effects of different anions (including SO 4 2- , HPO 4 2- , Cl - , NO 3 - , Br - , I - and ClO 4 - ) on the synthesis of HMUiO-66(Zr) in aqueous phase were studied, and a heat map was generated with various anions on the x-axis and reaction temperature on the y-axis. The color gradient from purple to red represents the intensity of the strongest diffraction peak of the UiO-66(Zr) structure at 7.4 0 . The visualized heat map can clearly compare the promoting effects of different anions on the crystallization of UiO-66(Zr). When no salt is present in the reaction, no UiO-66(Zr) product is formed within the reaction temperature range of 40 - 60 °C. While anions such as Cl - , NO 3 - , Br - , I - and ClO 4 - can promote the formation of crystalline UiO-66(Zr) within the reaction temperature range of 40 - 60 °C. A large number of literature reports indicate that Zr6 or Hf 6 The formation of clusters is a key step in the mild synthesis of Zr-based and Hf-based MOFs, unlike direct synthesis methods that usually require harsh conditions above 100 °C. The anions of the salts may promote the formation of clusters and lower the energy barrier through a solubilization mechanism, enabling UiO-66(Zr) to continue crystallizing in the aqueous phase under milder conditions. Among these anions, NO 3 - 、Br - 、I - and ClO 4 - salting-in ions promote the crystallization of UiO-66(Zr) better than the salting-out ion Cl - . This is because the salting-out ion reduces the solubility of BDC in the aqueous phase due to the Hofmeister effect, resulting in slow growth and weak crystallinity of the product. Finally, other salting-out ions such as SO 4 2- and HPO 4 2- only produce amorphous products. This is because SO 4 2- and HPO 4 2- have strong coordination interactions with Zr, much stronger than the interaction between Zr and the carboxyl group of the BDC ligand, thus preventing the formation of UiO-66(Zr). Figure 6 In B, when using NO 3 - as the additive medium and toluene as the oil phase, all HMUiO-66(Zr) synthesized at different temperatures formed a uniform mesoporous BJH pore size distribution curve. At 60 °C, due to the weakened interaction between the PEO layer of the F127 micelle and water, the hydrophobicity of the PEO layer was enhanced, resulting in larger mesopore sizes. This change promoted more hydrophobic toluene to penetrate into the micelle core, promoting their swelling and ultimately forming larger mesopores. Figure 6 In C, when using NO 3 - as the additive medium and without introducing toluene, for the BJH pore size distribution curves of all HMUiO-66(Zr) synthesized at different temperatures, HMUiO-66(Zr) could not form micelle-templated large mesopores between 40 - 60 °C. Figure 6 In D-I are the TEM images of HMUiO-66(Zr), further showing that when using toluene as the oil phase, large mesopores were formed in HMUiO-66(Zr) ( Figure 6 in D-F of Figure 6In (G-I). These observations prompted us to propose a mechanism in which the oil phase plays a key role in the formation of mesopores in HMMOFs. Generally, the F127 molecules in the micelles undergo slow dynamic exchange with the free F127 molecules in the aqueous phase. The soft template is unstable during the slow crystallization of MOFs at the micelle interface, resulting in the dynamic dissociation of F127 molecules from the micelles, preventing the formation of mesopores. Even when a small amount of toluene is added as the oil phase, toluene enters the hydrophobic core of the micelles, significantly improving the stability of the emulsion. This stability stems from the strong hydrophobic interaction between toluene and the hydrophobic PPO layer of the micelles, thus expanding the synthesis window of soft template self-assembly even at high reaction temperatures.

[0061] Figure 7 is a schematic diagram for the preparation of HMUiO-66(Zr). The salt phase and toluene oil phase act synergistically to regulate the mesopore size and structure of HMMOFs. When using NO 3 - salting-in ions, the hydrophobic interaction between toluene and the PEO of the F127 micelles allows toluene to regulate the size of the nanoemulsion, thereby controlling the mesopore size of HMUiO-66(Zr) ( Figure 7 in a). Further confirmation shows that Cl - salting-out ions increase the hydrophobicity of the micelles, enabling more toluene to enter the micelle core and promoting the formation of larger mesopores in HMUiO-66(Zr) ( Figure 7 in b). Figure 8 In (A-L) are the SEM and TEM images of HMUiO-66(Zr). When using NO 3 - salting-in ions, as the toluene volume increases from 30 μL to 100 μL, the mesopore entrances and internal mesopore sizes of HMUiO-66(Zr) gradually increase ( Figure 8 in (A-C) and (E-G)). Figure 8 In (M-P) are the N 2 adsorption isotherm and BJH pore size distribution curves of HMUiO-66(Zr). Calculations show that the average mesopore diameter can be adjusted between 6.8 - 10.3 nm ( Figure 8 in (M-O)). In contrast, when using Cl - salting-out ions, the degree of emulsion swelling increases significantly, forming open, branched mesopores with a pore size of up to about 18.1 nm ( Figure 8 in D, H, L, P). Figure 9 is the SEM image of HMUiO-66(Zr). Various salt ions, including NO 3 - , Br - , I - and ClO 4 -, it has limited impact on mesopore expansion. Adding excessive toluene cannot form larger mesopores, indicating that the swelling of the nanoemulsion has reached its limit. Figure 10 is the SEM image of HMUiO-66(Zr). Different chloride salts (KCl, LiCl, MgCl 2 , CaCl 2 ) also formed large and open mesopores similar to those of NaCl. Figure 11 is the XRD pattern of HMUiO-66(Zr) synthesized by adding various salt ions. The introduction of toluene oil phase did not destroy the crystal structure of HMUiO-66(Zr). Figures 12 - 15 are the SEM image, N 2 adsorption-desorption curve, and BJH pore size distribution curve of HMUiO-66(Zr) synthesized by adding various anions and carefully adjusting the volume of toluene, respectively. The entire process can continuously and precisely control the mesopore size to meet the requirements of specific pore sizes for different applications.

[0062] 1.2 NAD + @Synthesis of dehydrogenase@HMUiO-66(Zr) probe

[0063] At room temperature, 0.5 mL of HMUiO-66(Zr) suspension (4 mg / mL) was mixed with 0.5 mL of NAD + solution (2 mg / mL). The mixture was shaken at 25 °C for 1 hour. Next, 0.5 mL of GDH, 3-HBDH, L-LDH, and D-LDH solutions (2 mg / mL, dissolved in 20 mM Tris-HCl buffer, pH 7.4) were added to 0.5 mL of NAD + @HMUiO-66(Zr) suspension, and shaken at 25 °C for 1 hour. The enzyme-loaded NAD + @HMUiO-66(Zr) was collected by centrifugation, washed twice with water, and then dispersed in 2 mL of Tris-HCl buffer (20 mM, pH 7.4) for further use.

[0064] Figure 16 shows the effect of the mesopore size of HMUiO-66(Zr) on the loading capacity of (A) GDH, (B) 3-HBDH, (C) L-LDH, and (D) D-LDH, as well as their colorimetric responses to 25 μM Glu, Hb, L-LAc, and D-LAc. Different mesopore-sized HMUiO-66(Zr) were used to immobilize NAD + and dehydrogenases. Since the three-dimensional sizes of GDH, 3-HBDH, L-LDH, and D-LDH are approximately 4.4×5.1×6.5 nm, 3.8×4.4×5.2 nm, 4.5×5.2×6.3 nm, and 4.1×5.5×6.5 nm, respectively, sufficient space is required to accommodate the dehydrogenases and NAD+ Fixation. When the mesopore size increased from micropores to 10.6 nm, the loading amounts of GDH, 3-HBDH, L-LDH, and D-LDH increased significantly from 38, 31, 27, and 28 mg / g to 227, 207, 195, and 169 mg / g, respectively. When the mesopore size of HMUiO-66(Zr) expanded from micropores to 10.6 nm, the colorimetric responses for detecting Glu, Hb, L-LAc, and D-LAc (25 μM) increased significantly..

[0065] 1.3 Response Kinetics of Hierarchical Porous Metal-Organic Framework Sensing Probe

[0066] Add 140 μL of Glu solutions with different concentrations into a 96-well plate containing Tris-HCl buffer (20 mM, pH 7.4), and then add 20 μL of NAD + @GDH@HMUiO-66(Zr) suspension (1000 mg / mL). Then, add 40 μL of the chromogenic solution into the plate. Incubate the mixture at 37 °C for 20 minutes. Then, at fixed time intervals, detect the absorbance at 570 nm using a multifunctional microplate reader. The chromogenic solution was prepared by mixing 10 mL of PMS (4 mM, dissolved in Tris-HCl buffer (100 mM, pH 8)) with 10 mL of MTT (10 mM, dissolved in Tris-HCl buffer (100 mM, pH 8)), and it was used immediately after mixing. Subsequently, the same method was adopted to study the response kinetics of NAD + @3-HBDH@HMUiO-66(Zr), NAD + @L-LDH@HMUiO-66(Zr) and NAD + @D-LDH@HMUiO-66(Zr) probes.

[0067] Figure 17 Are the incubation time, Glu concentration, Hb concentration, L-LAc concentration, and D-LAc concentration on (A) NAD + @GDH@HMUiO-66(Zr) probe, (B) NAD + @3-HBDH@HMUiO-66(Zr) probe, (C), NAD + @L-LDH@HMUiO-66(Zr) and (D) NAD +Effect of @D-LDH@HMUiO-66(Zr) probe on absorbance intensity. The detection performance of the probe was characterized in detail. It can be seen from the response kinetics that as the incubation time prolonged, the absorbance intensity of these sensors at 570 nm gradually increased, and Glu, Hb, L-LAc, and D-LAc rapidly reached saturation at approximately 20, 10, 20, and 20 min, respectively. Therefore, in order to balance the consistency of array operation and the need for rapid detection of diabetic acidosis, 20 min was set as the equilibration time for subsequent sensing detection.

[0068] 1.4 Quantitative Analysis of Hierarchical Porous Metal-Organic Framework Sensing Probe

[0069] Add 140 μL of Glu standard solutions with different concentrations to a 96-well plate containing Tris-HCl buffer (20 mM, pH 7.4), and then add 20 μL of NAD + @GDH@HMUiO-66(Zr) suspension (1000 mg / mL). Then, add 40 μL of chromogenic solution to the plate. Incubate the mixture at 37 °C for 20 minutes, and detect the absorbance at 570 nm using a multifunctional microplate reader. The chromogenic solution was prepared by mixing 10 mL of PMS (4 mM, dissolved in Tris-HCl buffer (100 mM, pH 8)) with 10 mL of MTT (10 mM, dissolved in Tris-HCl buffer (100 mM, pH 8)), and used immediately after mixing to generate a standard curve for Glu. Subsequently, the same method was used to generate standard curves for Hb, L-LAc, and D-LAc using NAD + @3-HBDH@HMUiO-66(Zr), NAD + @L-LDH@HMUiO-66(Zr) and NAD + @D-LDH@HMUiO-66(Zr) probes.

[0070] Figure 18 is (A) NAD + @GDH@HMUiO-66(Zr) probe, (B) NAD + @3-HBDH@HMUiO-66(Zr) probe, (C), NAD + @L-LDH@HMUiO-66(Zr) and (D) NAD +Linear fitting curves of the absorbance of the @D-LDH@HMUiO-66(Zr) probe varying with the concentrations of Glu, Hb, L-LAc, and D-LAc. The colorimetric response shows that the increase in absorbance at 570 nm has a good linear correlation with the concentration changes of the four metabolic biomarkers. Using the 3σ rule, the limits of detection (LOD) of these probes for Glu, Hb, L-LAc, and D-LAc were determined to be 6.4 μM, 2.2 μM, 35 μM, and 24 μM, respectively, which are much lower than the concentration ranges of the metabolic biomarkers in the serum of patients with diabetic acidosis.

[0071] 1.5 Colorimetric Sensing Detection of Hierarchical Porous Metal-Organic Framework Sensing Array in the Presence of Interferents

[0072] Interference substances (potassium (K + ), sodium (Na + ), chloride (Cl - ), urea, cholesterol, creatinine, and bovine serum albumin (BSA)) at different concentrations were dissolved in the NAD + @GDH@HMUiO-66(Zr) probe solution, equilibrated for 20 minutes, and the probe solution was subjected to colorimetric determination to obtain the luminescence intensity at λ = 570 nm. In addition, interference substances (K + , Na + , Cl - , urea, cholesterol, creatinine, and BSA) at a concentration of 100 μM were dissolved in the probe solution, and different concentrations of Glu solution were added, equilibrated for 20 minutes, and the probe solution was subjected to colorimetric determination to obtain the luminescence intensity at λ = 570 nm. Subsequently, high-concentration Hb, L-LAc, and D-LAc solutions were dissolved in the NAD + @GDH@HMUiO-66(Zr) probe solution, equilibrated for 20 minutes, and the probe solution was subjected to colorimetric determination to obtain the luminescence intensity at λ = 570 nm. Subsequently, the same method was used to study the selectivity, anti-interference ability, and cross-interference of the NAD + @3-HBDH@HMUiO-66(Zr), NAD + @L-LDH@HMUiO-66(Zr), and NAD + @D-LDH@HMUiO-66(Zr) probes.

[0073] Figure 19 Four of these probes were integrated into a sensing array to simultaneously detect multiple metabolic biomarkers related to diabetic acidosis and construct a 4-input AND-NOR logic circuit by examining the color change patterns to quickly and accurately classify different types of diabetic acidosis based on the correlations between the biomarkers. Figure 20 Shows the effects of various possible co-existing interferents on (A) NAD +@GDH@HMUiO-66(Zr) probe, (B) NAD + @3-HBDH@HMUiO-66(Zr) probe, (C), NAD + @L-LDH@HMUiO-66(Zr) and (D) NAD + Effect of @D-LDH@HMUiO-66(Zr) probe sensing response. K + , Na + , Cl - , urea, cholesterol, creatinine and BSA responses were negligible, while after introducing the corresponding metabolic biomarkers, the absorbance at 570 nm increased significantly. Figure 21 is the effect of simultaneous addition of Glu and other interfering substances on NAD + @GDH@HMUiO-66(Zr) probe sensing response. Among them, (1) Glu, (2) Glu + K + , (3) Glu + Na + , (4) Glu + Cl - , (5) Glu + Urea, (6) Glu + Chol, (7) Glu + Cre, (8) Glu + BSA. (B) Effect of simultaneous addition of Hb and other interfering substances on NAD + @3-HBDH@HMUiO-66(Zr) probe sensing response. Among them, (1) Hb, (2) Hb + K + , (3) Hb + Na + , (4) Hb + Cl - , (5) Hb + Urea, (6) Hb + Chol, (7) Hb + Cre, (8) Hb + BSA. (C) Effect of simultaneous addition of L-LAc and other interfering substances on NAD + @L-LDH@HMUiO-66(Zr) probe sensing response. Among them, (1) L-LAc, (2) L-LAc + K + , (3) L-LAc + Na + , (4) L-LAc + Cl - , (5) L-LAc + Urea, (6) L-LAc + Chol, (7) L-LAc + Cre, (8) L-LAc + BSA. (D) Effect of simultaneous addition of D-LAc and other interfering substances on NAD + @D-LDH@HMUiO-66(Zr) probe sensing response. Among them, (1) D-LAc, (2) D-LAc + K + , (3) D-LAc + Na + , (4) D-LAc + Cl -, (5) D-LAc + Urea, (6) D-LAc + Chol, (7) D-LAc + Cre, (8) D-LAc + BSA. When these interfering substances coexist with Glu, Hb, L-LAc, and D-LAc, there is no obvious effect on the sensing response. Figure 22 is (A) the crosstalk analysis of the array's response to different Glu concentrations in the presence of high concentrations of Hb, L-LAc, and D-LAc. (B) the crosstalk analysis of the array's response to different Hb concentrations in the presence of high concentrations of Glu, L-LAc, and D-LAc. (C) the crosstalk analysis of the array's response to different L-LAc concentrations in the presence of high concentrations of Glu, Hb, and D-LAc. (D) the crosstalk analysis of the array's response to different D-LAc concentrations in the presence of high concentrations of Glu, Hb, and L-LAc. In the presence of Glu, L-LAc, and D-LAc, the sensing array effectively responded to changes in the Hb concentration, while high concentrations of Glu, L-LAc, and D-LAc did not cause an obvious colorimetric response. Similarly, due to the high specificity of enzyme recognition, no crosstalk interference was observed when detecting Hb, L-LAc, and D-LAc.

[0074] Application Example 1

[0075] 2.1 Hierarchical Porous Metal-Organic Framework Sensing Array for Quantitative Detection of Glu, Hb, L-LAc and D-LAc Biomarkers in Serum of Diabetic Acidosis Patients

[0076] Human serum samples were diluted 30-fold, 20-fold, 10-fold, and 10-fold with Tris-HCl buffer (20 mM, pH 7.4) for the detection of Glu, Hb, L-LAc, and D-LAc to ensure that the biomarker concentrations in the serum samples fell within the linear detection range of the sensing array. 140 μL of serum was added to a 96-well plate containing Tris-HCl buffer (20 mM, pH 7.4), and then 20 μL of NAD + @GDH@HMUiO-66(Zr) suspension (1000 mg / mL) was added. Then, 40 μL of the chromogenic solution was added to the plate. The mixture was incubated at 37 °C for 20 minutes, and the absorbance at 570 nm was measured using a multifunctional microplate reader. The absorbance intensity was converted into glucose concentration through the calibration curve for Glu quantification. Subsequently, using the same method, the concentrations of Hb, L-LAc, and D-LAc in the serum were detected using NAD + @3-HBDH@HMUiO-66(Zr), NAD + @L-LDH@HMUiO-66(Zr), and NAD + @D-LDH@HMUiO-66(Zr) probes.

[0077] Figure 23An integrated array was used to detect Glu, Hb, L-LAc, and D-LAc in (A) healthy human serum, (B) simulated DKA serum, (C) simulated L-lactic acidosis serum, and (D) simulated D-lactic acidosis serum, respectively. The absorbance responses of the array to Glu, Hb, and L-LAc biomarkers in healthy human serum samples were enhanced, indicating that the probe could effectively detect the corresponding biomarkers in real serum samples. By converting the absorbance enhancement into biomarker concentration, the levels of Glu, Hb, and L-LAc were determined to be approximately 5505, 220, and 916 μM, respectively, while almost no D-LAc was detected in healthy serum samples. To further evaluate the effectiveness of the array, 6000 μM Glu and 4000 μM Hb were additionally added to healthy serum samples to simulate DKA conditions. We observed that the absorbance responses of the array to Glu and Hb in the simulated DKA serum samples were significantly enhanced compared with those in healthy serum samples. The detected values of Glu and Hb in the simulated DKA samples were approximately 11704 μM and 4120 μM, respectively, and the recovery rates were 102.7% and 98.1%, respectively, further verifying the accuracy of the array. Similarly, the array effectively detected the increased levels of L-LAc and D-LAc in the simulated L-lactic acidosis and simulated D-lactic acidosis serum samples, indicating its ability to rapidly distinguish different types of diabetic acidosis based on the relevant changes in the levels of Glu, Hb, L-LAc, and D-LAc. Table 1 below shows the recovery of alkaline phosphatase (ALP) added additionally in real healthy serum samples and simulated DKA, L-lactic acidosis, and D-lactic acidosis samples determined by the integrated array.

[0078] Table 1

[0079] In summary, the present invention utilizes the formation of templating agent-directed metal-organic frameworks, and the synergistic effect between salts and the oil phase to hydrothermally synthesize HMUiO-66(Zr). The synergistic effect of salt ions and the oil phase on the soft-template self-assembly of HMUiO-66(Zr) under mild conditions was confirmed. The anions enabled the crystallization reaction temperature of HMUiO-66(Zr) to be as low as 40 °C, while the oil phase stabilized the micelle template by strengthening the micelle template structure and preventing surfactant dissociation. The synergistic effect of the salt phase and the oil phase can precisely control the size and structure of the mesopores, and the adjustable pore size range is 6.8 - 18.1 nm. The adjustable mesoporous structure of HMUiO-66(Zr) provides an efficient and versatile platform for applications, especially in biosensing and medical diagnosis. Taking HMUiO-66(Zr) as a representative platform, its open and adjustable mesoporous channels can effectively accommodate dehydrogenases and NAD +Cofactors significantly enhance their enzyme immobilization ability. These probes have high loading efficiency and sensitive biosensing ability. By integrating these probes into an array and using a 4-input AND-NOR logic gate combination to simultaneously detect and analyze changes in these metabolic biomarkers, different types of diabetic acidosis can be rapidly screened through the relevant changes in metabolic biomarkers, demonstrating their potential in providing a critical time window for timely treatment of acute and severe diseases.

[0080] The above are only the preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various changes can be made to the above embodiments of the present invention. That is, all simple, equivalent changes and modifications made in accordance with the claims and the content of the specification of the present invention application fall within the scope of the claims of the present invention patent. Those not described in detail in the present invention are all conventional technical contents.

Claims

1. A method for preparing a hierarchical porous metal organic framework sensing probe with adjustable pore size, characterized in that: The preparation method comprises the following steps: S1, under the synergistic effect of salt and hydrophobic oil phase solvent, hierarchical porous metal organic framework nanoparticles with open large cage-like mesoporous channels and microporous crystal walls are obtained; S2, NAD + The cofactor is loaded into the hierarchical porous metal organic framework nanoparticles to obtain a hierarchical porous metal organic framework nanoparticle with mesoporous channels and fixed with the cofactor; S3, glucose dehydrogenase, β-hydroxybutyrate dehydrogenase, L-lactate dehydrogenase and D-lactate dehydrogenase are loaded into the mesoporous channels of the hierarchical porous metal organic framework nanoparticles fixed with cofactors, and a plurality of hierarchical porous metal organic framework sensing probes NAD fixed with dehydrogenases with adjustable pore sizes are obtained. + @GDH@HMUiO-66(Zr), NAD + @3-HBDH@HMUiO-66(Zr), NAD + @L-LDH@HMUiO-66(Zr) and NAD + @D-LDH@HMUiO-66(Zr).

2. The preparation method according to claim 1, characterized in that: The step S1 comprises: S11, using a template to direct the formation of a metal organic framework and taking advantage of the synergistic effect between salt and a hydrophobic oil phase solvent, a hierarchical porous metal organic framework with adjustable mesopore size is generated under mild conditions by a hydrothermal method; S12, hierarchical porous metal-organic framework nanoparticles with tunable pore sizes were obtained by activation and de-templating.

3. The preparation method according to claim 2, characterized in that: In step S11, the mild condition refers to a reaction temperature of 40-60°C.

4. The preparation method according to claim 1 or 2, characterized in that: The mesopore size can be adjusted by adjusting the feed amount of the hydrophobic oil phase solvent and / or the type of salt in step S1.

5. The preparation method according to claim 1, characterized in that: The mesopore diameter of the hierarchical porous metal organic framework nanoparticles is continuously adjustable between 6.2-18.1 nm.

6. The preparation method according to claim 1, characterized in that: In step S1, the salt includes sulfate, phosphate, nitrate, chloride, bromide, or iodide, and the hydrophobic oil phase solvent includes toluene, triethylbenzene, trimethylbenzene, or xylene.

7. A sensor probe prepared by a hierarchical porous metal organic framework obtained by the preparation method according to any one of claims 1 to 6, characterized in that: The dehydrogenase and cofactor were separately immobilized in the mesopores of the hierarchical porous metal-organic framework nanoparticles.

8. An application of the hierarchical porous metal organic framework sensing probe according to claim 7, characterized in that: Using the specific recognition of metabolic biomarkers by dehydrogenase, dehydrogenase catalyzes the oxidation of glucose, β-hydroxybutyrate, L-lactic acid, and D-lactic acid in the mesopores of HMUiO-66(Zr), converting equal amounts of NAD + Converted into NADH, at the same time, NADH interacts with the electron transfer agent of PMS to catalyze the MTT colorimetric reaction, thereby achieving rapid and intuitive detection of metabolic markers.

9. The use according to claim 8, characterized in that: The detection responses of glucose, β-hydroxybutyrate, L-lactic acid and D-lactic acid solutions in the concentration range of 0-500 μM were measured using a graded porous metal-organic framework sensing probe, and a linear fitting analysis was performed.

10. The use according to claim 8, characterized in that: Four hierarchical porous metal-organic framework sensing probes NAD + @GDH@HMUiO-66(Zr), NAD + @3-HBDH@HMUiO-66(Zr), NAD + @L-LDH@HMUiO-66(Zr) and NAD + @D-LDH@HMUiO-66(Zr) was integrated into a sensor array to simultaneously analyze glucose, β-hydroxybutyrate, L-lactic acid, and D-lactic acid through colorimetric changes. When exposed to the corresponding metabolites, each probe produced a different color response, thereby achieving accurate screening of diabetic acidosis types.

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