A gold nanoparticle-modified magnetic covalent organic framework material and its preparation method and application

Abstract

The present invention relates to the field of bioanalysis and detection technology, and specifically to a gold nanoparticle-modified magnetic covalent organic framework material, its preparation method, and application. The gold nanoparticle-modified magnetic covalent organic framework material exhibits both glucose oxidase-like activity and peroxidase-like activity under acidic conditions. In the presence of the substrate glucose, the gold nanoparticle-modified magnetic covalent organic framework material oxidizes the glucose to generate hydrogen peroxide through self-cascade catalysis, and then utilizes the peroxidase-like activity to oxidize the chromogenic substrate TMB to generate oxTMB, which is then colorimetrically detected based on the color depth of the substrate. In addition, the generated oxTMB reacts with the fluorescence of the gold nanoparticle-modified magnetic covalent organic framework material itself to produce an inner filter effect, quenching the autofluorescence, allowing fluorescence detection to be performed based on the fluorescence change. Rapidly detecting glucose content through the colorimetric / fluorescence dual mode provides a new approach for convenient and accurate glucose content detection.

Description

Technical Field

[0001] The present invention relates to the technical field of biological analysis and detection, and in particular to a gold nanoparticle-modified magnetic covalent organic framework material, a preparation method and an application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Glucose is an important source of energy, but excessive intake or abnormal metabolism can lead to a variety of diseases. Glucose is associated with a variety of conditions, including diabetes, hypertension, obesity, and cardiovascular disease. Therefore, measuring glucose concentration is crucial for monitoring disease progression.

[0004] Currently, commercial blood glucose monitors are primarily categorized into two main methods: electrochemical and reflectance. However, the electrochemical method suffers from complex detection systems, expensive instrumentation, and the need for specialized personnel. Reflectance testing, on the other hand, suffers from poor anti-interference capabilities of test strips and insufficient accuracy. Enzyme-based cascade catalytic colorimetry is a novel glucose detection method that offers advantages such as ease of operation and independence from specific instrumentation. However, it also suffers from disadvantages such as low sensitivity. This is because natural glucose oxidase or natural peroxidase are easily inactivated during the reaction, necessitating the synthesis of substances with glucose oxidase- or peroxidase-like activity.

[0005] In recent years, the designability and modifiability of covalent organic frameworks have attracted much attention in the enzyme-like field. However, the covalent organic framework-based enzyme-like materials reported so far usually only have one type of enzyme-like activity, such as glucose oxidase-like activity or peroxidase-like activity. There are few reports on materials with both glucose oxidase-like and peroxidase-like activities.

[0006] In addition, the enzyme-based cascade catalytic colorimetric method uses colorimetry for single-mode detection, which has the disadvantage of low accuracy. Compared with the single-mode detection strategy, the dual-mode detection results are more reliable. Summary of the Invention

[0007] In order to overcome the above problems, the present invention provides a gold nanoparticle-modified magnetic covalent organic framework material and a preparation method and application thereof.

[0008] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0009] The first aspect of the present invention provides a gold nanoparticle-modified magnetic covalent organic framework material (Fe3O4@COF-Au NPs), which uses a magnetic covalent organic framework material (Fe3O4@COF NPs) as a matrix and is loaded with gold nanoparticles (Au NPs);

[0010] Among them, the magnetic covalent organic framework material is a core-shell structure, with Fe3O4 nanoparticles (Fe3O4NPs) as the core and a covalent organic framework material (COF) as the shell; the covalent organic framework material is a Schiff base type covalent organic framework material in which 1,3,5-tris(4-aminophenyl)benzene and 2,5-dimethoxyterephthalaldehyde are connected by a carbon-nitrogen double bond.

[0011] In one or more embodiments, the gold nanoparticles (Au NPs) have a particle size of 3 to 10 nm.

[0012] In one or more embodiments, the particle size of the Fe3O4 nanoparticles (Fe3O4NPs) is 200-400 nm.

[0013] In one or more embodiments, the thickness of the shell covalent organic framework material is 100-300 nm.

[0014] In one or more embodiments, the covalent organic framework (COF) has a repeating structural unit having a structure represented by formula (I):

[0015]

[0016] Formula (I).

[0017] The second aspect of the present invention provides a method for preparing the gold nanoparticle-modified magnetic covalent organic framework material according to the first aspect, comprising the following steps:

[0018] (1) 1,3,5-tris(4-aminophenyl)benzene and 2,5-dimethoxyterephthalaldehyde were dissolved in a first organic solvent, mixed evenly for the first time, and then Fe3O4 nanoparticles were added. After mixing evenly for a second time, acetic acid aqueous solution was added as a catalyst to hydrothermally synthesize a magnetic covalent organic framework material;

[0019] (2) The magnetic covalent organic framework material is dispersed in a second organic solvent, HAuCl4 solution is added, mixed evenly for a third time, and then NaBH4 solution is added and stirred to react to obtain a gold nanoparticle-modified magnetic covalent organic framework material.

[0020] In one or more embodiments, in step (1), the method for preparing Fe3O4 nanoparticles comprises the following steps:

[0021] The trivalent iron salt, sodium acetate and sodium citrate were dissolved in ethylene glycol, mixed evenly, and then hydrothermally synthesized Fe3O4 nanoparticles.

[0022] Preferably, the ferric salt includes ferric chloride, ferric sulfate or ferric acetate, preferably ferric chloride.

[0023] Preferably, the molar ratio of the ferric salt, sodium acetate and sodium citrate is (7-8): (40-50): (2-3), preferably 7.5:44:2.3.

[0024] Preferably, the concentration of the ferric iron salt in ethylene glycol is 0.117-0.133 mol / L, preferably 0.125 mol / L.

[0025] Preferably, the temperature of the hydrothermal reaction is 180-220° C., preferably 200° C.; the time of the hydrothermal reaction is 12-15 h, preferably 14 h.

[0026] In one or more embodiments, in step (1), the molar ratio of 1,3,5-tris(4-aminophenyl)benzene to 2,5-dimethoxyterephthalaldehyde is 2:(2.5-4), preferably 2:3.

[0027] In one or more embodiments, in step (1), the first organic solvent is a mixture of butanol and 1,4-dioxane, wherein the volume ratio of butanol to 1,4-dioxane is (0.8-1.2):1, preferably 1:1.

[0028] In one or more embodiments, in step (1), the concentration of 2,5-dimethoxyterephthalaldehyde in the first organic solvent is 0.0125-0.02 mol / L, preferably 0.015 mol / L.

[0029] In one or more embodiments, in step (1), the concentration of Fe3O4 nanoparticles in the first organic solvent is 2.5-3.5 g / L, preferably 3 g / L.

[0030] In one or more embodiments, in step (1), the hydrothermal reaction temperature is 65-80°C, preferably 70°C; and the hydrothermal reaction time is 40-60 hours, preferably 48 hours. At this temperature, both the synthesis of the covalent organic framework material and the formation of a core-shell structured magnetic covalent organic framework material can be ensured.

[0031] In one or more embodiments, in step (2), the second organic solvent is tetrahydrofuran.

[0032] In one or more embodiments, in step (2), the concentration of the magnetic covalent organic framework material in the second organic solvent is 3-4 g / L, preferably 3.3 g / L.

[0033] In one or more embodiments, in step (2), the concentration (mass fraction) of the HAuCl4 solution is 0.08-0.12%, preferably 0.1%.

[0034] In one or more embodiments, in step (2), the temperature of adding the HAuCl4 solution is 38-42°C, preferably 40°C.

[0035] In one or more embodiments, in step (2), the concentration of the NaBH4 solution is 0.4-0.6 mM, preferably 0.5 mM.

[0036] In one or more embodiments, in step (2), NaBH4 solution is added and the stirring reaction time is 20 to 30 hours, preferably 24 hours.

[0037] The third aspect of the present invention provides the use of the gold nanoparticle-modified magnetic covalent organic framework material described in the first aspect or the gold nanoparticle-modified magnetic covalent organic framework material prepared by the preparation method described in the second aspect in detecting glucose content.

[0038] In one or more embodiments, when detecting the glucose content, the pH value of the solution is 3-5 and the temperature is 25-50°C.

[0039] The fourth aspect of the present invention provides the use of the gold nanoparticle-modified magnetic covalent organic framework material described in the first aspect or the gold nanoparticle-modified magnetic covalent organic framework material prepared by the preparation method described in the second aspect in the preparation of products for detecting glucose content.

[0040] In one or more embodiments, the product comprises a reagent or a kit.

[0041] The fifth aspect of the present invention provides a product for detecting glucose content, comprising the gold nanoparticle-modified magnetic covalent organic framework material described in the first aspect or the gold nanoparticle-modified magnetic covalent organic framework material prepared by the preparation method described in the second aspect.

[0042] In one or more embodiments, the product comprises a reagent or a kit.

[0043] The beneficial effects of the present invention are:

[0044] (1) The gold nanoparticle-modified magnetic covalent organic framework material (Fe3O4@COF-Au NPs) provided by the present invention has both glucose oxidase-like activity and peroxidase-like activity under acidic conditions. In the presence of the substrate glucose, the Fe3O4@COF-Au NPs catalyze the oxidation of glucose to generate hydrogen peroxide (H2O2) through self-cascade catalysis, and then utilize the peroxidase-like activity to oxidize the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) to generate oxTMB, which can be colorimetrically detected based on the color depth of the substrate. In addition, the generated oxTMB will produce an inner filter effect with the fluorescence of the Fe3O4@COF-Au NPs themselves, quenching their own fluorescence, and fluorescence detection can be performed based on the fluorescence change. Therefore, the gold nanoparticle-modified magnetic covalent organic framework material (Fe3O4@COF-Au NPs) provided by the present invention can realize a self-cascade reaction of dual enzyme activities at the same pH, successfully realizing the rapid detection of glucose content in a colorimetric / fluorescence dual mode, and providing a new approach for convenient and accurate detection of glucose content.

[0045] (2) The gold nanoparticle-modified magnetic covalent organic framework material (Fe3O4@COF-Au NPs) provided by the present invention can realize colorimetric / fluorescence dual-mode rapid detection of glucose content. The linear ranges of fluorescence and colorimetry are 0.001 mM~2 mM and 0.05 mM~2.5 mM, respectively. The detection limits of fluorescence and colorimetry are as low as 106 nM and 5 μM. Therefore, it has broad application prospects in glucose content detection.

[0046] (3) The present invention first synthesized a core-shell magnetic covalent organic framework material (Fe3O4@COF NPs). A highly ordered porous structure exists on the surface of the covalent organic framework material (COF). Due to the pore size restriction effect on the surface of the covalent organic framework material (COF) carrier and the surface -NH2 as the binding site for HAuCl4, the gold nanoparticles (Au NPs) obtained by NaBH4 reduction can be in situ fixed to the shell of the magnetic covalent organic framework material (Fe3O4@COF NPs), and the size of the gold nanoparticles (Au NPs) is limited to less than 5 nm. At the same time, the gold nanoparticles (Au NPs) in situ fixed to the magnetic covalent organic framework material (Fe3O4@COF NPs) are uniformly dispersed on the surface of the covalent organic framework material (COF); uniformly dispersed and ultra-small gold nanoparticles (Au NPs) are the prerequisite for glucose oxidase-like activity. Compared with the method that requires the addition of a stabilizer or a protective agent to achieve uniform dispersion of gold nanoparticles (Au NPs), the in situ immobilization method of the present invention does not limit the activity of the enzyme.

[0047] (4) Materials with only a single glucose oxidase-like activity generally have better activity under neutral or alkaline pH conditions, while materials with only a single peroxidase-like activity generally have better activity under acidic pH conditions, requiring a two-step colorimetric detection method. Compared with materials with only a single glucose oxidase-like activity or a single peroxidase-like activity, the gold nanoparticle-modified magnetic covalent organic framework material (Fe3O4@COF-Au NPs) provided by the present invention has both glucose oxidase-like and peroxidase-like activities under acidic conditions, and only requires a single reaction to achieve rapid and accurate detection of glucose content. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0049] Figure 1 The scanning electron microscopy images and transmission electron microscopy images of Fe3O4@COF-Au NPs, where A and B are scanning electron microscopy images of Fe3O4@COF-Au NPs at different scales; C and D are transmission electron microscopy images of Fe3O4@COF-Au NPs at different scales;

[0050] Figure 2 FI-TR spectra, XRD patterns and fluorescence spectra of Fe3O4@COF-Au NPs, where A is the FI-TR spectra, B is the XRD pattern, and C is the fluorescence spectrum;

[0051] Figure 3 The XPS graph of Fe3O4@COF-Au NPs, where A is the XPS total spectrum and B is the Fe 2+ , C is the high-resolution XPS spectrum of N 1s, and D is the high-resolution XPS spectrum of Au 4f;

[0052] Figure 4 Figure 3 shows the results of the peroxidase-like activity study of Fe3O4@COF-Au NPs, where A is the peroxidase-like activity of Fe3O4@COF-AuNPs; B is a comparison of the peroxidase-like activities of Fe3O4@COF, Fe3O4, and Fe3O4@COF-Au NPs; C is the fluorescence spectrum of the terephthalic acid (TA) capture ·OH experiment; D is the UV-visible absorption spectrum of the tert-butyl alcohol (TBA), p-benzoquinone (PBQ), and tryptophan (Try) free radical scavenging experiment; the illustrations in A, B, and D are the colorimetric solutions of different reaction systems, respectively;

[0053] Figure 5To optimize the concentrations of Fe3O4@COF-Au NPs, TMB, and H2O2, A is the optimization of the Fe3O4@COF-Au NPs concentration in the range of (0-70 μg / mL) at the same TMB and H2O2 concentrations; B is the quantification of A; C is the optimization of the TMB concentration in the range of (0-0.5 mM) at the same Fe3O4@COF-Au NPs and H2O2 concentrations; D is the quantification of C; E is the optimization of the Fe3O4@COF-Au NPs and TMB with H2O2 concentration in the range of (0-20 mM), and F is the quantification of E;

[0054] Figure 6 The kinetic test results of Fe3O4@COF-Au NPs are shown, where A is the Michaelis-Menten curve of Fe3O4@COF-Au NPs for H2O2, B is the Lineweaver-Burk double reciprocal plot of Fe3O4@COF-Au NPs for H2O2; C is the Michaelis-Menten curve of Fe3O4@COF-Au NPs for TMB; D is the Lineweaver-Burk double reciprocal plot of Fe3O4@COF-Au NPs for TMB;

[0055] Figure 7 Figure 3 is the research results of glucose oxidase-like activity of Fe3O4@COF-Au NPs, where A is a schematic diagram of glucose oxidase-like activity of Fe3O4@COF-Au NPs; B is the UV-visible absorption spectrum of glucose oxidase-like activity of Fe3O4@COF-Au NPs; the inset in B is the colorimetric solution for glucose oxidase-like study;

[0056] Figure 8 The effect of pH on the dual enzyme activity of Fe3O4@COF-Au NPs; A is the result of the effect on peroxidase-like activity, and B is the result of the effect on glucose oxidase-like activity;

[0057] Figure 9 The effect of temperature on the dual enzyme activity of Fe3O4@COF-Au NPs; A is the result of the effect on peroxidase-like activity, and B is the result of the effect on glucose oxidase-like activity;

[0058] Figure 10 The process of dual-mode detection of glucose by Fe3O4@COF-Au NPs;

[0059] Figure 11Figure 3. Detection of glucose by Fe3O4@COF-Au NPs. (A) Schematic diagram of the cascade detection of glucose by Fe3O4@COF-Au NPs. (B) UV-visible absorption spectra after adding different concentrations of glucose (50 μM~2.5 mM, from bottom to top). (C) The linear relationship between absorbance at 650 nm and glucose concentration. (B) The inset shows the colorimetric solution with different concentrations of glucose added. (C) The regression equation is: y=0.2537x+0.1455.

[0060] Figure 12 These are the results of Fe3O4@COF-Au NPs detecting glucose, where A is a schematic diagram of Fe3O4@COF-Au NPs detecting glucose in fluorescence mode; B is the fluorescence spectrum of Fe3O4@COF-Au NPs after adding different concentrations of glucose (0 mM~2 mM); C is the linear relationship between the fluorescence intensity of Fe3O4@COF-Au NPs at 510 nm and the glucose concentration. The regression equation in C is: y=-738.2381x+2486.5190. DETAILED DESCRIPTION

[0061] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0062] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0063] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0064] Example 1

[0065] Synthesis of gold nanoparticle-modified magnetic covalent organic framework materials (Fe3O4@COF-Au NPs):

[0066] 1.1 Synthesis of Fe3O4 nanoparticles (Fe3O4NPs):

[0067] 2.04 g FeCl3·6H2O, 3.6 g anhydrous sodium acetate, and 0.6 g sodium citrate were dissolved in 60 mL ethylene glycol. The mixed solution was stirred at 1200 rpm for 1.5 h, then transferred to a 100 mL stainless steel autoclave and heated at 200 °C for 14 h. After cooling to room temperature, the product was magnetically separated and washed three times with anhydrous ethanol and deionized water, respectively, and dried in vacuum at 30 °C for 12 h to obtain Fe3O4 nanoparticles (Fe3O4NPs).

[0068] 1.2 Synthesis of magnetic covalent organic framework materials (Fe3O4@COF NPs):

[0069] 42 mg of 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 34.8 mg of 2,5-dimethoxyterephthalaldehyde (DMTP) were dissolved in a mixed solution of 8 mL of butanol and 8 mL of 1,4-dioxane, and ultrasonically mixed for 5 min to ensure uniform mixing of the solution; 36 mg of Fe3O4NPs were added to the mixed solution, and ultrasonication was continued for 5 min. Subsequently, 0.2 mL of acetic acid was added, and the mixture was stirred at 1200 rpm at room temperature for 2 h. Then, 1.8 mL of acetic acid aqueous solution (12 mol / L) was added, and the mixture was reacted at 70 °C for 48 h; after cooling to room temperature, the product was magnetically separated, washed three times with acetone and tetrahydrofuran, respectively, and dried in vacuum at 30 °C for 12 h to obtain magnetic covalent organic framework material (Fe3O4@COF NPs).

[0070] 1.3 Synthesis of gold nanoparticle-modified magnetic covalent organic framework materials (Fe3O4@COF-Au NPs):

[0071] 20 mg of Fe3O4@COF NPs were dissolved in 6 mL of tetrahydrofuran (THF) and ultrasonicated for 5 min to uniformly mix the solution; 1 mL of HAuCl4 (mass fraction 0.1%) was added dropwise at 40 °C and stirred at 1500 rpm for 2 h; then 2 mL of NaBH4 aqueous solution (0.5 mM) was added; after stirring overnight, the product was collected by magnet, washed three times with water and ethanol respectively, and vacuum dried for 12 h to obtain gold nanoparticle-modified magnetic covalent organic framework material (Fe3O4@COF-Au NPs).

[0072] Characterization of gold nanoparticle-modified magnetic covalent organic framework materials (Fe3O4@COF-Au NPs):

[0073] Figure 1 A and B are scanning electron micrographs of Fe3O4@COF-Au NPs at different scales. Figure 1As can be seen in the figure, the magnetic covalent organic framework material (Fe3O4@COF NPs) has a core-shell structure, with Fe3O4 nanoparticles (Fe3O4NPs) as the core and a covalent organic framework material (COF) as the shell. The inner core Fe3O4 is about 300 nm in size, and the outer COF shell is 200 nm. The -NH2 on the COF surface serves as a binding site for HAuCl4, and NaBH4 reduction produces small-sized Au NPs that are evenly distributed on the surface. Transmission electron microscopy (TEM) Figure 1 In Figures C and D, it can be seen that the size of Au NPs is around 5 nm and they are evenly distributed, which is a necessary condition for having glucose oxidase-like activity.

[0074] Figure 2 FI-TR spectrum, XRD pattern and fluorescence spectrum of Fe3O4@COF-Au NPs; FI-TR spectrum ( Figure 2 In A), Fe3O4@COF NPs are at 577 cm -1 The strong absorption peak at 1622 cm -1 The corresponding -C=N- characteristic absorption at 1676 cm indicates that DMTP and TAPB condense to form an imine bond; and the -CHO of DMTP -1 and -NH2 of TAPB at 2870 cm -1 and 2934 cm -1 There are weak stretching vibration bands everywhere; similarly, the FT-IR spectrum of Fe3O4@COF-Au NPs retains all the above characteristic peaks. Therefore, the in-situ loading of Au NPs does not destroy the COF structure. XRD pattern ( Figure 2 In Figure B), after the Au NPs were loaded, new peaks at 38.35, 44.45, and 64.79 appeared, corresponding to the (111), (200), and (220) diffraction peaks of Au, respectively. Figure 2 As can be seen in Figure C, Fe3O4@COF-Au NPs have strong fluorescence at 510 nm.

[0075] Figure 3 The XPS graph of Fe3O4@COF-Au NPs is shown in Figure 2. Figure 3 It can be seen that both Fe3O4@COF NPs and Fe3O4@COF-Au NPs contain C, N, and O elements. Figure 3 Medium BFe 2+ The spectrum shows that the magnetic nanoparticles Fe3O4 are well wrapped by COF; N 1s ( Figure 3 Middle C) Two deconvoluted peaks at 398.10 and 398.93 are attributed to the residual -NH2 group and -C=N- group, respectively. Figure 3 The two peaks of D correspond to the 4f of Au(0) 7 / 2 and 4f 5 / 2 .

[0076] Figures 1 to 3 The results above demonstrate the successful preparation of Fe3O4@COF-Au NPs.

[0077] Example 2

[0078] Study on the peroxidase-like activity of Fe3O4@COF-Au NPs:

[0079] Experimental method: 100 μL Fe3O4@COF-Au NPs (1 mg / mL), 100 μL TMB (10 mM), and 150 μL H2O2 (100 mM) were added to 1650 μL acetate buffer solution (pH = 4, 0.2 M) and reacted for 3 min. The UV-visible absorption spectrum in the range of 500-800 nm and the absorbance at 652 nm were then recorded.

[0080] The peroxidase-like activity of Fe3O4@COF-Au NPs was studied by oxidizing the chromogenic substrate TMB in the presence of H2O2. Figure 4 As shown in Figure A, only in the presence of H2O2 is TMB oxidized to the blue substrate oxTMB, which has a clear UV absorption peak at 652 nm. Figure 4 In B, it was found that Fe3O4 and Fe3O4@COF also have peroxidase-like activity, but the absorbance value of Fe3O4@COF-Au NPs at 652 nm is much greater than that of Fe3O4 and Fe3O4@COF, indicating that the activity of Fe3O4@COF-Au NPs is much higher than the former two. This is because the small-sized Au NPs formed by in situ reduction are deposited on the surface of Fe3O4@COF, providing more reactive sites. It is generally believed that the oxidation of TMB is attributed to the ROS produced by the reaction of H2O2 and Fe3O4@COF-AuNPs. Different reactive oxygen scavengers were used to explore the types of ROS produced during the reaction. Figure 4 As shown in C, tert-butyl alcohol, p-benzoquinone and tryptophan act as hydroxyl radicals (·OH), superoxide anions (O 2·- ) and singlet oxygen ( 1 O2) scavenger. Figure 4 As can be seen in Figure D, after the presence of tert-butyl alcohol and p-benzoquinone, the absorbance of oxTMB at 652 nm decreased significantly, and the color of the solution changed from dark blue to almost colorless. Next, using terephthalic acid (TA) as a typical probe of ·OH, 2-hydroxyterephthalic acid with a high blue fluorescence signal can be generated. Figure 4As shown in Figure C, only when Fe3O4@COF-AuNPs, H2O2, and TA are present can 2-hydroxyterephthalic acid be generated, with a characteristic fluorescence peak at 428 nm. The above results prove that after the Au NPs are in situ fixed on the Fe3O4@COF shell by NaBH4 reduction method, the peroxidase activity is improved, which can catalyze H2O2 to generate active intermediates ·OH and O 2·- .

[0081] Example 3

[0082] Kinetic test of Fe3O4@COF-Au NPs:

[0083] The concentrations of Fe3O4@COF-Au NPs, TMB and H2O2 were optimized. Steady-state kinetic measurements were performed in acetate buffer (pH = 4, 0.2 M) by changing the TMB concentration and fixing the H2O2 concentration (and vice versa). The catalytic rate of Fe3O4@COF-Au NPs was calculated and evaluated.

[0084] pass Figure 5 Fe3O4@COF-Au NPs 40 μg / mL, TMB 0.3 mM and H2O2 10 mM can be obtained as the most suitable concentrations in the catalytic reaction.

[0085] Based on this condition, the steady-state kinetics were calculated. By fitting the data, the typical Michaelis-Menten curve of Fe3O4@COF-Au NPs was obtained, and the Michaelis constant ( K m ) and the maximum rate of enzymatic reaction ( V max ).in, K m Represents the affinity of the enzyme to the substrate. The lower the value, the stronger the affinity. Under different H2O2, the calculation results are K m and V max 0.45 mM and 9.73 × 10 -8 M·s -1 ( Figure 6 A and B).

[0086] Table 1 shows the comparison of kinetic parameters of Fe3O4@COF-Au NPs and HRP. It can be seen from Table 1 that the kinetic parameters of Fe3O4@COF-Au NPs and HRP are significantly different from those of natural enzyme HRP. K m Compared with Fe3O4@COF-Au NPs, K mAnd high adsorption capacity, 8 times that of HRP, high affinity indicates excellent peroxidase activity. Similarly, for different concentrations of TMB as substrate, the calculation results are K m and V max 0.25 mM and 9.83 × 10 -8 M·s -1 ( Figure 6 C and D). Compared with other materials with peroxidase activity, the materials have comparable or better activity.

[0087] Table 1 Kinetic parameters of Fe3O4@COF-Au NPs and HRP

[0088]

[0089] Example 4

[0090] Study on the glucose oxidase activity of Fe3O4@COF-Au NPs:

[0091] Experimental method: 100 μL of Fe3O4@COF NPs-Au NPs (1 mg / mL) and 200 μL of glucose (0.1 M) were added to 1400 μL of acetate buffer (pH = 4, 0.2 M). After incubation at room temperature for 2 h, 200 μL of TMB (10 mM) and 100 μL of HRP (100 μg / mL) were added. The absorbance of the mixture was then recorded at 652 nm.

[0092] The results are as follows Figure 7 As shown in Figure B, in the presence of HRP and TMB, only the Fe3O4@COF NPs-Au NPs / glucose system has a high UV absorption peak at 650 nm. This proves that Fe3O4@COF NPs-Au NPs exerts glucose oxidase-like activity to catalyze glucose to produce H2O2, and then TMB is oxidized to oxTMB ( Figure 7 Middle A).

[0093] Example 5

[0094] The effects of pH and temperature on the dual enzyme activity of Fe3O4@COF NPs-Au NPs were studied. Figure 8 and 9 As shown, from Figure 8 As can be seen from Figure A, Fe3O4@COF NPs-Au NPs have excellent peroxidase-like activity in a wide pH range of pH = 3~6. Figure 8As shown in Figure B, Fe3O4@COF NPs-Au NPs exhibit excellent glucose oxidase-like activity within the pH range of 3–5. These results indicate that the peroxidase-like and glucose oxidase-like activities of Fe3O4@COF NPs-Au NPs have overlapping optimal pH ranges. Simultaneous dual enzyme activity at the same pH avoids the errors associated with a two-step assay, resulting in more accurate results and easier operation.

[0095] from Figure 9 It can be seen that Fe3O4@COF NPs-Au NPs have good peroxidase-like activity and glucose oxidase-like activity at temperatures of 40 ℃ and 25 ℃.

[0096] Example 6

[0097] Dual-mode glucose detection:

[0098] The detection process is as follows Figure 10 shown.

[0099] 100 μL of Fe3O4@COF NPs-Au NPs (1 mg / mL) and 200 μL of glucose at different concentrations were added to 1500 μL of acetate buffer solution (pH = 4, 0.2 M). After incubation at room temperature for 2 h, 200 μL of TMB (10 mM) was added. After incubation at 40 °C for 20 min, the absorbance of the mixture at 652 nm and the fluorescence intensity (E) at 510 nm were recorded. x =470 nm, E m =510nm) and used to draw absorbance-concentration curves and fluorescence intensity-concentration curves.

[0100] Fe3O4@COF NPs-Au NPs can exert peroxidase-like activity and glucose oxidase-like activity at the same pH, and a self-cascade catalytic detection system for glucose can be established. The color-developing substrate TMB is used to detect the content of glucose as the analyte by the color depth of the system ( Figure 11 Middle A).

[0101] like Figure 11 As shown in B and C, the absorbance at 650 nm gradually increases with the increase of glucose concentration. When the glucose concentration is between 0 and 2.5 mM, it changes linearly with the absorbance, and the regression coefficient R 2 =0.9903, and the detection limit can reach 5 μM (S / N=3).

[0102] Similarly, in the fluorescence mode, as the glucose concentration increases, the quenching effect of oxTMB on Fe3O4@COF NPs-AuNPs increases, and the fluorescence intensity at 510 nm gradually decreases ( Figure 12 (A and B). When the glucose concentration was between 0.001 and 2 mM, the fluorescence intensity showed a linear change, and the regression coefficient R 2 =0.9923, the detection limit can reach 106 nM (S / N=3) ( Figure 12 Middle C).

[0103] These results indicate that Fe3O4@COF NPs-Au NPs possess excellent dual enzyme activity and excellent detection performance under fluorescence-UV dual-mode detection. They can achieve self-cascade catalysis without the participation of natural enzymes, thus avoiding the harsh storage conditions of natural enzymes.

[0104] Example 7

[0105] Actual sample testing:

[0106] Glucose at different concentrations (0.05 mM, 0.50 mM, and 2 mM) was added to Wahaha drinking water, and colorimetric and fluorescence detection were performed.

[0107] 100 μL of Fe3O4@COF NPs-Au NPs (1 mg / mL) and 200 μL of glucose at different concentrations were added to 1500 μL of acetate buffer solution (pH = 4, 0.2 M). After incubation at room temperature for 2 h, 200 μL of TMB (10 mM) was added. After incubation at 40 °C for 20 min, the absorbance of the mixture at 652 nm and the fluorescence intensity (E) at 510 nm were recorded. x =470 nm, E m =510nm),

[0108] As shown in Table 2, the colorimetric method had recoveries ranging from 98.75% to 109.45%, with a relative standard deviation of less than 3.27%. The fluorescence method had recoveries ranging from 103.28% to 107.85%, with a relative deviation of less than 2.56%. This method demonstrates high recovery, good selectivity, and low relative standard deviation, making it suitable for glucose analysis.

[0109] Table 2 Dual-mode detection of glucose by Fe3O4@COF NPs-Au NPs

[0110]

[0111] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Application of a gold nanoparticle-modified magnetic covalent organic framework material in the preparation of a product for detecting glucose content, characterized in that: The invention uses a magnetic covalent organic framework material as a matrix and is loaded with gold nanoparticles; the particle size of the gold nanoparticles is 3 to 10 nm; The magnetic covalent organic framework material is a core-shell structure, with Fe3O4 nanoparticles as the core and a covalent organic framework material as the shell; the covalent organic framework material is a Schiff base type covalent organic framework material in which 1,3,5-tris(4-aminophenyl)benzene and 2,5-dimethoxyterephthalaldehyde are connected by a carbon-nitrogen double bond; When the colorimetric / fluorescence dual mode is used to detect glucose content, the pH value of the solution is 3-5 and the temperature is 25-50°C.

2. The use of the gold nanoparticle-modified magnetic covalent organic framework material according to claim 1 in preparing a product for detecting glucose content, characterized in that: The particle size of Fe3O4 nanoparticles is 200~400 nm; The thickness of the shell covalent organic framework material is 100~300 nm.

3. The use according to claim 1 or 2, characterized in that The preparation method of the gold nanoparticle-modified magnetic covalent organic framework material comprises the following steps: (1) 1,3,5-tris(4-aminophenyl)benzene and 2,5-dimethoxyterephthalaldehyde were dissolved in a first organic solvent, mixed evenly for the first time, and then Fe3O4 nanoparticles were added. After mixing evenly for a second time, acetic acid aqueous solution was added as a catalyst to hydrothermally synthesize a magnetic covalent organic framework material; (2) The magnetic covalent organic framework material is dispersed in a second organic solvent, HAuCl4 solution is added, mixed evenly for a third time, and then NaBH4 solution is added and stirred to react to obtain a gold nanoparticle-modified magnetic covalent organic framework material.

4. The use according to claim 3, characterized in that In step (1), the preparation method of Fe3O4 nanoparticles includes the following steps: The trivalent iron salt, sodium acetate and sodium citrate were dissolved in ethylene glycol, mixed evenly, and then hydrothermally synthesized Fe3O4 nanoparticles.

5. The use according to claim 3, characterized in that In step (1), the molar ratio of 1,3,5-tris(4-aminophenyl)benzene to 2,5-dimethoxyterephthalaldehyde is 2:(2.5-4); In step (1), the first organic solvent is a mixture of butanol and 1,4-dioxane, wherein the volume ratio of butanol to 1,4-dioxane is (0.8-1.2):1; In step (1), the concentration of 2,5-dimethoxyterephthalaldehyde in the first organic solvent is 0.0125-0.02 mol / L; In step (1), the concentration of Fe3O4 nanoparticles in the first organic solvent is 2.5-3.5 g / L; In step (1), the temperature of the hydrothermal reaction is 65-80°C; the time of the hydrothermal reaction is 40-60 h.

6. The use according to claim 3, characterized in that In step (2), the second organic solvent is tetrahydrofuran; In step (2), the concentration of the magnetic covalent organic framework material in the second organic solvent is 3-4 g / L; In step (2), the concentration of HAuCl4 solution is 0.08~0.12%; In step (2), the temperature of the HAuCl4 solution added is 38-42 °C; In step (2), the concentration of the NaBH4 solution is 0.4~0.6mM; In step (2), NaBH4 solution is added and the reaction is stirred for 20 to 30 hours.

7. A product for detecting glucose content, characterized in that: The invention relates to a magnetic covalent organic framework material modified with gold nanoparticles, wherein the magnetic covalent organic framework material is used as a matrix and loaded with gold nanoparticles; the particle size of the gold nanoparticles is 3 to 10 nm; The magnetic covalent organic framework material is a core-shell structure, with Fe3O4 nanoparticles as the core and a covalent organic framework material as the shell; the covalent organic framework material is a Schiff base type covalent organic framework material in which 1,3,5-tris(4-aminophenyl)benzene and 2,5-dimethoxyterephthalaldehyde are connected by a carbon-nitrogen double bond; When the colorimetric / fluorescence dual mode is used to detect glucose content, the pH value of the solution is 3-5 and the temperature is 25-50°C.

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