Hyodeoxycholic acid detection method based on enzyme-free electrochemical sensing

Abstract

The invention relates to the technical field of electrochemical analysis and detection, and discloses a hyodeoxycholic acid detection method based on non-enzyme electrochemical sensing. According to the method, renewable beta-cyclodextrin (beta-CD) is taken as a matrix, an alkali metal potassium coordinated cyclodextrin metal organic framework material (K-CD-MOFs) is synthesized through an improved methanol gas phase diffusion method, and the alkali metal potassium coordinated cyclodextrin metal organic framework material (K-CD-MOFs) is loaded on a reduced graphene oxide (rGO) substrate; and the rGO-K-CD-MOFs nanocomposite material with high specific surface area, excellent hydrophilicity and biocompatibility is prepared. Neutral red is used as a redox probe, and the change of current in the system is detected through differential pulse voltammetry. Researches show that the detection performance is greatly optimized through the synergistic effect of rGO and K-CD-MOFs, the composite material has a wide linear detection range in the concentration range of 5.0-100.0 [mu] M, the sensitivity reaches 0.10 [mu] A.[ mu] M <-1 >, the detection limit (LOD, S / N = 3) is 1.97 [mu] M, and the composite material has excellent selectivity on hyodeoxycholic acid (HDCA).

Description

Technical Field

[0001] The invention relates to the technical field of electrochemical analysis and detection, and discloses a method for detecting hyodeoxycholic acid based on non-enzyme electrochemical sensing. Background Art

[0002] In recent years, bile acids, as functional feed additives, have played a key role in improving animal liver metabolic function and promoting the development of green farming. In the pig farming industry, hyodeoxycholic acid (HDCA) accounts for approximately 60% of the total bile acid content in feed, and its accurate detection has become a critical component in ensuring animal health. Current detection technologies mainly rely on non-electrochemical methods such as ultraviolet spectrophotometry (UV-Vis), thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC). However, these methods generally have significant drawbacks such as high instrument purchase costs, complex sample pretreatment, and strict operational requirements. Against this backdrop, electrochemical sensing technology is considered the detection solution with the greatest industrial potential due to its portability, rapid response, and ease of operation. It is worth noting that although existing enzyme-based electrochemical sensors have made significant progress, the cost of immobilized enzymes accounts for approximately 40-60% of the total sensor cost, and their activity is easily affected by ambient temperature. Therefore, the development of electrochemical biosensors based on non-enzymatic recognition mechanisms, particularly specific detection systems for HDCA, has become a key research direction to overcome existing technological bottlenecks. Bile acids, due to their lack of electrochemically active sites in their fatty alcohol structure, are difficult to detect directly using conventional voltammetry. This characteristic has prompted researchers to focus on the development of indirect electrochemical oxidation strategies.

[0003] Metal-organic frameworks (MOFs), a class of porous crystalline materials formed by the self-assembly of metal nodes and organic ligands, have become a research hotspot in the field of materials science due to their high specific surface area, tunable pore structure, and surface functionalization potential. However, traditional MOFs are mostly constructed using rigid ligands containing benzene rings and transition metal ions. The potential risk of metal dissolution and the biotoxicity of organic ligands significantly limit their application in food testing. To address this bottleneck, cyclodextrin metal-organic frameworks (CD-MOFs) based on food-grade raw materials have emerged. Taking β-cyclodextrin (β-CD) as an example, its unique truncated cone-shaped cavity structure (inner diameter approximately 0.65 nm, height approximately 0.78 nm) can form host-guest inclusion complexes with molecules of specific sizes through intermolecular interactions.

[0004] The present invention relates to the field of electrochemical analysis and detection technology, and discloses a liquid material of a reduced graphene-modified cyclodextrin metal-organic framework for electrochemical detection of hyodeoxycholic acid. The method uses renewable β-cyclodextrin (β-CD) as a matrix, synthesizes an alkali metal potassium-coordinated cyclodextrin metal-organic framework material (K-CD-MOFs) by an improved methanol vapor diffusion method, and loads it on a reduced graphene oxide (rGO) substrate to prepare an rGO-K-CD-MOFs nanocomposite material with high specific surface area, excellent hydrophilicity and biocompatibility. Neutral red is used as a redox probe, and the change in current in the system is detected by differential pulse voltammetry. Studies have shown that the synergistic effect of rGO and K-CD-MOFs greatly optimizes the detection performance. The composite material exhibits a wide linear detection range in the concentration range of 5.0-100.0 μM, with a sensitivity of 0.10 μA·μM-1 and a detection limit (LOD, S / N=3) of 1.97 μM. It has excellent selectivity for hyodeoxycholic acid (HDCA). Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a non-enzymatic electrochemical method for detecting hyodeoxycholic acid.

[0006] The technical solution adopted by the present invention to solve its technical problem is:

[0007] A non-enzymatic electrochemical method for detecting hyodeoxycholic acid is described. The method uses renewable β-CD as a matrix, synthesizes alkali metal potassium-coordinated cyclodextrin metal-organic frameworks (K-CD-MOFs) via a modified methanol vapor diffusion method, and loads them onto a reduced graphene oxide (rGO) substrate to prepare rGO-K-CD-MOFs nanocomposites with high specific surface area, excellent hydrophilicity, and biocompatibility. Neutral red is used as a redox probe, and changes in the current in the system are detected by differential pulse voltammetry. Furthermore, the method specifically includes the following steps:

[0008] Step 1: Synthesis of K-CD-MOFs: β-cyclodextrin and potassium hydroxide were dissolved in a methanol / water mixed solvent, magnetically stirred at 25°C for 30 minutes, and then ultrasonically treated (40kHz, 300W) for 30 minutes to promote molecular dispersion. The mixed solution was filtered through a polytetrafluoroethylene filter membrane and transferred to a 50mL beaker, which was placed in a sealed container containing 50mL of methanol. It was allowed to stand for crystallization for 7 days under light-proof conditions to obtain white block crystals. It was washed with anhydrous ethanol three times in sequence to remove unreacted products, and finally treated in a vacuum drying oven at 40°C for 24 hours. The obtained crystals were ground into a uniform powder with a particle size of ≤5μm using an agate mortar for use.

[0009] Step 2: Synthesis of GO: Graphene oxide was prepared by a modified Hummer's method. Graphite powder, ammonium persulfate, and phosphorus pentoxide were sequentially placed in a concentrated sulfuric acid solution (10 mL) and subsequently stirred at 80°C for 4 hours. The mixture was washed with deionized water until the pH of the filtrate became neutral. The filtered solid was dried in a vacuum atmosphere for 2 hours to obtain pre-oxidized graphite. The pre-oxidized graphite was then placed in a concentrated sulfuric acid solution (24 mL) in an ice bath and stirred. Potassium permanganate was slowly added under the same conditions. The mixture was then heated to 40°C and maintained for 2 hours. Subsequently, the temperature was increased and maintained for 30 minutes. After cooling to room temperature, the reaction was terminated by adding deionized water (200 mL) and hydrogen peroxide solution. The mixture was washed four times with dilute hydrochloric acid solution (1:10 by volume) and washed with deionized water until neutral to remove residual metal ions. After 2 hours of sonication at low temperature (4°C, 40 kHz), a graphene oxide dispersion with a concentration of approximately 3 mg / mL was obtained.

[0010] Step 3: Preparation of rGO-K-CD-MOFs composite material: First, the graphene oxide dispersion obtained in step 2 was freeze-dried to obtain a flocculent powder. The graphene oxide was then dissolved at a certain concentration and sonicated for 1 hour (40kHz, 300W) to obtain a pale yellow transparent graphene oxide aqueous solution. The K-CD-MOF powder obtained in step 1 was dissolved in 20mL of water and stirred evenly. The mixture was then mixed with 20mL of the graphene oxide solution. The entire solution was continuously stirred and sonicated for 1 hour. Then, hydrazine monohydrate solution and ammonia solution were added to adjust the solution's pH to 10. The mixture was refluxed in a 95°C oil bath for 1 hour under the condition of a silicone oil seal to isolate oxygen.

[0011] Step 4: Dialysis: The reaction solution, which remained stable and free of precipitation, was immediately dialyzed against aqueous ammonia using a dialysis membrane for 6 hours to remove ammonia and hydrazine molecules. Finally, a stable rGO-K-CD-MOFs dispersion was successfully prepared and stored in the dark.

[0012] Step 5: Preparation of rGO-K-CD-MOFs Hyodeoxycholic Acid (HDCA) Sensor: The present invention uses a three-electrode system for detection: a Pt electrode is used as the counter electrode and working electrode, and a Hg / HgO electrode is used as the reference electrode. Differential pulse voltammetry (DPV) and chronoamperometry (CA) are used for determination on an electrochemical workstation. First, the rGO-K-CD-MOFs dispersion is mixed with deionized water, and the NR solution is added and vortexed for 2 minutes. At this time, the β-CD cavity forms a host-guest inclusion complex with the NR through hydrophobic interaction. The mixture is centrifuged (8000 rpm, 15 min) to remove unincluded NR molecules. The precipitate is washed multiple times with borate buffer and dried in a vacuum drying oven for 12 hours to obtain the rGO-K-CD-MOFs@NR inclusion complex. 10 mg of the inclusion complex is transferred to a beaker with 20 mL of deionized water, assuming it is a blank solution. HDCA standard solution was gradually added to the test system. Since HDCA is more hydrophobic than NR, HDCA molecules will replace NR molecules to form rGO-K-CD-MOFs@HDCA inclusion complex. After each addition, the system was allowed to stand for 10 seconds to allow the substitution equilibrium, and differential pulse voltammetry tests were performed to record the current data.

[0013] Furthermore, in the preparation method of the present invention, wherein:

[0014] Preferably, in step 1, the molar masses of β-CD and KOH are 1 mmol and 8 mmol, respectively, the ratio of methanol / water mixed solvent is 8:2, the total volume is 20 ml, and the diameter of the polytetrafluoroethylene filter membrane pore is 0.45 μm.

[0015] Preferably, in step 2, the masses of graphite powder, ammonium persulfate and phosphorus pentoxide are 2g, 2g and 2g respectively, wherein the graphite powder is 325 mesh, the concentration of concentrated sulfuric acid solution is 98%, and the mass of added potassium permanganate is 3g.

[0016] Preferably, in step 2, the mixture is heated to 35° C. and maintained for 2 hours, wherein the temperature is increased to 95° C. and maintained for 30 minutes.

[0017] Preferably, the concentration and volume of the hydrogen peroxide solution in step 2 are 30% and 6 mL, respectively.

[0018] Preferably, in step 3, graphene oxide is dissolved at a concentration of 5 mg / mL, wherein the volumes of hydrazine monohydrate solution and ammonia solution are 10 μL and 35 μL, respectively.

[0019] Preferably, the concentration of the ammonia solution in step 4 is 0.1M.

[0020] Preferably, the voltage range of the differential pulse voltammetry in step 5 is from -0.4V to 0.4V, the pulse height is 50mV, and the constant voltage of the chronoamperometry is 0.4V.

[0021] Preferably, in step 5, the volumes of the rGO-K-CD-MOFs dispersion, deionized water, and NR solution are 3 mL, 17 mL, and 2 mL, respectively, and the concentrations of the rGO-K-CD-MOFs dispersion and NR solution are 5 mg / mL and 10 mM, respectively.

[0022] The rGO-K-CD-MOFs composite material prepared by the preparation method described above was used to detect hyodeoxycholic acid (HDCA).

[0023] The preparation method as described above is used in the preparation of hyodeoxycholic acid (HDCA) sensor.

[0024] The advantages and positive effects achieved by the present invention are:

[0025] Compared with the existing technology, the improved methanol vapor diffusion method is used to prepare K-CD-MOFs materials with food-grade safety characteristics under mild conditions, overcoming the defects of using toxic organic solvents and high temperature and high pressure conditions in the traditional MOFs preparation process; K-CD-MOFs is innovatively compounded with rGO to construct a material with high specific surface area (16.297m 2 / g) and a three-dimensional heterostructure with excellent conductivity, with a current response four times higher than that of pure β-CD-based materials; through the synergistic effect of rGO and K-CD-MOFs, the electron cloud distribution and surface properties were optimized, significantly improving the material's adsorption and recognition performance for targets.

[0026] The non-enzymatic detection system constructed based on the competitive displacement principle showed excellent linearity in the range of 5.0-100.0 μM (R 2 >0.99), sensitivity up to 0.10μA·μM -1 The detection limit is as low as 1.97 μM (S / N = 3), approximately three times higher than existing electrochemical sensors. It is highly specific for hyodeoxycholic acid (HDCA) and can effectively distinguish structural analogs (HCA, TUDCA, etc.). It has a fast response time (reaching steady state in 2.3 seconds) and excellent stability. Using food-grade raw materials and a mild preparation process, it has good biocompatibility. It requires no complex pretreatment steps and can be directly used for testing biological samples such as serum and bile. Its low cost and simple operation provide new insights into clinical bile acid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1(a) SEM image of rGO-K-CD-MOFs in the present invention, (b) HRTEM image of rGO-K-CD-MOFs (the inset is a SAED image), (c) lattice spacing image of rGO-K-CD-MOFs, (d) SEM image of rGO-K-CD-MOFs and scanning element distribution map of C, O, and K;

[0028] Figure 2 (a) FTIR spectra of rGO-K-CD-MOFs, K-CD-MOFs, rGO-CD, and rGO in the present invention. (b) XRD spectra of rGO-K-CD-MOFs, rGO-CD, β-CD, and rGO. (c) Raman spectra and (d) thermogravimetric spectra of rGO-K-CD-MOFs, rGO-CD, and rGO.

[0029] Figure 3 (a) Full XPS spectrum of rGO-K-CD-MOFs, (b) high-resolution C1s spectrum, (c) high-resolution O1s spectrum, and (d) high-resolution K2p spectrum in the present invention;

[0030] Figure 4 DSC spectra of rGO-K-CD-MOFs, HDCA, NR, a mixture of rGO-K-CD-MOFs and HDCA (rGO-K-CD-MOFs / HDCA), an inclusion compound (rGO-K-CD-MOFs@HDCA), a mixture of rGO-K-CD-MOFs and NR (rGO-K-CD-MOFs / NR), and an inclusion compound (rGO-K-CD-MOFs@NR) in the present invention;

[0031] Figure 5 (a) Differential pulse voltammetry spectra of the rGO-K-CD-MOFs@NR inclusion complex of the present invention before and after the addition of analyte (HDCA, 5μM-100μM), (b) fitting curve with analyte concentration as the independent variable and peak current value as the dependent variable, (c) corresponding graph of the rGO-K-CD-MOFs@NR inclusion complex when the steady-state current is reached after the addition of analyte 5μM HDCA, (d) current response values ​​of the rGO-K-CD-MOFs@NR inclusion complex in the presence of HDCA and interferents (HCA, TUDCA, MCA), (e) differential pulse voltammetry response of the same rGO-K-CD-MOFs@NR solution to 5μM HDCA collected from 5 repeated measurements, (f) differential pulse voltammetry response of rGO-K-CD-MOFs to 100μM HDCA at different storage times (0-6 months). DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to the following examples. The following examples are descriptive rather than restrictive, and the scope of protection of the present invention cannot be limited by the following examples.

[0033] The various experimental operations involved in the specific embodiments are all routine techniques in the field. For parts not specifically annotated in this document, ordinary technicians in this field can refer to various commonly used reference books, scientific literature or related instructions, manuals, etc. before the filing date of this invention to implement them.

[0034] A non-enzymatic electrochemical method for the detection of hyodeoxycholic acid (HDCA) was developed. Alkali potassium-coordinated cyclodextrin metal-organic frameworks (K-CD-MOFs) were synthesized using a modified methanol vapor diffusion method using renewable β-CD as a matrix. These K-CD-MOFs were then loaded onto a reduced graphene oxide (rGO) substrate to create an rGO-K-CD-MOFs nanocomposite with high surface area, excellent hydrophilicity, and biocompatibility. Neutral red was used as a redox probe, and current changes in the system were monitored by differential pulse voltammetry.

[0035] Preferably, the method specifically includes the following steps:

[0036] Step 1. Synthesis of K-CD-MOFs: 1.135 g of β-cyclodextrin (β-CD, 1 mmol) and 0.448 g of potassium hydroxide (KOH, 8 mmol) were dissolved in 20 mL of a methanol / water mixture (8:2 by volume, containing 20% ​​deionized water). The mixture was magnetically stirred at 25°C for 30 minutes, followed by sonication (40 kHz, 300 W) for 30 minutes to promote molecular dispersion. The mixture was filtered through a 0.45 μm polytetrafluoroethylene filter and transferred to a 50 mL beaker, which was then placed in a sealed container containing 50 mL of methanol. Crystallization was allowed to proceed for 7 days in the dark, yielding white blocky crystals. The mixture was then washed three times with anhydrous ethanol to remove unreacted products and finally dried in a vacuum oven at 40°C for 24 hours. The resulting crystals were ground in an agate mortar to a uniform powder with a particle size of ≤5 μm for later use.

[0037] Step 2. Synthesis of GO: Graphene oxide was prepared by a modified Hummer's method. Graphite powder (2 g, 325 mesh), ammonium persulfate (2 g), and phosphorus pentoxide (2 g) were sequentially placed in a concentrated sulfuric acid solution (98%, 10 mL) and subsequently stirred at 80°C for 4 hours. The mixture was washed with deionized water until the pH of the filtrate became neutral. The filtered solid was dried in a vacuum atmosphere for 2 hours to obtain pre-oxidized graphite. 0.6 g of the pre-oxidized graphite was then placed in a concentrated sulfuric acid solution (98%, 24 mL) in an ice bath and stirred. 3 g of potassium permanganate was slowly added under the same conditions. The mixture was then heated to 35°C for 2 hours. Subsequently, the temperature was raised to 95°C and maintained for 30 minutes. After cooling to room temperature, the reaction was terminated by adding deionized water (200 mL) and hydrogen peroxide solution (30%, 6 mL). The mixture was washed four times with dilute hydrochloric acid solution (volume ratio 1:10) and washed with deionized water until neutral to remove residual metal ions. After 2 h of ultrasonic treatment at low temperature (4 °C, 40 kHz), a graphene oxide dispersion with a concentration of about 3 mg / mL was obtained.

[0038] Step 3, Preparation of rGO-K-CD-MOFs Composite Material: The prepared graphene oxide dispersion was freeze-dried to obtain a flocculent powder, and then the graphene oxide was dissolved at a concentration of 5 mg / mL and ultrasonicated for 1 hour (40 kHz, 300 W) to obtain a pale yellow transparent graphene oxide aqueous solution. K-CD-MOF (1 g) powder was dissolved in 20 mL of water, stirred evenly, and then mixed with 20 mL of graphene oxide solution. The entire solution was continuously stirred and ultrasonicated for 1 hour, and then 10 μL of hydrazine monohydrate solution and 35 μL of ammonia solution were added to adjust the pH of the solution to 10. Under the condition of sealing with silicone oil to isolate oxygen, reflux in a 95°C oil bath for 1 hour.

[0039] Step 4, Dialysis: The reaction mixture was immediately dialyzed against a 0.1 M ammonia solution for 6 hours, three times, to remove ammonia and hydrazine molecules. Finally, a stable rGO-K-CD-MOFs dispersion was successfully prepared and stored at room temperature in the dark.

[0040] Step 5. Preparation of rGO-K-CD-MOFs hyodeoxycholic acid (HDCA) sensor: Since the solubility of hyodeoxycholic acid in water is very low, a 1 mM bile acid stock solution was prepared by dissolving 4 mg of hyodeoxycholic acid in 10 mL of anhydrous ethanol. The prepared solution was further used for electrochemical detection. The electrochemical performance of the sensor was investigated using a three-electrode system. All measurements were performed on a Zahner Zennium electrochemical workstation, which was connected to a single-chamber cell with three electrodes (where the counter electrode and working electrode were Pt electrodes, respectively, and the Hg / HgO electrode was used as the reference electrode), including differential pulse voltammetry and chronoamperometry. The differential pulse voltammetry voltage range was -0.4 V to 0.4 V, with a pulse height of 50 mV. The CA constant voltage was 0.4 V. First, 3 mL of rGO-K-CD-MOFs dispersion (5 mg / mL) was mixed with 17 mL of deionized water, 2 mL of NR (10 mM) solution was added, and vortexed for 2 minutes. At this point, the β-CD cavity forms a host-guest inclusion complex with NR through hydrophobic interaction. The mixture was centrifuged (8000 rpm, 15 min) to remove unincluded NR molecules. The precipitate was washed three times with borate buffer and dried in vacuo at 40°C for 12 hours to obtain the rGO-K-CD-MOFs@NR inclusion complex. 10 mg of the inclusion complex was transferred to a beaker with 20 mL of deionized water, which was assumed to be a blank solution. HDCA standard solution (0-100 μM) was gradually added to the test system. Since HDCA is more hydrophobic than NR, HDCA molecules will replace NR molecules to form rGO-K-CD-MOFs@HDCA inclusion complexes. After each addition, the system was allowed to stand for 10 seconds to allow the displacement to equilibrate. Differential pulse voltammetry was performed to record the current data.

[0041] The rGO-K-CD-MOFs composite material prepared by the preparation method described above was used to detect hyodeoxycholic acid (HDCA).

[0042] The preparation method as described above is used in the preparation of hyodeoxycholic acid (HDCA) sensor.

[0043] Specifically, the relevant preparation and detection are as follows:

[0044] 1 Experimental part

[0045] 1.1 Experimental materials

[0046] The experimental raw materials are shown in Table 1.

[0047] Table 1 Experimental materials and reagents

[0048]

[0049]

[0050]

[0051] 1.2 Experimental instruments

[0052] The experimental instruments are shown in Table 2.

[0053] Table 2 Experimental instruments

[0054]

[0055]

[0056] 1.3 Characterization and analysis methods

[0057] Scanning electron microscopy analysis: The microstructure and morphology of the composites were observed using a Thermo Fisher Scientific Quattro S scanning electron microscope (SEM).

[0058] 1.3.1 Scanning Electron Microscopy (SEM)

[0059] Scanning Electron Microscope Analysis: The microstructure of rGO-K-CD-MOFs was observed using a field-emission high-resolution scanning electron microscope (FESEM) on an FEI Apreo microscope. Magnifications ranged from 1,000 to 10,000x. Energy Dispersive X-ray Spectroscopy (EDS) was also used to analyze the elemental distribution of the samples.

[0060] 1.3.2 Transmission Electron Microscopy (TEM)

[0061] In order to determine the microstructure and composition of the samples, a transmission electron microscope (TEM) model JEOL Ltd. (JEOL) was used to observe the samples at magnifications of 400,000 and 800,000 times, and selected electron diffraction (SAED) was tested.

[0062] 1.3.3 X-ray diffraction analysis (XRD)

[0063] X-ray diffraction (XRD) analysis of the sample powder was performed using a Rigaku Smartlab SE diffractometer under Cu Kα radiation. The diffraction angle was 5-40° and the diffraction rate was 5.0° / min.

[0064] 1.3.4 X-ray Photoelectron Spectroscopy (XPS)

[0065] To understand the valence states of the elements in the samples, X-ray photoelectron spectroscopy was performed using a Thermo Fisher Scientific 250Xi (USA) instrument. The X-ray source was a Mg Kα monochromatic source.

[0066] 1.3.5 Raman spectroscopy (RAMAN)

[0067] Raman spectra were collected on an optical Raman microscope using an excitation wavelength of 532 nm.

[0068] 1.3.6 Fourier Transform Infrared Spectroscopy (FT-IR)

[0069] Using a Bruker VECTOR 22 from 4000 to 400 cm -1 Fourier transform infrared spectroscopy (FT-IR) was performed.

[0070] 1.3.7 Differential Scanning Calorimetry (DSC)

[0071] Differential scanning calorimetry (DSC 250, America) was used, and heating was performed from 0° C. to 450° C. at a rate of 5° C. / min. The heat flow was monitored during the heating process.

[0072] 1.3.8 Thermogravimetric Analyzer (TGA)

[0073] Thermogravimetric tests were performed using a NETZSCH STA 449F5 thermogravimetric analyzer (TGA) in the temperature range of 0–700 °C at a flow rate of 40 mL / min and a heating rate of 10 °C / min under a nitrogen atmosphere.

[0074] 1.4 Electrochemical performance analysis

[0075] The electrochemical performance of the sensor was investigated using a three-electrode system. All measurements were performed on a Zahner Zennium electrochemical workstation connected to a single-chamber cell with three electrodes (the counter and working electrodes were Pt wire electrodes, respectively, and a Hg / HgO electrode served as the reference electrode). These measurements included differential pulse voltammetry and chronoamperometry. The differential pulse voltammetry voltage range was -0.4 V to 0.4 V, with a pulse height of 50 mV. The chronoamperometry constant voltage was 0.4 V.

[0076] 2 Results and Discussion

[0077] 2.1 Morphology analysis of rGO-K-CD-MOFs composites

[0078] The crystal morphology of K-CD-MOFs was studied and it was found that ( Figure 1 (a)), which presents typical rod-shaped crystal characteristics, and the length of the grain is about 1μm. In the final prepared rGO-K-CD-MOFs composite material, MOFs crystals grow in situ on the wrinkled surface of rGO to form a stable three-dimensional heterogeneous structure. This structural advantage is reflected in two aspects: on the one hand, the conductive network of rGO can promote the efficiency of electron transfer, and on the other hand, the high specific surface area and active sites of MOFs are expected to synergistically improve the adsorption performance of target molecules such as neutral red and hyodeoxycholic acid. Through high-resolution transmission electron microscopy (HR-TEM) images such as Figure 1 (b) reveals the structural characteristics of the composite material: the high electron density area (dark area) corresponds to the K-CD-MOFs crystal, which is in sharp contrast to the low electron density rGO substrate (bright area). This morphological feature is in good agreement with the SEM observation results. Figure 1 (c) The crystallographic characteristics of the material can be clearly identified. The (200) crystal plane of K-CD-MOFs is measured to have a lattice spacing of 1.4 nm, which is consistent with the data of its single crystal XRD standard card. The (200) diffraction ring appears in the selected area electron diffraction (SAED) pattern ( Figure 1 (b) Inset), which provides direct evidence for the growth of K-CD-MOFs on the rGO surface. The element distribution and types of rGO-K-CD-MOFs were observed under EDS diffraction. Figure 1 (d) It can be seen that C, O, and K are evenly distributed.

[0079] 2.2 Physicochemical structure analysis of rGO-K-CD-MOFs composites

[0080] FT-IR characterization results ( Figure 2 (a) reveals the evolution of the chemical structure of the material. rGO at 3400 cm -1 There is a broad hydroxyl stretching vibration peak at 1107 cm -1 The COC vibration peak at 3400 cm indicates that there are epoxy groups on the surface of the material, which confirms that the graphene oxide is not completely reduced. -1 The hydroxyl peak intensity in the region is significantly increased, which is directly related to the superposition effect of 21 hydroxyl groups in the β-CD molecule. -1 The newly appeared glycosidic bond characteristic vibration peak at 441 cm -1 The KO characteristic vibration peak appears at 1641 cm -1 The peak at is narrower than that of K-CD-MOFs; and the intensity of the OH peak is significantly lower than that of rGO-CD, which may be due to the formation of KO bonds, resulting in a weakening of the intensity of OH stretching vibration. This indicates that rGO and K-CD-MOFs have a strong interaction, such as π-π stacking, hydrogen bonding, or charge transfer, which changes the vibration mode of the KO bond.

[0081] X-ray diffraction analysis ( Figure 2 (b) reveals the evolution of the crystal structure of the material. rGO prepared by hydrazine hydrate reduction at 2θ = 19° (d 002=0.48nm), which proves that some oxygen-containing groups are effectively removed. The diffraction peak still exists in a broadened form in the composite material, indicating that the rGO sheets have nanoscale stacking disorder. This structural characteristic creates favorable conditions for subsequent composite reactions by exposing edge active sites. β-CD has a typical crystal structure and shows multiple sharp and high-intensity diffraction peaks in the XRD spectrum. The characteristic peaks at 2θ of 12°, 15°, 20°, etc. are an intuitive reflection of the orderliness of the β-CD crystal structure. Compared with rGO and β-CD, the XRD spectrum of rGO-CD shows diffraction peaks related to β-CD in addition to the (002) peak of rGO, but the peak intensity is reduced and some peaks are broadened. This shows that β-CD is successfully composited with rGO. However, the composite process may combine through interactions such as van der Waals forces, π-π stacking or hydrogen bonds, affecting the original crystal order of β-CD and causing changes in its diffraction peaks. The (002) peak position of rGO shifts slightly because the insertion of β-CD changes the electron cloud distribution and interaction between rGO layers, thereby affecting the interlayer spacing. The XRD spectrum of rGO-K-CD-MOFs combines the characteristic diffraction peaks of rGO, β-CD and MOFs. In addition to the (002) peak of rGO and some characteristic peaks of β-CD, sharp diffraction peaks corresponding to the crystal structure of MOFs appear at new angles, such as at 2θ=6.4° (d 200 =1.4 nm), which is significantly higher than the diffraction peak of rGO-CD, indicating that the K element exists in the target compound and has good crystallinity, which clearly confirms that K-CD-MOFs are successfully loaded into the rGO system.

[0082] Raman spectroscopy Figure 2 (c) By analyzing the intensity ratio of D peak and G peak (I D / I G ), which can provide a deeper understanding of the structural order and defect conditions. D / I G The ratio is 1.79, the highest among the three. This may be due to the interaction between cyclodextrin and rGO in K-CD-MOFs, which introduces more defects or disordered structures into the structure of rGO, changes the electron cloud distribution and lattice structure of rGO, and makes the D peak intensity relatively increased, indicating that rGO-K-CD-MOFs has a higher structural disorder and defect density. D / I G The ratio is 1.71, which is the lowest among the three. This shows that when only cyclodextrin is introduced into the composite with rGO, it may be combined through weaker interactions, such as van der Waals force or hydrogen bond, without significantly destroying the original structure of rGO. Therefore, its structural disorder and defect level are relatively low. D / IG The ratio is 1.74, which is between rGO-K-CD-MOFs and rGO-CD. This is because rGO itself will inevitably produce some defects during the preparation process, such as edge defects and vacancy defects.

[0083] Thermogravimetric curves such as Figure 2 As shown in (d), the mass loss of rGO-CD at 100°C is caused by the evaporation of crystalline water molecules in the β-CD cavity, and the mass loss at 200°C-600°C is due to the thermal degradation of β-CD. The thermogravimetric curve of rGO-K-CD-MOFs has an 8% mass loss between 30°C and 100°C, which is due to the loss of residual solvent in the crystal framework after reaching the boiling point of methanol / water at 100°C; at the same time, there is a 63% mass loss in the range of 200°C-600°C due to the oxidative decomposition of β-CD. The weight loss curves of rGO-CD and rGO-K-CD-MOFs are very similar, but there are also certain differences, which may be due to the K + The introduction of induced structural changes.

[0084] The surface chemical state of rGO-K-CD-MOFs composites was systematically analyzed by X-ray photoelectron spectroscopy ( Figure 3 (a)). Full spectrum analysis confirmed that the material is composed of three elements: C, O, and K. The high-resolution spectrum further revealed the chemical bonding characteristics of each component. C1s fine spectrum ( Figure 3 (b) Peak fitting shows three typical characteristic peaks: the main peak at 284.6 eV corresponds to the C-C / C=C bond of graphitized carbon, and the sp 2 Hybrid carbon skeleton and cyclic carbon structure of β-CD; the 286.5eV secondary peak is attributed to the CO bond, which originates from the β-CD hydroxyl group and the oxygen bridge structure of the metal coordination in MOFs; the 288.5eV weak peak corresponds to the carbonyl group (C=O), indicating that part of the carbon skeleton undergoes oxidative reconstruction during the composite process, which is related to the electron transfer effect during the coordination process of MOFs. O1s spectrum ( Figure 3 (c)) shows a double peak structure at 531.8eV and 533.2eV, corresponding to the metal-oxygen coordination bond (KO) and the surface adsorbed hydroxyl group (-OH), respectively. The appearance of the KO bond confirms that K + The strong coordination effect between the organic ligand and the MOFs is consistent with the theoretical prediction of the octahedral coordination mode in the MOFs structure. Figure 3 (d)) at 293.5eV(2p 3 / 2 ) and 296.5eV(2p 1 / 2 ) shows a typical double peak, and its spin-orbit splitting energy (Δ=3.0eV) and binding energy position are similar to those of K 0 →K +The oxidation state transition characteristics are consistent. Combined with the O1s spectrum analysis, it can be seen that K element is mainly K + It participates in the construction of MOFs skeleton and stabilizes the three-dimensional network structure through KOC coordination bonds.

[0085] Differential scanning calorimetry analysis results ( Figure 4 ) reveals the structural dependence of the material's thermodynamic behavior. rGO-K-CD-MOFs exhibits a broad endothermic peak at 94.7°C and 263.2°C (curve 1), indicating that the dehydration process leads to the loss of water and partial breakage of coordination bonds within the MOFs framework, respectively. The dehydration peak of the inclusion complex rGO-K-CD-MOFs@NR (curve 5) shifts to a lower temperature by 10.3°C (to 84.4°C), and a new endothermic peak appears at 322.8°C. This characteristic peak is a unique signal of the host-guest composite structure formed after the NR molecules enter the MOFs pores. The DSC curve of the physical mixture rGO-K-CD-MOFs / NR (curve 4) completely coincides with the endothermic peaks of pure NR (curve 3) at 99.6°C and 305.6°C (peak temperature deviation <1.2°C), confirming that there is no chemical interaction between the two. When HDCA replaces NR (curve 7), rGO-K-CD-MOFs@HDCA produces a new characteristic endothermic peak at 335.7°C, which is 12.9°C higher than that of rGO-K-CD-MOFs@NR. This is directly related to the enhanced host-guest binding energy caused by the larger molecular size of HDCA. The endothermic peaks of the physical mixture rGO-K-CD-MOFs / HDCA (curve 6) and pure HDCA (curve 2) at 198.6°C completely overlap (peak temperature deviation <0.9°C), further confirming the specificity of the replacement process.

[0086] 2.3 Analysis of electrochemical performance of rGO-K-CD-MOFs composite materials for detecting HDCA

[0087] Sensitivity is the core performance indicator of electrochemical sensors and directly determines their ability to detect trace amounts of target substances. In this study, the quantitative response characteristics of the rGO-K-CD-MOFs@NR sensing system to HDCA were evaluated by differential pulse voltammetry. Figure 5 As shown in (a), when the concentration of HDCA in the solution increases from 0 μM to 100 μM, the peak value of the differential pulse voltammetry current response gradually increases, and the increase is significantly dose-dependent with the HDCA concentration. Figure 5 (b) The linear response equation obtained is y = 0.10x + 4.16, and the correlation coefficient R 2 is 0.9947, so the sensitivity is 0.10μA·μM -1 Under the condition of S / N=3, the calculated detection limit was 1.97 μM.

[0088] Response kinetics is a key indicator for evaluating the practicality of electrochemical sensors. Figure 5 As shown in (c), after the introduction of 5μM HDCA, the rGO-K-CD-MOFs@NR sensor system reached 95% of the steady-state current response within 2.3 seconds, which is significantly faster than the previous sensor for detecting bile acid, indicating that it can quickly respond to the addition of HDCA and meet the response speed requirements in actual detection. The differential pulse voltammetry response of 5μM HDCA was measured 5 times on the same rGO-K-CD-MOFs@NR sensor system to evaluate the stability. Figure 5 (e) The results show that the peak current of the differential pulse voltammetry response changes very little, with a relative standard deviation (RSD) of 1.28%.

[0089] Anti-interference ability is a core indicator for evaluating the practicality of sensors. Given that structural analogs (such as hyocholic acid HCA, tauroursodeoxycholic acid TUDCA, and muricholic acid MCA) often coexist in biological samples, this study systematically investigated the selectivity of rGO-K-CD-MOFs@NR by chronoamperometry ( Figure 5 (d) Experiments showed that at a concentration of 5 μM, HDCA stimulation elicited a significant current response (ΔI = 3.65 μA), while equivalent concentrations of HCA, TUDCA, and MCA only caused weak signal fluctuations. This demonstrates that the rGO-K-CD-MOFs@NR inclusion complex has very strong specificity for HDCA and has great potential and prospects for application in HDCA sensors.

[0090] On the other hand, the stability of rGO-K-CD-MOFs was evaluated by differential pulse voltammetry response to 100 μM HDCA over a period of 6 months. The solution was sealed and stored at room temperature, and the measurement was repeated every 30 days ( Figure 5 (f)). The results show that the oxidation current decreased by 9.4% after 6 months, indicating that rGO-K-CD-MOFs have good stability.

[0091] 3. Summary

[0092] In summary, the present invention uses renewable β-cyclodextrin (β-CD) as a matrix, synthesizes alkali metal potassium coordinated cyclodextrin metal organic framework materials (K-CD-MOFs) by a modified methanol vapor diffusion method, and loads them on a reduced graphene oxide (rGO) substrate to successfully prepare rGO-K-CD-MOFs nanocomposites with high specific surface area, excellent hydrophilicity and biocompatibility. Neutral red is used as a redox probe, and the change in current in the system is detected by differential pulse voltammetry. The study found that the synergistic effect of rGO and K-CD-MOFs significantly improved the detection performance: the composite material exhibits a wide linear detection range in the concentration range of 5.0-100.0μM, with a sensitivity of 0.10μA·μM-1 and a detection limit (LOD, S / N=3) of 1.97μM, which is 4 times higher than the current response of pure β-CD-based materials. Interference resistance experiments evaluated by constant current (CA) demonstrated that the sensor exhibited significant selectivity for common coexisting compounds (such as hyocholic acid (HCA), tauroursodeoxycholic acid (TUDCA), and muricholic acid (MCA). The sensor also exhibited excellent stability (≥6 months at room temperature), with a detection signal retention of 90.6%. This work not only provides a new method for the specific detection of HDCA, but the proposed rGO-K-CD-MOFs composite strategy also offers new insights into the application of graphene-based composites in biosensing, demonstrating significant application value in food safety monitoring and livestock product quality control.

[0093] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for preparing an enzyme-free hyodeoxycholic acid electrochemical sensor based on rGO-K-CD-MOFs nanocomposite material, characterized in that: The following steps are involved: Step 1: Synthesis of K-CD-MOFs: β-cyclodextrin and potassium hydroxide were dissolved in a methanol / water mixed solvent, magnetically stirred at 25°C for 30 minutes, and then ultrasonically treated (40kHz, 300W) for 30 minutes to promote molecular dispersion. The mixed solution was filtered through a polytetrafluoroethylene filter membrane and transferred to a 50mL beaker, which was placed in a sealed container containing 50mL of methanol. It was allowed to stand for crystallization for 7 days under light-proof conditions to obtain white block crystals. It was washed with anhydrous ethanol three times in sequence to remove unreacted products, and finally treated in a vacuum drying oven at 40°C for 24 hours. The obtained crystals were ground into a uniform powder with a particle size of ≤5μm using an agate mortar for use. Step 2: Synthesis of GO: Graphene oxide was prepared by a modified Hummer's method. Graphite powder, ammonium persulfate, and phosphorus pentoxide were sequentially placed in a concentrated sulfuric acid solution (10 mL) and subsequently stirred at 80°C for 4 hours. The mixture was washed with deionized water until the pH of the filtrate became neutral. The filtered solid was dried in a vacuum atmosphere for 2 hours to obtain pre-oxidized graphite. The pre-oxidized graphite was then placed in a concentrated sulfuric acid solution (24 mL) in an ice bath and stirred. Potassium permanganate was slowly added under the same conditions. The mixture was then heated to 40°C and maintained for 2 hours. Subsequently, the temperature was increased and maintained for 30 minutes. After cooling to room temperature, the reaction was terminated by adding deionized water (200 mL) and hydrogen peroxide solution. The mixture was washed four times with dilute hydrochloric acid solution (1:10 by volume) and washed with deionized water until neutral to remove residual metal ions. After 2 hours of sonication at low temperature (4°C, 40 kHz), a graphene oxide dispersion with a concentration of approximately 3 mg / mL was obtained. Step 3: Preparation of rGO-K-CD-MOFs composite material: First, the graphene oxide dispersion obtained in step 2 was freeze-dried to obtain a flocculent powder. The graphene oxide was then dissolved at a certain concentration and sonicated for 1 hour (40kHz, 300W) to obtain a pale yellow transparent graphene oxide aqueous solution. The K-CD-MOF powder obtained in step 1 was dissolved in 20mL of water and stirred evenly. The mixture was then mixed with 20mL of the graphene oxide solution. The entire solution was continuously stirred and sonicated for 1 hour. Then, hydrazine monohydrate solution and ammonia solution were added to adjust the solution's pH to 10. The mixture was refluxed in a 95°C oil bath for 1 hour under the condition of a silicone oil seal to isolate oxygen. Step 4: Dialysis: The reaction solution, which remained stable and free of precipitation, was immediately dialyzed against aqueous ammonia using a dialysis membrane for 6 hours to remove ammonia and hydrazine molecules. Finally, a stable rGO-K-CD-MOFs dispersion was successfully prepared and stored in the dark. Step 5: Preparation of rGO-K-CD-MOFs Hyodeoxycholic Acid (HDCA) Sensor: The present invention uses a three-electrode system for detection: a Pt electrode is used as the counter electrode and working electrode, and a Hg / HgO electrode is used as the reference electrode. Differential pulse voltammetry (DPV) and chronoamperometry (CA) are used for determination on an electrochemical workstation. First, the rGO-K-CD-MOFs dispersion is mixed with deionized water, and the NR solution is added and vortexed for 2 minutes. At this time, the β-CD cavity forms a host-guest inclusion complex with the NR through hydrophobic interaction. The mixture is centrifuged (8000 rpm, 15 min) to remove unincluded NR molecules. The precipitate is washed multiple times with borate buffer and dried in a vacuum drying oven for 12 hours to obtain the rGO-K-CD-MOFs@NR inclusion complex. 10 mg of the inclusion complex is transferred to a beaker with 20 mL of deionized water, assuming it is a blank solution. HDCA standard solution was gradually added to the test system. Since HDCA is more hydrophobic than NR, HDCA molecules will replace NR molecules to form rGO-K-CD-MOFs@HDCA inclusion complex. After each addition, the system was allowed to stand for 10 seconds to allow the substitution equilibrium, and differential pulse voltammetry tests were performed to record the current data.

2. The method according to claim 1, characterized in that In step 1, the molar masses of β-CD and KOH are 1 mmol and 8 mmol, respectively. The ratio of the methanol / water mixed solvent is 8:2, the total volume is 20 ml, and the diameter of the polytetrafluoroethylene filter membrane pores is 0.45 μm.

3. The method according to claim 1, characterized in that In step 2, the masses of graphite powder, ammonium persulfate and phosphorus pentoxide are 2g, 2g and 2g respectively, wherein the graphite powder is 325 mesh, the concentration of concentrated sulfuric acid solution is 98%, and the mass of potassium permanganate added is 3g.

4. The method according to claim 1, wherein In step 2, the mixture was heated to 35° C. and maintained for 2 hours, wherein the temperature was increased to 95° C. and maintained for 30 minutes.

5. The method according to claim 1, wherein In step 2, the concentration and volume of the hydrogen peroxide solution are 30% and 6 mL, respectively.

6. The method according to claim 1, characterized in that In step 3, graphene oxide is dissolved at a concentration of 5 mg / mL, wherein the volumes of hydrazine monohydrate solution and ammonia solution are 10 μL and 35 μL, respectively.

7. The method according to claim 1, characterized in that The concentration of the ammonia solution in step 4 is 0.1M.

8. The method according to claim 1, characterized in that The voltage range of the differential pulse voltammetry in step 5 is -0.4 V to 0.4 V, the pulse height is 50 mV, and the constant voltage of the chronoamperometry is 0.4 V.

9. The method according to claim 1, characterized in that In step 5, the volumes of the rGO-K-CD-MOFs dispersion, deionized water, and NR solution were 3 mL, 17 mL, and 2 mL, respectively, and the concentrations of the rGO-K-CD-MOFs dispersion and NR solution were 5 mg / mL and 10 mM, respectively.

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