A low-trigger-potential electrochemiluminescence immunosensor, its preparation method, and its application.

Abstract

This invention discloses a low-trigger-potential electrochemiluminescence immunosensor, its preparation method, and its application, relating to the field of biodetection. It includes a three-electrode system comprising a working electrode, a reference electrode, and a counter electrode. The working electrode comprises: a conductive substrate; a vertically ordered mesoporous silica film formed on the surface of the conductive substrate, the vertically ordered mesoporous silica film having nanochannels; gold-copper alloy nanoparticles formed within the nanochannels; an epoxy-modified layer formed on the outer surface of the vertically ordered mesoporous silica film; a placental growth factor-specific antibody covalently bound to the epoxy-modified layer; and bovine serum albumin covalently bound to regions of the epoxy-modified layer not bound by the placental growth factor-specific antibody. This invention has the advantages of achieving efficient electrochemiluminescence at low potentials and improving detection sensitivity and stability.

Description

Technical Field

[0001] This invention relates to the field of biological detection, specifically to a low-trigger-potential electrochemiluminescence immunosensor, its preparation method, and its application. Background Technology

[0002] Placental growth factor (PlGF) is a crucial factor secreted by placental trophoblasts that regulates angiogenesis and reflects the health of placental development and function. This factor plays an irreplaceable role in perinatal medicine, including preeclampsia screening and diagnosis of fetal growth restriction. Currently, commonly used clinical methods for detecting placental growth factor include enzyme-linked immunosorbent assay (ELISA), flow cytometry, and radioimmunoassay. However, these traditional methods generally suffer from limitations such as high equipment costs, cumbersome procedures, long testing cycles, and high skill requirements for laboratory personnel, making them unsuitable for meeting the practical needs of real-time clinical testing and large-scale screening.

[0003] Electrochemiluminescence (ECL), a novel detection method combining electrochemistry and chemiluminescence, has shown broad application prospects in the field of biosensing due to its advantages such as low background signal, convenient operation, and wide linear response range. Luminol, a commonly used electrochemiluminescent agent, possesses advantages such as low toxicity, high quantum yield, and good water solubility. However, efficient electrochemiluminescence in conventional luminol-hydrogen peroxide systems typically requires relatively negative potentials (e.g., -1V). These high-potential conditions not only easily induce electrochemical interference but may also cause damage to the structure of biorecognition molecules, thereby affecting the sensitivity and stability of detection.

[0004] To address these issues, researchers have attempted to develop various materials to lower the electrochemiluminescence trigger potential of the luminol-hydrogen peroxide system. Existing technologies have reported electrochemiluminescence immunosensors that confine gold-palladium alloy nanoparticles within silica films, primarily for tumor marker detection. Another approach utilizes gold-palladium alloy nanoparticles and glucose oxidase to construct an electrochemiluminescent enzyme sensor for glucose detection. However, none of these technologies address the detection of placental growth factors, and existing low-potential electrochemiluminescence sensors for the luminol-hydrogen peroxide system suffer from low detection sensitivity and poor structural stability. Summary of the Invention

[0005] This invention aims to address one of the technical problems in related technologies to a certain extent. To this end, this invention provides a low-trigger-potential electrochemiluminescence immunosensor, its preparation method, and its application, which has the advantages of achieving efficient electrochemiluminescence at low potentials and improving detection sensitivity and stability.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A low-trigger-potential electrochemiluminescence immunosensor includes a three-electrode system, comprising a working electrode, a reference electrode, and a counter electrode. The working electrode includes: Conductive substrate; A vertically ordered mesoporous silica film is formed on the surface of the conductive substrate, and the vertically ordered mesoporous silica film has nanochannels. Gold-copper alloy nanoparticles are formed within the nanochannels; An epoxy-modified layer is formed on the outer surface of the vertically ordered mesoporous silica film; Placental growth factor-specific antibodies, which are covalently bound to the epoxy-modified layer; Bovine serum albumin, which is covalently bound to the region of the epoxy-modified layer that is not bound by the placental growth factor-specific antibody.

[0007] The low-trigger-potential electrochemiluminescence immunosensor proposed in this application significantly reduces the electrochemiluminescence trigger potential by confining gold-copper alloy nanoparticles within the nanochannels of a vertically ordered mesoporous silica film, acting as a co-reactant promoter in the luminol-hydrogen peroxide system, thus avoiding damage to biorecognition molecules. Combined with covalent immobilization of placental growth factor-specific antibodies and blocking with bovine serum albumin, it achieves ultrasensitive and highly selective quantitative detection of placental growth factors in biological samples, effectively solving the problems of high cost, complex operation, and insufficient sensitivity of existing detection methods.

[0008] Optionally, the molar ratio of gold to copper in the gold-copper alloy nanoparticles is 1:5 to 1:1.

[0009] Optionally, the conductive substrate is selected from one of indium tin oxide electrode, glassy carbon electrode, fluorine-doped tin oxide electrode, gold electrode, screen-printed electrode, graphite electrode, or carbon fiber electrode.

[0010] Furthermore, the present invention also provides a method for preparing a low-trigger-potential electrochemiluminescence immunosensor, wherein the low-trigger-potential electrochemiluminescence immunosensor includes the aforementioned low-trigger-potential electrochemiluminescence immunosensor, and the preparation method includes the following steps: S1, A vertically ordered mesoporous silica film is formed on the surface of a conductive substrate to obtain a modified electrode, wherein the vertically ordered mesoporous silica film has nanochannels. S2, gold-copper alloy nanoparticles are formed within the nanochannels; S3, an epoxy-modified layer is formed on the outer surface of the vertically ordered mesoporous silica film; S4, covalently bind placental growth factor-specific antibodies to the epoxy-modified layer; S5, covalently bind bovine serum albumin to the region of the epoxy-modified layer that is not bound by the placental growth factor-specific antibody.

[0011] Optionally, step S2 includes: immersing the modified electrode in an electrodeposition solution containing a gold precursor and a copper precursor, and applying a potential of -1.0 to 0V to the modified electrode to generate and confine the gold-copper alloy nanoparticles in situ within the nanochannel; the gold precursor is selected from at least one of chloroauric acid, gold nitrate, or a gold complex; the copper precursor is selected from at least one of copper chloride, copper sulfate, or a copper complex; the concentration of the gold precursor in the electrodeposition solution is 0.01 to 1 mmol / L, and the concentration of the copper precursor is 0.05 to 1 mmol / L.

[0012] Optionally, step S3 includes: immersing the modified electrode in an epoxy silane solution and reacting it in the dark for 0.2 to 5 hours; wherein the epoxy silane is selected from at least one of γ-glycidoxypropyltrimethoxysilane, 3-(2,3-epoxypropoxy)propyltrimethoxysilane or 2-(3,4-epoxycyclohexyl)ethyltriethoxysilane, and the concentration of the epoxy silane solution is 2.0 to 3.0 mM.

[0013] Optionally, step S4 includes: immersing the modified electrode in a buffer solution containing placental growth factor-specific antibodies and incubating it at a temperature of 0–5°C for 30–90 min, so that the placental growth factor-specific antibodies are covalently bound to the epoxy-modified layer; the concentration of the buffer solution is 1–10 μg / mL, and the pH of the buffer solution is 6.5–7.5.

[0014] Optionally, step S5 includes: immersing the modified electrode obtained in step S4 in a bovine serum albumin solution for 5 to 20 minutes, so that bovine serum albumin covalently binds to the region on the epoxy-modified layer that is not bound by the placental growth factor-specific antibody; the mass concentration of the bovine serum albumin solution is 0.2% to 2%.

[0015] Furthermore, this invention also provides an application of a low-trigger-potential electrochemiluminescence immunosensor, which, using the aforementioned low-trigger-potential electrochemiluminescence immunosensor, includes the following steps: S10, the working electrode of the low trigger potential electrochemiluminescence immunosensor is brought into contact with and incubated with the test sample containing placental growth factor, so that the placental growth factor in the test sample binds to the placental growth factor specific antibody. S20, the combined working electrode, reference electrode and counter electrode are placed in a detection solution containing luminol and hydrogen peroxide; S30, perform electrochemical scanning on the detection solution within the potential range of -0.5 to 0V to detect the electrochemiluminescence signal; S40, based on the detected electrochemiluminescence signal intensity, determine the concentration of placental growth factor in the sample to be tested.

[0016] Optionally, the detection linear range of the low trigger potential electrochemiluminescence immunoassay sensor is 10 fg / mL to 10 ng / mL, and the detection limit is not higher than 5.4 fg / mL; when the concentration of each interfering substance in the sample to be tested does not exceed 1 mM and the total concentration does not exceed 0.5 M, it is determined that the quantitative detection of placental growth factor is not affected by the interfering substances.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) For the first time, gold-copper alloy nanoparticles were applied to a low-trigger potential electrochemiluminescence system of luminol-hydrogen peroxide. The gold-copper alloy nanoparticles have different electronic structures and catalytic mechanisms, and exhibit excellent synergistic catalytic effects at low potentials. They can efficiently catalyze hydrogen peroxide and dissolved oxygen to produce ROS at ultra-low potentials of -0.5 to 0V, significantly enhancing the electrochemiluminescence signal while reducing damage to biorecognition molecules.

[0018] (2) By confining gold-copper alloy nanoparticles within nanochannels, the stability and dispersibility of the nanoparticles are improved, and aggregation and inactivation are avoided. Compared with single metal confinement, the electrochemiluminescence stability is significantly increased, and the electrochemiluminescence signal is significantly enhanced. At the same time, the size sieving, electrostatic repulsion, and hydrophilicity of the vertically ordered mesoporous silica film are utilized to block interference from macromolecular pollutants in complex samples such as serum, thereby improving the sensor's anti-contamination ability and stability.

[0019] (3) The first low-trigger potential electrochemiluminescence immunosensor for placental growth factor was constructed, which achieved ultra-high sensitivity detection of placental growth factor with a detection limit as low as 5.4 fg / mL and a linear range of 10 fg / mL to 10 ng / mL. The sensitivity is improved by 2 to 3 orders of magnitude compared with the existing methods, providing a new technical means for perinatal medical applications such as preeclampsia screening.

[0020] (4) The sensor is simple to manufacture and low in cost. It uses the electrodeposition method to synthesize gold-copper alloy nanoparticles in situ without the need for complex equipment. The entire detection process does not require complex sample pretreatment and is easy to operate, making it suitable for clinical point-of-care testing applications.

[0021] (5) The sensor has good selectivity, reproducibility and stability, and has strong anti-interference ability against interference such as glucose, inorganic salts and common cytokines. The electrochemiluminescence signal remains stable after 8 consecutive uses, and the signal retention rate is >87% after 5 days of storage at 4℃.

[0022] These features and advantages of the present invention will be disclosed in detail in the following specific embodiments and accompanying drawings. The preferred embodiments or means of the present invention will be shown in detail in conjunction with the accompanying drawings, but are not intended to limit the technical solutions of the present invention. In addition, each of these features, elements and components appearing in the following text and drawings is a plurality of, and different symbols or numbers are used for convenience of representation, but all represent parts with the same or similar construction or function. Attached Figure Description

[0023] The present invention will be further described below with reference to the accompanying drawings: Figure 1 This is a schematic diagram showing the statistical results of the electrochemiluminescence intensity of different modified electrodes in the preparation example in a phosphate buffer solution containing luminol and hydrogen peroxide; Figure 2 This is a schematic diagram showing the statistical results of the cyclic voltammetric responses of different modified electrodes in sulfuric acid or phosphate buffered solution (PBS) in the preparation example; Figure 3 A schematic diagram illustrating the statistical results for verifying the catalytic mechanism of the AuCu NPs@VMSF / ITO electrode in the preparation example; Figure 4 This is a schematic diagram showing the statistical results of the electrochemiluminescence and electrochemical performance characterization of different electrodes in the preparation example; Figure 5 A schematic diagram showing the comparison of electrochemiluminescence intensity of electrodes prepared with precursor solutions of different gold-copper ratios in the preparation example, and the statistical results of placental growth factor antibody and its incubation time. Figure 6 The images show the ECL intensity and linear relationship of the BSA / AbPlGF / AuCu NPs@O-VMSFNF / ITO electrode after incubation with different concentrations of PlGF in the preparation example. Figure 7 This is a schematic diagram showing the experimental results of the selectivity, repeatability, and stability of the BSA / Ab / AuCu NPs@O-VMSF / ITO electrode in the preparation example. Detailed Implementation

[0024] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described are intended to explain the present invention and should not be construed as limiting the invention.

[0025] The terms "an embodiment," "example," or "trademark" used in this specification refer to a particular feature, structure, or characteristic described in connection with the embodiment itself that may be included in at least one embodiment disclosed in this patent. The phrase "in an embodiment" appearing in various places throughout the specification does not necessarily refer to the same embodiment.

[0026] Example: Existing methods for detecting placental growth factors, such as enzyme-linked immunosorbent assay (ELISA), flow cytometry, and radioimmunoassay, suffer from drawbacks including high cost, complex operation, and high skill requirements for laboratory personnel. Furthermore, the luminol electrochemiluminescence system requires a relatively negative emission potential, which may lead to electrochemical interference or damage to biorecognition molecules, reducing detection sensitivity. While existing technologies utilize nanomaterials to lower the electrochemiluminescence trigger potential, these primarily employ AuPd alloy systems and do not address the detection of placental growth factors. Research on the use of gold-copper alloys for low-potential electrochemiluminescence in luminol-hydrogen peroxide systems has not been reported.

[0027] In response, this application proposes a low-trigger-potential electrochemiluminescence immunosensor, comprising a three-electrode system, which includes a working electrode, a reference electrode, and a counter electrode. The working electrode includes: Conductive substrate; A vertically ordered mesoporous silica film is formed on the surface of the conductive substrate, and the vertically ordered mesoporous silica film has nanochannels. Gold-copper alloy nanoparticles are formed within this nanochannel; An epoxy-modified layer is formed on the outer surface of the vertically ordered mesoporous silica film; Placental growth factor-specific antibodies are covalently bound to the epoxy-modified layer; Bovine serum albumin is covalently bound to regions of the epoxy-modified layer that are not bound by the placental growth factor-specific antibody.

[0028] The low-trigger-potential electrochemiluminescence immunosensor in this embodiment is a device that utilizes the principle of electrochemiluminescence to excite a luminescent signal at a low potential and detects specific biomolecules through an immune recognition mechanism. This sensor is typically used for the quantitative detection of target analytes in biological samples. The three-electrode system, in electrochemical measurements, consists of a working electrode, a reference electrode, and a counter electrode. The working electrode is the site of the electrochemical reaction, the reference electrode provides a stable potential reference, and the counter electrode provides the current loop. The conductive substrate is the basic material providing the conductive platform for the electrochemiluminescence immunosensor. This substrate can effectively transport electrons, thereby supporting subsequent electrochemical reactions. The vertically ordered mesoporous silica film is a silica film with highly ordered nanochannels. This film is typically formed on the surface of a conductive substrate through a self-assembly method. Its nanochannels can be used to confine nanoparticles and provide a large specific surface area and good biocompatibility. Gold-copper alloy nanoparticles refer to nano-sized alloy particles composed of gold and copper elements. These nanoparticles act as co-reactant promoters in the electrochemiluminescence system, catalyzing the generation of reactive oxygen species from hydrogen peroxide and dissolved oxygen, thereby enhancing the electrochemiluminescence signal of luminol and achieving low trigger potential luminescence. The epoxy-modified layer refers to a chemically modified layer containing epoxy groups introduced onto the outer surface of a vertically ordered mesoporous silica film. The epoxy-modified layer can provide active sites and immobilize biorecognition molecules, such as antibodies, through covalent bonds. Placental growth factor-specific antibodies are antibody molecules that can specifically recognize and bind to placental growth factor (PlGF). This antibody, as a biorecognition element, is used to capture placental growth factor in the sample. Bovine serum albumin is a commonly used biomolecule used to block areas on the surface of the immunosensor that are not bound by specific antibodies. By blocking non-specific sites, the adsorption of non-specific antibodies can be effectively reduced, improving the selectivity and sensitivity of the sensor.

[0029] Furthermore, the low trigger potential electrochemiluminescence immunosensor employs a three-electrode system for electrochemical measurements. The working electrode surface is modified with various functional materials. The reference electrode is typically a silver / silver chloride electrode or a saturated calomel electrode, providing a stable potential reference. The counter electrode is typically a platinum wire electrode or a graphite electrode, providing a current loop to ensure the smooth progress of the electrochemical reaction.

[0030] Specifically, the working electrode includes a conductive substrate. The conductive substrate can be made of various materials with good conductivity. For example, a glassy carbon electrode can be used, which has good conductivity and chemical stability. A gold electrode can also be used, as its surface is easy to modify and it has excellent conductivity. Alternatively, a screen-printed electrode can be used, which has relatively low preparation cost and is suitable for large-scale production. Based on this, a vertically ordered mesoporous silica film with nanochannels is formed on the surface of the conductive substrate. The formation of the vertically ordered mesoporous silica film can be achieved through various methods. For example, the sol-gel method can be used, controlling the hydrolysis and condensation process of the precursor to form a silica film with a mesoporous structure on the surface of the conductive substrate. Evaporation-induced self-assembly can also be used, driving the self-assembly of surfactants and silicon source precursors through solvent evaporation to form an ordered mesoporous structure. These methods can all prepare silica films with vertically aligned nanochannels, which will not be elaborated further here. Gold-copper alloy nanoparticles can be formed in situ within the nanochannels of the vertically ordered mesoporous silica film. For example, a conductive substrate modified with a silica film is immersed in a solution containing gold and copper salt precursors. Gold and copper ions are co-reduced within nanochannels to form alloy nanoparticles via chemical reduction or electrochemical deposition. In the chemical reduction method, reducing agents such as sodium borohydride or ascorbic acid can be added. In the electrochemical deposition method, metal ions are deposited within the nanochannels by applying an appropriate potential to form a gold-copper alloy. Furthermore, an epoxy-modified layer is formed on the outer surface of this vertically ordered mesoporous silica film. The epoxy-modified layer can be formed on the outer surface of the vertically ordered mesoporous silica film using a silane coupling agent. For example, a silica film electrode modified with gold-copper alloy nanoparticles is immersed in a solution containing epoxy-containing silanes. The silane coupling agent reacts with the hydroxyl groups on the silica surface, thereby introducing epoxy groups onto the outer surface of the film. This reaction is typically carried out under light-protected conditions to ensure the activity of the epoxy groups. Placental growth factor-specific antibodies are covalently bound to this epoxy-modified layer. Placental growth factor-specific antibodies can be covalently immobilized on an epoxy-modified layer. For example, an electrode containing an epoxy-modified layer is immersed in a buffer solution containing placental growth factor-specific antibodies. The epoxy groups undergo a ring-opening reaction with the amino or hydroxyl groups on the antibody molecule, forming stable covalent bonds. This binding process is typically incubated at low temperature for a period of time to ensure antibody activity and binding efficiency. Finally, bovine serum albumin (BSA) is covalently bound to regions on the epoxy-modified layer not bound by the placental growth factor-specific antibody to block the epoxy groups on the electrode surface that are not bound by the antibody. For example, after antibody binding is complete, the electrode is incubated in a bovine serum albumin solution.BSA molecules react with the remaining epoxy groups, occupying non-specific binding sites, thereby effectively reducing the non-specific adsorption of other non-target molecules in the sample onto the electrode surface, and improving the specificity and signal-to-noise ratio of the sensor.

[0031] The molar ratio of gold to copper in gold-copper alloy nanoparticles is 1:5 to 1:1.

[0032] In this embodiment, the relative content of gold and copper in the gold-copper alloy nanoparticles is defined. The molar ratio measures how the proportion of alloy components directly affects the crystal structure, electronic properties, distribution of surface active sites, and catalytic efficiency of electrochemical reactions. When the molar ratio of gold to copper is controlled within the range of 1:5 to 1:1, the alloy nanoparticles can form specific alloy phases or solid solution structures, thereby optimizing their catalytic performance in electrochemiluminescence reactions. For example, copper can provide more active sites, while gold helps improve the stability and conductivity of the alloy. By controlling the molar ratio, the catalytic oxidation ability of gold-copper alloy nanoparticles on electrochemiluminescent reagents such as luminol can be modulated, thus affecting the intensity and trigger potential of the electrochemiluminescence signal. Specifically, this molar ratio can be controlled by adjusting parameters such as the concentration ratio of gold and copper precursors in the electrodeposition solution, electrodeposition voltage, current density, or deposition time.

[0033] In summary, the electrochemiluminescence immunosensor utilizes gold-copper alloy nanoparticles as a key component; therefore, the composition ratio of these nanoparticles significantly impacts the sensor's electrochemiluminescence performance, catalytic activity, and stability. An inappropriate composition ratio may lead to insufficient electrochemiluminescence signal intensity, excessively high trigger potential, or poor stability, thus affecting the sensor's overall detection efficiency. By precisely controlling the molar ratio of gold to copper in the gold-copper alloy nanoparticles within the range of 1:5 to 1:1, the catalytic activity of the nanoparticles in the electrochemiluminescence reaction can be optimized, effectively promoting the oxidation of electrochemiluminescent reagents such as luminol and generating a stronger and more stable electrochemiluminescence signal. Simultaneously, optimizing this molar ratio also helps to reduce the trigger potential of the electrochemiluminescence reaction.

[0034] The conductive substrate is selected from one of the following: indium tin oxide electrode, glassy carbon electrode, fluorine-doped tin oxide electrode, gold electrode, screen-printed electrode, graphite electrode, or carbon fiber electrode.

[0035] In this embodiment, the conductive substrate can be an indium tin oxide (ITO) electrode, which possesses excellent conductivity and optical transparency, and is commonly used in electrochemical sensors requiring photoelectric coupling or transparent substrates. Its surface is easily chemically modified. Alternatively, the conductive substrate can be a glassy carbon electrode, a commonly used inert electrode material with excellent conductivity and good chemical stability, suitable for various electrochemical analyses. Furthermore, the conductive substrate can be a fluorine-doped tin oxide (FTO) electrode, which combines good conductivity and optical transparency with relatively low cost, high chemical stability, and mechanical strength, making it a good alternative to ITO electrodes. In other embodiments, the conductive substrate can be a gold electrode, which possesses excellent conductivity, chemical stability, and biocompatibility. Its surface is easily functionalized through thiol chemistry to achieve biomolecule immobilization. Simultaneously, the conductive substrate can also be a screen-printed electrode, which has advantages such as low cost and ease of integration, suitable for portable or on-site rapid detection devices. Moreover, the conductive substrate can be a graphite electrode, which has good conductivity, a large specific surface area, and low cost. Its surface can be activated through oxidation or other methods to enhance its bonding with the modified layer. In addition, the conductive substrate can also be a carbon fiber electrode, which has high conductivity, good mechanical strength and biocompatibility, making it suitable for miniaturization and in vivo detection applications.

[0036] The conductive substrates selected in this application all possess excellent conductivity and chemical stability, providing stable electron transport channels for the electrochemiluminescence reaction. The surface properties of these substrate materials also facilitate the subsequent formation of vertically ordered mesoporous silica films and the immobilization of functional components such as gold-copper alloy nanoparticles, epoxy-modified layers, placental growth factor-specific antibodies, and bovine serum albumin. This solves problems such as weak electrochemiluminescence signals, high background noise, or shortened sensor lifespan caused by inappropriate selection of conductive substrates, thereby improving the sensor's sensitivity, stability, and detection accuracy.

[0037] This embodiment also proposes a method for fabricating a low-trigger-potential electrochemiluminescence immunosensor, which includes the following steps: First, a vertically ordered mesoporous silica film is formed on the surface of a conductive substrate to obtain a modified electrode, wherein the vertically ordered mesoporous silica film has nanochannels; second, gold-copper alloy nanoparticles are formed within the nanochannels; next, an epoxy-modified layer is formed on the outer surface of the vertically ordered mesoporous silica film; subsequently, a placental growth factor-specific antibody is covalently bound to the epoxy-modified layer; finally, bovine serum albumin is covalently bound to the region of the epoxy-modified layer that is not bound by the placental growth factor-specific antibody.

[0038] In this embodiment, a vertically ordered mesoporous silica film is first formed on the surface of a conductive substrate to obtain a modified electrode. The vertically ordered mesoporous silica film has nanochannels to construct the nanostructure framework of the sensor. The conductive substrate can be selected from one of the following: indium tin oxide electrode, glassy carbon electrode, fluorine-doped tin oxide electrode, gold electrode, screen-printed electrode, graphite electrode, or carbon fiber electrode, to provide good conductivity and mechanical support. The formation of the vertically ordered mesoporous silica film is usually achieved by a template method, for example, using a surfactant as a structure directing agent to form an ordered mesoporous structure on the surface of the conductive substrate through a sol-gel process. Subsequently, the template is removed by calcination or other methods, thereby forming a large number of vertically arranged nanochannels in the film. The nanochannels not only increase the surface area of ​​the electrode but also provide a regular space for the confined growth of subsequent functional materials, ensuring the uniform distribution and efficient utilization of active sites.

[0039] Secondly, gold-copper alloy nanoparticles are formed within nanochannels to introduce electrochemiluminescence active centers into the sensor. These nanoparticles are key components for achieving low-trigger-potential electrochemiluminescence signals, and their formation can be achieved through methods including, but not limited to, electrochemical deposition, chemical reduction, or photoreduction. For example, by immersing a modified electrode in a solution containing gold and copper precursors, gold and copper ions are reduced and co-deposited in situ within the nanochannels under specific electrochemical conditions, forming alloy nanoparticles with a specific molar ratio of gold to copper. The confinement effect of the nanochannels helps control the size and morphology of the nanoparticles, preventing their aggregation and thus maintaining their high specific surface area and excellent electrochemical activity.

[0040] Next, an epoxy-modified layer is formed on the outer surface of a vertically ordered mesoporous silica film to provide active sites for the covalent coupling of biomolecules. The epoxy-modified layer is typically achieved through a silanization reaction, in which the modified electrode is immersed in a solution containing epoxy-based silanes. One end of the epoxy-based silane molecule (such as an alkoxysilane group) can undergo a condensation reaction with the hydroxyl groups on the silica film surface to form a covalent bond, while the other end (the epoxy group) is exposed. As a highly reactive chemical group, it can undergo ring-opening reactions with the amino, hydroxyl, or thiol groups of proteins (such as antibodies and bovine serum albumin) to form covalent bonds, ensuring the robustness and directionality of subsequent biomolecule immobilization.

[0041] Subsequently, placental growth factor-specific antibodies were covalently bound to the epoxy group-modified layer to achieve the sensor's specific recognition function. By immersing the modified electrode in a buffer solution containing placental growth factor-specific antibodies, the active groups such as amino groups on the antibody molecules undergo a covalent coupling reaction with the epoxy groups on the epoxy group-modified layer. This covalent binding method is more stable than physical adsorption, preventing antibody detachment during detection and ensuring the long-term stability and accuracy of the sensor. Simultaneously, the abundant active sites provided by the epoxy group-modified layer enable high-density antibody immobilization, improving the sensor's capture efficiency.

[0042] Finally, bovine serum albumin (BSA) was covalently bound to areas on the epoxy-modified layer that were not bound by placental growth factor-specific antibodies, thus blocking remaining non-specific binding sites on the electrode surface. After immobilization with placental growth factor-specific antibodies, unreacted epoxy groups or other exposed areas may still exist on the electrode surface. These areas may adsorb non-target proteins from the sample, leading to increased background signal and decreased detection specificity. By introducing BSA, an inert protein, it can covalently bind to or physically adsorb onto these remaining active sites, thereby blocking non-specific binding sites, significantly reducing background interference, and improving the sensor's signal-to-noise ratio and detection accuracy.

[0043] The specific steps for forming gold-copper alloy nanoparticles within nanochannels include immersing a micelle-free modified electrode in an electrodeposition solution containing gold and copper precursors, applying a potential of -1.0 to 0V to the modified electrode, and continuously electrodepositing for 1 to 10 seconds to allow the gold-copper alloy nanoparticles to be generated and confined in situ within the nanochannels.

[0044] In this embodiment, the modified electrode refers to an electrode on which a vertically ordered mesoporous silica film has been formed on the surface of a conductive substrate. The modified electrode is immersed in an electrodeposition solution containing a gold precursor and a copper precursor. The gold precursor can be at least one of chloroauric acid, gold nitrate, or a gold complex, which provides gold ions in solution. The copper precursor can be at least one of copper chloride, copper sulfate, or a copper complex to provide copper ions. These precursors ensure good reduction activity and solubility under electrodeposition conditions, thereby providing a sufficient source of metal ions for the formation of the gold-copper alloy.

[0045] During electrodeposition, a potential of -1.0 to 0 V is applied to the modified electrode to ensure that gold and copper ions are simultaneously reduced, thereby forming a gold-copper alloy within the nanochannel. By controlling the potential, the co-deposition process of gold and copper can be regulated, promoting alloy formation and avoiding unnecessary side reactions. In-situ formation refers to the formation of gold-copper alloy nanoparticles directly within the nanochannel through electrochemical reactions, rather than through external introduction. Confinement refers to the confinement of nanoparticles within the geometry of the nanochannel to control their size, morphology, and prevent aggregation.

[0046] The preferred concentrations of the gold precursor and the copper precursor in the electrodeposition solution are 0.01–1 mmol / L and 0.05–1 mmol / L, respectively, to ensure moderate deposition rates of gold and copper under given potential conditions. This results in the formation of gold-copper alloy nanoparticles with good composition and morphology, which are then uniformly distributed within the nanochannels. The in-situ generated and confined gold-copper alloy nanoparticles serve as highly efficient electrocatalysts, enhancing the catalytic efficiency and signal intensity of the electrochemiluminescence immunosensor. This leads to lower trigger potentials and higher detection sensitivity, addressing the issue of insufficient control over nanoparticle formation and providing a foundation for the accurate detection of placental growth factors.

[0047] The step of forming an epoxy group modification layer on the outer surface of a vertically ordered mesoporous silica film includes immersing the modified electrode in an epoxy silane solution for 0.2–5 hours in the dark. In this step, the modified electrode refers to an electrode on which the formation of a vertically ordered mesoporous silica film on the conductive substrate surface and the in-situ generation and confinement of gold-copper alloy nanoparticles within the nanochannels have been completed. The vertically ordered mesoporous silica film on its surface has abundant hydroxyl groups, which can undergo coupling reactions with epoxy silanes. The epoxy silane solution is used to introduce epoxy groups onto the surface of the modified electrode. These epoxy groups have high reactivity and can covalently couple with the amino or hydroxyl groups on the placental growth factor-specific antibody in subsequent steps, thereby achieving stable antibody immobilization. The light-protected operation prevents unnecessary polymerization, degradation, or oxidation reactions of the epoxy silane under light conditions, thus ensuring the integrity and activity of the epoxy groups and improving the quality and stability of the modification layer. Meanwhile, controlling the reaction time within the range of 0.2 to 5 hours ensures that the epoxy silanes are fully coupled to form a uniform and dense epoxy-modified layer, avoiding insufficient modification due to too short a reaction time or excessive polymerization due to too long a reaction time.

[0048] The epoxy silane is selected from at least one of γ-glycidoxypropyltrimethoxysilane, 3-(2,3-epoxypropoxy)propyltrimethoxysilane, or 2-(3,4-epoxycyclohexyl)ethyltriethoxysilane. These epoxy silane molecules all contain epoxy and silane groups. The silane groups can form stable siloxane bonds with hydroxyl groups on the surface of vertically ordered mesoporous silica films through hydrolysis and condensation reactions, thereby immobilizing the epoxy groups on the electrode surface. The epoxy groups serve as active sites for covalent binding with subsequent antibodies. These epoxy silanes provide high reactivity, good stability, and suitable steric hindrance to optimize antibody immobilization. Furthermore, the concentration of the epoxy silane solution is limited to 2.0–3.0 mM (mM is an abbreviation for millimoles per liter). Within this concentration range, it is ensured that the epoxy silane molecules form a uniform, dense, and sufficiently active modified layer on the electrode surface, avoiding incomplete modification due to too low a concentration or molecular stacking due to too high a concentration.

[0049] In summary, by selecting specific epoxy silanes and strictly controlling their solution concentration, reaction time, and light-protection conditions, a highly uniform, dense modification layer with sufficient active epoxy groups can be formed on the outer surface of a vertically ordered mesoporous silica film. This provides an ideal interface for the covalent binding of placental growth factor-specific antibodies, improving antibody immobilization efficiency and stability while minimizing non-specific adsorption. This achieves the goal of enhancing the sensitivity, specificity, and long-term stability of the low-trigger-potential electrochemiluminescence immunosensor, ultimately making the detection of placental growth factors more accurate and reliable.

[0050] Step S4 includes: immersing the modified electrode in a buffer solution containing placental growth factor-specific antibodies and incubating it at a temperature of 0–5°C for 30–90 min, so that the placental growth factor-specific antibodies are covalently bound to the epoxy-modified layer; the concentration of the buffer solution is 1–10 μg / mL and the pH of the buffer solution is 6.5–7.5.

[0051] In this embodiment, the modified electrode is immersed in a buffer solution containing placental growth factor-specific antibodies for incubation to promote the covalent binding reaction between the antibody and the epoxy group-modified layer. The incubation process ensures that the antibody molecules can fully contact the electrode surface and chemically bond with the epoxy groups. The buffer solution maintains a stable pH in the reaction system, providing a suitable chemical environment for the antibody, thereby ensuring its biological activity and binding efficiency. The incubation temperature is set in a low-temperature range of 0–5°C to inhibit non-specific adsorption and degradation of the placental growth factor-specific antibody. At lower temperatures, the thermal motion of molecules decreases, which helps reduce random adsorption of the antibody in non-target regions and delays antibody inactivation, thereby maximizing the preservation of the antibody's biological activity and structural integrity, ensuring its efficient covalent binding with the epoxy group-modified layer. An incubation time of 30–90 minutes ensures sufficient binding of the placental growth factor-specific antibody and avoids the potential decrease in antibody activity or increase in non-specific adsorption that may result from prolonged incubation. Within this timeframe, the antibody and the epoxy-modified layer can form a sufficient number of stable covalent bonds, achieving the ideal binding density. This provides ample binding sites for subsequent immune reactions, ensuring the sensor's detection performance. The concentration of placental growth factor-specific antibody in the buffer solution is set at 1–10 μg / mL to optimize the antibody binding density on the electrode surface. Too low an antibody concentration may result in insufficient binding sites on the electrode surface, affecting sensor sensitivity; while too high a concentration may cause antibody molecules to accumulate on the electrode surface, increasing steric hindrance and even inducing non-specific adsorption, thereby reducing the sensor's specificity and detection performance. Therefore, this concentration range is beneficial for achieving uniform antibody coverage on the electrode surface, ensuring optimal immune recognition efficiency. Furthermore, in this embodiment, the pH of the buffer solution is controlled within a weakly acidic to neutral range of 6.5–7.5 to maintain the conformational stability and biological activity of the placental growth factor-specific antibody. Under these pH conditions, the active groups on the antibody molecules (such as amino and carboxyl groups) can exist in an ionized state, thereby promoting efficient covalent coupling reactions between them and the epoxy groups on the epoxy-modified layer. At the same time, this pH range avoids antibody denaturation and inactivation under extremely acidic or alkaline conditions, ensuring the functional integrity of the antibody during the fixation process.

[0052] Step S5 includes: immersing the modified electrode obtained in step S4 in a bovine serum albumin solution for 5 to 20 minutes, so that bovine serum albumin is covalently bound to the region on the epoxy-modified layer that is not bound by the placental growth factor-specific antibody; the mass concentration of the bovine serum albumin solution is 0.2% to 2%.

[0053] In this embodiment, the modified electrode treated in step S4 is immersed in a bovine serum albumin solution for incubation to block the active sites on the epoxy-modified layer that are not occupied by placental growth factor-specific antibodies. Bovine serum albumin (BSA), a widely used biological blocking agent, contains abundant amino and carboxyl functional groups in its molecular structure. These groups can covalently react with the remaining epoxy groups on the epoxy-modified layer to form stable chemical bonds, thereby covering and passivating potential non-specific binding sites. This blocking treatment reduces the non-specific adsorption of non-target substances from the sample onto the sensor surface during subsequent detection, thus reducing background signal, improving the sensor's signal-to-noise ratio, and ensuring the accuracy and specificity of the detection results. Setting the incubation time within the range of 5 to 20 minutes ensures that the bovine serum albumin molecules have sufficient time to fully react with the active sites on the epoxy-modified layer, forming stable covalent bonds, thereby achieving the desired blocking effect. This avoids the potential for excessive adsorption of bovine serum albumin (BSA) on the electrode surface or the formation of an excessively thick nonspecific layer due to prolonged incubation, thus preventing interference with the binding of the target analyte (i.e., placental growth factor) to the specific antibody. Within this time range, an optimal balance can be achieved between blocking efficiency and overall sensor performance. Maintaining the BSA solution concentration within the range of 0.2% to 2% provides a sufficient number of BSA molecules to saturate all unoccupied active sites on the epoxy-modified layer, ensuring complete blocking of the active sites. Simultaneously, it avoids the formation of an excessively thick physical adsorption layer of BSA on the electrode surface due to excessively high concentrations.

[0054] This application also proposes an application of a low-trigger-potential electrochemiluminescence immunosensor for detecting the concentration of placental growth factor in a sample, the method comprising the following steps: S10, the working electrode of the low trigger potential electrochemiluminescence immunosensor is brought into contact with the test sample containing placental growth factor and incubated, so that the placental growth factor in the test sample binds to the placental growth factor specific antibody. S20, the combined working electrode, reference electrode and counter electrode are placed in a detection solution containing luminol and hydrogen peroxide; S30, perform electrochemical scanning on the detection solution within the potential range of -0.5 to 0V to detect the electrochemiluminescence signal; S40, based on the detected electrochemiluminescence signal intensity, determine the concentration of placental growth factor in the sample to be tested.

[0055] In this embodiment, step S10 enables the specific binding between placental growth factor in the sample to be tested and placental growth factor-specific antibodies immobilized on the surface of the sensor's working electrode. The sensor's working electrode is immersed in a solution containing the sample to be tested, or the sample is allowed to flow across the electrode surface via microfluidics. The incubation process is typically carried out at a specific temperature to ensure sufficient antigen-antibody reaction, thereby achieving effective capture of the target analyte and ensuring that the subsequent signal originates from the target analyte (i.e., placental growth factor). Step S20 is used to construct the environment for generating the electrochemiluminescence signal. After the antigen-antibody binding is completed, the working electrode, reference electrode, and counter electrode are placed together in a detection solution containing luminol and hydrogen peroxide. Luminol, as an electrochemiluminescence substrate, reacts with hydrogen peroxide after being oxidized on the electrode surface to generate a luminescent signal. The reference electrode provides a stable potential reference, and the counter electrode provides a current loop. Together, they constitute a three-electrode system, providing precise control for the electrochemical reaction. In step S30, this step is the excitation and detection process of the electrochemiluminescence signal. Electrochemical scanning of the detection solution is performed within a potential range of -0.5 to 0 V using equipment such as an electrochemical workstation. This potential range oxidizes luminol and triggers its reaction with hydrogen peroxide, thereby generating electrochemiluminescence. Electrochemical scanning can employ cyclic voltammetry, square wave voltammetry, or potentiostatic methods. By monitoring changes in luminescence intensity with potential or time, the electrochemiluminescence signal related to the analyte concentration is obtained. Step S40 is a quantitative analysis based on the detected electrochemiluminescence signal intensity. In practical applications, a series of placental growth factor standard samples with known concentrations are typically prepared in advance, and their corresponding electrochemiluminescence signal intensities are obtained using the same detection method, thus establishing a standard curve. By comparing the detected electrochemiluminescence signal intensity of the test sample with this standard curve, the concentration of placental growth factor in the test sample can be accurately determined.

[0056] In summary, this application provides an operable detection procedure for placental growth factors using the above-described detection method. First, placental growth factor-specific antibodies immobilized on the working electrode surface achieve specific capture of placental growth factors in the sample, ensuring detection accuracy. Subsequently, in a detection solution containing luminol and hydrogen peroxide, electrochemical scanning within a low trigger potential range efficiently excites the electrochemiluminescence signal. This low trigger potential operation not only reduces background interference and improves the signal-to-noise ratio but also helps protect the stability of bioactive substances. Finally, by quantitatively analyzing the intensity of the electrochemiluminescence signal, the concentration of placental growth factors in the sample can be accurately and sensitively determined. This method fully utilizes the low trigger potential characteristics of the sensor, providing a reliable and convenient detection method for the clinical diagnosis and research of placental growth factors.

[0057] The detection linear range of the low trigger potential electrochemiluminescence immunosensor is 10 fg / mL to 10 ng / mL, and the detection limit is not higher than 5.4 fg / mL. When the concentration of each interfering substance in the sample does not exceed 1 mM and the total concentration does not exceed 0.5 M, the quantitative detection of placental growth factor is not affected by the interfering substances.

[0058] In this embodiment, the detection linear range refers to the range within which the sensor can accurately quantify the concentration of placental growth factor. By precisely controlling the loading and distribution of gold-copper alloy nanoparticles within the nanochannels, and the fixed density of placental growth factor-specific antibodies, sufficient signal response is ensured at low concentrations, while the signal is not saturated at high concentrations, ultimately achieving this broad linear range. The detection limit refers to the lowest concentration of placental growth factor that the sensor can reliably detect. This ultra-low detection limit is achieved by optimizing the catalytic activity of the gold-copper alloy nanoparticles, improving the binding efficiency of placental growth factor-specific antibodies, and effectively blocking non-specific adsorption (e.g., via bovine serum albumin) to reduce background signal.

[0059] When the concentrations of each interfering substance in the sample do not exceed 1 mM and the total concentration does not exceed 0.5 M, the quantitative detection of placental growth factor is unaffected by the interfering substances, demonstrating the sensor's specificity and anti-interference capability for placental growth factor detection in complex biological samples. This performance relies on the highly selective recognition capability of placental growth factor-specific antibodies, ensuring that they bind only to the target analyte. Simultaneously, the synergistic effect of the epoxy-modified layer and bovine serum albumin passivates the areas of the electrode surface not occupied by the antibody, minimizing non-specific adsorption and thus suppressing the influence of other proteins, ions, or small molecules in the sample on the electrochemiluminescence signal.

[0060] In summary, the low-trigger-potential electrochemiluminescence immunoassay sensor of this application achieves an extremely low detection limit and a wide linear detection range when detecting placental growth factors. Specifically, by optimizing components such as the conductive substrate of the working electrode, the vertically ordered mesoporous silica film, the gold-copper alloy nanoparticles, the epoxy-modified layer, the placental growth factor-specific antibody, and bovine serum albumin, the sensor can generate a sufficiently strong signal for reliable detection even at low concentrations, while maintaining a good linear relationship at high concentrations, thus covering a wide concentration range required for clinical diagnosis. Furthermore, the specific binding of the antibody and the blocking effect of bovine serum albumin enhance the sensor's resistance to common interfering substances in complex biological samples, ensuring that the quantitative detection results of placental growth factors remain accurate and reliable even in the presence of certain concentrations of interfering substances, thereby improving the practicality and clinical application value of the detection.

[0061] Preparation Example: This preparation example provides a method for preparing a low-trigger-potential electrochemiluminescence immunosensor.

[0062] Mesoporous silica nanochannel films (VMSF) were grown on indium tin oxide (ITO) electrodes, and gold-copper nanoparticles were confined within the channels via in-situ deposition. Using this modified electrode, placental growth factor antibodies were covalently immobilized on its outer surface to prepare an immunoelectrode. The process of detecting placental growth factor in serum samples using electrochemiluminescence was then described.

[0063] (1) Preparation of vertically oriented mesoporous silica thin film / indium tin oxide electrode by electrochemical-assisted self-assembly: A silica nanoporous membrane was grown on the electrode surface using an electrochemically assisted self-assembly method: 1.585 g of cetyltrimethylammonium bromide and 3050 μL of tetraethoxysilane were added to a mixed solution of 20 mL ethanol and 20 mL sodium nitrate (0.1 mol / L, pH = 2.6) and stirred at room temperature for 2.5 h to obtain a precursor solution containing aminosilane. A three-electrode system was used, with an indium tin oxide (ITO) electrode as the working electrode, a platinum electrode as the counter electrode, and a silver / silver chloride electrode as the reference electrode. The ITO electrode was immersed in the aforementioned precursor solution, and a constant current density of –0.7 mA / cm² was applied to it for 10 s. The working electrode was then quickly removed, washed with a large volume of flowing ultrapure water, dried with nitrogen, and aged overnight at 120 °C to obtain ITO modified with a silica nanoporous membrane containing micelles (i.e., SM@VMSF / ITO).

[0064] (2) Preparation of AuCu NPs@VMSF / ITO: The electrode obtained in step 1 was immersed in a 0.1 mol / L hydrochloric acid-ethanol solution and stirred for 5 min to remove micelles, resulting in VMSF / ITO with open channels. The VMSF / ITO was then immersed in a solution containing gold and copper precursors (containing 0.5 mmol chloroauric acid and 0.5 mmol copper chloride, with a supporting electrolyte of 0.1 mol / L potassium chloride), and a constant voltage of -0.5 V was applied for 4 s to prepare AuCu NPs@VMSF / ITO.

[0065] By changing the precursor solution in step 2, separate precursor solutions containing 0.5 mmol chloroauric acid and 0.5 mmol copper chloride were prepared, while keeping other steps unchanged. Electrodes loaded with copper nanoparticles or palladium nanoparticles were obtained and denoted as AuNPs@VMSF / ITO or Cu NPs@VMSF / ITO.

[0066] (3) Preparation of the immunosensor: The obtained AuCu NPs@VMSF / ITO were used as the substrate electrode, and the antibody and VMSF layer were covalently crosslinked using γ-glycidoxypropyltrimethylsilane (GPTMS) as the crosslinking agent.

[0067] The specific experimental steps are as follows: AuCu NPs@VMSF / ITO were immersed in an ethanol solution of γ-glycidyl etheroxypropyltrimethylsilane and reacted at room temperature for 1 hour. After the reaction was completed, the electrode was washed with deionized water to obtain the AuCu NPs@O-VMSF / ITO electrode. "O-VMSF" specifically refers to the film modified with an epoxy silane (such as GPTMS).

[0068] Next, PlGF antibody (10 μg / mL, 40 μL) was drop-coated onto the surface of the AuCu NPs@O-VMSF / ITO electrode and incubated at 4 °C for 60 min. Unbound antibody was removed by thorough washing with phosphate buffer (0.01 mol / L, pH=7.4), and the electrode was dried to obtain Ab / AuCu NPs@O-VMSF / ITO. Finally, the electrode prepared in the previous step was incubated with bovine serum albumin solution (1%, 40 μL) at room temperature for 15 min to block non-specific sites. Finally, the electrode was thoroughly washed with phosphate buffer (0.01 mol / L, pH=7.4) and dried to obtain the immunosensor, named BSA / Ab / AuCu NPs@O-VMSF / ITO electrode, i.e., bovine serum albumin / antibody / gold-copper nanoparticles@vertically oriented mesoporous silica film / indium tin oxide electrode.

[0069] In this preparation example, Figure 1 The electrochemiluminescence signals of different modified electrodes in a phosphate buffer solution (0.01 M, pH=7.4) containing luminol (100 μM) and hydrogen peroxide (1 mM) are represented. Figure 1 Part (A) describes the electrochemiluminescence intensity (ECL)-time plots of the following electrodes: indium tin oxide (ITO), indium tin oxide modified with vertically ordered mesoporous silica film (VMSF / ITO), indium tin oxide modified with gold nanoparticles with vertically ordered mesoporous silica film (Au NPs@VMSF / ITO), indium tin oxide modified with copper nanoparticles with vertically ordered mesoporous silica film (Cu NPs@VMSF / ITO), and indium tin oxide modified with gold-copper alloy nanoparticles with vertically ordered mesoporous silica film (AuCu NPs@VMSF / ITO). Figure 1 Part (B) describes the electrochemiluminescence intensity statistics of an indium tin oxide electrode (AuCu NPs@VMSF / ITO) modified with a vertically ordered mesoporous silica film modified with gold-copper alloy nanoparticles, scanned 8 times after repeated rinsing. Figure 1 Section (C) describes the electrochemiluminescence intensity statistics of a copper nanoparticle-modified indium tin oxide electrode (Cu NPs@ITO) after eight scans following repeated rinsing. The detection solutions were all phosphate buffer solutions (0.01 M, pH = 7.4) containing luminol (100 μM) and hydrogen peroxide (1 mM). The electrochemiluminescence analyzer had a potential scan rate of 100 mV / s and a photomultiplier tube (PMT) voltage of 600 V.

[0070] Figure 2 The diagram illustrates the statistical results of the cyclic voltammetry (CV) responses of the following electrodes prepared in sulfate or phosphate buffer solutions: an indium tin oxide electrode modified with gold nanoparticles and vertically ordered mesoporous silica film (Au NPs@VMSF / ITO electrode), an indium tin oxide electrode modified with copper nanoparticles and vertically ordered mesoporous silica film (Cu NPs@VMSF / ITO electrode), and an indium tin oxide electrode modified with gold-copper alloy nanoparticles and vertically ordered mesoporous silica film (AuCuNPs@VMSF / ITO electrode). Figure 2 Part (A) shows the cyclic voltammetry curves of the Au NPs@VMSF / ITO electrode and the AuCu NPs@VMSF / ITO electrode in a sulfuric acid buffer solution (0.1M). Figure 2 (B) shows the cyclic voltammetry curves of Cu NPs@VMSF / ITO and AuCu NPs@VMSF / ITO electrodes in phosphate buffer solution (0.1M), with a scan rate of 50 mV / s. "vs.Ag / AgCl" refers to "relative to the silver / silver chloride electrode," i.e., the Ag / AgCl reference electrode.

[0071] Figure 3 The diagram shows the catalytic mechanism verification of the indium tin oxide electrode (AuCu NPs@VMSF / ITO electrode) modified with a vertically ordered mesoporous silica film modified with gold-copper alloy nanoparticles in the preparation example. Figure 3 Section (A) describes the electrochemiluminescence intensity-potential relationship of the AuCuNPs@VMSF / ITO electrode in 0.01M phosphate buffer solution (pH 7.4) containing or without 1mM hydrogen peroxide and 100μM luminol. The scan rate was 100mV / s, and the photomultiplier tube (PMT) voltage was 600V. Figure 3 Part (B) describes the ECL response of the AuCu NPs@VMSF / ITO electrode after standing at room temperature for 50 s in a phosphate buffer solution containing only hydrogen peroxide (H2O2) for 50 s (left) and after electrolyzing hydrogen peroxide with a voltage of -0.5V for 50 s (right), the applied voltage was removed and 100 μM luminol was added alone. Figure 3Section (C) describes the time-signal relationship of the AuCu NPs@VMSF / ITO electrode in nitrogen atmosphere, air atmosphere and oxygen atmosphere, and in 0.01M phosphate buffer solution (pH 7.4) containing 100μM luminol and 1mM hydrogen peroxide. Figure 3 Section (D) describes the statistical results of the ratio of test signal to baseline signal for the AuCu NPs@VMSF / ITO electrode in different radical scavengers. The test signal refers to the electrochemiluminescence intensity measured after the addition of a radical scavenger, while the baseline signal refers to the electrochemiluminescence intensity measured without the addition of a radical scavenger. It should be noted that a blank horizontal axis represents a blank control group without any radical scavenger. The electrochemiluminescence analyzer had a potential scan rate of 100 mV / s and a photomultiplier tube (PMT) voltage of 600 V.

[0072] Figure 4 The electrochemiluminescence and electrochemical performance characterization of different electrodes in the preparation examples are presented. Figure 4 Section (A) presents the statistical results of electrochemiluminescence (ECL) intensity for different electrodes under conditions containing 100 μM luminol and 1 mM hydrogen peroxide (H₂O₂). The electrochemiluminescence analyzer has a potential scan rate of 100 mV / s. -1 The photomultiplier tube (PMT) voltage is set to 600V. Figure 4 Part (B) shows different electrodes in the presence of 2.5 mM potassium ferricyanide (Fe(CN)6). 3- / 4- Cyclic voltammetry (CV) curves in 0.1 M potassium chloride (KCl) solution. Figure 4 Section (C) presents the electrochemical impedance spectroscopy (EIS) curves of different electrodes in a 0.1 M potassium chloride (KCl) solution containing 2.5 mM potassium ferricyanide. In the EIS test, a small-amplitude sinusoidal perturbation is applied to the reaction system, and then the response signal is measured. The data results generally include the real part (denoted as Z' or Re) and the imaginary part (denoted as Z'' or -Im). Figure 4 In section (C), the horizontal axis represents the real part of the impedance, and the vertical axis represents the imaginary part of the impedance.

[0073] Figure 5 This indicates the optimization of the precursor solution ratio in the preparation example, as well as the optimization of the incubation time of the placental growth factor (PlGF) antibody and PlGF. Figure 5Part (A) presents the electrochemiluminescence (ECL) intensity of AuCu NPs@VMSF / ITO electrodes obtained by electrodeposition in precursor solutions containing different gold-copper ratios, measured in a phosphate buffer solution (0.01 M, pH=7.4) containing luminol (100 μM) and hydrogen peroxide (H₂O₂, 1 mM). This figure allows for analysis of the effect of different gold-copper ratios on the electrode's ECL performance, thereby enabling optimization of the gold-copper ratio in the precursor solution. Figure 5 Section B shows a schematic diagram illustrating the relationship between the incubation time of the PlGF antibody and the electrochemiluminescence intensity. In the experiment, the concentration of the PlGF antibody was fixed at 10 μg / mL, and the concentration of PlGF was fixed at 1 ng / mL. This diagram helps determine the optimal antigen incubation time to improve the sensitivity and accuracy of the detection. Figure 5 Part (C) is a schematic diagram showing the relationship between the incubation time of PlGF and the electrochemiluminescence intensity.

[0074] Figure 6 The electrochemiluminescence intensity and linear relationship of BSA / AbPlGF / AuCu NPs@O-VMSFNF / ITO electrode after incubation with different concentrations of PlGF are shown. Figure 6 Part (A) shows the electrochemiluminescence intensity graphs for different concentrations of PlGF, visually illustrating the ECL intensity response of the electrode as the PlGF concentration changes. Specifically, in flow injection analysis mode, a series of PlGF standard solutions (including a blank control) of various concentrations were sequentially introduced into the detection system. All samples of different concentrations underwent the same optimized incubation time (based on...). Figure 5 (Results) An antigen-antibody binding reaction was performed to ensure consistent binding efficiency. The horizontal axis represents detection time, and the vertical axis represents electrochemiluminescence intensity. Each independent electrochemical intensity response peak corresponds to a complete detection event. The graph clearly shows that the intensity of the electrochemical intensity response peak decreases as the PlGF concentration increases from 0. This result indicates that, under the same incubation conditions, the electrode exhibits a concentration-dependent electrochemical intensity response to different concentrations of the target analyte, confirming the sensor's good detection capability for PlGF. Therefore, this graph provides a preliminary observation of the electrode's detection capability for different concentrations of PlGF. Figure 6 Section (B) shows the calibration curve of electrochemiluminescence intensity versus the logarithm of PlGF. Error bars represent the standard deviation of three measurements. The electrolyte solution is a phosphate buffer solution (0.01 M, pH = 7.4) containing luminol (100 μM) and hydrogen peroxide (1 mM). This calibration curve can be used for the quantitative analysis of PlGF concentration in unknown samples; the corresponding PlGF concentration is calculated from the curve by measuring the ECL intensity.

[0075] Figure 7Section A shows the ECL intensity ratio (I / I0) of the electrode before (I0) and after (I) incubation with different substances. The more significant the deviation of the ratio from 1, the better the selectivity of the electrode for that substance. For example, in this figure, the intensity ratio before and after incubation with placental growth factor is 0.5. That is, after incubation with placental growth factor, the ECL signal decreased by half. This represents the specific response of the electrode to placental growth factor (due to the antibody capturing PlGF, which hinders electron transfer or luminescence efficiency, resulting in a decrease in signal). The concentration of PlGF is 1 ng / mL, the concentration of alpha-fetoprotein (AFP) and interleukin-6 (IL-6) is 10 ng / mL, and the concentrations of glucose (Glu), sodium ions (Na⁺), potassium ions (K⁺), ascorbic acid (AA), and uric acid (UA) are 1 mM. Even when the concentrations of alpha-fetoprotein (AFP) and interleukin-6 are higher than those of placental growth factor, the I / I0 value is close to 1.0 (that is, the electrochemiluminescence intensity hardly changes). This figure can assess the selectivity of the electrode for the target PlGF, that is, determine whether the electrode can specifically recognize PlGF in a complex system without being affected by other substances. Figure 7 (B) represents the parallel detection of the ECL signal of 1 ng / mL PlGF using five sensing electrodes. Parallel experiments can be used to evaluate the repeatability of the electrodes. If the ECL signal fluctuations of the five electrodes are small, it indicates that the electrodes have good repeatability and the detection results are reliable. Figure 7 (C) shows the change in ECL intensity ratio of the BSA / AbPlGF / AuCu NPs@O-VMSFNF / ITO electrode after storage for different numbers of days, measured before and after the addition of 1 ng / mL PlGF. The error bars represent the standard deviation of three measurements. This graph can assess the stability of the electrode. If the change in ECL intensity ratio is small with increasing storage days, it indicates that the electrode is stable during storage and can be stored and used for a long time.

[0076] Test Example: In the detection of placental growth factor (PlGF), the aforementioned BSA / Ab / AuCu NPs@O-VMSF / ITO electrode was incubated with 50 μL of placental growth factor at different concentrations at 4°C for 90 min. The electrode was then slowly rinsed with phosphate buffer solution (0.01 M, pH=7.4) to remove unbound PlGF. The resulting electrode was designated PlGF / BSA / Ab / AuCu NPs@O-VMSF / ITO. Here, Ab refers to antibody. The obtained electrode was subjected to ECL detection using a phosphate buffer solution (0.01 M, pH=7.4) containing 100 μM luminol and 1 mM H2O2. Electrochemiluminescence was triggered by continuous CV scans, with a CV scan potential range of 0 to -0.5 V and a scan rate of 0.1 V / s. In actual sample analysis, the pre-existing PlGF in fetal bovine serum samples was quantified using the standard addition method. Fetal bovine serum was diluted 50-fold with phosphate buffer (0.01 M, pH = 7.4) before testing.

[0077] The principle behind this detection process is that gold-copper alloy nanoparticles can efficiently catalyze the reduction of hydrogen peroxide and dissolved oxygen at low potentials, generating a large number of reactive oxygen species. These reactive oxygen species can oxidize luminol anions, causing them to produce excited-state products, which in turn emit a strong electrochemiluminescence signal. Compared with existing technologies where luminol requires a relatively negative potential (e.g., -1V) to emit light efficiently, this sensor achieves low trigger potential luminescence, avoiding damage to biorecognition molecules. When placental growth factor in the sample binds to antibodies (Ab) to form an immune complex, a steric hindrance effect occurs, hindering the diffusion of luminol and hydrogen peroxide to the electrode surface, resulting in a decrease in the intensity of the electrochemiluminescence signal.

[0078] Finally, the concentration of placental growth factor in the sample was determined based on the degree of decrease in the detected electrochemiluminescence signal intensity. The sensor exhibits a linear detection range of 10 fg / mL to 10 ng / mL for placental growth factor, with a detection limit of 5.4 fg / mL. Furthermore, due to the size sieving, electrostatic repulsion, and hydrophilicity of the vertically ordered mesoporous silica film, as well as the antigen-antibody specific binding characteristics, the sensor demonstrates excellent resistance to interference from glucose, inorganic salts, and common cytokines. The quantitative detection of placental growth factor remains unaffected when the concentration of each interfering substance does not exceed 1 mM and the total concentration does not exceed 0.5 M, exhibiting good selectivity and stability.

[0079] The above are merely specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that the present invention includes, but is not limited to, the contents described in the accompanying drawings and the specific embodiments above. Any modifications that do not depart from the functional and structural principles of the present invention will be included within the scope of the claims.

Claims

1. A low-trigger-potential electrochemiluminescence immunosensor, characterized in that, The system includes a three-electrode system, comprising a working electrode, a reference electrode, and a counter electrode. The working electrode comprises: Conductive substrate; A vertically ordered mesoporous silica film is formed on the surface of the conductive substrate, and the vertically ordered mesoporous silica film has nanochannels. Gold-copper alloy nanoparticles are formed within the nanochannels; An epoxy-modified layer is formed on the outer surface of the vertically ordered mesoporous silica film; Placental growth factor-specific antibodies, which are covalently bound to the epoxy-modified layer; Bovine serum albumin, which is covalently bound to the region of the epoxy-modified layer that is not bound by the placental growth factor-specific antibody; The gold-copper alloy nanoparticles are formed by electrodeposition in the nanochannels of the vertically ordered mesoporous silica film via an electrodeposition solution containing gold and copper precursors, and the gold-copper alloy nanoparticles are confined within the nanochannels. The sensor is used to detect placental growth factor in a potential range of -0.5 to 0V, with a detection linear range of 10 fg / mL to 10 ng / mL and a detection limit not higher than 5.4 fg / mL.

2. The low trigger potential electrochemiluminescence immunosensor according to claim 1, characterized in that, The molar ratio of gold to copper in the gold-copper alloy nanoparticles is 1:5 to 1:

1.

3. The low trigger potential electrochemiluminescence immunosensor according to claim 1, characterized in that, The conductive substrate is selected from one of the following: indium tin oxide electrode, glassy carbon electrode, fluorine-doped tin oxide electrode, gold electrode, screen-printed electrode, graphite electrode, or carbon fiber electrode.

4. A method for preparing a low-trigger-potential electrochemiluminescence immunosensor, characterized in that, The low-trigger-potential electrochemiluminescence immunosensor comprises the low-trigger-potential electrochemiluminescence immunosensor according to any one of claims 1 to 3, and the preparation method comprises the following steps: S1, A vertically ordered mesoporous silica film is formed on the surface of a conductive substrate to obtain a modified electrode, wherein the vertically ordered mesoporous silica film has nanochannels. S2, gold-copper alloy nanoparticles are formed within the nanochannels; S3, an epoxy-modified layer is formed on the outer surface of the vertically ordered mesoporous silica film; S4, covalently bind placental growth factor-specific antibodies to the epoxy-modified layer; S5, covalently bind bovine serum albumin to the region of the epoxy-modified layer that is not bound by the placental growth factor-specific antibody; Step S2 includes: immersing the modified electrode in an electrodeposition solution containing a gold precursor and a copper precursor, and applying a potential of -1.0 to 0V to the modified electrode to generate and confine the gold-copper alloy nanoparticles in situ within the nanochannel; the gold precursor is selected from at least one of chloroauric acid, gold nitrate, or a gold complex; the copper precursor is selected from at least one of copper chloride, copper sulfate, or a copper complex; the concentration of the gold precursor in the electrodeposition solution is 0.01 to 1 mmol / L, and the concentration of the copper precursor is 0.05 to 1 mmol / L.

5. The preparation method according to claim 4, characterized in that, Step S3 includes: immersing the modified electrode in an epoxy silane solution and reacting it in the dark for 0.2 to 5 hours; wherein the epoxy silane is selected from at least one of γ-glycidoxypropyltrimethoxysilane, 3-(2,3-epoxypropoxy)propyltrimethoxysilane or 2-(3,4-epoxycyclohexyl)ethyltriethoxysilane, and the concentration of the epoxy silane solution is 2.0 to 3.0 mM.

6. The preparation method according to claim 4, characterized in that, Step S4 includes: immersing the modified electrode in a buffer solution containing placental growth factor-specific antibodies and incubating it at a temperature of 0–5°C for 30–90 min, so that the placental growth factor-specific antibodies are covalently bound to the epoxy-modified layer; the concentration of the buffer solution is 1–10 μg / mL and the pH of the buffer solution is 6.5–7.

5.

7. The preparation method according to claim 4, characterized in that, Step S5 includes: immersing the modified electrode obtained in step S4 in a bovine serum albumin solution for 5 to 20 minutes, so that bovine serum albumin covalently binds to the region on the epoxy-modified layer that is not bound by the placental growth factor-specific antibody; the mass concentration of the bovine serum albumin solution is 0.2% to 2%.

8. An application of a low-trigger-potential electrochemiluminescence immunosensor, characterized in that, The low trigger potential electrochemiluminescence immunosensor according to any one of claims 1-3 comprises the following steps: S10, the working electrode of the low trigger potential electrochemiluminescence immunosensor is brought into contact with and incubated with the test sample containing placental growth factor, so that the placental growth factor in the test sample binds to the placental growth factor specific antibody. S20, the combined working electrode, reference electrode and counter electrode are placed in a detection solution containing luminol and hydrogen peroxide; S30, perform electrochemical scanning on the detection solution within the potential range of -0.5 to 0V to detect the electrochemiluminescence signal; S40, based on the detected electrochemiluminescence signal intensity, determine the concentration of placental growth factor in the sample to be tested.

9. The application of the low trigger potential electrochemiluminescence immunosensor according to claim 8, characterized in that, The detection linear range of the low trigger potential electrochemiluminescence immunoassay sensor is 10 fg / mL to 10 ng / mL, and the detection limit is not higher than 5.4 fg / mL. When the concentration of each interfering substance in the sample to be tested does not exceed 1 mM and the total concentration does not exceed 0.5 M, it is determined that the quantitative detection of placental growth factor is not affected by the interfering substances.

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