A preparation method of a redox probe based on metal organic framework derived nanosheet, and products and applications thereof

Abstract

The present application relates to a kind of preparation method of redox probe based on metal organic framework derived nanosheet and its product and application, belong to the technical field of preparation of redox probe.The present application is mainly to form super-thin metal organic framework material by self-assembly of metal ion (Cu 2+ Or Co 2+ ) and organic ligand, continue to react with ascorbic acid (AA), chloroauric acid (HAuCl4) to obtain metal-UMOFNs@Au NPs, then mixed with alpha-fetoprotein antibody (anti-AFP), carcinoembryonic antibody (anti-CEA) and glucose oxidase (GOD) after incubation to obtain redox probe (metal-UMOFNs@Au NP-Ab2-GOD).In addition, the redox probe prepared by the present application can be used to prepare sandwich-type electrochemical immunosensor capable of simultaneously detecting CEA and / or AFP, and the immunosensor has higher sensitivity, good selectivity and stability.

Description

Technical Field

[0001] This invention belongs to the field of redox probe preparation technology, and relates to a method for preparing a redox probe based on metal-organic framework-derived nanosheets, as well as its products and applications. Background Technology

[0002] Cancer causes millions of deaths annually and is considered one of the world's deadliest diseases. Therefore, the detection of tumor markers in blood or tissues has attracted increasing interest in clinical cancer screening and early diagnosis. However, the combined assay of multiple tumor markers can facilitate the screening and diagnosis of certain cancer-related diseases. Compared to single-biomarker analysis, the analysis of multiple biomarkers not only reduces resources, analysis time, and cost but also provides larger sample volume output, higher analytical throughput, and improved analytical efficiency. Therefore, it is necessary to develop a rapid and sensitive analytical method to simultaneously measure multiple tumor markers. To date, various detection methods have been developed, such as luminescent electrochemical sensors, photoelectrochemical (PEC) immunosensors, enzyme-linked immunosorbent assays (ELISA), and electrochemical immunoassays. Among these, electrochemical immunosensors are widely used due to their high specificity, simple instrumentation, low cost, and high sensitivity. Despite some progress in this field, exploring new methods and strategies to further improve sensitivity and multiplexing capabilities remains a challenge.

[0003] To obtain independent signals for each biomarker in multi-nanocell samples, researchers have discovered that different types of materials can be used as signal recognition tags, such as dye-doped nanoparticles, metal ions, and redox probes. Redox probes can rapidly transfer electrons, providing distinct redox peaks in electrochemical scans and are widely used in electrochemical sensors. In recent years, several immunosensors labeled with redox probes have been proposed, capable of simultaneously detecting multiple analytes. Even so, due to the occurrence of several immune reactions on the same electrode surface, insufficient reaction space may lead to low signal intensity and detection limits compared to traditional single-analyte detection. Therefore, improving the intensity and sensitivity of the detection signal is an important research area for this type of sensor.

[0004] Metal-organic frameworks (MOFs) are porous materials formed through the self-assembly of metals or metal clusters with ligands. Compared with traditional mesoporous or microporous nanomaterials (zeolites, silica, etc.), MOFs have attracted widespread attention due to their exceptionally high surface area, tunable pore size, high chemical stability, and good electrochemical activity. Furthermore, MOFs not only possess large specific surface areas and active sites, but their ligands and surfaces can also be modified through in-situ or post-functionalization. These advantages make MOFs ideal active electrode materials. Recently, many electrochemical sensors based on electrodes modified with different MOFs have been reported. Liu et al. reported a non-enzymatically labeled electrochemical immunosensor using the Cu3(BTC)2 metal-organic framework; Yuan's group directly used the luminescent material [Ru(dcbpy)3]. 2+ MOFs with good ECL performance were synthesized as ligands for the detection of N-terminal pro-B-type natriuretic peptide (NT-proBNP); Zhang et al. developed a novel trace heavy metal ion (Pb) detection method based on Fe(III)-based MOFs. 2+ And As 3+ Electrochemical sensing platforms for detection. However, to date, there are still no reports focusing on using MOF derivatives as signal tags to label different secondary antibodies (Ab2) for the simultaneous electrochemical detection of multiple tumor markers based on a dual-catalytic amplification strategy.

[0005] Furthermore, two-dimensional (2D) MOFs possess unique physical and chemical properties, which are caused by electronic effects resulting from their thickness, high specific surface area, and high surface-to-volume atomic ratio. Compared to 3D MOFs, 2D MOFs exhibit ultrathin thickness, rapid mass transfer, and excellent electron transfer capabilities. Similar to other 2D nanomaterials, 2D MOF nanosheets offer new opportunities in fundamental research and applications as excellent materials for electrochemical analysis. Summary of the Invention

[0006] In view of this, one objective of the present invention is to provide a method for preparing a redox probe based on metal-organic framework-derived nanosheets; a second objective of the present invention is to provide a redox probe based on metal-organic framework-derived nanosheets; a third objective of the present invention is to provide an application of a redox probe based on metal-organic framework-derived nanosheets in a chemiimmunosensor; a fourth objective of the present invention is to provide a method for preparing a chemiimmunosensor; a fifth objective of the present invention is to provide a chemiimmunosensor; and a sixth objective of the present invention is to provide an application of a sandwich electrochemical immunosensor in the simultaneous detection of AFP and / or CEA.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] 1. A method for preparing a redox probe based on metal-organic framework-derived nanosheets, the method being as follows:

[0009] (1) Preparation of metal-UMOFNs@Au NPs: Disperse metal-UMOFNs in water and add ascorbic acid (AA). Stir to form a uniform colloidal suspension. Add 1% chloroauric acid (HAuCl4) solution and continue stirring to allow it to react fully. Then centrifuge to separate the precipitate. After washing and drying, metal-UMOFNs@Au NPs are obtained.

[0010] (2) Preparation of biocoupled redox probe (metal-UMOFNs@Au NP–Ab2–GOD): Dissolve metal-UMOFNs@Au NPs in water to form a homogeneous suspension, add 10 ng / mL of alpha-fetoprotein antibody (anti-AFP) or carcinoembryonic antibody (anti-AFP) and stir overnight, then add 1 mg / mL of glucose oxidase (GOD) and incubate. Centrifuge to obtain redox probe (metal-UMOFNs@Au NP–Ab2–GOD);

[0011] The metal-UMOFNs mentioned in step (1) are Cu-UMOFNs or Co-UMOFNs, wherein the Cu-UMOFNs are prepared by the following method: Cu(NO3)2·3H2O is dissolved in a mixed solution of N,N-dimethylformamide (DMF) and acetonitrile to form solution A. Phthalic acid (BDC) is dissolved in a mixed solution of N,N-dimethylformamide (DMF) and acetonitrile, and then polyvinylpyrrolidone (PVP) is added to dissolve and form solution B. Solution A and solution B are mixed and sonicated for 2-5 min. The mixture is heated at 135°C for 24 h to form a precipitate. After centrifugation, washing and drying are performed to obtain Cu-UMOFN.

[0012] The Co-UMOFNs are prepared as follows: phthalic acid (BDC) is dissolved in a mixed solution of N,N-dimethylformamide (DMF), ethanol and water, and then CoCl2·6H2O is added to completely dissolve it. Triethylamine (TEA) is then added, and the mixture is sonicated to form a colloidal suspension. The suspension is continuously sonicated in an autoclave for 8 hours, centrifuged, washed and dried to obtain Co-UMOFNs.

[0013] Preferably, in the preparation process of Cu-UMOFN, the mass-to-volume ratio of Cu(NO3)2·3H2O, N,N-dimethylformamide (DMF), and acetonitrile in solution A is 0.3:10:30 (g:mL:mL), the mass-to-volume ratio of phthalic acid (BDC), N,N-dimethylformamide (DMF), acetonitrile, and polyvinylpyrrolidone (PVP) in solution B is 0.3:30:10:0.9 (g:mL:mL:g), the volume ratio of solution A to solution B is 1:1, the centrifugation speed is 10000–14000 rpm, the time is 5–8 min, and the drying is carried out in air at 55–65°C.

[0014] Preferably, in the preparation process of Co-UMOFNs, the volume ratio of N,N-dimethylformamide (DMF), ethanol and water in the mixed solution is 32:2:2, and the molar volume ratio of phthalic acid (BDC), the mixed solution, CoCl2·6H2O and triethylamine (TEA) is 0.75:36:0.75:0.8 (mmol:mL:mmol:mL). The washing is performed using deionized water and ethanol, respectively.

[0015] Preferably, in step (1), the mass ratio of the metal-UMOFNs to ascorbic acid (AA) is 10:50; the mass-volume ratio of the metal-UMOFNs to chloroauric acid (HAuCl4) solution is 10:1 (mg:mL); and the drying is carried out under vacuum conditions at 55-65°C.

[0016] In step (2), the mass-volume ratio of the metal-UMOFNs@Au NPs, anti-AFP and glucose oxidase (GOD) is 5:0.5:1, mg:mL:mL, and the incubation is specifically incubated at 4°C for 4 hours.

[0017] 2. Redox probes based on metal-organic framework-derived nanosheets prepared according to the above.

[0018] Preferably, the redox probe based on metal-organic framework-derived nanosheets (metal-UMOFNs@AuNP–Ab2–GOD) is a redox probe based on copper organic framework-derived nanosheets (Cu-UMOFNs@Au NP–Ab2–GOD) or a redox probe based on cobalt organic framework-derived nanosheets (Co-UMOFNs@Au NP–Ab2–GOD).

[0019] 3. Based on the above, the application of redox probes based on metal-organic framework-derived nanosheets in the preparation of electrochemical immunosensors.

[0020] 4. A method for preparing a sandwich-type electrochemical immunosensor, the method comprising the following steps:

[0021] (1) Preparation of immunosensor: Au@Fe3O4 NPs were dispersed in Nafion solution to form a suspension, which was then drop-coated onto the surface of a glassy carbon electrode (GCE). After drying at room temperature, the suspension was placed in a mixed solution of alpha-fetoprotein antibody (anti-AFP) and carcinoembryonic antibody (anti-CEA) with a concentration of 10 ng / mL. After incubation at 4°C for 6 h, the mixture of alpha-fetoprotein antibody (anti-AFP) and carcinoembryonic antibody (anti-CEA) (Ab1) was immobilized by NH2-Au affinity, thus preparing an immunosensor.

[0022] (2) Preparation of an immunosensor for incubating antigen: A 1 wt.% bovine serum albumin (BSA) solution was drop-coated onto the surface of the immunosensor prepared in step (1). After reacting at room temperature for 2 h to block non-specific binding sites, an antigen mixture of alpha-fetoprotein antigen (AFP) and carcinoembryonic antigen (CEA) was added and incubated at room temperature for 30 min to form an immunosensor for incubating antigen.

[0023] (3) The redox probes (Cu-UMOFNs@Au NP–Ab2–GOD) and the redox probes (Co-UMOFNs@Au NP–Ab2–GOD) based on the metal copper organic framework derived nanosheets are mixed in equal mass ratio, then dissolved in PBS buffer solution at pH=7.4 and drop-coated onto the surface of the immunosensor prepared in step (2) for incubation of the antigen. The sandwich electrochemical immunosensor can be obtained after incubation for 30 min.

[0024] After each step of the preparation method is completed, unreacted substances are removed by washing with PBS buffer solution at pH 7.4 and a concentration of 0.1 mol / L.

[0025] Preferably, the mixed solution in step (1) contains equal masses of alpha-fetoprotein antibody (anti-AFP) and carcinoembryonic antibody (anti-CEA).

[0026] Preferably, the antigen mixture in step (2) contains equal masses of alpha-fetoprotein antigen (AFP) and carcinoembryonic antigen (CEA).

[0027] Preferably, the mass ratio of the total mass of alpha-fetoprotein antibody (anti-AFP) and carcinoembryonic antibody (anti-CEA) in the mixed solution, the total mass of alpha-fetoprotein antigen (AFP) and carcinoembryonic antigen (CEA) in the antigen mixture, and the mass ratio of the mixed probe is 10:500:10.ng:μL:mg.

[0028] 5. The sandwich electrochemical immunosensor prepared according to the above preparation method.

[0029] 6. The application of the sandwich electrochemical immunosensor described above in the detection of alpha-fetoprotein antigen (AFP) and / or carcinoembryonic antigen (CEA).

[0030] The beneficial effects of this invention are as follows: This invention discloses a method for preparing a redox probe based on metal-organic framework-derived nanosheets, mainly by using metal ions (Cu... 2+ or Co 2+ The metal-organic framework (UMOFNs) is formed by self-assembly with organic ligands to form ultrathin UMOFNs (which have the advantages of large specific surface area, good conductivity, and easy functionalization). Further reaction with ascorbic acid (AA) and chloroauric acid (HAuCl4) yields metal-UMOFNs@Au NPs (which have high electrochemical activity and conductivity). These are then mixed with anti-AFP antibodies, anti-CEA antibodies, and glucose oxidase (GOD) and incubated to obtain a redox probe (metal-UMOFNs@Au NP–Ab2–GOD). The redox probe (metal-UMOFNs@Au NP–Ab2–GOD) prepared in this invention has properties such as large specific surface area, multiple active sites, and good conductivity, and can be used to prepare an electrochemical immunosensor. This electrochemical immunosensor exhibits high detection sensitivity, wide linear range, low detection limit, good selectivity, and good stability, thus enabling simultaneous detection of AFP and CEA.

[0031] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0032] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0033] Figure 1 A is a flowchart of the preparation of Cu-UMOFNs@Au NP–Ab2–GOD in Example 1 and Co-UMOFNs@AuNP–Ab2–GOD in Example 2; B is a flowchart of the preparation of the sandwich electrochemical immunosensor in Example 3.

[0034] Figure 2Transmission electron microscopy (TEM) images of different 2D ultrathin nanosheets, where A represents Cu-UMOFN, B represents Co-UMOFN, C represents Cu-UMOFNs@Au NPs, and D represents Co-UMOFNs@Au NPs.

[0035] Figure 3 In the diagram, A and B represent solutions containing 0.1 mol / L KCl at a concentration of 5 mmol / L [Fe(CN)6]. 3- / 4- Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to characterize the immunosensor modification process in solution, where a is a bare glassy carbon electrode (GCE), b is Au@Fe3O4NPs / GCE, c is Ab1 / Au@Fe3O4NPs / GCE (Ab1: a mixed solution of anti-AFP and anti-CEA), d is BSA / Ab1 / Au@Fe3O4NPs / GCE, and e is Ag / BSA / Ab1 / Au@Fe3O4NPs / GCE (Ag: a mixed solution of AFP and CEA).

[0036] Figure 4 In the figure, A and B represent the cyclic voltammetry (CV) and differential pulse voltammetry (DPV) characteristics of the chemiimmunoassay sensor in 0.1 mol / L PBS buffer solution at pH 7.4, respectively (where a is the blank sample (zero analyte), b is 1 ng / mL CEA, c is 1 ng / mL AFP, and d is a mixture of 1 ng / mL AFP and 1 ng / mL CEA in PBS (pH 7.4) containing 4.0 mmol / L glucose). C and D represent the cyclic voltammetry (CV) and differential pulse voltammetry (DPV) responses in 5 mL of 0.1 mol / L PBS buffer solution at pH 7.4 with and without glucose (4 mmol / L), respectively.

[0037] Figure 5 The results of DPV detection after incubating the sandwich electrochemical immunosensor prepared in Example 3 with a mixture of AFP and CEA antigens and a bioconjugated redox probe are shown. A is the differential pulse spectrum of the electrochemical immunosensor for AFP and CEA detection, and B is the calibration curve of the electrochemical immunosensor for AFP and CEA.

[0038] Figure 6 This is for the specific detection of the sandwich electrochemical immunosensor targeting CEA and AFP prepared in Example 3. Detailed Implementation

[0039] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0040] Example 1

[0041] A redox probe based on Cu-organic framework-derived nanosheets (Cu-UMOFNs@Au NP–Ab2–GOD) is prepared by the following method:

[0042] (1) Preparation of Cu-UMOFNs: Cu(NO3)2·3H2O was dissolved in a mixed solution of N,N-dimethylformamide (DMF) and acetonitrile to form solution A (where the mass-volume ratio of Cu(NO3)2·3H2O, N,N-dimethylformamide (DMF) and acetonitrile was 0.3:10:30, g:mL:mL). Phthalic acid (BDC) was dissolved in a mixed solution of N,N-dimethylformamide (DMF) and acetonitrile, and then polyvinylpyrrolidone (PVP) was added to form solution B (where phthalic acid...). The mass-to-volume ratio of acid (BDC), N,N-dimethylformamide (DMF), acetonitrile, and polyvinylpyrrolidone (PVP) was 0.3:30:10:0.9 (g:mL:mL:g). Solution A and solution B were mixed (volume ratio 1:1), sonicated for 2 min, and heated at 135 °C for 24 h to form a precipitate. After centrifugation (14000 rpm for 8 min), the precipitate was washed (three times sequentially with DMF and methanol) and dried (in air at 60 °C) to obtain Cu-UMOFNs.

[0043] (2) Preparation of Cu-UMOFNs@Au NPs: Cu-UMOFNs were dispersed in water and ascorbic acid (AA) was added (the mass ratio of Cu-UMOFNs to ascorbic acid (AA) was 10:50). The mixture was stirred to form a uniform colloidal suspension. A 1% chloroauric acid (HAuCl4) solution (the mass-volume ratio of Cu-UMOFNs to chloroauric acid (HAuCl4) solution was added dropwise and the mixture was stirred until homogeneous. The precipitate after centrifugation was washed and dried (under vacuum conditions at 60℃) to obtain Cu-UMOFNs@Au NPs.

[0044] (3) Preparation of redox probe (Cu-UMOFNs@Au NP–Ab2–GOD): Cu-UMOFNs@Au NPs were dissolved in water to form a uniform suspension. Anti-AFP at a concentration of 10 g / mL was added and stirred overnight. Glucose oxidase (GOD) at a concentration of 1 mg / mL was then added (the mass-volume ratio of Cu-UMOFNs@Au NPs, anti-AFP and glucose oxidase (GOD) was 5:0.5:1, mg:mL:mL). The mixture was then incubated (specifically at 4℃ for 4 h). After centrifugation, the redox probe (Cu-UMOFNs@Au NP–Ab2–GOD) based on Cu organic framework derived nanosheets was obtained.

[0045] The process for preparing the redox probe (Cu-UMOFNs@Au NP–Ab2–GOD) based on Cu-organic framework-derived nanosheets in Example 1 above is as follows: Figure 1 As shown in Figure A.

[0046] Example 2

[0047] A redox probe based on metal-Co organic framework-derived nanosheets (Co-UMOFNs@Au NP–Ab2–GOD) is prepared by the following method:

[0048] (1) Preparation of Co-UMOFNs: Dissolve phthalic acid (BDC) in a mixed solution of N,N-dimethylformamide (DMF), ethanol and water (the volume ratio of N,N-dimethylformamide (DMF), ethanol and water is 32:2:2), add CoCl2·6H2O and let it dissolve completely, then add triethylamine (TEA) (the molar volume ratio of phthalic acid (BDC), mixed solution, CoCl2·6H2O and triethylamine (TEA) is 0.75:36:0.75:0.8, mmol:mL:mmol:mL), sonicate to dissolve and form a colloidal suspension, continuously sonicate in an autoclave for 8 hours, centrifuge, wash (washed 3 times with deionized water and ethanol), and dry to obtain Co-UMOFNs.

[0049] (2) Preparation of Co-UMOFNs@Au NPs: Co-UMOFNs were dispersed in water and ascorbic acid (AA) was added (the mass ratio of Co-UMOFNs to ascorbic acid (AA) was 10:50). The mixture was stirred to form a uniform colloidal suspension. A 1% chloroauric acid (HAuCl4) solution (the mass-volume ratio of Co-UMOFNs to chloroauric acid (HAuCl4) solution was added dropwise and the mixture was stirred until homogeneous. The precipitate after centrifugation was washed and dried (under vacuum conditions at 60℃) to obtain Co-UMOFNs@Au NPs.

[0050] (3) Preparation of redox probe (Co-UMOFNs@Au NP–Ab2–GOD): Co-UMOFNs@Au NPs were dissolved in water to form a uniform suspension. Anti-CEA at a concentration of 10 g / mL was added and stirred overnight. Glucose oxidase (GOD) at a concentration of 1 mg / mL was then added (the mass-volume ratio of Co-UMOFNs@Au NPs, anti-CEA and glucose oxidase (GOD) was 5:0.5:1, mg:mL:mL). The mixture was then incubated (specifically at 4℃ for 4 h). Centrifugation yielded the redox probe (Co-UMOFNs@Au NP–Ab2–GOD) based on metal-Co organic framework derived nanosheets.

[0051] The procedure for preparing the redox probe (Co-UMOFNs@Au NP–Ab2–GOD) based on metal-Co organic framework-derived nanosheets in Example 2 above is as follows: Figure 1 As shown in Figure A.

[0052] Example 3

[0053] A sandwich electrochemical immunosensor is prepared using a redox probe based on Cu organic framework-derived nanosheets (Cu-UMOFNs@Au NP–Ab2–GOD) prepared in Example 1 and a redox probe based on Co organic framework-derived nanosheets (Co-UMOFNs@Au NP–Ab2–GOD) prepared in Example 2. The specific preparation method is as follows:

[0054] (1) Preparation of immunosensor: Au@Fe3O4 NPs suspension was dispersed in Nafion solution, dropped onto the surface of glassy carbon electrode (GCE), dried at room temperature, and then placed in a mixed solution consisting of 1 mL of 10 ng / mL anti-AFP antibody solution and 1 mL of 10 ng / mL anti-CEA antibody solution (where the mass concentration ratio of anti-AFP antibody to anti-CEA antibody in the mixed solution is 1:1). After incubation at 4℃ for 6 h, the anti-AFP antibody and anti-CEA antibody can be immobilized by NH2-Au affinity to form fixed antibody (Ab1), thereby preparing the immunosensor;

[0055] (2) Preparation of an immunosensor for incubation of antigen: A 1 wt.% bovine serum albumin (BSA) solution was drop-coated onto the surface of the immunosensor prepared in step (1). After reacting at room temperature for 2 h to block non-specific binding sites, 500 μL of an antigen mixture of alpha-fetoprotein antigen (AFP) and 500 μL of carcinoembryonic antigen (CEA) was added (the mass concentration ratio of alpha-fetoprotein antigen (AFP) to carcinoembryonic antigen (CEA) in the antigen mixture was 1:1). The mixture was incubated at room temperature for 30 min to form an immunosensor for incubation of antigen.

[0056] (3) Mix the redox probe (Cu-UMOFNs@Au NP–Ab2–GOD) (10mg) based on Cu organic framework derived nanosheets prepared in Example 1 and the redox probe (Co-UMOFNs@Au NP–Ab2–GOD) (10mg) based on Co organic framework derived nanosheets prepared in Example 2 in an equal mass ratio to form a mixed probe. Then, dissolve it in PBS buffer solution at pH=7.4 and drop it onto the surface of the immunosensor prepared in step (2) to incubate for 30 min to obtain a sandwich electrochemical immunosensor.

[0057] After each step of the above preparation method is completed, unreacted substances are removed by washing with PBS buffer solution with pH=7.4 and a concentration of 0.1mol / L.

[0058] In Example 3, the sandwich electrochemical immunosensor was fabricated using the Cu-UMOFNs@Au NP–Ab2–GOD redox probe prepared in Example 1 and the Co-UMOFNs@Au NP–Ab2–GOD redox probe prepared in Example 2. The procedure is as follows: Figure 1 B is shown in the figure.

[0059] Performance testing

[0060] 1. Material characterization using transmission electron microscopy (TEM)

[0061] Figure 2 These are transmission electron microscopy (TEM) images of different 2D ultrathin nanosheets, where A represents Cu-UMOFN, B represents Co-UMOFN, C represents Cu-UMOFNs@Au NPs, and D represents Co-UMOFNs@Au NPs. Figure 2 TEM images A and B show that the Cu-UMOFN and Co-UMOFN nanosheets are ultrathin and almost transparent. Figure 2 TEM images C and D show that after gold nanoparticles (Au NPs) are reduced in situ to form Cu-UMOFNs@Au NPs or Co-UMOFNs@Au NPs, Au NPs with a particle size of approximately 20 nm are uniformly distributed on the surface of the Cu-UMOFN or Co-UMOFN nanosheets. The formation of Cu-UMOFNs@AuNPs and Cu-UMOFNs@AuNPs not only improves the conductivity of metal-organic framework (MOF) materials, but also allows for the immobilization of biological antibodies via Au-NH2.

[0062] 2. The fabrication process of the electrochemical immunosensor was characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS).

[0063] Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used in a solution containing 0.1 mol / L KCl and 5 mmol / L [Fe(CN)6]. 3- / 4- The modification process of the immunosensor in solution was characterized, as follows: Figure 3 As shown in Figures A and B, where a is a bare glassy carbon electrode (GCE), b is Au@Fe3O4NPs / GCE, c is Ab1 / Au@Fe3O4NPs / GCE (Ab1: a mixed solution of anti-AFP and anti-CEA), d is BSA / Ab1 / Au@Fe3O4NPs / GCE, and e is Ag / BSA / Ab1 / Au@Fe3O4NPs / GCE (Ag: a mixed solution of AFP and CEA). Figure 3As shown in Figure A, when Au@Fe3O4 NPs are modified onto a bare glassy carbon electrode (GCE), an Au@Fe3O4 NPs-modified glassy carbon electrode (GCE) is formed, namely Au@Fe3O4NPs / GCE. The peak current value is significantly enhanced because the Au@Fe3O4 NPs composite material has excellent conductivity and electron transfer performance, leading to a significant increase in peak current. However, when Ab1 (a mixed solution of anti-AFP and anti-CEA), BSA, and a mixed solution of AFP and CEA antigens are sequentially immobilized on the surface of Au@Fe3O4NPs / GCE, the peak current gradually decreases. This is because the antibodies Ab1 (a mixed solution of anti-AFP and anti-CEA), BSA, and antigens (a mixed solution of AFP and CEA antigens) are all biological proteins that hinder electron transfer, thus preventing electrons from transferring from the solution to the electrode surface. Additionally, the EIS detection results corresponding to the CV figure are as follows... Figure 3 As shown in Figure B, the change in the Rct value is consistent with the change in the CV current, indicating that the present invention has indeed successfully constructed an immune sensor.

[0064] 3. To investigate whether the electrochemical signal in multiplex immunoassays partially originates from nonspecific interactions between the immunosensor and the nanotag using cyclic voltammetry (CV) and differential pulse voltammetry (DPV).

[0065] Figure 4 Figures A and B show the cyclic voltammetry (CV) and differential pulse voltammetry (DPV) characteristics of the chemiimmunoassay sensor in 0.1 M PBS buffer at pH 7.4, respectively (where a is a blank sample (zero analyte), b is 1 ng / mL CEA, c is 1 ng / mL AFP, and d is a mixture of 1 ng / mL AFP and 1 ng / mL CEA in PBS (pH 7.4) containing 4.0 mmol / L glucose). Figures C and D show the cyclic voltammetry (CV) and differential pulse voltammetry (DPV) responses in 5 mL of 0.1 mol / L PBS buffer at pH 7.4 with and without glucose (4 mmol / L), respectively. Figure 4 As can be seen from curves a in sections A and B, almost no redox peaks were observed in the zero analyte sample; however, the addition of CEA and AFP alone resulted in the observation of corresponding redox peaks (e.g., ...). Figure 4 (Curves c and d of A and B are shown in the figure); when the two target biomarkers CEA and AFP are added to the sample simultaneously, two pairs of redox peaks can be obtained simultaneously (e.g., curves c and d of A and B in the figure); Figure 4 (See curve d for A and B). This indicates that the measurable signal in the electrochemical sensor prepared in this invention does not originate from the non-specific adsorption of the nanotag, but rather from the specific interaction between the antigen and antibody.

[0066] To further evaluate the cross-reactivity and crosstalk between several immunoassays at adjacent sites, several control assays were performed on individual analytes (AFP or CEA) using the same immunosensor and nanotag. Each target analyte produced only one corresponding voltammetric peak, such as... Figure 4 Curves b and c in sections A and B are shown. Two voltammetric peaks are only produced when both target analytes (AFP and CEA) are present in the sample, with a potential difference of 250 mV (Epa) between the two pairs of oxidation peaks. Figure 4 (Curve d in A and B is shown in Figure 1). Therefore, the immunosensor can simultaneously detect two tumor markers based on the position of the corresponding oxidation peaks. In multiplex immunoassays, there is almost no crosstalk or cross-reactivity between the two analytes.

[0067] In addition, the dual catalytic amplification performance of the immune sensor was investigated using CV and DPV experiments. Figure 4 C and D represent the cyclic voltammetry (CV) and differential pulse voltammetry (DPV) responses in 5 mL of 0.1 mol / L PBS buffer solution at pH 7.4 with and without glucose (4 mmol / L), respectively. Both CV and DPV show corresponding peak current signals (curve a), indicating the successful establishment of the effective electroactivity of the Cu(Co)-UMOFNs@Au NP–Ab2–GOD bioconjugate. The two pairs of redox peaks in CV show a catalytic process; when 20 μL of 0.1 mol / L glucose is added to PBS, the cathode peak decreases while the anodic peak increases significantly. Furthermore, a significant increase in the peak current of DPV was observed, with the response current being more than three times higher than without glucose. These findings indicate that the constructed sandwich immunosensor possesses strong electrocatalytic activity towards glucose, and its sensitivity can be significantly improved by adding an appropriate amount of glucose to the test matrix. The electrochemical measurement and bioelectrocatalytic principle are as follows:

[0068] Glucose + GOD (ox) → gluconolacton + GOD (red) (1)

[0069] GOD (red) + O2 → GOD (ox) + H2O2 (2)

[0070] Cu (II)-UMOFNs + e → Cu (I)-UMOFNs (3)

[0071] Cu (Ⅰ)-UMOFNs + 2H2O2 → Cu (Ⅱ)-UMOFNs + O2 + 2H2O (4)

[0072] 4. Quantitative detection of AFP and CEA

[0073] DPV detection was performed on the sandwich electrochemical immunosensor prepared in Example 3, which used a chemical immunosensor combined with a hybrid probe formed by the redox probe ((Cu-UMOFNs@Au NP–Ab2–GOD) prepared in Example 1) and the redox probe ((Co-UMOFNs@Au NP–Ab2–GOD) prepared in Example 2). The results are as follows: Figure 5 As shown, A is the differential pulse spectrum of the sandwich electrochemical immunosensor prepared in Example 3 for the detection of AFP and CEA, and B is the calibration curve of the sandwich electrochemical immunosensor prepared in Example 3 for AFP and CEA. Figure 5 As shown in Figure A, the response signals of AFP and CEA are approximately 0.061V and 0.41V, respectively, and the signals do not interfere with each other. Furthermore, the current response value increases with increasing mixed antigen concentration. The results show that the peak current of the calibration curve exhibits a good linear relationship with the logarithm of the target concentration (e.g., ...). Figure 5 As shown in Figure B), the regression equation for AFP is I = 25.89logc AFP +122.28, R 2 =0.9958, the regression equation for CEA is I = 17.82logc CEA +118.16, R 2 =0.9958, where I is the current response signal in microamps (μA) and c is the concentration of AFP or CEA in ng / mL. The detection range for the target antigen AFP is 0.001–100 ng / mL, with a detection limit of 0.28 pg / mL; the detection range for the target antigen AFP is 0.001–80 ng / mL, with a detection limit of 0.31 pg / mL.

[0074] 5. Selectivity and stability testing of the immune sensor

[0075] The specificity of the sandwich electrochemical immunosensor prepared in Example 3 was evaluated using prostate-specific antigen (PSA), human immunoglobulin G (IgG), BSA, and ovarian cancer antigen (CA-125) as potential interfering substances. The results are as follows: Figure 6 As shown. From Figure 6 It can be seen that the sandwich electrochemical immunosensor prepared in Example 3 is most sensitive to CEA and AFP, with the highest current value, while the signals of other interfering substances are extremely weak, indicating that the immunosensor has good selectivity for the specific recognition of CEA and AFP in electrochemical detection.

[0076] A stability study was conducted to evaluate the stability of the sandwich electrochemical immunosensor prepared in Example 3. The specific method was as follows: the electrochemical immunosensor prepared in Example 3 was stored at 4°C and detected every 3 days. After 3 weeks, the response signals of the sandwich electrochemical immunosensor prepared in Example 3 to AFP and CEA were 89.6% and 88.2% of their initial values, respectively. These results indicate that the electrochemical immunosensor prepared in Example 2 has good stability.

[0077] 6. Immunosensors are used for the detection of clinical samples.

[0078] Serum samples were measured using both chemiluminescent immunoassay (CMIA) and the sandwich electrochemical immunosensor (multiplexed immunoassay (ng / mL)) prepared in Example 3 to explore the application effect of the immunosensor in clinical sample detection. The results, including accuracy and measurement bias, are shown in Table 1. Table 1 shows that there was no significant difference between the two methods compared to the clinical standard CMIA (relative error 10%). This indicates that the electrochemical immunosensor proposed in this invention has the potential for early detection of clinical biomarkers.

[0079] Table 1. Application of immunosensors in human serum samples

[0080]

[0081] In summary, this invention is the first to report a sandwich electrochemical immunosensor using MOFs as redox probes to accurately measure multiple tumor markers. The sandwich immunosensor of this invention can detect CEA or AFP individually, or simultaneously, exhibiting advantages such as a wide linear range, low detection limit, good selectivity, and good stability. The nanoscale ultrathin sheet-like MOF material (metal-UMOFNs@Au NPs) prepared in this invention can serve as an ideal platform for biomolecule immobilization in sandwich electrochemical adsorption sensors; metal-UMOFNs@Au NPs possess excellent catalytic activity, biocompatibility, and chemical stability; furthermore, they can be directly used as redox mediators, generating distinguishable electrical signals based on reversible redox reactions. This sandwich electrochemical immunosensor provides a novel "differential electrical signal strategy" for the clinical detection of multiple tumor markers and also offers a new concept for the development of multi-component immunosensors. Therefore, metal-UMOFNs@Au NPs can represent a new paradigm of multifunctional materials because they combine three different functional roles in a single material: catalyst, redox mediator, and loading platform. Furthermore, in our detection technique, an enzyme-assisted signal amplification mechanism is integrated into the detection process to improve the sensitivity of the immunosensor.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a redox probe based on metal-organic framework-derived nanosheets, characterized in that, The preparation method is as follows: (1) Preparation of metal-UMOFNs@Au NPs: After dispersing metal-UMOFNs in water, ascorbic acid was added and stirred to form a uniform colloidal suspension. After adding 1% chloroauric acid solution, stirring was continued to allow it to react fully. Then, centrifugation was performed. The precipitate after centrifugation was washed and dried to obtain metal-UMOFNs@Au NPs. (2) Preparation of biocoupled redox probe: Dissolve metal-UMOFNs@Au NPs in water to form a uniform suspension, add 10 ng / mL of alpha-fetoprotein antibody or carcinoembryonic antibody and stir overnight, add 1 mg / mL of glucose oxidase and incubate, centrifuge to obtain redox probe; The metal-UMOFNs mentioned in step (1) are Cu-UMOFNs or Co-UMOFNs, wherein the Cu-UMOFNs are prepared by the following method: Cu(NO3)2·3H2O is dissolved in a mixed solution of N,N-dimethylformamide and acetonitrile to form solution A. Phthalic acid is dissolved in a mixed solution of N,N-dimethylformamide and acetonitrile, and then polyvinylpyrrolidone is added to dissolve it to form solution B. Solution A and solution B are mixed and sonicated for 2~5 min. The mixture is heated at 135℃ for 24 h to form a precipitate. After centrifugation, washing and drying are performed to obtain Cu-UMOFN. The Co-UMOFNs are prepared as follows: phthalic acid is dissolved in a mixed solution of N,N-dimethylformamide, ethanol and water, and then CoCl2·6H2O is added to completely dissolve it. Triethylamine is then added, and the mixture is sonicated to form a colloidal suspension. The suspension is continuously sonicated in an autoclave for 8 hours, centrifuged, washed and dried to obtain Co-UMOFNs.

2. The preparation method according to claim 1, characterized in that, In the preparation of Cu-UMOFN, the mass-to-volume ratio of Cu(NO3)2·3H2O, N,N-dimethylformamide, and acetonitrile in solution A is 0.3:10:30 (g:mL:mL), and the mass-to-volume ratio of phthalic acid, N,N-dimethylformamide, acetonitrile, and polyvinylpyrrolidone in solution B is 0.3:30:10:0.9 (g:mL:mL:g). The volume ratio of solution A to solution B is 1:

1. The centrifugation speed is 10000~14000 rpm and the time is 5~8 min. The drying is carried out in air at 55~65℃.

3. The preparation method according to claim 1, characterized in that, In the preparation of Co-UMOFNs, the volume ratio of N,N-dimethylformamide, ethanol and water in the mixed solution is 32:2:2, and the molar volume ratio of phthalic acid, mixed solution, CoCl2·6H2O and triethylamine is 0.75:36:0.75:0.8 (mmol:mL:mmol:mL). The washing is performed with deionized water and ethanol, respectively.

4. The preparation method according to claim 1, characterized in that, In step (1), the mass ratio of the metal-UMOFNs to ascorbic acid is 10:50; the mass-volume ratio of the metal-UMOFNs to chloroauric acid solution is 10:1 (mg:mL); and the drying is carried out under vacuum conditions at 55~65℃. In step (2), the mass-volume ratio of the metal-UMOFNs@Au NPs, anti-AFP and glucose oxidase is 5:0.5:1, mg:mL:mL, and the incubation is specifically incubated at 4°C for 4 h.

5. A redox probe based on metal-organic framework-derived nanosheets prepared by the preparation method according to any one of claims 1 to 4.

6. The application of the redox probe based on metal-organic framework-derived nanosheets according to claim 5 in the preparation of electrochemical immunosensors.

7. A method for preparing a sandwich-type electrochemical immunosensor, characterized in that, The preparation method includes the following steps: (1) Preparation of immunosensor: Au@Fe3O4NPs were dispersed in Nafion solution to form a suspension, which was then drop-coated onto the surface of a glassy carbon electrode. After drying at room temperature, the suspension was placed in a mixed solution of alpha-fetoprotein antibody and carcinoembryonic antibody, both with a concentration of 10 ng / mL. After incubation at 4°C for 6 h, the mixture of alpha-fetoprotein antibody and carcinoembryonic antibody was immobilized by NH2-Au affinity, thus preparing an immunosensor. (2) Preparation of an immunosensor for incubating antigen: A 1 wt.% bovine serum albumin (BSA) solution was drop-coated onto the surface of the immunosensor prepared in step (1). After reacting at room temperature for 2 h to block non-specific binding sites, an antigen mixture formed by alpha-fetoprotein antigen and carcinoembryonic antigen was added and incubated at room temperature for 30 min to form an immunosensor for incubating antigen. (3) The redox probe based on copper-based organic framework nanosheets and the redox probe based on cobalt-based organic framework nanosheets as described in claim 6 are mixed in equal mass ratio to form a mixed probe. Then, the mixed probe is dissolved in a PBS buffer solution at pH 7.4 and drop-coated onto the surface of the immunosensor prepared in step (2) for incubation for 30 min to obtain a sandwich electrochemical immunosensor. After each step of the preparation method is completed, unreacted substances are removed by washing with PBS buffer solution at pH 7.4 and a concentration of 0.1 mol / L.

8. The sandwich electrochemical immunosensor prepared by the preparation method according to claim 7.

9. The application of the sandwich electrochemical immunosensor according to claim 8 in the preparation of products for detecting alpha-fetoprotein antigen and / or carcinoembryonic antigen.

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