Preparation method of enhanced electrochemiluminescence immunosensor

Abstract

The application discloses a preparation method of an enhanced electrochemiluminescence (ECL) immunosensor and belongs to the technical field of biosensors. The application is a sandwich type enhanced ECL immunosensor which is simple to prepare, excellent in performance and good in stability. The technical scheme is that two-dimensional RuMOF nanosheets directly integrated with a luminescent ligand are used as a sensing substrate and a light source, and MoS2 nanospheres with excellent planar electron transport and catalytic characteristics are used as an ECL signal enhancement catalyst, and a high-performance sensing interface is constructed through the synergistic coupling of the two. The application is simple to prepare, good in reproducibility and excellent in performance of the prepared finished product sensor.

Description

Technical Field

[0001] This invention belongs to the field of biosensing technology, and particularly relates to a method for preparing an enhanced electrochemiluminescence immunosensor. Background Technology

[0002] Currently, electrochemiluminescence (ECL) sensors are widely used in biomedical detection, and enhanced electrochemiluminescence (ECL) sensors are a key tool for improving the detection performance of disease biomarkers. However, in practical applications, the singularity of catalysts often restricts further breakthroughs in sensor performance, especially for high-sensitivity detection of complex biomarkers such as hepatitis B surface antigen (HBsAg), which places higher demands on the diversity of catalysts and their synergistic enhancement mechanisms. Currently, most research focuses on optimizing single catalyst systems, such as the ECL enhancement strategies based on noble metals or single metal oxides reported in patents CN1675539A and CN1918471. While these strategies can achieve some signal amplification, they often struggle to simultaneously achieve high catalytic activity, good electron transport characteristics, and tunable band structure, leading to bottlenecks in sensor sensitivity, detection limit, and stability. Furthermore, the limited interfacial coupling efficiency between traditional catalysts and luminescent materials also limits the full potential of ECL performance, resulting in limited improvement in sensor sensitivity and a high detection limit. In addition, the immobilization process of luminescent materials (such as ruthenium bipyridine) in traditional methods is complex and prone to leakage or deactivation, which affects the stability and reproducibility of the sensor. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing an enhanced electrochemiluminescence immunosensor.

[0004] To achieve the above-mentioned objective, this invention provides a method for preparing an enhanced electrochemiluminescence immunosensor, comprising the following steps: Preparation of S1 and 2D RuMOF nanosheets: A one-step hydrothermal or solvothermal method was used, with Ru(dcbpy)3²⁺ as the organic ligand, reacting with a metal salt in a reaction vessel under mild conditions (e.g., 40-100°C). After centrifugation, washing, and drying, two-dimensional RuMOF nanosheets were obtained. This method utilizes the direct coordination ability of the six carboxyl groups on Ru(dcbpy)3²⁺ to integrate the luminescent center in situ and firmly into the MOF framework. The bipyridine ruthenium derivative Ru(dcbpy)3²⁺ was used. 2+ As an organic ligand for MOFs, the six carboxyl groups in its structure directly participate in coordination, which not only simplifies the synthetic steps but also allows Ru(dcbpy)3 to be used as an organic ligand. 2+The inherent superior luminescence properties of RuMOF nanosheets were successfully constructed by in-situ integration into the MOF framework, resulting in highly efficient luminescence performance. This direct ligand integration strategy avoids the cumbersome subsequent luminescence labeling process, improves material stability and uniformity, and opens up new avenues for the design of high-performance ECL sensing substrates. Preparation of S2 and MoS2 nanosphere catalysts: MoS2 nanospheres with multi-level structures and rich edge active sites were synthesized by hydrothermal method using molybdenum source and sulfur source as precursors under high temperature and high pressure. Further utilization of MoS2 nanospheres Catalytic properties, coupled with 2D RuMOF nanosheets In this sandwich sensor structure, RuMOF serves as a high-intensity luminescent substrate, while MoS2 acts as a highly efficient ECL catalyst. The steric hindrance regulation effect of Mo atoms in MoS2, combined with the electronic properties of S atoms, significantly accelerates the electron transfer process on the RuMOF surface and effectively promotes the free radical reaction during luminescence, thereby achieving a significant enhancement of the ECL signal. Preparation of S3 and Ab2-MoS2 bioconjugates: MoS2 nanospheres were activated with glutaraldehyde to activate the carboxyl groups, and then coupled with the detection antibody (Ab2) via a Schiff base reaction to form Ab2-MoS2 bioconjugates. Non-specific sites were blocked with bovine serum albumin (BSA) for later use. S4. Construction of composite sensing interface: The 2D RuMOF nanosheet dispersion prepared in step S1 is drop-coated onto the pretreated and clean working electrode surface and dried to form a uniform film layer; then the MoS2 nanosphere dispersion prepared in step S2 is drop-coated onto the RuMOF film layer, and RuMOF / MoS2 composite electrode is formed by electrostatic adsorption or covalent bonding. S5. Immunorecognition interface assembly: On the RuMOF / MoS2 composite electrode obtained in step 4, the following operations are performed sequentially: a) Fixation of capture antibody: Use EDC / NHS to activate the carboxyl groups on the surface of the composite electrode, add capture antibody (Ab1) solution, and incubate to fix Ab1; b) Blocking: Add BSA solution and incubate to block non-specific binding sites on the electrode; c) Antigen binding: Add a sample solution containing the target antigen HBsAg and incubate to allow the target antigen HBsAg to specifically bind to Ab1; d) Signal probe binding: The Ab2-MoS2 bioconjugate solution prepared in step S3 is added dropwise and incubated to form a complete “electrode-RuMOF / MoS2-Ab1-antigen-Ab2-MoS2” sandwich structure.

[0005] Compared with the prior art, the beneficial effects of the present invention are: Innovative Material Design: This method pioneered the direct use of Ru(dcbpy)3²⁺, a substance with intrinsic luminescence properties, as a synthetic ligand for MOFs, achieving integration of luminescence function and framework structure. This approach avoids the problems of easy detachment and uneven distribution of luminescent molecules found in traditional physical mixing methods, thus improving the material's uniformity and luminescence stability.

[0006] Highly efficient synergistic enhancement mechanism: By constructing a RuMOF / MoS2 heterointerface, dual signal amplification is achieved. MoS2 not only acts as a highly efficient catalyst to promote the oxidation of co-reactants and generate more reaction intermediates, but its unique two-dimensional electronic structure can also significantly accelerate interfacial electron transfer, thereby synergistically enhancing the ECL luminescence intensity of RuMOF.

[0007] Excellent detection performance: The sensor built based on this technology exhibits an extremely wide linear range (1.0 × 10⁻¹) for the detection of HBsAg. 4 Up to 1.0×10⁻ 6 mg·mL⁻¹ and an extremely low limit of detection (3.7 × 10⁻¹). 5 The sensitivity is far superior to most ECL sensors based on traditional catalytic materials (such as gold nanoparticles and graphene), with a concentration of mg·mL⁻¹.

[0008] Simple and stable preparation: The entire material synthesis and sensor assembly process is characterized by mild conditions, clear steps, good reproducibility, ease of operation and large-scale preparation, and the resulting sensor has excellent repeatability and long-term stability. Attached Figure Description

[0009] Figure 1 This is a flowchart illustrating the structure and fabrication process of the enhanced electrochemiluminescence immunosensor of the present invention.

[0010] Figure 2 Transmission electron microscopy image of 2D RuMOF nanosheets prepared for the example.

[0011] Figure 3 Scanning electron microscope image of MoS2 nanospheres prepared for the example.

[0012] Figure 4 The image shows the ECL response curves of the sensor to different concentrations of HBsAg in the application example, along with the corresponding calibration curves. Detailed Implementation

[0013] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0014] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual images. They should not be construed as limiting the scope of this patent. To better illustrate the embodiments of the present invention, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual dimensions of the product. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0015] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," "inner," and "outer" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present patent. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0016] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0017] The structure of the enhanced electrochemiluminescence immunosensor prepared in this invention is as follows: Figure 1 As shown, its preparation process mainly includes the following steps: Synthesis of 2D RuMOF nanosheets: A certain amount of Ru(dcbpy)3²⁺ and zinc nitrate were dissolved in a mixed solvent of N,N-dimethylformamide (DMF) and water. After ultrasonic dissolution, the solution was transferred to a high-pressure reactor and reacted at 80°C for 24 hours. After natural cooling, the product was collected by centrifugation, washed several times with DMF and ethanol, and then vacuum dried to obtain 2D RuMOF nanosheets. The transmission electron microscopy image of the 2D RuMOF nanosheets prepared in this step is shown below. Figure 2 As shown.

[0018] Synthesis of MoS2 nanospheres: Sodium molybdate and thiourea were dissolved in deionized water, stirred until dissolved, and then transferred to a reaction vessel. The mixture was reacted at 200°C for 24 hours. After cooling, the mixture was centrifuged, washed, and dried to obtain MoS2 nanosphere powder. A scanning electron microscope image of the MoS2 nanospheres prepared in this step is shown below. Figure 3 As shown.

[0019] Preparation of the Ab2-MoS2 signal probe: 4 mg of MoS2 nanospheres were dispersed in 1 mL of PBS buffer (pH 7.4), and 0.25% glutaraldehyde was added. The mixture was activated at 4°C for 12 hours to complete carboxyl activation. The cross-linking agent was then removed, and the mixture was shaken overnight at room temperature. The activated MoS2 was then mixed with 1 mL of 1.0 × 10⁻⁶ PBS buffer. -6 mg·mL -1 Ab2 was mixed with MoS2 and an amide bond was formed via a Schiff base reaction, allowing Ab2 to bind to the MoS2 surface. The bioconjugate was collected by centrifugation and washed with PBS (pH=7.4) to remove unbound Ab2. The final product was stored at 4°C for later use. To reduce non-specific binding of the Ab2-MoS2 complex to other proteins, 100 µL of 1% bovine serum albumin was mixed with the complex and shaken at room temperature for 30 minutes. Subsequently, it was washed with PBS to remove unreacted BSA, and the final product was dispersed in 1 mL of PBS (pH=7.4) for later use. Through these steps, the Ab2-MoS2 complex was successfully prepared, laying the foundation for the subsequent construction and application of sensors.

[0020] Assembly of ECL immune sensor: 1. Polish the glassy carbon electrode sequentially with 0.3μm and 0.05μm alumina powder, then ultrasonically clean it in ethanol and water respectively, and dry it with nitrogen.

[0021] 2. Take 10 μL of a 1 mg mL⁻¹ 2D RuMOF nanosheet dispersion and drop it onto the electrode surface, then dry it at room temperature.

[0022] 3. Add 10 μL of a 0.5 mg mL⁻¹ MoS₂ nanosphere dispersion to the modified electrode and dry to form a RuMOF / MoS₂ composite interface.

[0023] 4. Add 20 μL of a 2:1 EDC / NHS mixed activation solution to the electrode to activate the carboxyl groups. Then add 20 μL of a 1.0 × 10⁻⁻⁻⁶ solution. 8 HBsAg capture antibody Ab1 was injected at mg·mL⁻¹ to form a 2D RuMOF-Ab1 bioconjugate. 10 μL of 1% BSA was added to the 2DRuMOF-Ab1 modified electrode, and the electrode was incubated at 37°C for 30 min to block non-specific binding sites. Excess BSA was then removed with PBS.

[0024] 5. Add 10 μL of 1% BSA solution and incubate at 37°C for 30 minutes to block nonspecific sites, then rinse with PBS.

[0025] 6. Add 20 μL of different concentrations (1.0 × 10⁻⁶) dropwise. -14 -1.0×10 -6 mg·mL -1 The HBsAg standard solution was incubated at 4°C for 12 hours to ensure specific binding of HBsAg to Ab1, and then unreacted HBsAg was removed with PBS.

[0026] 7. Add 10 μL of 1.0×10 -6 mg·mL -1 The Ab2-MoS2 bioconjugate was dropped onto the electrode, incubated at 4°C for 120 minutes, and then thoroughly rinsed with PBS solution at pH 7.4 to remove unbound material, thus obtaining the finished sensor, which was stored at 4°C.

[0027] Figure 4 The diagram shows the ECL of the enhanced electrochemiluminescence immunosensor prepared in this invention for different concentrations of HBsAg, as well as the electrochemiluminescence table, electrochemical impedance spectroscopy, and CV curves.

[0028] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing an enhanced electrochemiluminescence immunosensor, characterized in that, Includes the following steps: Preparation of S1 and 2D RuMOF nanosheets: Two-dimensional RuMOF nanosheets were obtained by reacting Ru(dcbpy)3²⁺ with metal salt in a reaction vessel using a one-step hydrothermal or solvothermal method. Preparation of S2 and MoS2 nanosphere catalysts: MoS2 nanospheres with multi-level structures and rich edge active sites were synthesized by hydrothermal method using molybdenum source and sulfur source as precursors under high temperature and high pressure. Preparation of S3 and Ab2-MoS2 bioconjugates: MoS2 nanospheres were activated with glutaraldehyde to activate the carboxyl groups, and then coupled with Ab2 through a Schiff base reaction to form Ab2-MoS2 bioconjugates. S4. Construction of composite sensing interface: The 2D RuMOF nanosheet dispersion prepared in step S1 is drop-coated onto the pretreated and clean working electrode surface to form a uniform RuMOF film. Subsequently, the MoS2 nanosphere dispersion prepared in step S2 was modified onto the RuMOF film layer by electrostatic adsorption or covalent bonding to form a RuMOF / MoS2 composite electrode. S5. Immunorecognition interface assembly: On the RuMOF / MoS2 composite electrode obtained in step 4, the following operations are performed sequentially: a) Immobilization of capture antibodies: The carboxyl groups on the surface of the RuMOF / MoS2 composite electrode were activated using EDC / NHS, and Ab1 solution was added dropwise to immobilize Ab1 and form 2D RuMOF-Ab1 bioconjugates; b) Blocking: Add BSA solution to block non-specific binding sites on the electrode; c) Antigen binding: Add a sample solution containing the target antigen HBsAg to allow the target antigen HBsAg to bind specifically to Ab1; d) Signal probe binding: The Ab2-MoS2 bioconjugate solution prepared in step S3 was added dropwise to complete the construction of MOF and MoS2-mediated enhanced ECL system.

2. The method for preparing an enhanced electrochemiluminescence immunosensor according to claim 1, characterized in that, The metal salt mentioned in step S1 is Zn 2+ or Zr 4+ The reaction conditions are a temperature of 40-100°C.

3. The method for preparing an enhanced electrochemiluminescence immunosensor according to claim 1 or 2, characterized in that, Step S3 further includes: mixing 100 µL of 1% BSA into the prepared Ab2-MoS2 bioconjugate and shaking for 30 minutes at room temperature.

4. The method for preparing an enhanced electrochemiluminescence immunosensor according to claim 1 or 2, characterized in that, The concentration of Ab2 is 1.0 × 10⁻⁶. -6 mg·mL -1 .

5. The method for preparing an enhanced electrochemiluminescence immunosensor according to claim 4, characterized in that, The process of activating the carboxyl group in step S3 includes: dispersing 4 mg MoS2 in 1 mL PBS solution (pH=7.4), adding 0.25% glutaraldehyde, and reacting at 4°C for 12 hours to complete the activation of the carboxyl group.

6. The method for preparing an enhanced electrochemiluminescence immunosensor according to claim 1 or 2, characterized in that, In step S5, the EDC / NHS concentration is 2:1, and the Ab1 concentration is 1.0 × 10⁻⁶. -8 mg·mL -1 .

7. The method for preparing an enhanced electrochemiluminescence immunosensor according to claim 1 or 2, characterized in that, In step S5, the concentration of HBsAg is 1.0 × 10⁻⁶. -14 -1.0×10 -6 mg·mL -1 .

8. The method for preparing an enhanced electrochemiluminescence immunosensor according to claim 1 or 2, characterized in that, In step S5, the concentration of the Ab2-MoS2 bioconjugate solution is 1.0 × 10⁻⁶. -14 -1.0×10 -6 mg·mL -1 The incubation conditions are: 4°C, 120 minutes.

9. The method for preparing an enhanced electrochemiluminescence immunosensor according to claim 8, characterized in that, In step S5, after incubation, the mixture is thoroughly rinsed with PBS solution at pH 7.4 to remove unbound material.

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