Molybdenum disulfide modified platinum electrode, preparation method thereof, electrochemiluminescence immunosensor comprising the electrode and application

Abstract

The application discloses a molybdenum disulfide modified platinum electrode, a preparation method of the electrode, an electrochemiluminescence immunosensor containing the electrode and application of the electrode, and the molybdenum disulfide modified platinum electrode comprises a conductive substrate and a molybdenum disulfide nano coating layer formed on the surface of the conductive substrate by in-situ growth through an atomic layer deposition process. The application grows MoS2 on the electrode substrate by the atomic layer deposition technology to form a firm coating layer which is chemically bonded, solves the problem that a physical adsorption layer formed by a traditional drop coating method is easy to fall off, and greatly improves the reuse frequency and long-term stability of the sensor. The in-situ grown MoS2 coating layer can fully play the advantages of high catalytic activity and high specific surface area, and significantly enhances the ECL signal; and the application combines the signal amplification effect of MoS2, the specific recognition of the sandwich immunoassay and the efficient enrichment of the magnetic separation technology, and can realize trace detection of AFP.

Description

Technical Field

[0001] This invention relates to the fields of analytical chemistry and electrochemiluminescence sensor technology, and particularly to a molybdenum disulfide modified electrode based on atomic layer deposition technology, an electrochemiluminescence immunosensor containing the electrode, and the application of the sensor in the detection of tumor markers, especially alpha-fetoprotein. Background Technology

[0002] Alpha-fetoprotein (AFP) is a key biomarker for various tumors, including hepatocellular carcinoma, and its serum levels are closely related to the occurrence and development of these diseases. Highly sensitive and specific detection of AFP is crucial for early diagnosis, treatment monitoring, and prognostic assessment of tumors.

[0003] Electrochemiluminescence immunoassay (ECL) combines the high sensitivity of chemiluminescence with the controllability of electrochemical processes, making it a powerful detection technique in the field of bioanalysis. Among its key aspects, interface modification of the working electrode is crucial for enhancing sensor performance. Introducing nanomaterials onto the electrode surface can effectively increase specific surface area, improve electron transfer rate, and enhance catalytic activity, thereby significantly amplifying the ECL signal.

[0004] Molybdenum disulfide (MoS2), a typical two-dimensional layered nanomaterial, has shown great potential in constructing high-performance ECL sensors due to its excellent catalytic properties, good biocompatibility, and low cost. Studies have shown that MoS2 can effectively catalyze ECL co-reactants (such as tripropylamine) to generate more active free radicals, thereby significantly enhancing the ECL emission of luminescent materials (such as ruthenium bipyridine complexes).

[0005] Currently, MoS2 modification of electrode surfaces is mostly achieved using a drop-coating method, which involves mixing pre-synthesized MoS2 nanomaterials with polymeric binders (such as Nafion, DMF, etc.) and then dropping the mixture onto the electrode surface, followed by drying to form a film. However, this method has the following drawbacks: (1) the physically adsorbed coating has weak adhesion to the electrode substrate and is prone to detachment during use, resulting in poor sensor stability and short lifespan; (2) the insulating binder used hinders electron / mass transport at the electrode interface, reducing catalytic efficiency; and (3) the thickness, uniformity, and repeatability of the modified layer are difficult to control precisely, making it unsuitable for large-scale, standardized sensor production. Therefore, developing a new method for MoS2 electrode modification that is firmly bonded, stable in performance, and reproducible is crucial for promoting the practical application of high-performance ECL sensors. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing a molybdenum disulfide modified platinum electrode, its preparation method, an electrochemiluminescence immunosensor containing the electrode, and its application.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: In the first aspect of the present invention, a molybdenum disulfide modified electrode is provided, comprising a conductive substrate and a molybdenum disulfide nano-coating (MoS2 coating) formed in situ on the surface of the conductive substrate by an atomic layer deposition process.

[0008] Preferably, the conductive substrate is a platinum electrode, a gold electrode, a glassy carbon electrode, or a silicon wafer; more preferably, the conductive substrate is a platinum electrode.

[0009] Preferably, the thickness and morphology of the molybdenum disulfide nanocoating are controlled by controlling the number of atomic layer deposition cycles, wherein the number of cycles is 20 to 200, more preferably 100.

[0010] A second aspect of the present invention provides a method for preparing a molybdenum disulfide modified electrode as described above, comprising the following steps: S1. Cleaning and plasma activation pretreatment of the conductive substrate; S2. The pretreated conductive substrate is placed in the atomic layer deposition reaction chamber. MoCl5 is used as the molybdenum source, H2S as the sulfur source, and nitrogen as the carrier gas and purge gas. MoCl5 and H2S are alternately pulsed and the atomic layer deposition reaction is carried out at 400-500℃. The number of atomic layer deposition cycles is controlled to be 20 to 200 times, and a molybdenum disulfide nano-coating is grown in situ on the substrate surface.

[0011] Preferably, step S1 specifically involves: ultrasonically cleaning the conductive substrate sequentially in acetone, ethanol, and deionized water, drying it with nitrogen gas, and then subjecting it to oxygen plasma treatment.

[0012] Preferably, the process also includes a post-processing step: sealing the deposited electrode with bovine serum albumin (BSA) solution to reduce non-specific adsorption.

[0013] Preferably, an atomic layer deposition cycle includes: a MoCl5 pulse for 0.5-3 seconds, followed by a nitrogen purging for 10-60 seconds; and an H2S pulse for 0.5-3 seconds, followed by a nitrogen purging for 10-60 seconds.

[0014] Preferably, the reaction temperature in step S2 is 450°C. One atomic layer deposition cycle consists of: a MoCl5 pulse for 1 second, followed by nitrogen purging for 30 seconds; an H2S pulse for 1 second, followed by nitrogen purging for 30 seconds.

[0015] A third aspect of the present invention provides an electrochemiluminescence immunosensor, the sensor comprising a molybdenum disulfide modified electrode as described above, the molybdenum disulfide modified electrode being used as a working electrode.

[0016] Preferably, the sensor further includes: (a) Capture probe containing a biotinylated first antibody (Bio-Ab1). (b) A detection probe containing a secondary antibody labeled with an electrochemiluminescent marker; (c) Magnetic separation element comprising streptavidin-modified magnetic beads (Dynabeads M-280). The sensor is used to separate and detect the target antigen by binding the magnetic separation element with the capture probe, the target antigen, and the detection probe to form a magnetic sandwich immune complex (specifically, a magnetic sandwich immune complex of "magnetic beads-biotin-first antibody-target antigen-second antibody-luminescent label").

[0017] Preferably, the electrochemiluminescent label is a ruthenium complex, the second antibody labeled by the electrochemiluminescent label is denoted as (Ru-Ab2), and the target antigen is a tumor marker.

[0018] Preferably, the tumor marker is alpha-fetoprotein (AFP), the capture probe is a biotinylated anti-alpha-fetoprotein first antibody, and the detection probe is a ruthenium complex-labeled anti-alpha-fetoprotein second antibody. The sensor performs detection in a detection system containing tripropylamine co-reactant. During the detection process, a voltage is applied to the surface of the molybdenum disulfide modified electrode to excite the electrochemiluminescent label to generate an electrochemiluminescent signal.

[0019] More specifically, when the target antigen (AFP) is present, it forms a magnetic sandwich immune complex with the capture probe and the detection probe: a "magnetic bead-biotin-Ab1-antigen-Ab2-ruthenium" complex. This complex can be enriched on the surface of a MoS2-modified electrode under an external magnetic field. After applying a voltage, the ruthenium complex reacts with the co-reactant (TPA) via an ECL reaction, generating a light signal.

[0020] Preferably, the method for detecting alpha-fetoprotein using the sensor includes the following steps: 1) Mix and incubate the sample to be tested, the biotinylated anti-alpha-fetoprotein primary antibody, and the ruthenium complex-labeled anti-alpha-fetoprotein secondary antibody; 2) Add streptavidin-modified magnetic beads to the mixture obtained in step 1) and incubate to form a magnetic sandwich immune complex; 3) The magnetic sandwich immune complex is separated from the product obtained in step 2) by magnetic separation, washed and transferred to the detection system containing the molybdenum disulfide modified electrode. A magnetic field is applied to enrich the magnetic sandwich immune complex on the surface of the molybdenum disulfide modified electrode. 4) In a detection system containing tripropylamine (TPA), an excitation voltage (e.g., 1.1-1.2 V relative to the Ag / AgCl electrode) is applied to the molybdenum disulfide modified electrode, and the intensity of the generated electrochemiluminescence is detected. 5) The alpha-fetoprotein content in the sample to be tested is calculated based on the pre-established standard curve characterizing the relationship between electrochemiluminescence intensity and alpha-fetoprotein concentration.

[0021] A fourth aspect of the present invention provides the application of the electrochemiluminescence immunosensor described above in the preparation of tumor marker detection kits or detection devices.

[0022] A fifth aspect of the invention provides an electrochemiluminescence immunoassay apparatus for detecting tumor markers, particularly AFP, the apparatus comprising a sensor as described above, as well as a measuring cell, a counter electrode, and a reference electrode.

[0023] The beneficial effects of this invention are: Compared with the prior art, the present invention has the following outstanding advantages and significant beneficial effects: 1. Significantly improved electrode stability: This invention uses atomic layer deposition technology to grow MoS2 in situ on the electrode substrate, forming a chemically bonded and robust coating. This solves the problem of easy detachment of the physical adsorption layer in the traditional drop coating method, and greatly improves the number of times the sensor can be reused and its long-term stability.

[0024] 2. Excellent electrochemical performance: This invention avoids the use of polymer binders, eliminating their obstruction to interfacial electron / mass transport; the in-situ grown MoS2 coating can fully utilize its high catalytic activity and high specific surface area advantages, significantly enhancing the ECL signal; experimental data show that the 100-MoS2 / Pt electrode of this invention has a 31.6% higher ECL intensity than the bare platinum electrode.

[0025] 3. High detection sensitivity: This invention combines the signal amplification effect of MoS2, the specific recognition of sandwich immunoassay, and the efficient enrichment of magnetic separation technology to achieve trace detection of AFP. Experimental results show that the detection limit of the sensor of this invention is as low as 14.75 pg / mL, and the linear range is as wide as 0.08-1000 ng / mL, which fully meets the needs of clinical diagnosis.

[0026] 4. Precise and controllable preparation method with good reproducibility: ALD technology has digital and self-limiting growth characteristics. This invention can achieve atomic-level precision control of the thickness and morphology of the MoS2 coating by precisely controlling the number of cycles, ensuring high consistency of electrode performance and production repeatability of different batches, which is conducive to large-scale manufacturing.

[0027] 5. Accurate and reliable detection results: In the spike recovery experiment in artificial serum samples and the comparison experiment with the clinical gold standard method (Roche cobas e411), the sensor of the present invention showed extremely high detection accuracy (correlation coefficient R²=0.995), proving its great application potential in the field of clinical testing. Attached Figure Description

[0028] Figure 1 This is a schematic diagram illustrating the detection principle of the electrochemiluminescence immunoassay sensor of the present invention; Figure 2 The images shown are scanning electron microscope (SEM) image (a) and atomic force microscope (AFM) image of the 100-MoS2 / Pt electrode in this invention. Figure 3 This is a comparison of the Raman spectra of MoS2 / Pt electrodes with different cycle numbers in this invention; Figure 4 The X-ray photoelectron spectroscopy full spectrum (a), Mo 3d high-resolution spectrum (b), and S 2p high-resolution spectrum (c) of the 100-MoS2 / Pt electrode in this invention are shown. Figure 5 The images show a comparison of cyclic voltammetry (a: potassium ferricyanide system; b: Ru(bpy)3²⁺ / TPA system) and electrochemiluminescence response (c: Vt curve; d: ECL intensity) for different electrodes in this invention. Figure 6 Electrochemiluminescence response results of pure platinum electrode and 100-MoS2 / Pt electrode in this invention to pure TPA solution and TPA solution containing 10 mM Ru(bpy)3²⁺: (a): Acquisition voltage graph; (b): Comparison graph of calculated luminescence intensity. Figure 7 This represents the optimized detection conditions in this invention; Figure 8 The following are the AFP detection results in this invention: (a) is the fitted standard curve, and (b) is the luminescence intensity diagram of multiple tests on blank samples; Figure 9 The results of the selectivity (a) and stability / repeatability (b) tests of the sensor in this invention are shown. Figure 10 The results of the correlation analysis between the sensor of this invention and the Roche cobas e411 system for detecting AFP concentration in artificial serum samples are shown. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0030] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. For examples where specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments whose manufacturers are not specified, they are all commercially available products.

[0032] This invention provides a molybdenum disulfide modified platinum electrode, an electrochemiluminescence immunosensor containing the electrode, and its application in the detection of tumor markers, particularly alpha-fetoprotein. Figure 1 The diagram illustrates the detection principle of this invention. In this invention, Dynabeads M-280, coupled with a biotinylated primary antibody, is used as a capture probe, and a ruthenium-labeled secondary antibody is used as a detection probe. In the presence of AFP, a magnetic sandwich immune complex is formed through a specific immune reaction. This complex can be effectively immobilized on the electrode surface under the action of an external magnetic field and rapidly removed after the magnetic field is removed. This design not only ensures good repeatability of the detection but also meets the instrument's requirements for high-throughput detection. After magnetic enrichment, the ruthenium complex in the immune complex generates an ECL signal at 620 nm under the applied potential, thereby achieving highly sensitive quantitative analysis of AFP.

[0033] The above is the general concept of the present invention. Detailed embodiments are provided below to further illustrate the present invention.

[0034] Material source description Bis(2,2'-bipyridine)(4'-methyl-[2,2']bipyridine-4-carboxylic acid)ruthenium dichloride, denoted as Ru-COOH, was purchased from Suna Tech Inc. (Suzhou, China). DMF, molybdenum pentachloride, potassium ferricyanide, and potassium ferrocyanide were purchased from Sinopharm Chemical Reagent Co., Ltd. Glycine and bovine serum albumin were purchased from Merck. N-hydroxysuccinimide (NHS), N,N'-dicyclohexylcarbodiimide (DCC), Sulfo-NHS-LC-LC-biotin, and Dynabeads M-280 were purchased from Thermo Fisher Scientific. Follicle-stimulating hormone (FSH), carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), prostate-specific antigen (PSA), and AFP were purchased from Fapon Biotech Co., Ltd. (Guangdong, China). Anti-AFP antibody pairs: coating antibody (Ab1) was 2A7, and labeling antibody (Ab2) was 2F8; purchased from Fapon Biotech Co., Ltd. (Guangdong, China). Artificial serum samples were all purchased from Braceds Biotech Co., Ltd. (Shenzhen, China).

[0035] Instrument Characterization Description The morphology of the samples was characterized using a ZEISS Sigma 300 scanning electron microscope, and the elemental distribution was evaluated using energy-dispersive X-ray spectroscopy (EDS-XPS). The MoS2 / Pt electrode was characterized using a LabRam HR Evolution confocal Raman spectrometer, with a spectral range of 320–480 nm. The surface morphology of the MoS2 / Pt electrode was characterized using a MicroNano D-5A atomic force microscope. X-ray photoelectron spectroscopy was performed using an EscaLab 250Xi spectrometer with Al Kα X-rays as the light source. Electrochemical measurements were performed using a CHI 660D electrochemical workstation. ECL measurements were performed using a custom-designed ECL detection system.

[0036] Example 1: Preparation of molybdenum disulfide modified electrode 1. Substrate pretreatment: Platinum electrode with dimensions of 20.5×5×0.2mm was ultrasonically cleaned in acetone, ethanol and deionized water for 20 minutes in sequence, and then dried with nitrogen gas; then oxygen plasma treatment was performed for 5 minutes to clean and activate the surface, resulting in a pretreated substrate.

[0037] 2. Atomic Layer Deposition (ALD): The pretreated substrate is placed in the ALD reaction chamber and heated to 450℃. MoCl5 is used as the molybdenum source (the source bottle is heated to 200℃), H2S is used as the sulfur source, and nitrogen is used as both the carrier gas and the purge gas. MoCl5 and H2S are alternately pulsed to carry out the ALD reaction. The ALD cycle program is as follows: MoCl5 pulse for 1 second, N2 purge for 30 seconds; H2S pulse for 1 second, N2 purge for 30 seconds; the ALD cycle is controlled to be 100 times to deposit a MoS2 coating on the platinum sheet surface. The resulting molybdenum disulfide modified electrode is denoted as the 100-MoS2 / Pt electrode.

[0038] 3. Sealing treatment: Immerse the 100-MoS2 / Pt electrode in a 5wt% BSA solution and seal it at 37℃ for 24 hours. After removal, gently rinse with deionized water and store at 4℃ for later use.

[0039] I. Characterization of molybdenum disulfide modified electrodes Figure 2 Here are scanning electron microscope (SEM) images (a) and atomic force microscope (AFM) images of the 100-MoS2 / Pt electrode in this invention; as shown in the image (b). Figure 2 Figure (a) shows a uniform and dense MoS2 deposit on a platinum substrate with a consistent thickness distribution. Furthermore, EDS analysis confirmed the uniform distribution of Mo and S elements throughout the material, consistent with the successful synthesis of the two-dimensional MoS2 structure. Atomic force microscopy images revealed a macroscopically uniform surface morphology over a large scanning area, with no obvious agglomerates or defect regions observed, indicating excellent macroscopic uniformity of the coating. Within a 10 μm × 10 μm scanning range, the average roughness of the film was 12.7 nm, and the root mean square roughness was 15.5 nm. Cross-sectional thickness analysis revealed that the deposited MoS2 film possesses a uniformly distributed nanosheet / particle-connected nanostructure, constituting an electrode / electrolyte interface formed by ordered stacked sheets.

[0040] Figure 3Raman spectra of molybdenum disulfide-modified electrodes obtained with different ALD cycles were compared (in the figure, black: 50-MoS2 / Pt; red: 100-MoS2 / Pt; blue: 200-MoS2 / Pt). For bulk MoS2, characteristic E¹2ᵍ and A1ᵍ modes were observed at 379.01 and 404.84 cm⁻¹, respectively. The intensity of the A1ᵍ mode was significantly stronger than that of the E¹2ᵍ mode. Furthermore, the frequency difference (Δ) between these two peaks was measured to be approximately 25.83. cm⁻¹. This value is in excellent agreement with reported data for bulk MoS₂ (typically Δ is around 25-26 cm⁻¹). The dominant Alᵍ intensity and large frequency difference together confirm a multilayer structure, typically consisting of five or more layers. Compared to 50-MoS₂ / Pt (50 ALD cycles), 100-MoS₂ / Pt (100 ALD cycles) and 200-MoS₂ / Pt (200 ALD cycles) exhibit significantly higher Raman intensities, corresponding to higher MoS₂ material loading on their surfaces.

[0041] Figure 4 The X-ray photoelectron spectroscopy (XPS) full spectrum (a), Mo 3d high-resolution spectrum (b), and S 2p high-resolution spectrum (c) of the 100-MoS2 / Pt electrode in this invention are shown below. Figure 4 As shown in Figure (b), the core level peaks of Mo 3d5 / 2 and Mo 3d3 / 2 are located at binding energies of 229.1 eV and 232.3 eV, respectively, which is consistent with the presence of Mo in 100-MoS2 / Pt. Figure 4 In Figure (c), the S 2p spectrum shows two distinct peaks at 162.1 eV and 163.1 eV, which are attributed to S 2p3 / 2 and S 2p1 / 2, respectively, further confirming the formation of 100-MoS2 / Pt.

[0042] II. Electrochemical characteristics of molybdenum disulfide modified electrodes A series of molybdenum disulfide modified electrodes were prepared using different ALD cycles, and their electrochemical performance was systematically evaluated by cyclic voltammetry. Figure 5 Different colored curves represent different ALD cycle counts; a cycle count of 0 represents a bare platinum electrode. For example... Figure 5 As shown in Figure (a), the bare platinum electrode is in Fe(CN)6³⁻ / 4⁻ Clear and symmetrical redox pairs were observed in the solution, indicating efficient electron transfer at the electrode surface. The redox peak current decreased after MoS2 modification. Notably, the 100-MoS2 / Pt electrode exhibited a current response comparable to the bare Pt electrode, but with significantly broadened peaks and a positive shift in oxidation potential. Although CV results indicate that MoS2 itself has relatively low conductivity, its excellent catalytic activity, high specific surface area, and rapid interfacial electron transfer capability effectively compensate for this limitation, thereby significantly improving the ECL efficiency of the electrode.

[0043] Subsequently, CV tests were performed in a TPA solution containing 1 μM Ru(bpy)3²⁺, such as... Figure 5 (Figure b) The results show that the oxidation peak current of the 100-MoS2 / Pt electrode at approximately 1.1 V is significantly higher than that of the bare Pt electrode and other MoS2-modified electrodes with different ALD cycles, indicating that the 100-MoS2 / Pt electrode possesses excellent electrocatalytic activity. These findings confirm the successful deposition of MoS2 onto the platinum surface and demonstrate that the obtained 100-MoS2 / Pt composite electrode exhibits significantly improved catalytic performance. Therefore, 100-MoS2 / Pt was selected for subsequent experiments.

[0044] To comprehensively evaluate the catalytic performance of the electrodes, ECL tests were performed on bare Pt and 100-MoS2 / Pt electrodes in TPA solutions with and without Ru(bpy)3²⁺. The voltage-time curves recorded by a logarithmic amplifier circuit and the corresponding ECL intensities obtained through signal processing together reveal the ECL performance of the electrodes. The results are as follows: Figure 6 As shown, in pure TPA solution, the two electrodes produce comparable signals. However, when 10 mM Ru(bpy)3²⁺ is added to the TPA solution, the 100-MoS2 / Pt electrode exhibits a significantly stronger voltage response, with the ECL intensity increasing by 31.6% compared to the bare Pt electrode. This result further confirms the excellent ECL efficiency of the 100-MoS2 / Pt electrode, consistent with conclusions drawn from earlier cyclic voltammetry data.

[0045] III. Optimization of Detection Conditions Given the decisive role of detection conditions in the performance of ECL biosensors, this invention systematically optimizes key experimental parameters. Optimization was performed using 2 ng / mL antigen samples, focusing on TPA concentration, TPA solution pH, incubation time, and the concentrations of key reagent kit components (R1 and R2). Experimental results are as follows: Figure 7 As shown.

[0046] As a co-reactant, the concentration of TPA directly affects the ECL response of the immunosensor. The ECL intensity initially increases and then decreases with increasing TPA concentration, reaching a peak at 60 mM. Therefore, 60 mM was selected as the optimal working concentration. Furthermore, the pH of the TPA solution plays a crucial role in determining the immunosensor response. The peak current gradually increases as the pH increases from 5.5 to 7.40, and then decreases with further increases in pH. Therefore, pH 7.40 was determined as the optimal value.

[0047] After adding Dynabeads M-280 to the biotinylated immune complex, the ECL signal was monitored in real time. The signal rose sharply within 3-9 minutes, continued to increase at a slower rate within 9-15 minutes, and then essentially reached a plateau. This indicates that efficient binding between the magnetic beads and the biotinylated immune complex is achieved within 15 minutes, and the reaction is nearing completion. Therefore, a 15-minute incubation time was chosen as the optimal condition to balance the requirements of detection performance and high-throughput detection.

[0048] To improve detection sensitivity, the concentrations of key reagent kit components were tested using low, medium, and high concentration sample systems. The results showed that when R1 was fixed at 0.8 μg / mL, the luminescence signal increased as the R2 concentration increased from 0.6 to 1.0 μg / mL. When R2 was fixed at 0.8 μg / mL, the signal decreased slightly with increasing R1 concentration, but the overall fluctuation was small, indicating good system stability. Simultaneous increases in both R1 and R2 resulted in a signal decrease, indicating that excess R1 was not completely captured by the magnetic beads. In conclusion, with the magnetic bead concentration fixed at 0.72 mg / mL, an R1 of 0.8 μg / mL and an R2 of 1.0 μg / mL were selected as the optimal ratio. Under these conditions, all sample concentrations, especially the medium and high values, exhibited the highest luminescence response.

[0049] Example 2: Construction and Detection of Electrochemiluminescence Immunosensor 1. Preparation of biological probes: Preparation of the capture probe: Sulfo-NHS-LC-LC-Biotin was added to the anti-AFP monoclonal antibody (Ab1) solution at a molar ratio of 20:1, shaken at room temperature for 1 hour, and purified by Zeba desalting column to obtain the capture probe: the first biotinylated antibody, denoted as biotinylated antibody Bio-Ab1 (abbreviated as Bio-Ab1).

[0050] Preparation of detection probe: Ru-COOH was activated with DCC / NHS (mass ratio 1:1.1) to generate Ru-NHS ester, which was then added to anti-AFP monoclonal antibody (Ab2) at a molar ratio of 50:1 (Ru:Ab2). The reaction was carried out in a water bath at 37°C for 2 hours. The reaction was terminated with glycine and purified to obtain the detection probe: a second antibody labeled with an electrochemiluminescent marker, denoted as ruthenium-labeled antibody Ru-Ab2 (abbreviated as Ru-Ab2).

[0051] 2. Alpha-fetoprotein (AFP) test: 1) Take 30 μL of AFP standard or serum sample of different concentrations, add 40 μL of Bio-Ab1 (R1 reagent, final concentration 0.8 μg / mL) and 40 μL of Ru-Ab2 (R2 reagent, final concentration 1.0 μg / mL) in sequence, and incubate in a constant temperature shaker at 37℃ for 9 minutes to form immune complexes.

[0052] 2) Add 30 μL of streptavidin-modified magnetic beads (Dynabeads M-280, 0.72 mg / mL) and continue incubation at 37°C for 15 minutes.

[0053] 3) Place the reaction tube on the magnetic separation rack, let it stand for 1 minute, discard the supernatant, and repeat the magnetic separation washing 3 times with washing buffer; 4) The magnetic sandwich immune complex obtained after final washing is resuspended in buffer solution and transferred entirely to the measurement cell. The magnetic sandwich immune complex is enriched on the surface of the 100-MoS2 / Pt electrode prepared in Example 1 by means of the bottom of the measurement cell or the built-in magnet. A detection base solution containing 60 mM TPA (pH 7.40) was injected into the measuring cell. A three-electrode system was used (working electrode: 100-MoS2 / Pt electrode; reference electrode: Ag / AgCl; counter electrode: platinum wire). A potential of 1.1 V was applied on the electrochemical workstation to trigger the ECL reaction. Signal Acquisition and Processing: The ECL signal is detected using a photomultiplier tube and converted into a voltage signal by a logarithmic amplifier circuit. The baseline voltage 0.4 seconds before the applied voltage is used as the noise (S). noise The voltage value 0.4 seconds after the voltage is applied is the signal (S). target ), calculate ECL strength (Q ECL =S target -S noise ).

[0054] 5) Results Analysis: A standard curve was obtained by fitting the logarithm of the AFP standard concentration to the x-axis and the measured ECL intensity to the y-axis using the Rodbard model, as shown below. Figure 8 As shown in Figure (a), the equation of the curve is: y = 9.76559 × 10 6+(1677.28-9.76559×10 6 The concentration of AFP can be calculated by substituting the ECL intensity of the sample into the standard curve. The results show that the linear range for AFP detection by this sensor is 0.08-1000 ng / mL, with a detection limit as low as 14.75 pg / mL.

[0055] Compared to existing methods, the sensor proposed in this invention exhibits a significant advantage in linear range. Although its detection limit is slightly higher than that of purely electrochemical methods, it remains among the leading ECL sensing platforms, fully meeting the application requirements for high-precision bioanalysis. This superior performance is primarily attributed to the integration of MoS2, which serves as a multifunctional platform. The MoS2 nanostructure enhances biocompatibility and catalytic activity, while its large specific surface area promotes more efficient electrochemical processes. Furthermore, the rigid matrix of MoS2 stabilizes the Ru(bpy)3²⁺ complex, resulting in higher quantum yield and longer emission lifetime.

[0056] Example 3 Sensor Performance Evaluation 1. Selectivity: AFP was replaced with common interfering substances (CEA, FSH, CA19-9, PSA) at high concentrations (100 ng / mL) and detected according to the method in Example 2. Results are as follows: Figure 9 As shown in (a), it can be seen that the ECL signal generated by the interfering object is not significantly different from the blank background signal, indicating that the sensor of the present invention has extremely high specificity for AFP.

[0057] 2. Stability and repeatability: Three different batches of 100-MoS2 / Pt electrodes were used, each electrode being used to repeatedly detect a medium concentration of AFP (10 ng / mL) in 10 replicates. Results are as follows: Figure 9 As shown in (b), the RSD of 10 measurements for each electrode is less than 5%; the RSD of the measurements between the three electrodes is 3.79%, indicating that the sensor has excellent operational stability and fabrication reproducibility.

[0058] 3. Clinical Relevance: Using the sensor of this invention and the Roche cobas e411 immunoassay analyzer (the industry gold standard), 20 artificial serum samples with different concentrations of AFP were tested in parallel. Figure 10 As shown, the detection results of the two methods exhibit a very high linear correlation (R²=0.995), confirming the high accuracy and application potential of the sensor of the present invention in clinical sample detection.

[0059] The test results above show that, in this invention, by controlling the deposition cycle number using ALD, MoS2 coatings of different thicknesses can be prepared on the surface of a platinum electrode. This MoS2 coating, formed through in-situ growth, is firmly bonded to the substrate and exhibits good stability, thus meeting the requirements for reusability and high-throughput detection. Electrodes modified with different cycle numbers were systematically evaluated using CV, and the optimal modified electrode (100-MoS2 / Pt) was selected. Based on this electrode, an electrochemical immunosensing platform for large-scale screening and diagnosis of tumor markers can be constructed. In the detection system of this invention, streptavidin-modified magnetic beads are used to capture the target antigen, forming a sandwich-type immune complex, which significantly enhances the specificity and signal intensity of the detection. Compared with the bare platinum electrode, the 100-MoS2 / Pt modified electrode shows a 31.6% increase in ECL intensity under the same conditions, resulting in a higher signal-to-noise ratio and better detection sensitivity, with a detection limit as low as 14.75 pg / mL. Furthermore, when applied to artificial serum samples, the sensor showed a strong correlation with the detection results of the clinically mature Roche cobas e411 immunoassay analyzer, demonstrating promising clinical application prospects.

[0060] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A molybdenum disulfide modified electrode, characterized in that, It includes a conductive substrate and a molybdenum disulfide nanocoating formed on the surface of the conductive substrate by in-situ growth through an atomic layer deposition process.

2. The molybdenum disulfide modified electrode according to claim 1, characterized in that, The conductive substrate is a platinum electrode, a gold electrode, a glassy carbon electrode, or a silicon wafer. The thickness and morphology of the molybdenum disulfide nanocoating are controlled by adjusting the number of atomic layer deposition cycles, which ranges from 20 to 200.

3. A method for preparing a molybdenum disulfide modified electrode as described in claim 1 or 2, characterized in that, Includes the following steps: S1. Cleaning and plasma activation pretreatment of the conductive substrate; S2. The pretreated conductive substrate is placed in the atomic layer deposition reaction chamber. MoCl5 is used as the molybdenum source, H2S as the sulfur source, and nitrogen as the carrier gas and purge gas. MoCl5 and H2S are alternately pulsed and the atomic layer deposition reaction is carried out at 400-500℃. The number of atomic layer deposition cycles is controlled to be 20 to 200 times, and a molybdenum disulfide nano-coating is grown in situ on the substrate surface.

4. The method for preparing the molybdenum disulfide modified electrode according to claim 3, characterized in that, One atomic layer deposition cycle consists of: MoCl5 pulse for 0.5–3 seconds, followed by nitrogen purging for 10–60 seconds; H2S pulse for 0.5–3 seconds, followed by nitrogen purging for 10–60 seconds.

5. An electrochemiluminescence immunosensor, characterized in that, The sensor includes a molybdenum disulfide modified electrode as described in claim 1 or 2, wherein the molybdenum disulfide modified electrode is used as the working electrode.

6. The sensor according to claim 5, characterized in that, The sensor also includes: (a) A capture probe containing a biotinylated first antibody; (b) A detection probe containing a secondary antibody labeled with an electrochemiluminescent marker; (c) A magnetic separation element comprising streptavidin-modified magnetic beads; The sensor is used to separate and detect the target antigen by binding the magnetic separation element with the capture probe, the target antigen, and the detection probe to form a magnetic sandwich immune complex.

7. The sensor according to claim 6, characterized in that, in, The electrochemiluminescent label is a ruthenium complex, and the target antigen is a tumor marker.

8. The sensor according to claim 7, characterized in that, The tumor marker is alpha-fetoprotein (AFP), the capture probe is a biotinylated anti-AFP first antibody, and the detection probe is a ruthenium complex-labeled anti-AFP second antibody. The sensor performs detection in a detection system containing tripropylamine co-reactant. During the detection process, a voltage is applied to the surface of the molybdenum disulfide modified electrode to excite the electrochemiluminescent label to generate an electrochemiluminescent signal.

9. The sensor according to claim 8, characterized in that, The method for detecting alpha-fetoprotein using the aforementioned sensor includes the following steps: 1) Mix and incubate the sample to be tested, the biotinylated anti-alpha-fetoprotein primary antibody, and the ruthenium complex-labeled anti-alpha-fetoprotein secondary antibody; 2) Add streptavidin-modified magnetic beads to the mixture obtained in step 1) and incubate to form a magnetic sandwich immune complex; 3) The magnetic sandwich immune complex is separated from the product obtained in step 2) by magnetic separation, washed and transferred to the detection system containing the molybdenum disulfide modified electrode as described in claim 1 or 2. A magnetic field is applied to enrich the magnetic sandwich immune complex on the surface of the molybdenum disulfide modified electrode. 4) In a detection system containing tripropylamine, an excitation voltage is applied to a molybdenum disulfide modified electrode, and the intensity of the generated electrochemiluminescence is detected; 5) The alpha-fetoprotein content in the sample to be tested is calculated based on the pre-established standard curve characterizing the relationship between electrochemiluminescence intensity and alpha-fetoprotein concentration.

10. The use of an electrochemiluminescence immunosensor according to any one of claims 5-9 in the preparation of a tumor marker detection kit or detection device.

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