Colorimetric immunoassay method based on core-shell structure composite COF-based nano enzyme

Abstract

According to the colorimetric immunoassay method based on the core-shell structure composite COF-based nano enzyme, Fe3O4 (at) COF (at) Pt NPs has excellent dual-enzyme-like activity and magnetism, after the Fe3O4 (at) COF (at) Pt NPs is used for marking a target molecule monoclonal antibody, the Fe3O4 (at) COF (at) Pt NPs is used as a marking probe, colorimetric signal recognition detection on a target molecule can be effectively achieved, and the accuracy and the anti-interference capability of immunodetection are improved. Immunoassay of biomacromolecules, pathogens and the like such as proteins, viruses and bacteria can be realized by replacing recognition elements such as antibodies, and the method has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of immunoassay, and particularly relates to a preparation of a core-shell structure Fe3O4@COF@Pt nanoparticle, a Fe3O4@COF@Pt nanoparticle coupled antibody and a colorimetric immunoassay method based on a composite COF-based nanoparticle double-enzyme activity. BACKGROUND

[0002] In recent years, various detection techniques such as mass spectrometry (MS), PCR, electrochemical sensing and surface plasmon resonance have been used for pollutant analysis, among which enzyme-linked immunosorbent assay (ELISA) has become a general method for clinical biomarker detection due to its simple operation and high throughput. Although traditional colorimetric ELISA based on horseradish peroxidase (HRP) has high specificity, it is limited by poor enzyme stability and high cost. Nano-enzyme-linked immunoassay technology introduces functionalized nano-enzymes (such as fluorescent or magnetic modification), which not only overcomes the above limitations, but also makes nano-enzyme-linked immunoassay have more characteristics.

[0003] Nanocatalysts, as nanomaterials that mimic the activity of natural enzymes, have shown significant potential in catalysis and biosensing. Compared with natural enzymes, nanocatalysts have the advantages of low cost, high stability and adjustable activity. Since the first report of peroxidase-like activity of Fe3O4 nanoparticles, noble metal (such as Au, Pt), metal oxide / sulfide, covalent organic framework (COF) and single-atom materials have emerged as nanocatalysts. Among them, noble metal nanoparticles (NPs) have become a research hotspot due to their size effect and electronic structure advantages, but the problems of easy aggregation and insufficient substrate adsorption sites limit their catalytic efficiency. By loading noble metal nanoparticles on porous COFs carriers, the particle size can be effectively controlled and the substrate contact can be enhanced. The ordered pore structure and strong metal-support interaction of COFs can also improve the catalytic stability and activity. To solve the problem of nanocatalyst recovery, magnetic component Fe3O4 is introduced to achieve rapid separation, and the magnetic core itself also synergistically enhances the enzyme-like activity of the composite. This composite COF-based nanocatalyst with magnetic, high enzyme-like activity and stability provides a new strategy for the accurate detection of biomolecules in complex samples. In the future, the development of multifunctional composite nanocatalysts and the optimization of their synergistic effects will further improve the sensitivity and practicality of ELISA.

[0004] In summary, based on the need for the development of high-sensitivity and convenient ELISA detection methods, exploring the preparation of composite COF-based nanocatalysts with simple preparation and high catalytic activity, and establishing a sensitive and convenient ELISA detection method, is a problem that needs to be solved. SUMMARY

[0005] (I) Technical problems to be solved

[0006] The application provides a colorimetric immunoassay method based on a core-shell structure composite COF-based nanoscale enzyme.

[0007] (II) Technical solutions

[0008] To solve the above technical problems, the application provides a colorimetric immunoassay method based on a core-shell structure composite COF-based nanoscale enzyme, which comprises the following steps: preparing Fe3O4@COF@Pt NPs; using Fe3O4@COF@Pt NPs to label a single antibody Ab2 of a target molecule to prepare a signal probe Fe3O4@COF@Pt-Ab2; coating a single antibody Ab1 of the target molecule on an enzyme-labeled plate; combining the target molecule with Ab1 and Fe3O4@COF@Pt-Ab2 in sequence to form an immune sandwich complex, adding TMB, and using Fe3O4@COF@Pt NPs to catalyze the oxidation of TMB into oxidized TMB to generate a visible colorimetric signal at 652 nm, so as to realize colorimetric immunoassay.

[0009] Further, the preparation of Fe3O4@COF@Pt NPs comprises the following steps:

[0010] (1) Fe3O4 NPs preparation: measuring ethylene glycol in a beaker, sequentially adding poly(4-styrene sulfonic acid-co-maleic acid) sodium salt, iron chloride hexahydrate and sodium acetate trihydrate, stirring and dispersing, transferring the solution to a reaction kettle, heating to 200 DEG C, naturally cooling to room temperature after reaction, washing with water and ethanol for multiple times, vacuum drying, and storing at 4 DEG C for standby;

[0011] (2) Fe3O4@PEI NPs preparation: adding Fe3O4 NPs into a polyethyleneimine aqueous solution and ultrasonicating, then washing with water and ethanol for multiple times, vacuum drying for standby;

[0012] (3) Fe3O4@COF NPs preparation: dissolving Fe3O4@PEI and polyvinylpyrrolidone in acetonitrile, ultrasonicating and uniformly mixing, sequentially adding 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 2,5-difluoro-p-xylylene diformaldehyde (DFTA) and ultrasonicating and uniformly mixing, dropping glacial acetic acid, standing at room temperature, adding an ascorbic acid aqueous solution while stirring, then immediately heating to 80 DEG C, naturally cooling to room temperature after refluxing, washing with water and ethanol for multiple times, and vacuum drying for standby;

[0013] (4) Fe3O4@COF@Pt NPs preparation: weigh Fe3O4@COF into water, ultrasonic treatment to make it completely dispersed, then add ethanol, H2PtCl6 solution in turn, ultrasonic mixing, drop NaBH4 solution under stirring at room temperature until the solution turns from yellow to brown black, then continue stirring, after the reaction is completed, separate and enrich and wash with pure water for several times, disperse the obtained brown black product in water for standby.

[0014] Further, the preparation of Fe3O4@COF@Pt NPs specifically comprises the following steps:

[0015] (1) Fe3O4 NPs preparation: weigh 50 mL of ethylene glycol in a 100 mL beaker, add 1.25 g of poly(4-styrene sulfonic acid-co-maleic acid) sodium salt, 1.35 g of iron chloride hexahydrate, and 6.22 g of sodium acetate trihydrate in turn, and stir and disperse vigorously, then transfer the solution to a reaction kettle, heat to 200°C, and react for 10 h, after the reaction is completed, naturally cool to room temperature, wash with water and ethanol alternately for several times, vacuum dry at 60°C, and store at 4°C for standby;

[0016] (2) Fe3O4@PEI NPs preparation: add 100 mg of Fe3O4 NPs into 20 mL of 2.5 mg / mL polyethyleneimine aqueous solution and ultrasonic for 30 min, then wash with water and ethanol alternately for several times, and vacuum dry at 60°C for standby;

[0017] (3) Fe3O4@COF NPs preparation: dissolve 20 mg of Fe3O4@PEI and 240 mg of polyvinylpyrrolidone in 80 mL of acetonitrile, ultrasonic for 30 min to mix uniformly, add 21 mg of 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 18 mg of 2,5-difluoro-p-xylyleneglycol (DFTA) in turn and ultrasonic for 6 min to mix uniformly, drop 600 μL of glacial acetic acid, stand at room temperature for 2 h, add 1.6 mL of 0.5 mM ascorbic acid aqueous solution under stirring, then immediately heat to 80°C to react, reflux for 6 h, then naturally cool to room temperature, wash with water and ethanol alternately for several times, and vacuum dry at 60°C for standby;

[0018] (4) Fe3O4@COF@Pt NPs preparation: weigh 20 mg of Fe3O4@COF into 10 mL of water, ultrasonic treatment for 30 min to make it completely dispersed, then add 100 mL of ethanol, 10 mL of H2PtCl6 solution in turn, ultrasonic mixing for 1 min, drop 1.6 mg / mL NaBH4 solution under stirring at room temperature until the solution turns from yellow to brown black, then continue stirring for 2 h, after the reaction is completed, separate and enrich and wash with pure water for several times, disperse the obtained brown black product in 40 mL of water for standby.

[0019] Further, the preparation of Fe3O4@COF@Pt-Ab2 includes the following steps: the Fe3O4@COF@Pt NPs aqueous solution is magnetically separated, and is ultrasonically dispersed in PBS; Ab2 is added to the mixture, after incubation at room temperature on a vertical mixer, BSA is added for incubation to block non-specific binding sites; the Fe3O4@COF@Pt-Ab2 conjugate is magnetically separated, is washed with PBST for multiple times, and is dispersed in a PBST solution, and is stored at 4 DEG C for standby use.

[0020] Further, the preparation of Fe3O4@COF@Pt-Ab2 includes the following steps: the Fe3O4@COF@Pt NPs aqueous solution is magnetically separated, and is ultrasonically dispersed in PBS; Ab2 is added to the mixture, after incubation at room temperature on a vertical mixer, BSA is added for incubation to block non-specific binding sites; the Fe3O4@COF@Pt-Ab2 conjugate is magnetically separated, is washed with PBST for multiple times, and is dispersed in a PBST solution, and is stored at 4 DEG C for standby use.

[0021] (Three) beneficial effects

[0022] The present application provides a kind of colorimetric immunoassay method based on core-shell structure composite COF based nanozyme, Fe3O4@COF@Pt NPs has good peroxidase activity and oxidase activity, after being labeled target molecule monoclonal antibody, it can be used as labeled probe, can effectively realize the colorimetric identification detection of target molecule, improve the accuracy and anti-interference ability of immunodetection. By replacing antibody and other recognition elements, the immunodetection of biological macromolecules such as protein, virus and bacteria and pathogens can be realized, and has broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 SEM images of Fe3O4 NPs, Fe3O4@COF NPs and Fe3O4@COF@Pt NPs obtained in the embodiments of the present application;

[0024] In the figure: (a) is the SEM image of Fe3O4 NPs; (b) is the SEM image of Fe3O4@COF NPs; (c) is the SEM image of Fe3O4@COF@Pt NPs;

[0025] Figure 2 TEM images and element mapping images of Fe3O4 NPs, Fe3O4@COF NPs and Fe3O4@COF@Pt NPs obtained in the embodiments of the present application;

[0026] Figure: (a) is TEM image of Fe3O4 NPs; (b) is TEM image of Fe3O4@COF NPs; (c) is TEM image of Fe3O4@COF@Pt NPs; (d) is elemental mapping of Fe3O4@COF@Pt NPs;

[0027] Figure 3 HRTEM image, hysteresis loop, XRD pattern, XPS and FT-IR spectrum of the product obtained in the example of the present application;

[0028] Figure: (a) is HRTEM image of Fe3O4@COF@Pt NPs; (b) is hysteresis loop of Fe3O4 NPs, Fe3O4@PEI NPs, Fe3O4@COF NPs and Fe3O4@COF@Pt NPs; (c) is XRD pattern of Fe3O4@COF@Pt NPs; (d) is high resolution XPS spectrum of N1s; (e) is high resolution XPS spectrum of Pt4f; (f) is FT-IR spectrum of Fe3O4 NPs, Fe3O4@COF NPs and Fe3O4@COF@Pt NPs;

[0029] Figure 4 XRD pattern of Fe3O4 NPs;

[0030] Figure 5 XPS spectrum of Fe3O4@COF@Pt NPs;

[0031] Figure: (a) is full spectrum; (b) is Fe2p spectrum; (c) is C1s spectrum; (d) is O1s spectrum; (e) is F1s spectrum;

[0032] Figure 6 UV-vis absorption spectrum and Zeta potential of Fe3O4@COF@Pt NPs;

[0033] Figure: (a) is UV-vis spectrum of Fe3O4 NPs, Fe3O4@COF NPs, Fe3O4@COF@Pt NPs; (b) is Zeta potential of Fe3O4 NPs, Fe3O4@COF NPs, Fe3O4@COF@Pt NPs, Fe3O4@COF@Pt-Ab2;

[0034] Figure 7 Effect of different factors on peroxidase property of Au@CeO2@Pt NPs;

[0035] In the figure: (a) is pH; (b) is temperature; (c) is TMB concentration; (d) is H2O2 concentration;

[0036] Figure 8 The enzymatic kinetic curve and enzyme reaction related analysis diagram of Fe3O4@COF@Pt NPs;

[0037] In the figure: (a) is the UV-absorption spectrum of different systems; (b) is the fixed Michaelis-Menten curve and corresponding Lineweaver-Burk curve of different TMB concentrations under the concentration (8mM); (c) is the Michaelis-Menten curve and Lineweaver-Burk curve of different concentrations under the fixed TMB concentration (0.3mM); (d) is the electron paramagnetic resonance (EPR) spectrum of DMPO+Fe3O4@COF@Pt NPs; (e) is the EPR spectrum of DMPO+Fe3O4@COF@Pt NPs; (f) is the free radical scavenging experiment of isopropyl alcohol (IPA) and tryptophan (TRP) on and

[0038] Figure 9 The enzyme reaction related analysis diagram of Fe3O4@COF@Pt NPs;

[0039] In the figure: (a) is the UV-vis absorption spectrum of Fe3O4 NPs, Fe3O4@COF NPs and Fe3O4@COF@Pt NPs after reaction with single-component TMB chromogenic solution under the same reaction conditions; (b) is the comparison of UV-vis absorption intensity of Fe3O4@COF@Pt NPs before and after antibody coupling;

[0040] Figure 10 The basic principle schematic diagram of colorimetric immunoassay for detecting SEB based on core-shell structure composite COF-based nanoscale enzyme Fe3O4@COF@Pt NPs;

[0041] Figure 11 The response situation and standard curve of SEB colorimetric detection based on core-shell structure composite COF-based nanoscale enzyme Fe3O4@COF@Pt NPs;

[0042] In the figure: (a) is the relationship between SEB concentration and UV-vis absorption spectrum intensity in TMB oxidation reaction; (b) is the linear relationship between corresponding SEB concentration and OX absorbance of TMB at 652nm;

[0043] Figure 12 ​​Specificity and reproducibility test of the Fe3O4@COF@Pt NPs-based SEB colorimetric detection;

[0044] In the figure: (a) is specificity (blank sample is PBS buffer); (b) is the reproducibility of immunoassay. DETAILED DESCRIPTION

[0045] In order to make the purpose, content and advantages of the present application clearer, the specific embodiments of the present application are described in further detail below in combination with the drawings and examples.

[0046] The present embodiment proposes a colorimetric immunoassay method based on core-shell structure composite COF-based nanoscale enzyme, which specifically comprises the following steps:

[0047] 1. Preparation of Fe3O4@COF@Pt NPs

[0048] (1) Preparation of Fe3O4 NPs: weigh 50 mL of ethylene glycol in a 100 mL beaker, and then add 1.25 g of poly(4-styrene sulfonic acid-co-maleic acid) sodium salt, 1.35 g of iron chloride hexahydrate, and 6.22 g of sodium acetate trihydrate into the beaker, and stir and disperse them vigorously, then transfer the solution to a reaction kettle, heat it to 200°C, and react for 10 h, then naturally cool it to room temperature, and wash it with water and ethanol alternately for several times, and then dry it in a vacuum at 60°C, and store it at 4°C for standby;

[0049] (2) Preparation of Fe3O4@PEI NPs: add 100 mg of Fe3O4 NPs into 20 mL of 2.5 mg / mL polyethyleneimine aqueous solution, and ultrasonicate for 30 min, then wash it with water and ethanol alternately for several times, and then dry it in a vacuum at 60°C for standby;

[0050] (3) Preparation of Fe3O4@COF NPs: dissolve 20 mg of Fe3O4@PEI and 240 mg of polyvinylpyrrolidone in 80 mL of acetonitrile, and ultrasonicate for 30 min to mix them uniformly, then add 21 mg of 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 18 mg of 2,5-difluoro-p-xylylene diformic acid (DFTA) into the mixture, and ultrasonicate for 6 min to mix them uniformly, then drop 600 μL of glacial acetic acid into the mixture, and let it stand at room temperature for 2 h, then add 1.6 mL of 0.5 mM ascorbic acid aqueous solution into the mixture while stirring, then immediately heat it to 80°C, and react for 6 h, then naturally cool it to room temperature, and wash it with water and ethanol alternately for several times, and then dry it in a vacuum at 60°C for standby;

[0051] (4) Preparation of Fe3O4@COF@Pt NPs: 20 mg Fe3O4@COF was added into 10 mL water and ultrasonically treated for 30 min to make it completely dispersed, then 100 mL ethanol and 10 mL H2PtCl6 solution were added successively, and ultrasonically mixed for 1 min. At room temperature, 1.6 mg / mL NaBH4 solution was added dropwise with vigorous stirring until the solution changed from yellow to brown-black, and then the stirring was continued for 2 h. After the reaction was completed, the brown-black product was separated, washed with pure water for several times, and dispersed in 40 mL water for standby.

[0052] 2. Preparation of Fe3O4@COF@Pt NPs-Ab2 signal probe

[0053] 8 mL Fe3O4@COF@Pt NPs aqueous solution was magnetically separated and ultrasonically dispersed in 8 mL 10 mM PBS (pH = 7.2-7.4) for 20 min; 1 mL 1 mg / mL Ab2 was added to the mixture, which was incubated at room temperature on a vertical mixer for 2 h, then 500 μL 10 wt% BSA was added and incubated for 1 h to block the non-specific binding sites; Fe3O4@COF@Pt-Ab2 conjugate was magnetically separated, washed with PBST (1% Tween-20) for several times, and dispersed in 1% PBST solution for storage at 4°C.

[0054] 3. Characterization of Fe3O4@COF@Pt NPs, etc.

[0055] The above obtained products Fe3O4@COF@Pt NPs, etc. were characterized by SEM, TEM, element mapping, XRD, XPS and Zeta potential, etc.

[0056] (1) SEM, TEM and element mapping characterization

[0057] The size and morphology of Fe3O4@COF@Pt were characterized by scanning electron microscope (SEM) and transmission electron microscope (TEM). Figure 1 and Figure 2 It was shown that the Fe3O4 nanoparticles synthesized by hydrothermal method presented regular spherical shape with a particle size of about 150 nm. After the COF layer was coated on the magnetic particles by the dual-ligand assisted encapsulation strategy, the shape of the material remained unchanged, and the particle size increased to about 200 nm. In addition, a large number of Pt nanoparticles were observed on the nanoscale enzyme, and the metal nanoparticles were mainly distributed in the COF layer and did not affect the structure of Fe3O4@COF. Further element mapping analysis of Fe3O4@COF@Pt showed that Fe, O, C, N, F and Pt elements were uniformly distributed, the distribution of F element proved the successful coating of COF layer, and the presence of Pt element indicated that Pt NPs were successfully loaded on the surface of Fe3O4@COF.

[0058] (2) Characterization of HRTEM, XRD and XPS

[0059] Figure 3 The high-resolution transmission electron microscopy (HRTEM) image in the middle (a) shows the lattice fringes of Fe3O4@COF@Pt with an interlayer spacing of 0.226 nm, corresponding to the (111) plane of metallic platinum. X-ray diffraction (XRD) analysis also confirmed the successful preparation of Fe3O4@COF@Pt. Figure 3 The middle (c) and Figure 4 indicate that the main peak at 35.39° can be attributed to the (311) plane of Fe3O4 (PDF #89-0688), while the main peak at 40.06° corresponds to the (111) plane of platinum (Pt) (PDF #87-0640), which is consistent with the results of HRTEM. In addition, diffraction peaks at 5.60°, 7.42° and 9.70° can also be identified, corresponding to the (200), (210) and (220) plane diffraction of the COF shell, respectively. The composition and chemical valence of the material were characterized by X-ray photoelectron spectroscopy (XPS) analysis. Figure 5 is a full spectrum, indicating that the composite material is mainly composed of Fe, O, N, C, F and Pt elements, which is consistent with the previous analysis. As shown in Figure 3 indicated in the middle (d), the peaks at 398.60 eV and 400.30 eV in the N1s spectrum correspond to the C=N bond and the -NH2 bond, respectively, and the presence of the C=N bond verifies the successful preparation of TAPB-DFTACOF. As shown in Figure 3 indicated in the middle (d), the peaks at 71.3 eV and 74.4 eV in the Pt4f spectrum belong to Pt4f7 / 2 and Pt4f5 / 2, indicating that Pt exists in zero valence state. The chemical bond composition of COF was verified by Fourier transform infrared spectroscopy (FT-IR). As shown in Figure 3 indicated in the middle (f), the infrared spectrum of the prepared sample shows that, compared with bare Fe3O4, the synthesized Fe3O4@COF appears new peaks at 1675 cm⁻¹ and 1621 cm⁻¹, which correspond to the stretching vibration of the -CHO group and the stretching vibration of the C=N bond in the COF shell, respectively. Next, the magnetic properties of Fe3O4@COF@Pt were further tested. As shown in Figure 3As shown in (b), the saturation magnetization values ​​of Fe3O4, Fe3O4@PEI, Fe3O4@COF, and Fe3O4@COF@Pt are 60.66, 59.27, 26.98, and 21.86 emu / g, respectively. With the gradual decrease in the proportion of Fe3O4 during material preparation, the saturation magnetization value of the material also gradually decreases. However, relatively speaking, the composite material still possesses excellent magnetic properties and can effectively perform separation and enrichment functions. Furthermore, the products obtained during the nanozyme synthesis process were tested using a UV-Vis spectrophotometer, such as... Figure 6 As shown in (a), Fe3O4 exhibits an absorption peak at 404 nm, while the absorption peaks of Fe3O4@COF and Fe3O4@COF@Pt show a certain degree of red shift compared to the absorption peak of Fe3O4@COF@Pt. This indirectly proves the stepwise synthesis of the materials. Finally, the Zeta potential of the materials was measured using a nanoparticle size analyzer to verify the successful preparation of the materials and the dispersibility of the nanomaterials. Figure 6 As shown in (b), the gradual change in Zeta potential further confirms the successful synthesis of Fe3O4@COF@Pt and that the complex has good dispersibility, making it suitable for nano-enzyme-linked immunosorbent assay (ELISA).

[0060] 4. Peroxidase activity and mechanism analysis of Fe3O4@COF@Pt NPs

[0061] The enzyme-like activity of Fe3O4@COF@Pt NPs was determined through a series of comparative experiments. The optimal colorimetric analytical characteristics depend on factors such as substrate concentration, temperature, and pH. Figure 7 As shown in (a), the UV-Vis absorbance of the reaction solution reaches its maximum when the pH of the system is 5.0. Figure 7 (b) shows that the UV-Vis absorbance remains at a high level in the range of 0–60 °C, and the optimal catalytic activity is around 30 °C. All experiments were conducted at room temperature for ease of operation. Figure 5 (c) and (d) show that when TMB and At concentrations of 8 mM and 0.3 mM, the UV-Vis absorbance essentially reached its maximum.

[0062] To further elucidate the steady-state kinetics and catalytic mechanism of Fe3O4@COF@Pt NPs, while maintaining Changing the concentration of TMB while keeping the overall concentration constant. Maximum reaction rate (V max The higher the value of K, the stronger the enzyme's ability to catalyze the substrate, while the Michaelis constant (K) is the higher the value of K. m The smaller the value, the stronger the affinity between the enzyme and the substrate. m and V maxThe isosteady-state dynamic parameters were calculated using the Lineweaver-Burk double reciprocal curves of the Michaelis-Menten equations. Figure 8 As shown in the table below, compared with the kinetic parameters of other enzymes, the Ki of Fe3O4@COF@Pt NPs with TMB as substrate is... m The value is relatively small or comparable to other enzymes, indicating that it has a good affinity for TMB.

[0063]

[0064] V of Fe3O4@COF@Pt NPs with TMB as substrate max The values ​​are approximately 9.49 times and 3.27 times that of Fe3O4 NPs and HRP, respectively, indicating that Fe3O4@COF@Pt NPs have a stronger catalytic ability for TMB, demonstrating their outstanding ability to oxidize TMB like an enzyme.

[0065] EPR spectroscopy was used to capture hydroxyl radicals and superoxide anions. In the test, 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO), as a spin trap material, was able to trap... and Generate stable DMPO / With DMPO / The complexes have characteristic spectra with relative intensities of 1:2:2:1 and 1:1:1:1:1:1, respectively. For example... Figure 8 As shown in (d) and (e), after adding Fe3O4@COF@Pt, a distinct characteristic ESR signal can be observed, and DMPO / With DMPO / The signal intensity of the samples was significantly higher than that of the blank control group. This indicates that Fe3O4@COF@Pt can activate... and The generation of reactive oxygen species (ROS), namely and Furthermore, the results of electron spin resonance (ESR) were verified through free radical scavenging experiments using the selective scavengers isopropanol (IPA) and L-tryptophan (TRP), such as... Figure 8 As shown in (f). Add Then, Fe3O4@COF@Pt+ The absorbance of the +TMB system increased significantly compared to the control group when added When the selective scavenger IPA was used, the absorbance of the system decreased sharply, confirming the peroxidase-like activity of the nanozyme. Similarly, analysis also confirmed its oxidase-like activity. Combining the results of ESR and free radical scavenging experiments, it can be concluded that Fe3O4@COF@Pt generates [a specific substance] during the catalytic process. and .

[0066] Figure 9 Figure (b) shows that the catalytic activity of the Fe3O4@COF@Pt-Ab2 coupling is not significantly different compared to Fe3O4@COF@Pt NPs. Figure 9 As shown in (a), compared with Fe3O4 NPs or Fe3O4@COF NPs, in and Under acidic conditions (pH=5), the absorbance of TMB changes significantly upon the addition of Fe3O4@COF@Pt NPs, indicating the formation of the charge-transfer complex oxTMB. The combination of highly dispersed metal nanoparticles and the high loading capacity of COF materials is the reason for the enhanced enzyme catalytic activity of Fe3O4@COF@PtNPs. Figure 8 (a) shows Fe3O4@COF@Pt NPs in and The presence of these enzymes also allows them to catalyze other chromogenic agents (ABTS and OPD), indicating that the enzyme-mimicking activity of Fe3O4@COF@Pt NPs is substrate-independent. These results suggest that Fe3O4@COF@Pt NPs hold promise as ideal enzyme mimics.

[0067] 5. Establishment of a standard curve for SEB colorimetric detection based on a dual-enzyme-like Fe3O4@COF@Pt assay.

[0068] The SEB colorimetric detection principle based on Fe3O4@COF@Pt is as follows: Figure 10 As shown, Fe3O4@COF@Pt NPs were prepared, and then Fe3O4@COF@Pt NPs were coupled with SEB antibody Ab2 to prepare the signal probe Fe3O4@COF@Pt-Ab2. SEB monoclonal antibody Ab1 was coated onto an ELISA plate, and non-specific binding sites were blocked with BSA. When SEB sequentially binds to Ab1 and Fe3O4@COF@Pt-Ab2 to form an immune sandwich complex, the simulated peroxidase and oxidase activities of Fe3O4@COF@Pt NPs oxidize colorless TMB into blue oxidized TMB (oxTMB), thus achieving specific and sensitive detection of SEB in complex samples via colorimetric signal.

[0069] Under optimal experimental conditions, establish a standard curve between absorbance values ​​and SEB concentration. For example...Figure 11 As shown in FIG. 17 (a), as the concentration of SEB protein increased from 0 ng / mL to 1000 ng / mL, the colorimetric signal also increased, indicating that the absorbance was positively correlated with the concentration of SEB protein. As shown in FIG. 17 (b), the colorimetric signal of the SEB protein standard curve was stable and repeatable. Figure 11 OX The absorbance value of TMB was linearly related to lgC SEB (1-200 ng / ml), and the fitted linear equation was as follows: Y=0.1349+0.0353lgC SEB (R 2 =0.9924, C SEB represented the concentration of SEB protein, and Y represented the absorbance value at 652 nm), and the detection limit calculated according to 3σ / S (where S was the slope of the linear regression equation, and σ was the standard deviation of 8 blank samples) was 0.395 ng / mL. The above results confirmed that the colorimetric immunoassay had the characteristics of wide detection range and low LOD, and could realize the sensitive quantitative detection of SEB.

[0070] 6. Specificity and reproducibility determination based on colorimetric detection of Fe3O4@COF@Pt with double-enzyme-like activity

[0071] Specificity is the basis for the feasibility of immunoassay. Five proteins (Ricin, Abrin, Hum-IgG, Nucleocapsid Protein, and BSA) with a concentration of 100 ng / mL were selected as interferents. Figure 12 As shown in FIG. 22 (a), the reactions of the interferents were similar to the blank, and were significantly different from the target protein (20 ng / mL), indicating that the colorimetric immunoassay method had high specificity.

[0072] Reproducibility is another important factor for the performance of immunoassay. When detecting the target protein (20 ng / mL) with the same concentration, the reproducibility of the immunoassay was examined by 8 parallel experiments, Figure 12 As shown in FIG. 22 (b), the relative standard deviation (RSD) of the colorimetric signal was 3.05%, indicating that the method had good reproducibility.

[0073] 7. Detection of simulated samples

[0074] In order to verify the feasibility of the immunoassay method in practical application, tests were respectively carried out in simulated samples of tap water, milk, and coconut water, and the results were shown in the following table. The recovery rate of the nano-enzyme-linked immunosorbent assay was 93.6-115.4%, which proved that the immunoassay system of the application had good applicability and could be used for actual sample detection.

[0075]

[0076] ​According to the embodiment, the Fe3O4@COF@Pt NPs have good peroxidase activity and oxidase activity, the target molecule monoclonal antibody is labeled by the Fe3O4@COF@Pt NPs as a labeled probe, and the colorimetric signal recognition and detection of the target molecule SEB are effectively realized, and the method has the advantages of high sensitivity, simple operation, good reproducibility, strong anti-interference, etc. By replacing the antibody and other recognition elements, the immunodetection of biological macromolecules such as proteins, viruses and bacteria and pathogens can be realized, and the method has a wide application prospect.

[0077] The above only describes the preferred embodiments of the present application, and it should be pointed out that for ordinary skilled persons in the art, some improvements and modifications can be made without departing from the technical principles of the present application, and these improvements and modifications should also be considered as the protection scope of the present application.

Claims

1. A colorimetric immunoassay method based on core-shell structure composite COF-based nanoszyme, characterized in that, The colorimetric immunoassay method comprises the following steps: preparing Fe3O4@COF@Pt NPs; using Fe3O4@COF@Pt NPs to label a single Ab2 of a target molecule to prepare a signal probe Fe3O4@COF@Pt-Ab2; coating a single Ab1 of the target molecule on an enzyme-labeled plate; the target molecule is sequentially combined with Ab1 and Fe3O4@COF@Pt-Ab2 to form an immune sandwich complex, TMB is added, Fe3O4@COF@Pt NPs catalyzes the oxidation of TMB to oxidized TMB, a visible colorimetric signal is generated at 652 nm, and colorimetric immunoassay is realized.

2. The colorimetric immunoassay method based on core-shell structure composite COF-based nanoszyme according to claim 1, wherein, The preparation of Fe3O4@COF@Pt NPs comprises the following steps: (1) Fe3O4 NPs preparation: measure 50 mL of ethylene glycol in a 100 mL beaker, and then add 1.25 g of poly(4-styrene sulfonic acid-co-maleic acid) sodium salt, 1.35 g of iron chloride hexahydrate and 6.22 g of sodium acetate trihydrate, and stir and disperse; the solution is transferred to a reaction kettle and heated to 200 DEG C for reaction; after the reaction is completed, the solution is naturally cooled to room temperature, washed with water and ethanol for multiple times, vacuum dried at 60 DEG C, and stored at 4 DEG C for standby; (2) Fe3O4@PEI NPs preparation: ultrasonic Fe3O4 NPs in polyethyleneimine aqueous solution, and then washed with water and ethanol for multiple times, and vacuum dried for standby; (3) Fe3O4@COF NPs preparation: dissolve Fe3O4@PEI and polyvinylpyrrolidone in acetonitrile, ultrasonic mix uniformly, add 1,3,5-tris(4-aminophenyl)benzene and 2,5-difluoro-p-xylylene glycol in sequence and ultrasonic mix uniformly, drop in glacial acetic acid, stand at room temperature, add anti-hydroquinone aqueous solution while stirring, and then immediately heat to 80 DEG C for reaction; after reflux, the solution is naturally cooled to room temperature, washed with water and ethanol for multiple times, and vacuum dried for standby; (4) Fe3O4@COF@Pt NPs preparation: weigh Fe3O4@COF and add to water, ultrasonic treat to completely disperse, then add ethanol and H2PtCl6 solution in sequence, ultrasonic mix uniformly, drop in NaBH4 solution while stirring at room temperature until the solution turns from yellow to brown black, then continue to stir, after the reaction is completed, separate and enrich, wash with pure water for multiple times, and disperse the obtained brown black product in water for standby.

3. The colorimetric immunoassay method based on core-shell structure composite COF-based nanoszyme according to claim 2, wherein, The preparation of Fe3O4@COF@Pt NPs comprises the following steps: (1) Fe3O4 NPs preparation: measure 50 mL of ethylene glycol in a 100 mL beaker, and then add 1.25 g of poly(4-styrene sulfonic acid-co-maleic acid) sodium salt, 1.35 g of iron chloride hexahydrate and 6.22 g of sodium acetate trihydrate, and stir and disperse; the solution is transferred to a reaction kettle and heated to 200 DEG C for reaction; after the reaction is completed, the solution is naturally cooled to room temperature, washed with water and ethanol for multiple times, vacuum dried at 60 DEG C, and stored at 4 DEG C for standby; (2) Preparation of Fe3O4@PEI NPs: 100 mg Fe3O4 NPs were added into 20 mL 2.5 mg / mL polyethyleneimine aqueous solution and ultrasonicated for 30 min, then washed with water and ethanol for several times, and dried at 60°C under vacuum for later use; (3) Preparation of Fe3O4@COF NPs: 20 mg Fe3O4@PEI and 240 mg polyvinylpyrrolidone were dissolved in 80 mL acetonitrile and ultrasonicated for 30 min, then 21 mg 1,3,5-tris(4-aminophenyl)benzene and 18 mg 2,5-difluoro-tetrachlorobenzene were added in sequence and ultrasonicated for 6 min, 600 μL glacial acetic acid was added dropwise, and the mixture was left to stand at room temperature for 2 h, 1.6 mL 0.5 mM anti-ascorbic acid aqueous solution was added while stirring, then the mixture was heated to 80°C immediately, and reacted for 6 h under reflux, and then naturally cooled to room temperature, washed with water and ethanol for several times, and dried at 60°C under vacuum for later use; (4) Preparation of Fe3O4@COF@Pt NPs: 20 mg Fe3O4@COF was weighed into 10 mL water, and ultrasonicated for 30 min to make it completely dispersed, then 100 mL ethanol and 10 mL H2PtCl6 solution were added in sequence, and ultrasonicated for 1 min to mix, at room temperature, 1.6 mg / mL NaBH4 solution was added dropwise while stirring until the solution turned from yellow to brown black, then the stirring was continued for 2 h, after the reaction was completed, the product was separated, enriched, and washed with pure water for several times, and the obtained brown black product was dispersed in 40 mL water for later use.

4. The colorimetric immunoassay method based on core-shell structure composite COF-based nanoszyme according to claim 1, wherein, The preparation of Fe3O4@COF@Pt-Ab2 includes the following steps: the Fe3O4@COF@Pt NPs aqueous solution was magnetically separated and ultrasonically dispersed in PBS; Ab2 was added to the mixture, and after incubation at room temperature on a vertical mixer, BSA was added for incubation to block the non-specific binding sites; the Fe3O4@COF@Pt-Ab2 conjugate was magnetically separated, washed with PBST for several times, and dispersed in a PBST solution for storage at 4°C for later use.

5. The colorimetric immunoassay method based on core-shell structure composite COF-based nanzyme according to claim 4, wherein, The preparation of Fe3O4@COF@Pt-Ab2 specifically includes the following steps: 8 mL Fe3O4@COF@Pt NPs aqueous solution was magnetically separated and ultrasonically dispersed in 8 mL 10 mM PBS for 20 min; 1 mL 1 mg / mL Ab2 was added to the mixture, and after incubation at room temperature on a vertical mixer for 2 h, 500 μL 10 wt% BSA was added for incubation for 1 h to block the non-specific binding sites; the Fe3O4@COF@Pt-Ab2 conjugate was magnetically separated, washed with PBST for several times, and dispersed in a 1% PBST solution for storage at 4°C for later use.

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