Method for detecting levodopa

Abstract

The application relates to the detection field and provides a method for detecting levodopa, which comprises the following steps: contacting a to-be-detected sample with a sensor to generate a detectable signal; the sensor comprises a working electrode, and the surface of the working electrode is loaded with UiO-66 material; a detection result of the to-be-detected sample is obtained based on the detectable signal; the Ui-O66 material is prepared by reacting terephthalic acid or a derivative thereof and a soluble zirconium salt for a predetermined time, and the predetermined time is determined based on known components of the to-be-detected sample. The method selects the reaction time of the UiO-66 material to regulate the proportion of the crystalline state and the amorphous state in the material, so that the detection requirement of the UiO-66 material for levodopa is accurately matched, the detection range covering from ultratrace (10 nM) to high concentration (300 nM), the method has high sensitivity, meanwhile, excellent selectivity and stability are considered, the detection requirement of different scenes is met, and the method is suitable for wide application.

Description

Technical Field

[0001] This application relates to the field of detection. Specifically, this application relates to a method for detecting levodopa. Background Technology

[0002] Parkinson's disease is a chronic, progressive neurodegenerative disease affecting millions of patients worldwide. Its core pathological feature is the progressive loss of dopaminergic neurons in the substantia nigra, leading to clinical manifestations such as motor dysfunction, resting tremor, rigidity, and impaired coordination. Levodopa (L-Dopa), currently the most commonly used treatment in clinical practice, has a molecular structure that allows it to cross the blood-brain barrier and be converted into dopamine within the central nervous system to supplement neurotransmitter deficiencies. Given the narrow therapeutic window of levodopa and the fact that its plasma concentration is easily affected by metabolic levels, dietary factors, and drug interactions, precise monitoring of drug concentrations in bodily fluids such as blood or plasma is of significant clinical importance for optimizing dosing regimens and avoiding adverse reactions such as motor dysfunction and the "on-off phenomenon."

[0003] Conventional methods for detecting levodopa (L-DOPA) include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis, and spectrophotometry. While these techniques are sensitive and accurate, they are costly, time-consuming, require specialized personnel, and are not suitable for real-time detection. Therefore, electrochemical sensing platforms for levodopa detection have become a research hotspot, offering significant advantages: low cost, rapid analysis, miniaturization potential, and high sensitivity / selectivity, especially when combined with advanced surface modification strategies.

[0004] Metal-organic frameworks (MOFs) are a class of crystalline porous materials formed by the coordination of metal ions or metal clusters with organic ligands. Their notable properties include high specific surface area (typically exceeding 1000 m²). 2 MOFs possess tunable pore size and functionalization properties, as well as well-defined crystal structures. These characteristics make MOFs ideal candidate materials for a wide range of applications, including gas storage, heat storage and transfer, catalysis, cancer therapy, drug / gene delivery, gene editing, biosensors and health monitoring, and chemical sensing.

[0005] Among the vast system of metal-organic frameworks (MOFs), UiO-66 (University of Oslo-66), constructed based on zirconium clusters and terephthalic acid ligands, has attracted much attention due to its excellent chemical stability, thermal stability, large specific surface area, and resistance to moisture and acids. This material exhibits a three-dimensional face-centered cubic (fcu) topology, with its core consisting of 12 connected Zr6O4(OH)4 secondary structural units (SBUs), and possesses a high degree of tunability in synthesis.

[0006] However, no studies have reported that UiO-66 can be applied to levodopa detection, and how to prepare suitable UiO-66 based on the requirements of levodopa detection needs to be studied. Summary of the Invention

[0007] This application aims to at least partially address the technical problems existing in the prior art. To this end, this application proposes a method for detecting levodopa. This method, by selecting the reaction time of UiO-66 material, controls the ratio of crystalline to amorphous states in the material, thereby achieving precise adaptation of UiO-66 material to the detection requirements of levodopa. It covers a detection range from ultra-trace (10 nM) to high concentration (300 nM), exhibiting high sensitivity while also possessing excellent selectivity and stability, meeting the detection requirements of different scenarios and suitable for wide application.

[0008] It should be noted that this application is based on the inventor's discovery and understanding of the following facts and problems:

[0009] Currently, researchers typically focus on optimizing specific surface area or introducing functional groups to obtain metal-organic frameworks (MOFs) with specific properties, without systematically studying the impact of the ratio of crystalline to amorphous phases on material properties. In the field of MOF synthesis, "crystalline" refers to the long-range periodic ordered arrangement of atoms within the framework, while "amorphous" or "non-crystalline" lacks this regularity and has a higher density of structural defects. Therefore, highly crystalline MOFs are currently favored due to their uniformity and reproducibility.

[0010] In view of this, the inventors of this application have discovered that introducing amorphous regions or defects (such as missing ligands or missing nodes) into materials can significantly improve the electrochemical performance of the materials, and the mechanism of action includes:

[0011] (1) Improve porosity accessibility and molecular diffusion rate;

[0012] (2) Provides additional active sites for electron transfer or analyte binding;

[0013] (3) Enhance the conductivity of materials by optimizing the charge permeation network.

[0014] For UiO-66 material used to detect levodopa, an effective means of determining the electrochemical behavior of UiO-66 by adjusting the ratio of crystalline to amorphous states can be provided. This allows for the adjustment of the stability of UiO-66 material and its sensitivity and selectivity for levodopa detection. Although it is conventionally believed that an excessively high proportion of amorphous states leads to a decrease in the electrochemical performance of MOF materials, the inventors unexpectedly discovered that when the proportion of amorphous states in UiO-66 is higher than that of crystalline states, its levodopa detection sensitivity is significantly improved, enabling ultra-trace detection (detection limit as low as 10 nM). This breakthrough discovery provides a new material design approach for developing high-performance levodopa sensors.

[0015] Therefore, this application proposes a method for detecting levodopa. According to an embodiment of this application, the method includes: contacting a sample to be tested with a sensor to generate a detectable signal; the sensor includes a working electrode, the surface of which is loaded with UiO-66 material; obtaining a detection result of the sample to be tested based on the detectable signal; wherein the Ui-O66 material is prepared by reacting terephthalic acid or its derivatives with a soluble zirconium salt for a predetermined time, the predetermined time being determined based on the known composition of the sample to be tested.

[0016] Based on the known components in the sample to be tested, the detection requirements for levodopa can be determined, specifically in terms of sensitivity, selectivity, or stability. The inventors of this application have discovered that the ratio of crystalline to amorphous states varies under different reaction time conditions, resulting in differences in the material's stability, detection sensitivity, and selectivity. Therefore, based on different detection requirements, the appropriate reaction time for the UiO-66 material can be selected. This allows for precise adaptation of the UiO-66 material to the detection requirements of levodopa, covering a detection range from ultra-trace (10 nM) to high concentration (300 nM), exhibiting high sensitivity while also maintaining excellent selectivity and stability, meeting the detection requirements of different scenarios, and suitable for wide application.

[0017] According to embodiments of this application, the method for detecting levodopa may also have the following additional technical features:

[0018] According to an embodiment of this application, the predetermined time is determined based on the known components of the sample to be tested, according to a predetermined rule; the predetermined rule includes:

[0019] When the sample to be tested contains levodopa and at least one of the following substances: ascorbic acid, acetaminophen, uric acid, tyrosine and dopamine, the predetermined time is 10 hours to 26 hours;

[0020] When the levodopa content in the sample to be tested is not less than 50 nM, the predetermined time is 50 minutes to 26 hours;

[0021] When the levodopa content in the sample to be tested is not less than 30 nM and less than 50 nM, the predetermined time is 50 minutes to 14 hours.

[0022] When the levodopa content in the sample to be tested is less than 30 nM, the predetermined time is 50 minutes to 8 hours.

[0023] According to an embodiment of this application, the predetermined time is determined based on the known components of the sample to be tested, according to a predetermined rule;

[0024] The predetermined rules include:

[0025] When the sample to be tested contains levodopa and at least one of the following substances: ascorbic acid, acetaminophen, uric acid, tyrosine and dopamine, the predetermined time is 10 hours to 26 hours;

[0026] When the levodopa content in the sample to be tested is 50 nM to 300 nM, the predetermined time is 50 minutes to 26 hours.

[0027] When the levodopa content in the sample to be tested is not less than 30 nM and less than 50 nM, the predetermined time is 50 minutes to 14 hours.

[0028] When the levodopa content in the sample to be tested is not less than 10 nM and less than 30 nM, the predetermined time is 50 minutes to 8 hours.

[0029] According to an embodiment of this application, the predetermined time is determined based on the known components of the sample to be tested, according to a predetermined rule;

[0030] The predetermined rules include:

[0031] When the sample to be tested contains levodopa and at least one of the following substances: ascorbic acid, acetaminophen, uric acid, tyrosine and dopamine, the predetermined time is 12 hours or 24 hours;

[0032] When the levodopa content in the sample to be tested is 50 nM to 300 nM, the predetermined time is 1 hour, 6 hours, 12 hours or 24 hours.

[0033] When the levodopa content in the sample to be tested is not less than 30 nM and less than 50 nM, the predetermined time is 1 hour, 6 hours or 12 hours.

[0034] When the levodopa content in the sample to be tested is not less than 10 nM and less than 30 nM, the predetermined time is 1 hour or 6 hours.

[0035] According to an embodiment of this application, the predetermined time is determined based on the detected ambient temperature and / or humidity, according to a predetermined rule;

[0036] The predetermined rules include:

[0037] When the detection environment is high temperature, high humidity and / or low temperature, the predetermined time is 10 hours to 26 hours, the high temperature is 80℃ to 120℃, the high humidity is 75% to 95% RH, and the low temperature is 0℃ to 10℃.

[0038] According to embodiments of this application, the soluble zirconium salt includes one or more of ZrCl4, ZrO(NO3)2·xH2O, ZrOCl2·xH2O, and Zr(SO4)2·xH2O, where 1≤x≤8.

[0039] According to embodiments of this application, the derivatives of terephthalic acid include one or both of chloroterephthalic acid and sulfonic acid terephthalic acid.

[0040] According to an embodiment of this application, the reaction temperature is 100°C to 130°C.

[0041] According to embodiments of this application, the reaction is carried out in water and a polar amide solvent.

[0042] According to embodiments of this application, the polar amide solvent includes one or more of N,N-dimethylformamide, N,N-diethylformamide, and N,N-dimethylacetamide.

[0043] According to an embodiment of this application, the reaction includes: dissolving 1-4 mmol Zr(SO4)2·H2O and 1-4 mmol terephthalic acid in 70 mL-100 mL of water, adding 0.5 mL-2 mL of formic acid and mixing well, then adding 5 mL-15 mL of N,N-dimethylformamide and letting it stand for a predetermined time.

[0044] According to an embodiment of this application, the working electrode is selected from screen-printed electrodes.

[0045] According to embodiments of this application, the sample to be tested includes blood, plasma, saliva, or interstitial fluid.

[0046] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0047] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0048] Figure 1 The following diagrams illustrate the UiO-66 metal-organic framework (MOF): (A) Simplified front view; (B) Front view with channels, cavity filling, and unit cell labeled; (C) Top view with primary and secondary pore sizes labeled; (D) Top view with channels, cavity filling, and unit cell labeled. Orange spheres represent primary pore sizes, and green spheres represent secondary pore sizes; both can be used for molecular encapsulation and trapping.

[0049] Figure 2 The chemical structure of 1,4-phthalic acid ester (H2BDC) is shown.

[0050] Figure 3 Fourier transform infrared (FTIR) spectra of UiO-66 materials synthesized at four different reaction times are shown.

[0051] Figure 4 The XRD patterns of UiO-66 metal-organic framework materials prepared under two different synthesis times are shown: the product synthesized at 1 hour has lower crystallinity and is mainly amorphous, while the product synthesized at 24 hours has a highly crystalline structure and very few defects.

[0052] Figure 5 The transmission electron microscope (TEM) images of UiO-66 (1h) are shown, with the left and right images taken from different angles.

[0053] Figure 6 The transmission electron microscope (TEM) image of (A) UiO-66(1h) is shown. Figure 5 (a) Left image); (b) 3D reconstructed TEM image of UiO-66(1h); (c) Height distribution fitting result based on Boltzmann bending step function f(x) = y0+ h / 2 tanh(ξ / w) + αξ + βξ2 (where ξ = x - x0); (d) Height distribution fitting using Gaussian function f(x) = y0+a exp[-(x - x0)2 / b2] (parameters c, g, k and l); (e) Corresponding material height distribution fitting based on Lorentz function f(x) = y0+a / [b0+ (x-x0)2].

[0054] Figure 7 The transmission electron microscope (TEM) image of (A) UiO-66 (1h) is shown. Figure 5(a) Right image); (b) 3D reconstructed TEM image of UiO-66 (1h); (c) Height distribution fitted with Boltzmann bending step function f(x) = y0 + h / 2tanh(ξ / w) + αξ + βξ2 (where ξ = x - x0); (d) Height distribution fitted with Gaussian function f(x) = y0 + a exp[-(x - x0)2 / b2] (c, g, k and l parameters); (e) Height distribution fitted with Lorentz function f(x) = y0 + a / [b0 + (x-x0)2], corresponding to the above materials respectively.

[0055] Figure 8 Transmission electron microscope images of UiO-66 (6h) are shown, with the left and right images taken from different angles.

[0056] Figure 9 The transmission electron microscope (TEM) image of (A) UiO-66 (6h) is shown. Figure 8 (a) Left image); (b) 3D reconstructed TEM image of UiO-66 (6h); (c) Height distribution fitted with Boltzmann bending step function f(x) = y0 + h / 2 tanh(ξ / w) + αξ + βξ2 (where ξ = x - x0); (d) Height distribution fitted with Gaussian function f(x) = y0 + aexp[-(x - x0)2 / b2] (c, g, k and l parameters); (e) Height distribution fitted with Lorentz function f(x) = y0 + a / [b0 + (x-x0)2], corresponding to the above materials respectively.

[0057] Figure 10 The transmission electron microscope image of (A) UiO-66 (6h) is shown. Figure 8 (a) Right image); (b) Three-dimensional reconstructed transmission electron microscope image of UiO-66 (6h); (c) Height distribution fitted with Boltzmann bending step function f(x) = y0+h / 2 tanh(ξ / w) + αξ + βξ2 (where ξ = x - x0); (d) Height distribution fitted with Gaussian function f(x) = y0+ a exp[-(x - x0)2 / b2] (c, g, k and l parameters); (e) Height distribution fitted with Lorentz function f(x) = y0+ a / [b0+ (x-x0)2], corresponding to the above materials respectively.

[0058] Figure 11 The transmission electron microscope (TEM) images of UiO-66 (12h) are shown, with the left and right images taken from different angles.

[0059] Figure 12 The transmission electron microscope image of (A) UiO-66 (12h) is shown. Figure 11 (a) Left image); (b) Three-dimensional reconstructed transmission electron microscope image of UiO-66 (12h); (c) Height distribution fitted with Boltzmann bending step function f(x) = y0 + h / 2 tanh(ξ / w) + αξ + βξ2 (where ξ = x - x0); (d) Height distribution fitted with Gaussian function f(x) = y0 + a exp[-(x - x0)2 / b2] (c, g, k, and l parameters); (e) Height distribution fitted with Lorentz function f(x) = y0 + a / [b0 + (x-x0)2], corresponding to the above materials respectively.

[0060] Figure 13 The transmission electron microscope image of (A) UiO-66 (12h) is shown. Figure 11 (a) Right image); (b) Three-dimensional reconstructed transmission electron microscope image of UiO-66 (12h); (c) Height distribution fitted with Boltzmann bending step function f(x) = y0 + h / 2 tanh(ξ / w) + αξ + βξ2 (where ξ = x - x0); (d) Height distribution fitted with Gaussian function f(x) = y0 + a exp[-(x - x0)2 / b2] (c, g, k, and l parameters); (e) Height distribution fitted with Lorentz function f(x) = y0 + a / [b0 + (x-x0)2], corresponding to the above materials respectively.

[0061] Figure 14 The images shown are transmission electron microscope (TEM) images of the UiO-66 (24h), with the left and right images taken from different angles.

[0062] Figure 15 The transmission electron microscope image of (A) UiO-66 (24h) is shown. Figure 14(a) Right image); (b) Three-dimensional reconstructed transmission electron microscope image of UiO-66 (24h); (c) Height distribution fitted with Boltzmann bending step function f(x) = y0 + h / 2 tanh(ξ / w) + αξ + βξ2 (where ξ = x - x0); (d) Height distribution fitted with Gaussian function f(x) = y0 + a exp[-(x - x0)2 / b2] (c, g, k, and l parameters); (e) Height distribution fitted with Lorentz function f(x) = y0 + a / [b0 + (x-x0)2], corresponding to the above materials respectively.

[0063] Figure 16 The transmission electron microscope image of (A) UiO-66 (24h) is shown. Figure 14 (a) Left image); (b) Three-dimensional reconstructed transmission electron microscope image of UiO-66 (24h); (c) Height distribution fitted with Boltzmann bending step function f(x) = y0 + h / 2 tanh(ξ / w) + αξ + βξ2 (where ξ = x - x0); (d) Height distribution fitted with Gaussian function f(x) = y0 + a exp[-(x - x0)2 / b2] (c, g, k, and l parameters); (e) Height distribution fitted with Lorentz function f(x) = y0 + a / [b0 + (x-x0)2], corresponding to the above materials respectively.

[0064] Figure 17 FESEM microscopy images of the UiO-66 (1h) coated electrode surface are shown.

[0065] Figure 18 FESEM microscopy images of the electrode surface coated with UiO-66 (6h) are shown.

[0066] Figure 19 FESEM microscopy images of the electrode surface coated with UiO-66 (12h) are shown.

[0067] Figure 20 FESEM microscopy images of the electrode surface coated with UiO-66 (24h) are shown.

[0068] Figure 21 The stability evaluation of UiO-66 (1h) MOF based on particle size is shown under three temperature conditions (4±1°C, 25±1°C, and 35±1°C) and eight pH conditions (3, 4, 5, 6, 7.4, 8.5, 9, and 10). This figure was generated using GraphPadPrism software, and each subplot has a different style and parameter settings.

[0069] Figure 22 The stability evaluation of UiO-66 (6h) MOF based on particle size is shown under three temperature conditions (4±1°C, 25±1°C, and 35±1°C) and eight pH conditions (3, 4, 5, 6, 7.4, 8.5, 9, and 10). This figure was generated using GraphPadPrism software, and each subplot has a different style and parameter settings.

[0070] Figure 23 The stability evaluation of UiO-66 (12h) MOF based on particle size is shown under three temperature conditions (4±1°C, 25±1°C, and 35±1°C) and eight pH conditions (3, 4, 5, 6, 7.4, 8.5, 9, and 10). This figure was generated using GraphPadPrism software, and each subplot has a different style and parameter settings.

[0071] Figure 24 The stability evaluation of UiO-66 (24h) MOF based on particle size is shown under three temperature conditions (4±1°C, 25±1°C, and 35±1°C) and eight pH conditions (3, 4, 5, 6, 7.4, 8.5, 9, and 10). This figure was generated using GraphPadPrism software, and each subplot has a different style and parameter settings.

[0072] Figure 25 Cyclic voltammetry (CVs) curves of a UiO-66-based metal-organic framework (MOF) modified screen-printed electrode (SPE) synthesized with a fixed reaction time (1 h) are shown at a levodopa concentration gradient.

[0073] Figure 26 Cyclic voltammetry (CVs) curves of a UiO-66-based metal-organic framework (MOF) modified screen-printed electrode (SPE) synthesized with a fixed reaction time (6 h) are shown under a levodopa concentration gradient.

[0074] Figure 27 Cyclic voltammetry (CVs) curves of a UiO-66-based metal-organic framework (MOF) modified screen-printed electrode (SPE) synthesized with a fixed reaction time (12 h) are shown at a levodopa concentration gradient.

[0075] Figure 28 Cyclic voltammetry (CVs) curves of a UiO-66-based metal-organic framework (MOF) modified screen-printed electrode (SPE) synthesized with a fixed reaction time (24 h) are shown at a levodopa concentration gradient.

[0076] Figure 29Cyclic voltammetry (CVs) curves of a screen-printed electrode (SPE) modified with UiO-66 metal-organic framework (MOF) are shown in the presence of different concentrations of tyrosine, acetaminophen, ascorbic acid, uric acid, dopamine and mixtures thereof, with the MOF synthesis reaction time fixed at 1 hour.

[0077] Figure 30 Cyclic voltammetry (CVs) curves of a screen-printed electrode (SPE) modified with UiO-66 metal-organic framework (MOF) are shown in the presence of different concentrations of tyrosine, acetaminophen, ascorbic acid, uric acid, dopamine and mixtures thereof, with the MOF synthesis reaction time fixed at 24 hours. Detailed Implementation

[0078] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0079] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0080] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0081] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

[0082] The inventors of this application have discovered that by adjusting the ratio of crystalline to amorphous states, the stability of Uio-66 material and its sensitivity and selectivity for detecting levodopa can be modified. While it is conventionally believed that an excessively high proportion of amorphous states leads to a decrease in the electrochemical performance of MOF materials, the inventors unexpectedly found that when the proportion of amorphous states in UiO-66 is higher than that of crystalline states, its levodopa detection sensitivity is significantly improved, enabling ultra-trace detection (detection limit as low as 10 nM). This breakthrough discovery provides a new material design approach for developing high-performance levodopa sensors.

[0083] Therefore, this application proposes a method for detecting levodopa. According to an embodiment of this application, the method includes: contacting a sample to be tested with a sensor to generate a detectable signal; the sensor includes a working electrode, the surface of which is loaded with UiO-66 material; and obtaining a detection result of the sample to be tested based on the detectable signal, specifically, the detection result includes the levodopa content; wherein the Ui-O66 material is prepared by reacting terephthalic acid or its derivatives with a soluble zirconium salt for a predetermined time, the predetermined time being determined based on the known composition of the sample to be tested.

[0084] Based on the known components in the sample to be tested, the detection requirements for levodopa can be determined, specifically in terms of sensitivity, selectivity, or stability. The inventors discovered that the ratio of crystalline to amorphous states varies under different reaction time conditions, resulting in differences in the material's stability, detection sensitivity, and selectivity.

[0085] Specifically, UiO-66 materials with a higher proportion of amorphous state than crystalline state have higher detection sensitivity;

[0086] When the ratio of crystalline to amorphous states reaches equilibrium, the oxidation reaction of L-DOPA can be enhanced. UiO-66 material has both good stability, detection sensitivity and selectivity, especially excellent selectivity. It can still specifically detect L-DOPA in the presence of coexisting interfering substances such as ascorbic acid, acetaminophen, uric acid, tyrosine and dopamine, ensuring high selectivity in complex biological matrices.

[0087] Therefore, based on different detection requirements, the appropriate reaction time for the UiO-66 material can be selected. This allows for precise adaptation of the UiO-66 material to the detection requirements of levodopa, covering a detection range from ultra-trace (10 nM) to high concentration (300 nM), exhibiting high sensitivity while also ensuring excellent selectivity and stability, meeting the detection requirements of different scenarios and suitable for wide application.

[0088] It should be noted that the known components of the sample to be tested can be estimated by considering the sample's origin, such as predicting the components it contains and the approximate content of levodopa. Furthermore, the preset time can be determined based on these estimates.

[0089] It should also be noted that the ratio of crystalline to amorphous states in UiO-66 material can be determined by the peak shapes of the diffraction peaks in the XRD pattern. The sharper and clearer the diffraction peaks, the higher the proportion of crystalline state and the higher the crystallinity; the corresponding peaks can be called "crystalline peaks." Conversely, the wider and weaker the diffraction peaks, the higher the proportion of amorphous state and the lower the crystallinity; the corresponding peaks can be called "amorphous peaks." This allows for a qualitative assessment of the ratio of crystalline to amorphous states. Furthermore, based on the peak information of crystalline and amorphous peaks, such as peak area and half-peak width, the ratio of crystalline to amorphous states can be quantitatively compared.

[0090] In some embodiments, the detectable signal includes a time-current response curve. Based on the time-current response curve of the test sample and a series of time-current response curves of levodopa at known concentrations, the concentration of levodopa in the test sample can be calculated.

[0091] According to embodiments of this application, the sample to be tested includes blood, plasma, saliva, or interstitial fluid.

[0092] According to embodiments of this application, the soluble zirconium salt comprises one or more of ZrCl4, ZrO(NO3)2·xH2O, ZrOCl2·xH2O, and Zr(SO4)2·xH2O, where 1 ≤ x ≤ 8, for example, 1, 2, 3, 4, 5, 6, 7, and 8; the terephthalic acid derivative comprises one or two of chloroterephthalic acid and sulfonic acid terephthalic acid; the reaction is carried out in water and a polar amide solvent; the polar amide solvent comprises one or more of N,N-dimethylformamide, N,N-diethylformamide, and N,N-dimethylacetamide. Therefore, the UiO-66 material prepared by reacting the soluble zirconium salt with terephthalic acid or its derivatives possesses excellent stability, detection sensitivity, and selectivity.

[0093] According to an embodiment of this application, the predetermined time is determined based on the known components of the sample to be tested, according to a predetermined rule;

[0094] The predetermined rules include:

[0095] (1) When the sample to be tested contains levodopa and at least one of the following substances: ascorbic acid, acetaminophen, uric acid, tyrosine, and dopamine, the predetermined time is 10 hours to 26 hours (e.g., 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, or 26 hours). By controlling the reaction time to 10 hours to 26 hours, a highly selective UiO-66 material can be obtained, which can still specifically detect levodopa in the presence of coexisting interfering substances such as ascorbic acid, acetaminophen, uric acid, tyrosine, and dopamine, ensuring high selectivity in complex biological matrices. Among them, when the predetermined time is 10 hours to 14 hours, the crystalline and amorphous ratios in the obtained UiO-66 material are close, reaching an equilibrium state, which can enhance the levodopa oxidation reaction and obtain a more selective UiO-66 material.

[0096] (2) When the levodopa content in the sample to be tested is not less than 50 nM (e.g., 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM), the predetermined time is 50 minutes to 26 hours (e.g., 50 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours). Therefore, the levodopa content in the sample to be tested can be accurately determined.

[0097] (3) When the content of levodopa in the sample to be tested is not less than 30 nM and less than 50 nM (for example, it can be 30 nM, 32 nM, 35 nM, 36 nM, 38 nM, 40 nM, 42 nM, 45 nM, or 48 nM), the predetermined time is 50 minutes to 14 hours (for example, it can be 50 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, or 14 hours). Therefore, the levodopa content in the sample to be tested can be accurately determined. Wherein, a preset time of 1 hour to 12 hours results in a higher proportion of amorphous material in the obtained UiO-66 material, a lower detection limit, and higher sensitivity.

[0098] (4) When the content of levodopa in the sample to be tested is less than 30 nM (e.g., 10 nM, 15 nM, 20 nM, 25 nM), the predetermined time is 50 minutes to 8 hours (e.g., 50 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours). By controlling the predetermined time to 50 minutes to 8 hours, the proportion of amorphous state in the obtained UiO-66 material is higher than that of crystalline state, which exhibits a significantly improved detection sensitivity. This discovery breaks through the traditional concept that an excessively high proportion of amorphous state will lead to a decrease in electrochemical performance. Experimental results show that the detection limit of this UiO-66 material with a high proportion of amorphous state for levodopa can be as low as 10 nM, achieving an ultra-trace detection level. This special structure-performance relationship provides a new material design idea for the development of high-sensitivity biosensors.

[0099] According to an embodiment of this application, the predetermined time is determined based on the known components of the sample to be tested, according to a predetermined rule;

[0100] The predetermined rules include:

[0101] When the sample to be tested contains levodopa and at least one of the following substances: ascorbic acid, acetaminophen, uric acid, tyrosine and dopamine, the predetermined time is 10 hours to 26 hours, preferably 12 hours or 24 hours.

[0102] When the levodopa content in the sample to be tested is 50 nM to 300 nM, the predetermined time is 50 minutes to 26 hours, preferably 1 hour, 6 hours, 12 hours or 24 hours.

[0103] When the levodopa content in the sample to be tested is not less than 30 nM and less than 50 nM, the predetermined time is 50 minutes to 14 hours, preferably 1 hour, 6 hours or 12 hours.

[0104] When the levodopa content in the sample to be tested is not less than 10 nM and less than 30 nM, the predetermined time is 50 minutes to 8 hours, preferably 1 hour or 6 hours.

[0105] According to an embodiment of this application, the predetermined time is determined based on the ambient temperature and / or humidity being detected, according to predetermined rules. These predetermined rules include: when the environment being detected is high temperature, high humidity, and / or low temperature, the predetermined time is 10 to 26 hours; the high temperature is 80°C to 120°C; the high humidity is 75% to 95% RH; and the low temperature is 0°C to 10°C. By controlling the predetermined time to 10 to 26 hours, the crystalline and amorphous states of the obtained UiO-66 material system reach equilibrium, and the dominant process shifts from growth to stabilization and defect repair, resulting in enhanced structural stability and durability. This makes it suitable for long-term monitoring in harsh environments such as high humidity or high temperature, and also applicable to devices requiring a temporary lifespan, such as disposable sensors used in short-term intensive care monitoring. Furthermore, a predetermined time of 22 to 26 hours results in even stronger stability of the obtained UiO-66 material.

[0106] According to embodiments of this application, the reaction temperature is 100℃~130℃, for example 100℃, 105℃, 110℃, 115℃, 120℃, 125℃, and 130℃. This ensures the reaction system is under ideal solvothermal synthesis conditions, promoting effective coordination between metal ions (soluble zirconium salt) and organic ligands (terephthalic acid or its derivatives) while avoiding structural damage due to excessively high temperatures. Secondly, this temperature is beneficial for controlling crystallization kinetics, resulting in UiO-66 materials with both high specific surface area and stable structure. Finally, the mild reaction conditions ensure the controllability and repeatability of the synthesis process, laying the foundation for large-scale production.

[0107] According to embodiments of this application, the reaction comprises: dissolving 1-4 mmol Zr(SO4)2·H2O and 1-4 mmol terephthalic acid in 70 mL-100 mL of water, adding 0.5 mL-2 mL of formic acid and mixing well, then adding 5 mL-15 mL of N,N-dimethylformamide and allowing it to stand for a predetermined time. This facilitates the effective coordination of Zr(SO4)2·H2O and terephthalic acid, resulting in a Uio-66 material with both high specific surface area and stable structure, exhibiting high detection sensitivity and selectivity.

[0108] According to embodiments of this application, the base electrode includes a screen-printed electrode. Screen-printed electrodes (SPEs) have become an important platform for developing disposable, portable, and scalable electrochemical sensors. These electrodes are fabricated using thick-film printing technology, offering high repeatability and enabling low-cost, large-scale production. By loading the Uio-66 material prepared using the aforementioned method onto the screen-printed electrode, the stability of the working electrode can be effectively improved. Its application in the detection of levodopa exhibits high selectivity and high sensitivity, meeting various detection requirements.

[0109] For example, the sensor used to detect levodopa can be a portable biosensor for point-of-care testing.

[0110] According to the embodiments of this application, the method for detecting levodopa in this application also has the following advantages:

[0111] (1) Tunable MOF structure: The crystallinity can be adjusted by controlling the synthesis time, thereby optimizing the electrochemical activity.

[0112] (2) High sensitivity: The increased specific surface area and the abundance of defective active sites significantly enhanced the L-DOPA oxidation signal.

[0113] (3) Selective detection: effectively reduces interference from matrix components (such as ascorbic acid, acetaminophen, tyrosine, dopamine and uric acid).

[0114] (4) Low-cost manufacturing: Based on screen-printed electrode (SPE) technology, mass production can be achieved, which is suitable for point-of-care diagnostic scenarios.

[0115] It should be noted that the method for detecting levodopa can be used for non-diagnostic purposes, such as drug quality control of levodopa preparations; it can also be used for diagnostic purposes, and can be applied in the field of point-of-care medical diagnosis, especially for monitoring neurological diseases (such as Parkinson's disease).

[0116] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0117] Example 1

[0118] I. Synthesis of UiO-66 Metal-Organic Framework Materials

[0119] In a 100 mL reactor, 2.5 mmol of terephthalic acid (H₂BDC) and 2.5 mmol of zirconium sulfate hydrate (Zr(SO₄)₂·H₂O) were dissolved in 80 mL of ultrapure water, and 1 mL of formic acid was added as a regulator. Then, 10 mL of N,N-dimethylformamide (DMF) was injected into the mixture, and after continuous stirring, the suspension was placed at 115°C for 1, 6, 12, and 24 hours, respectively. After the reaction was completed, the system was cooled to room temperature under nitrogen protection by an ammonia stream, and the synthesized product (MOF suspension) was collected after degassing.

[0120] Short-time reaction (1 hour) mainly produces an amorphous phase, while long-time reaction (24 hours) yields a highly crystalline structure. The crystallinity and chemical structure of the material were systematically characterized using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and scanning electron microscopy (SEM).

[0121] II. Electrode Modification

[0122] UiO-66 suspensions (samples with different crystallization times of 1 hour, 6 hours, 12 hours, and 24 hours) were loaded onto the surface of a screen-printed electrode (SPE) using a drop-coating method. The specific operation is as follows:

[0123] 1. Add 2 μL of MOF suspension (concentration 3 mg·mL) -1 The sample was spotted onto the working electrode area and allowed to dry overnight. Subsequently, a polythionine conductive layer was constructed using electrochemical deposition: first, 60 μL of 0.25 mM thionine solution (prepared with pH 7.2 PBS) was added to the electrode surface, and a +1.2 V potential was applied for 2 minutes to activate the monomer; then, a 100 mV·s⁻¹ potential was applied... -1 The polythionine film was grown by performing 20 cyclic voltammetric scans within a potential window of -0.6 V to +0.1 V. To further stabilize the film, the electrode was placed in pH 7.2 PBS and voltammetric scans were performed at 0.1 V·s. -1 The rate was 40 cycles of cyclic voltammetry scanning in the range of -0.5 V to +0.1 V.

[0124] 2. Enzyme immobilization step: 1.4 mg tyrosinase and 10 μL bovine serum albumin (BSA, final concentration 1 mg / mL) were added. -1 90 μL of PBS and 1 μL of 2.5% glutaraldehyde solution were mixed. 2.5 μL of this mixture was drop-coated onto the modified electrode surface and cross-linked and cured at room temperature. Assuming no loss of enzyme activity, the enzyme loading on the electrode surface was equivalent to 58.8 units of activity. For serum sample detection, the electrode was immersed in 1% Nafion solution for 30 seconds and dried at room temperature for 15 minutes. This process was repeated three times to form a protective layer, obtaining the final working electrode. Nafion was chosen based on its good biocompatibility and suitability for sensing bodily fluid environments as reported in the literature. Compared to other candidate materials (such as o-phenylenediamine polymers), although Nafion-modified sensors exhibit biofouling properties similar to other materials, they show superior reproducibility.

[0125] Stable adhesion of the MOF film is ensured through an electrochemical activation procedure (e.g., cyclic voltammetry in PBS).

[0126] III. L-DOPA Detection

[0127] The working electrode prepared above was subjected to cyclic voltammetry analysis with the following parameters:

[0128] Cyclic voltammetry (CV) tests of the prepared working electrode were performed in a three-electrode system: the modified working electrode was used as the working electrode, a platinum wire as the counter electrode, and an Ag / AgCl electrode as the reference. The electrolyte was 0.1 M phosphate-buffered saline (PBS) at pH 7.4. The potential scan range was -1.2 V to +1.2 V, and the scan rate was 50 mV s. -1 By repeatedly performing CV tests with varying analyte concentrations, a linear relationship between the oxidation peak current and concentration was established, and quantification was performed based on this relationship.

[0129] The limit of detection (LOD) is calculated using the formula LOD = 3σ / slope, where σ is the standard deviation of the blank signal and the slope is taken from the calibration curve. The linear dynamic range is determined from the lowest detectable concentration to the concentration range where linearity begins to deviate.

[0130] Selectivity experiments were conducted in the presence of potential interfering substances, including common serum tyrosine, acetaminophen, ascorbic acid, uric acid, and dopamine, to verify the electrode's specificity. The experimental procedure was consistent with CV testing, comparing peak current changes before and after the addition of interfering substances.

[0131] Stability studies included: ① repeated CV tests for several consecutive days at a fixed concentration of the target analyte to assess signal retention; ② drying and storing the electrode at 4 °C, and testing its response again after a maximum of 4 weeks to examine long-term storage stability.

[0132] IV. Results and Analysis

[0133] 1. Crystallinity-dependent structural and morphological evolution of UiO-66 framework materials: analytical characterization and quantitative modeling

[0134] Zirconium (Zr)-based UiO-66 framework materials (such as...) Figure 1 As shown, this study aimed to systematically investigate the time-dependent evolution of crystallinity and its related structural characteristics through synthesis at a series of controllable reaction times (specifically 1 hour, 6 hours, 12 hours, and 24 hours). Characterization employed a combination of techniques, including Fourier transform infrared spectroscopy (FTIR), powder X-ray diffraction (PXRD), transmission electron microscopy (TEM), and 3D height map reconstruction, combined with multivariate mathematical modeling. The research objective was to establish a quantitative correlation between synthesis time, crystallinity, and functionally relevant material properties of metal-organic frameworks (MOFs), including porosity, surface uniformity, and defect distribution.

[0135] like Figure 3As shown, the FTIR spectra of all UiO-66 samples exhibit characteristic vibrational modes of the terephthalic acid carboxylic acid linker. Notably, at 1580 cm⁻¹... -1 Nearby (COO) - (asymmetric stretching vibration) and 1390 cm -1 Nearby (COO) - The bands of symmetric stretching vibrations can serve as sensitive indicators of the coordination status of Zr6 nodes. Short-duration samples (1 hour and 6 hours) exhibit broadened peaks, lower intensities, and unresolved shoulders, indicating the presence of numerous uncoordinated or weakly bound carboxylic acid groups. Long-duration samples (12 hours and 24 hours) show sharper, stronger peaks and a reduced full width at half maximum (FWHM), indicating a more homogeneous linker environment and more complete coordination. This spectroscopic evidence supports the time-dependent evolution of coordination and highlights the crucial role of thermal aging in achieving complete node-linker integration.

[0136] PXRD pattern ( Figure 4 The presence of UiO-66 crystal domains was confirmed, and the crystallinity increased with increasing reaction time. The 1-hour sample showed broadened, low-intensity diffraction peaks and increased baseline noise, typical characteristics of significant amorphous components, indicating low crystallinity and predominantly amorphous features; its peak positions were difficult to calibrate and did not conform to standard UiO-66. The 6-hour sample showed clearer peak development, but still lacked the sharpness and resolution of high-quality crystals. The 12-hour and 24-hour samples exhibited clear diffraction patterns highly consistent with the original UiO-66 simulation data, indicating the successful construction of the extended periodic network. The grain size was estimated using the Scherrer equation, and the calculated crystal domain size increased from approximately 49.4 nm (1 hour) to 76.5 nm (24 hours), confirming the gradual growth of the crystal over time. The 24-hour synthesized product exhibited a highly crystalline structure with very few defects.

[0137] TEM images ( Figure 5 Further differentiation was made between the morphological differences of low-crystallinity and high-crystallinity samples: the 1-hour sample consisted of irregular particles, exhibiting a faceted, isotropic morphology with high surface roughness; the 24-hour sample, on the other hand, displayed a faceted polyhedral morphology consistent with a face-centered cubic framework. (In the 3D height reconstruction image...) Figure 6 and Figure 7 In the 1-hour sample, discontinuous morphological features and inconsistent lattice order were observed, indicating incomplete or partially missing framework expansion. Notably, volume reconstruction revealed highly non-uniform distribution and pore deformation, consistent with structural collapse or defect formation in the early stages of nucleation.

[0138] Figures 6 to 16The study presents a comprehensive visualization and quantitative characterization of UiO-66 nanoparticles synthesized under different reaction durations (1 hour, 6 hours, 12 hours and 24 hours), providing key insights into the structural evolution, morphological maturity and surface uniformity of materials during solvothermal reactions.

[0139] TEM micrographs (in the initial stage of synthesis (1 hour)) Figure 6 (A) Figure 7 Image (A) shows grains with blurred boundaries and irregular shapes, exhibiting loosely defined grain characteristics. The relative amorphousness of these grains indicates that nucleation events are dominant, but crystal growth is insufficient. The corresponding 3D surface reconstruction image (A) Figure 6 (B) Figure 7 (B) further supports this conclusion: the height profile shows insufficient uniformity, flat-top structure, and poor crystal plane development. This type of morphology indicates the connection between Zr6 nodes and BDC (…). Figure 2 The time required for self-assembly to form a standard octahedral UiO-66 framework is insufficient. Figure 6 China (CE) and Figure 7 Statistical modeling of the high-order distribution in the middle (CE) provides a more in-depth analysis: Boltzmann S-shaped fitting ( Figure 6 (C) Figure 7 The middle (C) shows a gradual transition, reflecting high distribution heterogeneity and no clear boundary between particle edges; Gaussian fitting ( Figure 6 (D) Figure 7 The asymmetry of the middle (D) curve and its wide half-width at half-maximum (FWHM) indicate non-uniform surface features; the Lorentz curve ( Figure 6 Middle (E) Figure 7 The middle (E) exhibits an extended tail, which is a typical characteristic of localized defects and highly dispersed systems. Comprehensive analysis indicates incomplete framework formation, particle aggregation, and a high energy barrier for crystal nucleation.

[0140] After 6 hours of reaction, the morphology showed significant refinement. (TEM image) Figure 8 , Figure 9 (A) Figure 10 (A) shows that the nanoparticle boundaries are clearer and the edges are smoother, with some particles exhibiting initial signs of polyhedralization. Compared to the 1-hour sample, the particle size and shape are more uniform, indicating that the system has successfully transitioned from a nucleation-dominated to a growth-dominated kinetic stage. 3D reconstruction image ( Figure 9 (B) Figure 10 (B) shows increased height and more defined geometric features, with a clearer contrast and steeper slope in the height profile at the grain edges. This trend indicates more localized crystal growth, reduced surface defects, and improved grain integrity. Statistical fitting results are consistent with the above observations: Boltzmann fitting ( Figure 9 (C) Figure 10 The middle (C) shows a steeper transition between the surface and granular regions, indicating a narrower size distribution; Gaussian and Lorentz profiles ( Figure 9 Middle (DE), Figure 10 The narrower and more symmetrical DE indicates enhanced uniformity and reduced morphological irregularity.

[0141] The sample synthesized in 12 hours represents a higher stage of morphological development. TEM image ( Figure 11 , Figure 12 (A) Figure 13 The image (A) shows well-faceted grains with higher crystallinity, and many exhibit regular shapes close to the expected octahedral geometry of UiO-66, indicating that growth proceeded along a specific crystallographic direction. This anisotropic growth is a typical characteristic of MOFs, stemming from the directionality of metal-connector coordination. 3D image ( Figure 12 (B) Figure 13 (B) shows a significantly sharp transition in height and uniform particle height, with extremely low surface roughness in the interparticle regions. The morphological uniformity is achieved through excellent fitting of the Gaussian and Lorentz models. Figure 12 Middle (DE), Figure 13 As supported by (DE), the fitted curve at this point is close to the ideal distribution, with minimal asymmetry in the tail and peak. These parameters indicate that the growth-induced defects have been successfully terminated, and the system tends towards a uniform thermodynamically stable morphology. Boltzmann transition ( Figure 12 (C) Figure 13 The middle (C) is also sharper, reflecting a clear boundary between the background and the particle area—an indicator of low polydispersity.

[0142] The final stage of synthesis, lasting 24 hours, marks the completion of the crystallization process. TEM image ( Figure 14 , 15 (A) Figure 16 The image (A) shows highly uniform particles with sharp, clear edges. Many particles exhibit the expected octahedral geometry of UiO-66, consistent with the complete coordination of the Zr6O4(OH)4 cluster with the 1,4-phthalic acid linker. The results confirm that the system has reached equilibrium, and the dominant process has shifted from growth to stabilization and defect repair. (3D reconstruction image) Figure 15 In (B) and (B) of 16, the high crystallinity and uniform particle height are more clearly demonstrated. The surface height variation of these well-developed particles is minimal, indicating that agglomeration or surface roughness is negligible. Statistical model ( Figure 15 The results from CE16 and CE16 show that the Gaussian and Lorentz fits are almost perfect, with extremely narrow half-widths and highly symmetrical profiles; the Boltzmann transition exhibits a steep S-shaped change, corresponding to near monodispersity of the high-population population and complete spatial separation between particles and the substrate.

[0143] A clear morphological evolution trajectory was observed across all reaction durations: from vaguely defined nucleating seed crystals (1 hour), through planarization and growth (6-12 hours), to the final formation of fully crystalline octahedral particles (24 hours). This process conforms to the classic LaMer growth model, i.e., controlled growth and size focusing after nucleation. 3D morphology reconstruction and statistical modeling reinforced this kinetic path, reflecting reduced polydispersity and improved uniformity through quantitative indicators such as narrowing of the full width at half maximum (FWHM), enhanced Gaussian symmetry, and sharpening of the Boltzmann transition. Such multi-scale, statistically supported characterization is crucial for quality control in MOF synthesis, especially when target applications require high crystallinity and monodispersity. The synergistic effect of high-resolution imaging, 3D morphology reconstruction, and robust statistical fitting not only elucidates the formation kinetics of UiO-66 but also establishes a reproducible framework for the analysis of other MOF and nanoparticle systems.

[0144] 2. L-DOPA detection based on different crystal / amorphous UiO-66

[0145] This application focuses on the development and characterization of a series of UiO-66-based metal-organic frameworks (MOFs) synthesized and immobilized on screen-printed electrodes (SPEs) at different time intervals (1 hour, 6 hours, 12 hours, and 24 hours). The research objective is to optimize the crystal structure, morphological stability, and electrochemical performance of the UiO-66-modified electrodes to achieve selective and highly sensitive detection of L-DOPA. The aim is to elucidate the correlation between synthesis time and structure-electronic performance indicators, ultimately preparing a high-performance, miniaturized electrochemical biosensor suitable for real-time point-of-care detection. From a broader perspective, this research integrates interdisciplinary studies in materials science, analytical chemistry, neuroscience, and biomedical engineering. By combining crystal engineering principles with advanced electrochemical techniques and physiological relevance, this application fills a key translational gap in non-invasive disease monitoring. The exploration of the time dependence of MOF crystallization and its impact on electron transfer kinetics enriches the fundamental theory of nanostructured sensing interfaces. Furthermore, the application of screen-printed electrodes reflects the trend of scalable manufacturing technology converging with next-generation medical diagnostics.

[0146] Figure 17Field emission scanning electron microscopy (FESEM) images of UiO-66 synthesized in 1 hour and loaded on SPE are presented. Its surface morphology is significantly amorphous and blurry, indicating very few nucleation events in the early stages of MOF formation. From a crystallographic perspective, this disorder severely limits the accessibility of the micropores required for molecular adsorption. In electrochemical sensing scenarios, such disordered networks typically reduce surface conductivity and weaken electron transfer efficiency, and are therefore generally considered unfavorable for achieving high-sensitivity detection. However, the results of this application show the opposite trend: despite the underdeveloped morphology being considered poorly conductive, this system still achieved the best detection limit among similar configurations. This suggests that while morphology may hinder conductivity to some extent, it simultaneously provides unique advantages that can be efficiently utilized. For example, this structural feature is particularly advantageous in scenarios where binding affinity requirements are low or diffusion control is needed, such as sustained-release drug delivery platforms, or sensors designed to minimize nonspecific interactions. Therefore, although it is not the ideal morphology for high-sensitivity electrochemical detection in the traditional sense, it can achieve excellent sensitivity and open up new possibilities for specialized biomedical applications that require precise customization of surface interactions.

[0147] Figure 18 The UiO-66 / SPE synthesized over 6 hours is shown, exhibiting preliminary evidence of partial crystallization. The material initially displays a semi-ordered granular structure with moderate porosity. This transitional morphology simultaneously enhances surface area and accessibility to redox reactive sites. From a chemical engineering perspective, this structure may strike a balance between conductivity and mechanical stability, making it suitable for hybrid sensing systems requiring flexibility and responsiveness. For example, flexible biosensors integrated into wearable health monitoring devices could benefit from this intermediate morphology, as it maintains stability under mild mechanical deformation.

[0148] Figure 19 The key illustration shows the 12-hour synthesis of UiO-66 / SPE. Under these conditions, the nanocrystal structure is most uniform, with minimal particle aggregation and excellent dispersion. This optimized structure directly enhances the double-layer capacitance and electron mobility at the solid-liquid interface. Importantly, from a biomedical engineering perspective, this type of material offers superior analyte accessibility, making it ideal for detecting trace amounts of L-DOPA in interstitial fluid or saliva, thus supporting non-invasive diagnostic applications. Therefore, this synthesis duration represents a crucial turning point in MOF evolution, transitioning from structure formation to functionalization peaks.

[0149] Figure 20A case study of 24-hour synthesis was presented, where over-crystallization resulted in a dense, intergrowth structure. While this morphology is beneficial for long-term mechanical and chemical stability, it reduces the accessible surface area and hinders analyte diffusion. However, from an environmental sensing perspective, such robust structures can be used for long-term pollutant monitoring in harsh aquatic media, demonstrating an interdisciplinary approach from biomedicine to environmental monitoring.

[0150] Figure 21 The colloidal and thermal stability of the UiO-66 particles synthesized in 1 hour was emphasized. Significant fluctuations in particle size under different pH and temperature conditions indicate a fragile framework, making them susceptible to hydrolysis and thermal degradation. In biochemical applications, this instability makes them unsuitable for use in physiological environments with varying pH and temperature. However, from a pharmaceutical formulation perspective, this degradability could potentially be used as a controlled-release carrier in acidic tumor microenvironments.

[0151] Figure 22 The stability of the synthesized product after 6 hours showed some improvement but remained moderate. Partial crystallization imparted some toughness, but it could not completely resist thermal expansion or ionic interference. This intermediate stability characteristic may be suitable for devices requiring a short service life, such as disposable sensors used in short-term intensive care monitoring.

[0152] Figure 23 The synthesized UiO-66 particles exhibited excellent physicochemical stability over 12 hours. Their durability at physiological pH (6.8–7.4) and body-temperature-related temperatures (25–37 °C) makes them a preferred candidate for biosensing applications. From an interdisciplinary integration perspective, these properties are key to embedding these MOFs into microfluidic lab-on-a-chip systems or implantable devices, as long-term structural integrity under biofluid exposure is crucial.

[0153] Figure 24 It was confirmed that the UiO-66 framework structure synthesized over 24 hours was the most robust, but the porosity was reduced.

[0154] Figures 25 to 28 The cyclic voltammetric (CV) response results of four UiO-66 variants to increasing L-DOPA concentrations are presented in summary below:

[0155] Figure 25 The legend for the 1-hour sample shows different colored curves corresponding to levodopa solutions with concentrations ranging from 10 nM to 300 nM, characterizing the specific electrochemical response of the modified electrode. The oxidation and reduction peak currents show a regular increase with increasing substrate concentration, confirming the excellent sensitivity and electrocatalytic activity of this MOF material for levodopa detection. The experimental results show broad but poorly resolved redox peaks, indicating low electrochemical reversibility. The data reflect the sluggish electron transfer kinetics caused by amorphous and poorly connected pathways.

[0156] Figure 26 (6-hour sample) The different colored curves shown in the legend correspond to levodopa solutions with concentrations ranging from 10 nM to 400 nM, characterizing the specific electrochemical response of the modified electrode. The oxidation and reduction peak currents show a regular increase with increasing substrate concentration, confirming that this MOF material possesses excellent sensitivity and electrocatalytic activity for levodopa detection. Experimental results show improved redox peak resolution and higher peak currents, indicating enhanced analyte adsorption and diffusion. This finding is related to the observed semi-crystalline morphology, confirming the structure-function correlation.

[0157] Figure 27 The legend for the 12-hour sample shows different colored curves corresponding to L-DOPA solutions with concentrations ranging from 30 nM to 160 nM, characterizing the specific electrochemical response of the modified electrode. The sample exhibited the best performance, displaying a sharp redox peak, high current response, and a linear dependence on L-DOPA concentration. The results confirm the optimal synergy between porosity, crystallinity, and electrochemical accessibility.

[0158] Figure 28 (24-hour sample) The different colored curves shown in the legend correspond to levodopa solutions with concentrations ranging from 50 nM to 300 nM, characterizing the specific electrochemical response of the modified electrode. The oxidation and reduction peak currents show a regular increase with increasing substrate concentration, confirming that this MOF material has excellent sensitivity and electrocatalytic activity for levodopa detection. The figure shows strong but broader peaks, suggesting a saturation effect or diffusion limitation caused by dense crystal stacking. Although still functional, this configuration has limited advantages in the rapid detection of low-concentration analytes.

[0159] From a biosensor design perspective, the 12-hour sample achieved an ideal balance between sensitivity, specificity, and operational robustness. Furthermore, using L-DOPA as the target analyte linked this technology with neurology and psychopharmacology, building an interdisciplinary translational bridge.

[0160] Figure 29 and Figure 30 The specificity of the UiO-66-based sensor in the presence of structurally similar or electrochemically active interfering substances was investigated. Each colored curve represents the electrochemical response of the modified electrode to a specific concentration of analyte. The addition of different analyte concentrations did not cause significant changes in the oxidation and reduction peak currents, indicating that the developed sensor exhibits sensitivity and selectivity only for L-Dopa. (1-hour sample...) Figure 29 The 24-hour crystallization variant exhibited moderate selectivity, possibly due to disordered adsorption; in contrast, the 24-hour crystallization variant ( Figure 30MOFs exhibit superior selectivity due to pore size repulsion and electrostatic control—properties that can be further modulated through linker substitution or post-synthetic modification. These findings echo molecular recognition theory in biochemistry, reinforcing the potential applications of MOFs in enzyme-mimicking synthetic receptors or selective chemical sensors.

[0161] By preparing and immobilizing UiO-66 variants on SPE with different synthesis durations, a complex relationship between crystal structure, electrochemical properties, and potential biosensing applications was revealed. In particular, the exploration of synthesis durations provided important insights into the electrochemical behavior of the material and its applicability to L-DOPA detection.

[0162] From an electrochemical perspective, the UiO-66 / SPE synthesized in 12 hours shows the greatest potential in terms of sensitivity, linearity, and electrochemical reversibility. Its nanocrystal structure and well-dispersed composition enhance electron transfer kinetics and analyte accessibility, making it the optimal choice for low-concentration L-DOPA detection in real-time, non-invasive applications such as saliva or interstitial fluid analysis. This performance improvement is highly consistent with the expected behavior of electrochemical biosensors—higher crystallinity and optimized pore structure lead to superior capacitive behavior and high-resolution redox peaks.

[0163] From a biosensor design perspective, the 12-hour sample achieves an ideal balance between sensitivity, specificity, and robustness in real-world applications. However, the 1-hour and 6-hour samples perform better in terms of the limit of detection (LoD). Although the 1-hour sample is amorphous and structurally disordered, its higher surface area and looser packing structure may be more favorable for trace L-DOPA detection, thus exhibiting a relatively high LoD. Similarly, the 6-hour sample, due to its partial crystallization and moderate porosity, shows improved electron transfer kinetics and LoD compared to the highly crystalline 12-hour sample. While these materials are less advantageous in high-precision biosensing, they are beneficial in scenarios requiring rapid, low-concentration detection, such as point-of-care diagnostics or early disease screening devices.

[0164] In contrast, while the UiO-66 / SPE synthesized in 12 hours exhibits the best redox behavior and electrochemical resolution, it is prone to saturation at high analyte concentrations, reducing its applicability in rapid low-concentration detection. Its dense crystal structure, to some extent, inhibits analyte diffusion, leading to a decrease in its ability to detect low concentrations of L-DOPA (crucial for early diagnosis).

[0165] From a mathematical modeling perspective, the UiO-66 synthesized over 12 hours performs better due to its more stable and repeatable electrochemical response. Mathematical models incorporating parameters such as electrochemical reversibility, current response, and sensitivity tend to favor the 12-hour variant. These models highlight the material's ability to maintain high sensitivity consistently under various environmental conditions, making it an ideal choice for long-term applications in real-time monitoring systems.

[0166] In summary, while the 1-hour and 6-hour samples are more suitable for detecting low-concentration analytes due to their enhanced surface area and electrochemical activity, the 12-hour sample demonstrates superior overall electrochemical performance, robustness, and stability. Mathematical modeling further supports the 12-hour synthesis as the most promising candidate for biosensing applications requiring high consistency and performance, particularly in scenarios where measurement accuracy is critical over time. Table 1 summarizes the key experimental results for each synthesis duration, highlighting the balance between crystallinity, morphology, and electrochemical performance in L-DOPA detection. It also includes data on linear detection range, detection limit, stability, and specificity for each case, helping to clarify the performance of the UiO-66 / SPE sensor in different scenarios.

[0167] Table 1. Electrochemical performance of UiO-66 functionalized screen-printed electrodes (SPEs) for levodopa detection

[0168]

[0169] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A method for detecting levodopa, characterized in that, include: The sample to be tested is brought into contact with the sensor to generate a detectable signal; the sensor includes a working electrode, the surface of which is loaded with UiO-66 material. Based on the detectable signal, the detection result of the sample to be tested is obtained; in, The UiO-66 material is prepared by reacting terephthalic acid or its derivatives with a soluble zirconium salt for a predetermined time, the predetermined time being determined based on the known composition of the sample to be tested. The predetermined time is determined based on the known components of the sample to be tested, according to predetermined rules; The predetermined rules include: When the sample to be tested contains levodopa and at least one of the following substances: ascorbic acid, acetaminophen, uric acid, tyrosine and dopamine, the predetermined time is 10 hours to 26 hours; When the levodopa content in the sample to be tested is not less than 50 nM, the predetermined time is 50 minutes to 26 hours; When the levodopa content in the sample to be tested is not less than 30 nM and less than 50 nM, the predetermined time is 50 minutes to 14 hours. When the levodopa content in the sample to be tested is less than 30 nM, the predetermined time is 50 minutes to 8 hours.

2. The method according to claim 1, characterized in that, The predetermined time is determined based on the known components of the sample to be tested, according to predetermined rules; The predetermined rules include: When the sample to be tested contains levodopa and at least one of the following substances: ascorbic acid, acetaminophen, uric acid, tyrosine and dopamine, the predetermined time is 10 hours to 26 hours; When the levodopa content in the sample to be tested is 50 nM to 300 nM, the predetermined time is 50 minutes to 26 hours. When the levodopa content in the sample to be tested is not less than 30 nM and less than 50 nM, the predetermined time is 50 minutes to 14 hours. When the levodopa content in the sample to be tested is not less than 10 nM and less than 30 nM, the predetermined time is 50 minutes to 8 hours.

3. The method according to claim 1, characterized in that, The predetermined time is determined based on the known components of the sample to be tested, according to predetermined rules; The predetermined rules include: When the sample to be tested contains levodopa and at least one of the following substances: ascorbic acid, acetaminophen, uric acid, tyrosine and dopamine, the predetermined time is 12 hours or 24 hours; When the levodopa content in the sample to be tested is 50 nM to 300 nM, the predetermined time is 1 hour, 6 hours, 12 hours or 24 hours. When the levodopa content in the sample to be tested is not less than 30 nM and less than 50 nM, the predetermined time is 1 hour, 6 hours or 12 hours. When the levodopa content in the sample to be tested is not less than 10 nM and less than 30 nM, the predetermined time is 1 hour or 6 hours.

4. The method according to claim 1, characterized in that, The predetermined time is determined based on the detected ambient temperature and / or humidity, according to predetermined rules; The predetermined rules include: When the detection environment is high temperature, high humidity and / or low temperature, the predetermined time is 10 hours to 26 hours, the high temperature is 80℃ to 120℃, the high humidity is 75% to 95% RH, and the low temperature is 0℃ to 10℃.

5. The method according to claim 1, characterized in that, The soluble zirconium salt includes one or more of ZrCl4, ZrO(NO3)2·xH2O, ZrOCl2·xH2O and Zr(SO4)2·xH2O, where 1≤x≤8; The derivatives of terephthalic acid include one or both of chloroterephthalic acid and sulfonic acid terephthalic acid.

6. The method according to claim 1, characterized in that, The reaction temperature is 100℃~130℃; The reaction was carried out in water and a polar amide solvent; The polar amide solvent includes one or more of N,N-dimethylformamide, N,N-diethylformamide, and N,N-dimethylacetamide.

7. The method according to claim 1, characterized in that, The reaction includes: Dissolve 1-4 mmol Zr(SO4)2·H2O and 1-4 mmol terephthalic acid in 70 mL-100 mL of water, add 0.5 mL-2 mL of formic acid and mix well, then add 5 mL-15 mL of N,N-dimethylformamide and let stand for the predetermined time.

8. The method according to claim 1, characterized in that, The working electrode is selected from screen-printed electrodes.

9. The method according to claim 1, characterized in that, The samples to be tested include blood, plasma, saliva, or interstitial fluid.

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