An aperture-adjustable hierarchical porous metal-organic framework sensing probe, a preparation method and application thereof

Abstract

The application discloses a kind of adjustable aperture hierarchical porous metal organic framework sensing probe and its preparation method and its application, comprising 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 channel and microporous crystal wall are obtained;S2, NAD + cofactor is loaded into hierarchical porous metal organic framework nanoparticles;S3, glucose dehydrogenase, beta-hydroxybutyric acid dehydrogenase, L-lactic acid dehydrogenase and D-lactic acid dehydrogenase are loaded into the mesoporous channel of the organic framework nanoparticles, respectively, to obtain a variety of adjustable aperture hierarchical porous metal organic framework sensing probes with dehydrogenase immobilized. The probe can be used for rapid, accurate and simultaneous detection of glucose, beta-hydroxybutyric acid, L-lactic acid and D-lactic acid biomarkers in human serum samples, and is particularly suitable for screening and diagnosis of different types of diabetic acidosis.

Description

TECHNICAL FIELD

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

[0002] Dysregulation of metabolic pathways, such as carbohydrates, acids and lipids, is closely related to diseases such as diabetes, obesity, cardiovascular disease and cancer (Nat. Rev. Urol. 2019, 16, 339-362). Diabetic acidosis is an acute complication of diabetes, and the typical manifestations are diabetic ketoacidosis (DKA) or lactic acidosis (Pediatr. Nephrol. 2018, 33, 673-681). DKA is derived from insulin deficiency, leading to abnormal fat metabolism and excessive accumulation of ketone bodies (such as β-hydroxybutyrate, acetoacetate), thereby increasing blood acidity and damaging organ function (Anal. Chem. 2020, 92, 2291-2300). Although the treatment of diabetes has improved, this life-threatening disease is still the main cause of morbidity and mortality in type 1 and type 2 diabetes in some severe cases (Diabetic Med. 2015, 32, 14-23). Elevated levels of L-lactate (L-LAc) or D-lactate (D-LAc) exacerbate metabolic acidosis, leading to more pronounced acidosis and higher mortality in DKA patients, and poorer recovery (J. Crit. Care. 2023, 78, 154377).

[0003] Therefore, rapid, accurate and simultaneous detection of metabolic biomarkers, including glucose (Glu), β-hydroxybutyrate (Hb), L-lactate (L-LAc) and D-lactate (D-LAc), is crucial for diagnosing and assessing the severity of diabetic acidosis. SUMMARY

[0004] The purpose of the present application is to provide a hierarchical porous metal-organic framework sensing probe with adjustable pore size and a preparation method and application thereof, thereby solving the problem that the prior art still lacks a method capable of rapidly, accurately and simultaneously detecting metabolic biomarkers to screen the type of diabetic acidosis.

[0005] In order to solve the above problems, the present application adopts the following technical solutions:

[0006] According to a first aspect of the present application, a preparation method of a hierarchical porous metal-organic framework sensing probe is provided, which comprises the steps of: S1, obtaining hierarchical porous metal-organic framework nanoparticles (HMUiO-66(Zr)) with open large cage-like mesoporous channels and microporous crystal walls under the synergistic action of salt and hydrophobic oil phase solvent; S2, adding 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 the cofactor fixed therein; S3, glucose dehydrogenase (GDH), beta-hydroxybutyric acid dehydrogenase (3-HBDH), L-lactic acid dehydrogenase (L-LDH) and D-lactic acid dehydrogenase (D-LDH) are respectively loaded into the mesoporous channels of the hierarchical porous metal organic framework nanoparticle with the cofactor fixed therein to obtain a plurality of hierarchical porous metal organic framework sensing probes with dehydrogenase fixed therein and adjustable pore sizes 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 English full name of HMUiO-66(Zr) is Hierarchical Mesoporous UiO-66(Zr). UiO-66(Zr) is a very classic porous material in metal organic framework (MOFs), which has excellent chemical stability and long-term stability.

[0008] The preparation method of the hierarchical porous metal organic framework sensing probe provided by the application mainly has the technical effect that the size and structure of the mesoporous channels can be accurately controlled by using the synergistic effect of salt and hydrophobic oil phase solvent. The anion (such as Cl - , NO3 - , Br - , I - and ClO4 - ) of the salt can reduce the harsh reaction conditions required for MOF synthesis, so as to adapt to the mild conditions required for soft template self-assembly. At the same time, the hydrophobic oil phase solvent (such as toluene) can stabilize the micellar template by forming strong hydrophobic interaction with the PPO segment of the micelle, so as to prevent the surfactant molecules from being dissociated from the micelles into the solution due to the interaction between the metal precursor and the PEO segment during the MOF crystallization process.

[0009] According to the present application, the salt used in step S1 can be any one of NaCl, NaNO3, NaBr, Nal, NaClO4, KC1, LiCl, MgCl2 and CaCl2, and the hydrophobic oil phase solvent refers to a solvent with low polarity and strong lipophilicity, such as toluene, triethylbenzene, trimethylbenzene and the like. The salt can significantly reduce the reaction energy and promote the crystallization of the hierarchical porous metal organic framework nanoparticles in the aqueous phase. The highly hydrophobic oil phase has a stabilizing effect on the micelles and emulsions, preventing the surfactant molecules from prematurely dissociating during the slow growth of the hierarchical porous metal organic framework.

[0010] More preferably, the salt is sodium nitrate or sodium chloride, and the hydrophobic oil phase solvent is toluene. The reason is that NO3 - The crystallization of UiO-66(Zr) shows the strongest promoting effect, so that the reaction temperature can be reduced to only 40℃, and the highest crystallinity HMUiO-66(Zr) is produced; Cl - The salting-out ions increase the hydrophobicity of the micelles, allowing more toluene to enter the micelle core, and enabling the formation of HMUiO-66(Zr) with larger mesopore size. Toluene has the highest hydrophobicity among common oil phases (such as triethylbenzene, trimethylbenzene, xylene, toluene, etc.), and the inventors have fully demonstrated that toluene can maximize the expansion of mesopore size, so toluene is most preferred as the hydrophobic oil phase solvent for pore expansion.

[0011] Preferably, the step S1 comprises: S11, using a template agent (such as F127, etc.) to guide the formation of metal organic framework, using the synergy between salt and oil phase, generating hierarchical porous metal organic framework (HMUiO-66(Zr)-as) with adjustable mesoporous pore size by hydrothermal; S12, obtaining hierarchical porous metal organic framework nanoparticles (HMUiO-66(Zr)) by activation and demolding. More preferably, by adjusting the type of salt and the amount of toluene, the mesoporous pore size of the obtained HMUiO-66(Zr) is continuously adjustable between 6.8-18.1 nm. In the preferred embodiment, when using sodium nitrate salt, the toluene feeding amount is 30 μL, 70 μL and 100 μL respectively, and the pore size of HMUiO-66(Zr) is 6.8 nm, 8.9 nm and 10.3 nm respectively. Specifically, 100 mg of F127 is dissolved in 6 mL of deionized water. Then, HAc (125 μL, 2.19 mmol), toluene (30 μL, 70 μL and 100 μL) and NaNO3 (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 uniform solution. Then, BDC (166 mg, 1 mmol) is added and stirred for 20 minutes. Then, ZrO(NO3)2·2H2O (231 mg, 1 mmol) is added to the solution. The reaction mixture is stirred in a 60°C water bath for 24 hours. After that, the obtained sample is centrifuged and washed with water twice, then washed with DMF twice to remove residual unreacted ligand. Then, the synthesized sample is washed with ethanol and soaked in ethanol at 60°C for two days, replacing the ethanol every day. Finally, the sample is vacuum dried at 80°C for 24 hours. In the preferred embodiment, when using sodium chloride salt, 400 μL of toluene feeding amount can be used to synthesize HMUiO-66(Zr) with a pore size of about 18.1 nm. Specifically, 100 mg of F127 is dissolved in 6 mL of deionized water. Then, HAc (125 μL, 2.19 mmol), toluene (400 μL, 3.8 mmol) and NaCl (500 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 uniform solution. Then, BDC (166 mg, 1 mmol) is added and stirred for 20 minutes. Then, ZrO(NO3)2·2H2O (231 mg, 1 mmol) is added to the solution. The reaction mixture is stirred in a 60°C water bath for 24 hours. After that, the obtained sample is centrifuged and washed with water twice, then washed with DMF twice to remove residual unreacted ligand. Then, the synthesized sample is washed with ethanol and soaked in ethanol at 60°C for two days, replacing the ethanol every day. Finally, the sample is vacuum dried at 80°C for 24 hours.

[0012] Preferably, the step S2 comprises dissolving glucose dehydrogenase (GDH), beta-hydroxybutyric acid dehydrogenase (3-HBDH), L-lactate dehydrogenase (L-LDH) and D-lactate dehydrogenase (D-LDH) in Tris-HCl buffer (20 mM, pH 7.4), respectively, and adding NAD + @HMUiO-66(Zr), the dehydrogenase-immobilized hierarchical porous metal-organic framework 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)). Specifically, 0.5 mL of GDH, 3-HBDH, L-LDH and D-LDH solution (2 mg / mL, dissolved in 20 mM Tris-HCl buffer, pH 7.4) was added to 0.5 mL of NAD + @HMUiO-66(Zr) suspension, respectively, and shaken at 25 °C for 1 h. The enzyme-loaded NAD + @HMUiO-66(Zr) was washed twice with water and then dispersed in 2 mL of Tris-HCl buffer (20 mM, pH 7.4). With the increase of hierarchical porous metal-organic framework mesopore size from traditional microporous structure to 10.6 nm, the loading 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 was further increased to 18.1 nm of branched mesopore, the loading of GDH, 3-HBDH, L-LDH and D-LDH decreased to 153, 137, 132, and 114 mg / g, respectively. This decrease in loading is likely due to the excessive mesopore size, which reduces the number of mesopore cages available for enzyme immobilization. Therefore, the appropriate mesopore size helps the diffusion of the enzyme and immobilization into the material, thereby achieving higher immobilization efficiency.

[0013] According to the research of the present application, the mesopore size can be regulated by adjusting the amount of hydrophobic oil phase solvent and / or the type of salt. The mesopore size of HMUiO-66(Zr) is adjusted to match the space requirement of various dehydrogenases. It should be understood that the mesopore size is preferably 1-2 times larger than the size of the enzyme to meet the requirement of enzyme diffusion and fixation in the mesopore, at which the loading capacity of the enzyme is optimal; however, when the mesopore size is too large, for example, reaches 18.1 nm, the loading capacity decreases because the number of mesopores for fixing the enzyme decreases accordingly. Therefore, the HMUiO-66(Zr) with a suitable mesopore size matching the size of the enzyme can achieve optimal loading capacity and detection sensitivity.

[0014] It should be understood that UiO-66(Zr) is a classic MOFs structure that has attracted attention due to its excellent chemical stability and wide application. However, the traditional UiO-66(Zr) mainly has microporous structure, which is difficult to meet the needs of biomacromolecule (such as enzyme) immobilization, thus limiting its application in metabolic marker sensing probes. However, up to now, it has not been possible to synthesize UiO-66(Zr) with ordered mesopores, mainly due to the following difficulties: MOFs crystallization usually requires high temperature (> 90℃) and organic solvents, while micellar self-assembly requires aqueous phase and mild temperature (< 70℃), which are fundamentally incompatible with each other, making it extremely difficult to construct stable mesoporous structure in UiO-66(Zr).

[0015] The key point of the present application is that by discovering the salt-oil synergistic regulation mechanism, this technical problem is successfully solved: 1) the salt reduces the crystallization temperature of UiO-66(Zr), for example, NO3- promotes the crystallization of UiO-66(Zr), which can form stable crystal phase in aqueous phase at low temperature (40-60℃); 2) the oil phase can increase the stability of the micelles at high temperature when the crystal grows on the surface of the micelles; 3) the salt-oil synergistic effect greatly widens the synthesis window of HMUiO-66(Zr), making the mesopore size 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 application, 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), dehydrogenase and cofactor are respectively fixed in the mesopore of the hierarchical porous metal organic framework nanoparticle.

[0017] Another key point of the present application is the innovation of the probe application layer: 1) The adjustable mesoporous structure optimizes the enzyme immobilization and improves the sensing sensitivity: The microporous structure of traditional MOFs limits the entry of macromolecules (such as enzymes, cofactors), making it difficult to efficiently immobilize biological molecules. The HMUiO-66(Zr) prepared according to the method of the present application 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), improve the enzyme immobilization amount and activity, and the appropriate mesopore size matches the size of the enzyme, finally optimizing the sensitivity performance of the probe; 2) Multi-marker integrated detection, improving the monitoring ability of diabetic acidosis: Traditional biosensing is mainly based on single marker detection, which cannot realize comprehensive disease analysis. The present application integrates four different probes into a sensing array to realize the simultaneous detection of Glu, Hb, L-LAc and D-LAc. Through multi-marker correlation analysis, the progress and type of diabetic acidosis can be more accurately monitored. Compared with single marker detection, this strategy is more conducive to providing systematic disease diagnosis and improving the monitoring ability of diabetic acute complications.

[0018] According to a third aspect of the present application, a use of the above-mentioned hierarchical porous metal-organic framework sensing probe is provided, 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 are used as recognition centers for Glu, Hb, L-LAc and D-LAc, respectively, they have high specificity and do not interfere with the quantitative detection of corresponding metabolic biomarkers in complex biological samples. Accordingly, the hierarchical porous metal-organic framework sensing probe, through the dehydrogenase and NAD + cofactor constructed cascade response system is suitable for 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 for the biomarker to be detected and 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 a Tris-HCl buffer solution to form a probe solution, a Glu solution is added to the probe solution for incubation, and the absorbance intensity is measured using a multifunctional enzyme marker. Subsequently, the same method is used to respectively use NAD + @3-HBDH@HMUiO-66(Zr), NAD + @L-LDH@HMUiO-66(Zr) and NAD +D-LDH@HMUiO-66(Zr) probe, using multifunctional enzyme label meter to measure Hb, L-LAc and D-LAc light intensity.

[0021] Preferably, in the concentration range of 0-500 μM of Glu solution, using hierarchical porous metal organic framework sensing probe (NAD + @GDH@HMUiO-66(Zr)) to carry out colorimetric response to Glu, and linear fitting analysis of light intensity of the probe and Glu concentration for quantitative analysis of Glu. Subsequently, using the same method, respectively using NAD + @3-HBDH@HMUiO-66(Zr), NAD + @L-LDH@HMUiO-66(Zr) and NAD + D-LDH@HMUiO-66(Zr) to carry out colorimetric response to Hb, L-LAc and D-LAc, and linear fitting analysis of light intensity of the probe and Hb, L-LAc and D-LAc concentration for quantitative analysis of Hb, L-LAc and D-LAc.

[0022] Preferably, the detection lower limit of Glu, Hb, L-LAc and D-LAc solution is 6.4 μM, 2.2 μM, 35 μM and 24 μM, respectively.

[0023] Accordingly, the hierarchical porous metal organic framework 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)) can be used for clinical detection application, which can directly and quantitatively detect the content of Glu, Hb, L-LAc and D-LAc in human serum to screen the type of diabetic acidosis. Among them, first respectively using hierarchical porous metal organic framework 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)) to make standard curves of Glu, Hb, L-LAc and D-LAc metabolic biomarkers, then different diabetic patients or healthy human serum is added to the probe solution for incubation, and the multifunctional enzyme label is used to measure the light intensity, and through the standard curve, the light intensity is converted into the concentration of the corresponding metabolic biomarker, so as to realize quantitative detection in the serum sample.

[0024] The application proves the synergistic effect of the anion of the salt and the oil phase on the self-assembly of the HMUiO-66(Zr) soft template under mild conditions. The anion of the salt makes the HMUiO-66(Zr) crystallization reaction temperature as low as 40 DEG C, and the oil phase stabilizes the micellar template by strengthening the micellar template structure and preventing the surfactant from dissociating. The synergistic effect of the salt phase and the oil phase can accurately control the size and structure of the mesopore, and the pore size can be adjusted in the range of 6.8-18.1 nm. The adjustable mesoporous structure of the HMUiO-66(Zr) provides an efficient and universal platform for application, especially in biosensing and medical diagnosis. Taking the HMUiO-66(Zr) as a representative platform, the open and adjustable mesoporous channel can effectively accommodate dehydrogenase and NAD + cofactors, significantly enhancing the enzyme immobilization capacity. These probes have high loading efficiency and sensitive biosensing ability, and the detection limits of Glu, Hb, L-LAc and D-LAc are as low as 6.4 mu M, 2.2 mu M, 35 mu M and 24 mu M respectively. By integrating these probes into an array and using a 4-input AND-NOR logic gate combination, the changes of these metabolic biomarkers can be simultaneously detected and analyzed, so as to quickly and accurately screen the types of diabetic acidosis.

[0025] The application provides a pore size adjustable hierarchical porous metal organic framework sensing probe, a preparation method and application thereof, and has the following beneficial effects relative to the prior art:

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

[0027] 2) The content of Glu, Hb, L-Lac and D-LAc in human serum is quantitatively detected by colorimetry, and a simple, reliable and specific detection method is provided to screen the types of diabetic acidosis;

[0028] 3) The HMUiO-66(Zr) prepared according to the application provides a multifunctional platform, highlights the potential of the HMUiO-66(Zr) in biomolecule immobilization and disease diagnosis, and at the same time paves the way for wider application in biosensing and catalytic processes.

[0029] In summary, the present application relates to a hierarchical porous metal organic framework sensing probe with adjustable pore size, a preparation method and application thereof, the size and structure of the mesopore can be accurately controlled under the synergistic effect between anions and an oil phase, and a plurality of hierarchical porous metal organic framework sensing probes with adjustable pore size and fixed dehydrogenase are prepared under mild conditions. The hierarchical porous metal organic framework sensing probe is also particularly suitable for specific detection of Glu, Hb, L-Lac and D-LAc biomarkers in human serum samples, and provides a convenient, rapid and specific detection technology, which can be used for screening and diagnosis of different types of diabetic acidosis, and improves the monitoring ability of diabetic acute complications. BRIEF DESCRIPTION OF DRAWINGS

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

[0031] Figure 2 is a schematic diagram of (A) HMUiO-66(Zr) structure, (B) XRD pattern of HMUiO-66(Zr), (C) N2 adsorption isotherm and corresponding BJH pore size distribution curve (inset) of HMUiO-66(Zr), (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 Zr, O, C, Na and N element distribution maps according to the present application;

[0032] Figure 3 is (A) XRD pattern of HMUiO-66(Zr) and (B) XRD pattern of HMUiO-66(Zr) after soaking in aqueous solution at different pH values for 1 day according to the present application;

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

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

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

[0036] Figure 7 is a SEM image of HMUiO-66(Zr) samples synthesized according to the present application at (a) toluene with NO3 - salt anions. (b) toluene with CI - schematic representation of the preparation of HMMOFs by soft-templating self-assembly under the synergistic effect of (a) toluene with NO3

[0037] Figure 8 is (A-H) SEM images, (I-L) TEM images, (M-P) BJH pore size distribution curves and the corresponding N2adsorption isotherms (inset) of representative HMUiO-66(Zr) samples synthesized according to the present application using NaNO3mediated salt and (A, E, I, M) 30, (B, F, J, N) 70 and (C, G, K, O) 100 μL of different amounts of toluene or using NaCl mediated salt and (D, H, L, P) 400 μL of toluene;

[0038] Figure 9 is a SEM image of HMUiO-66(Zr) samples synthesized according to the present application using 400 μL of toluene and 8.5 mmol of salts (A) NaCl, (B) NaNO3, (C) NaBr, (D) Nal and (E) NaClO4H2O;

[0039] Figure 10 is a SEM image of HMUiO-66(Zr) samples synthesized according to the present application using 400 μL of toluene and 8.5 mmol of salts (A) KCl, (B) LiCl, (C) MgCl2and (D) CaCl2;

[0040] Figure 11are XRD patterns of (A) simulated UiO-66(Zr) and HMUiO-66(Zr) samples synthesized using NaNO3mediated salt and different amounts of toluene (a) 30, (b) 50, (c) 70, (d) 90 and (e) 100 μL; (B) simulated UiO-66(Zr) and HMUiO-66(Zr) samples synthesized using NaCl mediated salt and different amounts of toluene (a) 50, (b) 100, (c) 200, (d) 400 μL;

[0041] Figure 12 are SEM images of HMUiO-66(Zr) samples synthesized according to the present application using NaNO3mediated salt 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) N2adsorption curves, (F-J) hysteresis loop sections and (K-O) BJH pore size distribution plots of HMUiO-66(Zr) samples synthesized according to the present application using NaNO3mediated salt 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 μL;

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

[0044] Figure 15 are (A-D) N2adsorption curves, (E-H) hysteresis loop sections and (I-L) BJH pore size distribution plots of HMUiO-66(Zr) samples synthesized according to the present application 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;

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

[0046] Figure 17 are the effect of incubation time, Glu concentration, Hb concentration, L-LAc concentration and D-LAc concentration on (A) NADH production, (B) NADH production, (C) NADH production, (D) NADH production, (E) NADH production, (F) NADH production, (G) NADH production, (H) NADH production, (I) NADH production, (J) NADH production, (K) NADH production, (L) NADH production, (M) NADH production, (N) NADH production, (O) NADH production, (P) NADH production, (Q) NADH production, (R) NADH production, (S) NADH production, (T) NADH production, (U) NADH production, (V) NADH production, (W) NADH production, (X) NADH production, (Y) NADH production and (Z) NADH production studies according to the present application.+ @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 absorbance intensity study;

[0047] Figure 18 (A) NAD according to the present invention + @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 absorbance intensity study;

[0048] Figure 19 Four probes according to the present invention integrated into one sensing array to construct a 4-input AND-NOR logic gate circuit;

[0049] Figure 20 Various possible coexisting interferents on (A) NAD according to the present invention + @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 sensing response study;

[0050] Figure 21 (A) NAD while adding Glu and other interfering substances according to the present invention + @GDH@HMUiO-66(Zr) probe response study, 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) NAD while adding Hb and other interfering substances +@3-HBDH@HMUiO-66(Zr) probe response influence study, 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) NAD + @L-LDH@HMUiO-66(Zr) probe response influence study, 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) NAD + @D-LDH@HMUiO-66(Zr) probe response influence study, 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 are according to the present application (A) cross-talk analysis of array response to different Glu concentrations in the presence of high concentrations of Hb, L-LAc and D-LAc; (B) cross-talk analysis of array response to different Hb concentrations in the presence of high concentrations of Glu, L-LAc and D-LAc; (C) cross-talk analysis of array response to different L-LAc concentrations in the presence of high concentrations of Glu, Hb and D-LAc; (D) cross-talk analysis of array response to different D-LAc concentrations in the presence of high concentrations of Glu, Hb and L-LAc;

[0052] Figure 23 are according to the present application the use of the integrated array for 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. DETAILED DESCRIPTION

[0053] The preferred embodiments of the present application will be described below in conjunction with the accompanying drawings.

[0054] As shown in Figure 1A FIG. 1 is a flow chart of a preparation method of hierarchical porous metal organic framework sensing probes according to the present application, which comprises the following steps: using a template agent (such as F127) to guide the formation of metal organic framework, using the synergistic effect between salt and oil phase to generate hierarchical porous metal organic framework with adjustable mesoporous pore size through hydrothermal method under mild conditions; then, obtaining hierarchical porous metal organic framework nanoparticles UiO-66(Zr) with adjustable pore size through activation and template removal; loading NAD + cofactor into the hierarchical porous metal organic framework nanoparticles to obtain hierarchical porous metal organic framework nanoparticles with mesoporous channels and immobilized cofactor; loading GDH, 3-HBDH, L-LDH and D-LDH into the mesoporous channels of the hierarchical porous metal organic framework nanoparticles with immobilized cofactor, respectively, to obtain a plurality of hierarchical porous metal organic framework sensing probes with adjustable pore size and immobilized 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).

[0055] As shown in Figure 1B FIG. 2 is a hierarchical porous metal organic framework structure and sensing probes constructed therefrom and applications thereof according to the present application. (1) The hierarchical porous metal organic framework sensing probes prepared according to the above method have dehydrogenase and nicotinamide adenine dinucleotide NAD + cofactor in the mesoporous channels at the same time. Through the specific recognition of metabolic biomarkers by dehydrogenase, these enzymes catalyze the oxidation of Glu, Hb, L-LAc and D-LAc in the mesopores of HMUiO-66(Zr), and convert an equal amount of NAD + into NADH. At the same time, NADH interacts with the electron transfer agent PMS to catalyze the MTT colorimetric reaction, realizing rapid and intuitive detection of metabolites. (2) The above four probes are integrated into the same sensing array to simultaneously analyze Glu, Hb, L-LAc and D-LAc through colorimetric changes. Each probe produces a different color response when exposed to the corresponding metabolite, facilitating rapid and instant detection of these biomarkers through digital output (0 / 1) for negative or positive.

[0056] Example 1

[0057] 1.1 Synthesis of HMU-1 O-66 (Zr)

[0058] 100 mg of F127 was dissolved in 6 mL of deionized water at room temperature. Then, HAc (125 μL, 2.19 mmol), toluene (30 μL, 70 μL, and 100 μL), and NaNO3 (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. Next, BDC (166 mg, 1 mmol) was added and the mixture was stirred for 20 minutes. Subsequently, ZrO(NO3)2·2H2O (231 mg, 1 mmol) was added to the solution. The reaction mixture was stirred in a 60 °C water bath for 24 hours. The resulting sample was then centrifuged and washed twice with water, followed by twice with DMF to remove residual unreacted ligands. The synthesized sample was then washed with ethanol and immersed in ethanol at 60 °C for two days, changing the ethanol daily. Finally, the sample was vacuum dried at 80 °C for 24 hours.

[0059] Figure 2 In the presence of NaNO3 and toluene as the salt and oil phases, respectively, HMUiO-66(Zr) with typical open cage-like mesopores coupled with microporous crystal walls was synthesized. Figure 2 Image B is the wide-angle X-ray diffraction (XRD) pattern of HMUiO-66(Zr), which matches well with the simulated UiO-66(Zr) pattern, confirming the formation of the crystal framework. Figure 2 The C in the figure represents the N2 adsorption isotherm and its corresponding Barrett-Joyner-Halenda (BJH) pore size distribution. The sample exhibits a combination of Type I and Type IV isotherms, indicating the simultaneous presence of micropores and mesopores in HMUiO-66 (Zr). The total surface area of ​​BET is approximately 960 m². 2 / g, of which the mesoporous specific surface area is 281m³ 2 / g. The mesopore size of the adsorption branches was determined to be approximately 10.6 nm using the BJH method. Generally, cage-like mesoporous materials exhibit typical H2-type or H1-type hysteresis loops due to their relatively narrow mesopore connections. Interestingly, the adsorption branches of the HMUiO-66(Zr) sample showed a sharp step at a P / P0 ratio of 0.75, corresponding to the presence of mesopores around 10.6 nm. Within the P / P0 range of 0.6–0.85, the adsorption and desorption branches almost overlapped, indicating the absence of a significant 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, allowing N2 molecules to rapidly enter and exit without a significant hysteresis effect. Figure 2 The image in the middle (DE) is a scanning electron microscope (SEM) image of HMUiO-66(Zr), which consists of uniformly monodisperse spherical NPs with a diameter of 70-90 nm. Figure 2Figure 6C) at higher magnification. The surface of the HMUiO-66(Zr) NPs can be clearly distinguished with uniform and ordered mesoporous entrances Figure 2 Figure 6E). Figure 2 Figures 6F-H are transmission electron microscopy (TEM) images of HMUiO-66(Zr) further confirming that the HMUiO-66(Zr) is well dispersed with ordered large mesopores uniformly distributed throughout the NPs. The small size of the HMUiO-66(Zr) NPs allows for a high degree of openness of the mesopores, which facilitates the efficient diffusion of biomacromolecules within the HMUiO-66(Zr). This structural feature further explains the lack of hysteresis in the N2 adsorption isotherm. Figure 2 Figure 6I is a scanning TEM (STEM) and elemental mapping of the HMUiO-66(Zr) NPs, which reveals a uniform distribution of the Zr, C, and O elements, and the absence of Na and N elements, indicating that the NaNO3 salt was completely removed. Figure 3 Figures 6A-B are XRD patterns of the HMUiO-66(Zr) that exhibit excellent chemical and thermal stability, retaining its crystalline structure after exposure to solutions with pH values ranging from 1 to 12 Figure 3 Figure 6A). In addition, it exhibits significant long-term stability, retaining its integrity even after being left in acidic, neutral, and basic solutions for 14 days Figure 3 Figure 6B). Figure 4 is an FTIR pattern of the 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 a thermogravimetric pattern of the HMUiO-66(Zr). The thermal decomposition temperature of the HMUiO-66(Zr) is as high as 517°C, similar to the decomposition temperature of the HMUiO-66(Zr) reported previously, which calculated that the HMUiO-66(Zr) contains a large number of defects, with only 8.98 linkers connecting each Zr6cluster.

[0060] Figure 6 Figure 6A is a study of the effect of different anions, including SO4 2- , HPO4 2- , Cl - , NO3 - , Br - , I - , and ClO4 - , on the synthesis of HMUiO-66(Zr) in aqueous phase, with various anions as the x-axis and the reaction temperature as the y-axis, generating a heat map. The color gradient from purple to red indicates that 7.4 0The intensity of the strongest diffraction peak of the UiO-66(Zr) structure. The visualized heat map can clearly compare the degree of promotion of different anions on the crystallization of UiO-66(Zr). When there is no salt in the reaction, the reaction temperature in the range of 40-60℃ does not generate UiO-66(Zr) product. While Cl - , NO3 - , Br - , I - and ClO4 - anions can promote the formation of crystalline UiO-66(Zr) at the reaction temperature range of 40-60℃. A large number of literatures show that the formation of Zr6or Hf6cluster is a key step for the mild synthesis of Zr-based and Hf-based MOFs, while the direct synthesis method usually requires harsh conditions above 100℃. The anion of the salt can promote the formation of the cluster and reduce the energy barrier through the solubilization mechanism, so that UiO-66(Zr) continues to crystallize in the aqueous phase under milder conditions. Among these anions, the salt dissociation ions of NO3 - , Br - , I - and ClO4 - are better than the salt dissociation ions of Cl - for promoting the crystallization of UiO-66(Zr). This is because the salt dissociation ions reduce the solubility of BDC in the aqueous phase due to the Hofmeister effect, resulting in slow growth and weak crystallinity of the product. Finally, the other salt dissociation ions of SO4 2- and HPO4 2- only generate amorphous products. This is because SO4 2- and HPO4 2- have a strong coordination interaction with Zr, which is much stronger than the interaction between Zr and the carboxyl group of the BDC ligand, thereby preventing the formation of UiO-66(Zr). Figure 6 In B, when NO3 - was used as the additive medium and toluene was used as the oil phase, all HMUiO-66(Zr) synthesized at different temperatures formed uniform mesoporous BJH pore size distribution curves. At 60℃, due to the weakening of the interaction between the PEO layer of F127 micelles and water, the hydrophobicity of the PEO layer was enhanced, thereby obtaining larger mesopore size. This change promoted more hydrophobic toluene to penetrate into the micellar core, promoting their swelling, and ultimately forming larger mesopores. Figure 6 In C, when NO3 - was used as the additive medium and no toluene was introduced, the BJH pore size distribution curves of all HMUiO-66(Zr) synthesized at different temperatures failed to form micellar templated large mesopores between 40-60℃. Figure 6The TEM image of HMUiO-66(Zr) shows that large mesopores were formed in HMUiO-66(Zr) when toluene was used as the oil phase. Figure 6 (F127 micelles), but no large mesopores were observed when only F127 micelles were used. Figure 6 These observations led us to propose a mechanism in which the oil phase plays a crucial role in the formation of mesopores in HMMOFs. Generally, F127 molecules in the micelles undergo a slow, dynamic exchange with free F127 molecules in the aqueous phase. The soft template is unstable during the slow crystallization of MOFs at the micelle interface, leading to the dynamic dissociation of F127 molecules from the micelles and preventing mesopore formation. Even a small amount of toluene added as an oil phase allows it to enter the hydrophobic core of the micelles, significantly improving emulsion stability. This stability stems from the strong hydrophobic interaction between toluene and the hydrophobic PPO layer of the micelles, thereby expanding the synthetic window for soft template self-assembly even at high reaction temperatures.

[0061] Figure 7 This is a schematic diagram of the preparation of HMUiO-66(Zr). The salt phase and toluene oil phase work synergistically to regulate the mesopore size and structure of HMMOFs. When using NO3... - When salt-dissolved ions are present, the hydrophobic interaction between toluene and the PEO of F127 micelles allows toluene to modulate the size of the nanoemulsion, thereby controlling the mesopore size of HMUiO-66(Zr). Figure 7 (a) This further confirms Cl - Salting-out ions increased the hydrophobicity of the micelles, allowing more toluene to enter the micelle core and promoting the formation of larger mesopores in HMUiO-66(Zr). Figure 7 (b) Figure 8 The AL image shows SEM and TEM images of HMUiO-66(Zr). When using NO3... - When dissolving ions in salt solution, as the volume of toluene increases from 30 μL to 100 μL, the mesoporous inlet and internal mesoporous size of HMUiO-66(Zr) gradually increase. Figure 8 (AC and EG). Figure 8 The MP curve represents the N2 adsorption isotherm and BJH pore size distribution curve of HMUiO-66(Zr). Calculations show that the average mesopore diameter can be adjusted between 6.8 and 10.3 nm. Figure 8 (MO). In contrast, using Cl - When salt ions are released, the emulsion swells significantly, forming open, branched mesopores with a pore size of approximately 18.1 nm. Figure 8 (D, H, L, P). Figure 9 This is a SEM image of HMUiO-66(Zr). It shows various salt ions, including NO3-.- , Br - , I - and CIO4 - , the addition of excess toluene cannot form larger mesopores, indicating that the swelling of nanoemulsion has reached the limit. Figure 10 are SEM images of HMUiO-66(Zr). Different chloride salts (KCI, LiCI, MgCI2, CaCI2) also form large and open mesopores similar to NaCI. Figure 11 are XRD patterns of HMUiO-66(Zr) synthesized with the addition of various salt ions. The introduction of toluene oil phase does not destroy the crystal structure of HMUiO-66(Zr). Figure 12-15 are SEM images, N2 adsorption desorption curves and BJH pore size distribution curves of HMUiO-66(Zr) synthesized with the addition of various anions and careful adjustment of the volume of toluene. The whole process can continuously and accurately control the mesopore size to meet the requirements of different applications for specific pore sizes.

[0062] 1.2 NAD + @Dehydrogenase@Synthesis of HMUiO-66(Zr) probes

[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 h. Next, 0.5 mL of GDH, 3-HBDH, L-LDH and D-LDH solutions (2 mg / mL, dissolved in 20 mM Tris-HCI buffer, pH 7.4) were added to 0.5 mL of NAD + @HMUiO-66(Zr) suspension, respectively, and shaken at 25 °C for 1 h. The enzyme-loaded NAD + @HMUiO-66(Zr) was collected by centrifugation, washed twice with water, and then dispersed in 2 mL of Tris-HCI buffer (20 mM, pH 7.4) for the next step.

[0064] Figure 16 are the effects of mesopore size of HMUiO-66(Zr) on the loading capacity of (A) GDH, (B) 3-HBDH, (C) L-LDH and (D) D-LDH, and their colorimetric responses to 25 mM Glu, Hb, L-LAc and D-LAc. HMUiO-66(Zr) with different mesopore sizes was used to immobilize NAD +and dehydrogenases. Since the three-dimensional dimensions of GDH, 3-HBDH, L-LDH and D-LDH are about 4.4 x 5.1 x 6.5 nm, 3.8 x 4.4 x 5.2 nm, 4.5 x 5.2 x 6.3 nm and 4.1 x 5.5 x 6.5 nm, respectively, sufficient space is required to accommodate the dehydrogenases and NAD + When the mesopore size was increased from micropore to 10.6 nm, the loading 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) was enlarged from micropore to 10.6 nm, the colorimetric response for the detection of Glu, Hb, L-LAc and D-LAc (25 μΜ) was significantly increased.

[0065] 1.3 Response kinetics of hierarchical porous metal-organic framework sensing probes

[0066] Into a 96-well plate containing Tris-HCl buffer (20 mM, pH 7.4), 140 μL of Glu solution with different concentrations was added, and then 20 μL of NAD + @GDH@HMUiO-66(Zr) suspension (1000 mg / mL). Then, 40 μL of color developing solution was added to the plate. The mixture was incubated at 37 °C for 20 min. Then, the absorbance at 570 nm was detected at fixed time intervals using a multifunctional microplate reader. The color developing 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. Subsequently, the same method was used to investigate 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 absorbance intensity. The detection performance of the probe was characterized in detail. From the response kinetics, it can be seen that the absorbance intensity of these sensors at 570 nm gradually increased with the extension of incubation time, and Glu, Hb, L-LAc and D-LAc reached saturation rapidly at about 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 equilibrium time for subsequent sensor detection

[0068] 1.4 Quantitative analysis of hierarchical porous metal-organic framework sensing probes

[0069] 140 μL of different concentrations of Glu standard solution 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). Then, 40 μL of color developing solution was added to the plate. The mixture was incubated at 37°C for 20 min, and the absorbance at 570 nm was detected using a multifunctional enzyme marker. The color developing 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 was used immediately after mixing to generate a standard curve of Glu. Subsequently, the same method was used to generate standard curves of 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) probe to generate standard curves of Hb, L-LAc and D-LAc.

[0070] Figure 18 (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 absorbance of D-LDH@HMUiO-66(Zr) probe as a function of Glu, Hb, L-LAc and D-LAc concentration. The colorimetric response shows a good linear correlation between the increase of absorbance at 570 nm and the change of concentration of the four metabolic biomarkers. 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, using the 3σ rule, which are much lower than the concentration range of 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] Different concentrations of interfering substances (potassium (K + ), sodium (Na + ), chlorine (Cl - ), urea, cholesterol, creatinine and bovine serum albumin (BSA)) were dissolved in NAD + @GDH@HMUiO-66(Zr) probe solution, and the colorimetric determination of the probe solution was carried out after 20 minutes of equilibrium response to obtain the luminescence intensity at λ = 570 nm. In addition, 100 μM concentration of interfering substances (K + , Na + , Cl - , urea, cholesterol, creatinine and BSA) were dissolved in the probe solution, and the colorimetric determination of the probe solution was carried out after 20 minutes of equilibrium response to obtain the luminescence intensity at λ = 570 nm after adding different concentrations of Glu solution. Subsequently, high concentration of Hb, L-LAc and D-LAc solution was dissolved in NAD + @GDH@HMUiO-66(Zr) probe solution, and the colorimetric determination of the probe solution was carried out after 20 minutes of equilibrium response to obtain the luminescence intensity at λ = 570 nm. Subsequently, the same method was used to study NAD + @3-HBDH@HMUiO-66(Zr), NAD + @L-LDH@HMUiO-66(Zr) and NAD + @D-LDH@HMUiO-66(Zr) probe.

[0073] Figure 19 is a 4-input AND-NOR logic circuit that integrates the four probes into a sensing array, simultaneously detects multiple metabolic biomarkers related to diabetic acidosis and constructs a color change pattern by checking the color change pattern, and quickly and accurately classifies different types of diabetic acidosis according to the correlation between the biomarkers. Figure 20 is the effect of various possible coexisting interfering substances 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, Chol, Cre and BSA. The response of all can be ignored, while after introducing the corresponding metabolic biomarkers, the absorbance at 570 nm is obviously enhanced. Figure 21 Effect of (A) simultaneous addition of Glu and other interfering substances on NAD + Effect of GDH@HMUiO-66(Zr) probe sensing response. Wherein (1) Glu, (2) Glu+K + , (3) Glu+Na + , (4) Glu+Cl - , (5) Glu+Urea, (6) Glu+Chol, (7) Glu+Cre, (8) Glu+BSA. (B) simultaneous addition of Hb and other interfering substances on NAD + Effect of 3-HBDH@HMUiO-66(Zr) probe sensing response. Wherein (1) Hb, (2) Hb+K + , (3) Hb+Na + , (4) Hb+Cl - , (5) Hb+Urea, (6) Hb+Chol, (7) Hb+Cre, (8) Hb+BSA. (C) simultaneous addition of L-LAc and other interfering substances on NAD + Effect of L-LDH@HMUiO-66(Zr) probe sensing response. Wherein (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) simultaneous addition of D-LAc and other interfering substances on NAD + Effect of D-LDH@HMUiO-66(Zr) probe sensing response. Wherein (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. These interfering substances had no obvious influence on the sensing response when coexisting with Glu, Hb, L-LAc and D-LAc. Figure 22 (A) The cross-talk analysis of the array response to different Glu concentrations in the presence of high concentrations of Hb, L-LAc and D-LAc. (B) The cross-talk analysis of the array response to different Hb concentrations in the presence of high concentrations of Glu, L-LAc and D-LAc. (C) The cross-talk analysis of the array response to different L-LAc concentrations in the presence of high concentrations of Glu, Hb and D-LAc. (D) The cross-talk analysis of the array response to different D-LAc concentrations in the presence of high concentrations of Glu, Hb and L-LAc. The sensing array responded effectively to the concentration change of Hb in the presence of Glu, L-LAc and D-LAc, while high concentrations of Glu, L-LAc and D-LAc did not cause obvious colorimetric response. Similarly, no cross-talk interference was observed in the detection of Hb, L-LAc and D-LAc due to the high specificity of enzyme recognition.

[0074] Application Example 1

[0075] 2.1 Quantitative detection of Glu, Hb, L-LAc and D-LAc biomarkers in diabetic ketoacidosis patient serum using hierarchical porous metal-organic framework sensing array Figure 23

[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, respectively, 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). Next, 40 μL of color developing solution was added to the plate. The mixture was incubated at 37 °C for 20 min, and the absorbance at 570 nm was detected using a multifunctional enzyme label meter. The absorbance intensity was converted into glucose concentration by the calibration curve for Glu quantification. Subsequently, the same method was used to detect 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) probe to detect the concentrations of Hb, L-LAc and D-LAc in serum.

[0077] ​is the detection of 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 the integrated array. The enhanced absorbance response of the array to Glu, Hb and L-LAc biomarkers in the healthy human serum sample indicates that the probes can effectively detect the corresponding biomarkers in real serum samples. By converting the absorbance enhancement to biomarker concentration, the levels of Glu, Hb and L-LAc were determined to be about 5505, 220 and 916 μΜ, respectively, while almost no D-LAc was detected in the healthy serum sample. To further evaluate the effectiveness of the array, 6000 μΜ Glu and 4000 μΜ Hb were additionally added to the healthy serum sample to simulate DKA conditions. We observed that the absorbance response of the array to Glu and Hb in the simulated DKA serum sample was significantly enhanced compared to the healthy serum sample. The detected values of Glu and Hb in the simulated DKA sample were about 11704 μΜ and 4120 μΜ, respectively, with recoveries of 102.7% and 98.1%, respectively, further verifying the accuracy of the array. Likewise, the array effectively detected the elevated levels of L-LAc and D-LAc in the simulated L-lactic acidosis and simulated D-lactic acidosis serum samples, indicating that it can rapidly distinguish different types of diabetic acidosis according to the related changes in the levels of Glu, Hb, L-LAc and D-LAc. Table 1 below is 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]

[0080]

[0081] In summary, the present application utilizes the template-directed formation of metal-organic frameworks, the synergistic effect between salt and oil phase, and hydrothermal synthesis of HMUiO-66(Zr). The synergistic effect of salt ions and oil phase on the self-assembly of HMUiO-66(Zr) soft template under mild conditions is demonstrated. Anions lower the crystallization reaction temperature of HMUiO-66(Zr) to 40°C, while oil phase stabilizes the micellar template by strengthening the micellar template structure and preventing surfactant dissociation. The synergistic effect of salt phase and oil phase can precisely control the size and structure of mesopores, with a tunable pore size range of 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 diagnostics. Taking HMUiO-66(Zr) as a representative platform, its open and tunable mesoporous channels can effectively accommodate dehydrogenase and NAD+ The cofactor significantly enhances its enzyme immobilization ability. These probes have high loading efficiency and sensitive bioanalytical ability, by integrating these probes into an array, using a 4-input AND-NOR logic gate to combine simultaneous detection and analysis of these metabolic biomarkers, through the related changes of metabolic biomarkers to quickly screen different types of diabetic acidosis, show their potential in providing a critical time window for timely treatment of acute and severe diseases.

[0082] The above-mentioned is only the preferred embodiment of the present application, not to limit the scope of the present application, the above-mentioned embodiment of the present application can also be made various changes. That is, according to the claims and the content of the specification of the present application, simple, equivalent changes and modifications are made, all fall within the scope of the claims of the present application. The present application is not described in detail, all are conventional technical content.

Claims

1. A method for preparing a tunable-aperture hierarchical porous metal-organic framework sensing probe, characterized in that, The preparation method comprises the following steps: S1, under the synergistic effect of a salt and a hydrophobic oil phase solvent, obtaining hierarchical porous metal organic framework nanoparticles with open large cage mesoporous channels and microporous crystal walls, the salt being sodium nitrate, and the hydrophobic oil phase solvent being toluene; S2, loading NAD + loading the cofactor into the hierarchical porous metal-organic framework nanoparticles to obtain a hierarchical porous metal-organic framework nanoparticle with mesoporous channels and immobilized cofactor; S3, glucose dehydrogenase, β-hydroxybutyric acid dehydrogenase, L-lactic acid dehydrogenase and D-lactic acid dehydrogenase are respectively loaded into the mesoporous channels of the hierarchical porous metal organic framework nanoparticles fixed with cofactors, to obtain a plurality of pore size adjustable hierarchical porous metal organic framework sensor 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).

2. The production method according to claim 1, characterized by, The step S1 comprises: S11, using a template to guide the formation of metal organic framework, and using the synergistic effect between a salt and a hydrophobic oil phase solvent, generating hierarchical porous metal organic framework with adjustable mesopore size under mild conditions by a hydrothermal method; S12, obtaining hierarchical porous metal organic framework nanoparticles with adjustable pore size by activation and template removal.

3. The preparation method according to claim 2, characterized in that, In step S11, the mild conditions refer to a reaction temperature of 40-60℃.

4. The production method according to claim 1 or 2, characterized by, The mesopore size is adjustable by adjusting the feeding amount of the hydrophobic oil phase solvent in step S1.

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

6. A hierarchical porous metal-organic framework prepared probe prepared by the preparation method according to any one of claims 1-5, characterized in that, Dehydrogenase and cofactor are respectively fixed in the mesopores of the hierarchical porous metal organic framework nanoparticles.

7. Use of the hierarchically porous metal-organic framework sensing probe according to claim 6, characterized in that, Using the specific recognition of dehydrogenase to metabolic biomarkers, dehydrogenase catalyzes the oxidation of glucose, β-hydroxybutyric acid, L-lactic acid and D-lactic acid in the mesoporous of HMUiO-66(Zr), and equal amounts of NAD + are converted into NADH, at the same time, NADH interacts with the electron transfer agent of PMS, catalyzes the MTT colorimetric reaction, so as to realize the rapid and intuitive detection of metabolic markers.

8. Use according to claim 7, characterized in that, In the concentration range of 0-500 μM of glucose, β-hydroxybutyric acid, L-lactic acid and D-lactic acid solution, respectively, the hierarchical porous metal organic framework sensing probe is used for detection response, and linear fitting analysis is performed.

9. Use according to claim 7, characterized in that, Four hierarchically 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) were integrated into a sensing array for simultaneous analysis of glucose, β-hydroxybutyric acid, L-lactic acid and D-lactic acid by colorimetric change. Each probe produced a different color response when exposed to the corresponding metabolite, enabling accurate screening of the type of diabetic acidosis.

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