FRET (Fluorescence Resonance Energy Transfer) fluorescent probe with controllable configuration as well as preparation method and application method thereof

Abstract

The invention relates to an FRET fluorescent probe with a controllable configuration and a preparation method and an application method thereof, the FRET fluorescent probe is an FRET probe system based on a sparse biotinylation and limited streptavidin coupling strategy, and the specific system can detect QDs modified by a small amount of biotin on the surface, limited streptavidin and labeled receptor dye and a detection antibody of a small amount of biotin. A controllable self-assembly structure is formed through biotin-streptavidin action, so that the distance between donors and receptors is highly consistent, the structural uniformity and energy transfer efficiency are improved, background signals are reduced, and the predictability and reproducibility of detection signals are improved. The multi-donor-single receptor structure can amplify signals and improve sensitivity, and is suitable for detection of low-abundance targets such as HER2 and the like. In addition, the system can regulate and control the space structure, reduce background interference and improve specificity, fluorescence signals can respond continuously along with dynamic change of a target, modularization and platform compatibility are achieved, the system adapts to various antibodies and platforms, the detection process is simple and convenient, and real-time monitoring is facilitated.

Description

Technical Field

[0001] The present application relates to the field of biomedical imaging detection technology, and in particular to a FRET fluorescent probe with a controllable configuration, a preparation method thereof, and an application method thereof. Background Art

[0002] Human epidermal growth factor receptor 2 (HER2), a key transmembrane tyrosine kinase receptor protein of the ErbB receptor family, plays a key role in the development and progression of various malignant tumors. Numerous studies have demonstrated that HER2 is abnormally expressed in a variety of tumors, including breast cancer, gastric cancer, and ovarian cancer. Gene amplification or protein overexpression is closely associated with high tumor invasiveness, rapid progression, high risk of metastasis, and poor prognosis. For example, approximately 15%–20% of breast cancer patients are HER2-positive. These patients often face challenges such as accelerated tumor proliferation, increased recurrence rates, and treatment resistance. Therefore, HER2 has become an important biomarker for accurate breast cancer classification, prognostic assessment, and personalized treatment planning.

[0003] Currently, several methods are used clinically for HER2 detection. Immunohistochemistry, fluorescence in situ hybridization (FISH), enzyme-linked immunosorbent assay (ELISA), and chemiluminescence immunoassay are commonly used. Some commercial kits have also attempted to incorporate fluorescent probes for rapid detection. However, these technologies have encountered several challenges in practical application. Firstly, the operational procedures are complex, requiring high technical skills and experimental conditions. Secondly, the detection process is heavily dependent on the type of tissue or blood sample, and different sample types may lead to variability in test results. Furthermore, these methods have limited response ranges, poor sensitivity, and insufficient dynamic tracking capabilities, making it difficult to continuously monitor HER2 expression levels as the disease progresses or during treatment. In particular, these detection methods cannot meet clinical needs in areas such as early cancer screening, efficacy assessment, and personalized treatment. Therefore, there is a need to develop a new, more sensitive probe system that can be used for continuous detection at multiple time points.

[0004] Quantum dots (QDs), with their uniquely narrow emission spectra, high fluorescence intensity, and excellent photostability, are ideal energy donors in fluorescence resonance energy transfer (FRET) systems. However, existing FRET probe technologies still face numerous challenges. During the current construction of FRET probes, the donor-acceptor distance is difficult to precisely control. The random nature of the connection between QDs and antibodies and acceptor dyes means that a single QD may be conjugated to multiple antibodies or dyes, forming complexes with high structural heterogeneity. This makes the FRET distance both unpredictable and unreproducible. Furthermore, the random nature of the coupling probability further impacts the probe's performance stability. Although some covalent FRET probes achieve target response through structural design, the increased rigidity of the donor-acceptor configuration after covalent attachment limits the ability to control the spatial conformation. This leads to issues such as easily activated signals, high background signals, and irreversible or uncontrollable responses, making it difficult to meet the practical application requirements of high-sensitivity and dynamic detection.

[0005] In summary, the development of fluorescent probes with adjustable spatial configuration, strong signal amplification capability, gradual and partially reversible FRET response has important technological innovation significance for realizing dynamic visual monitoring of tumor markers such as HER2. Summary of the Invention

[0006] The purpose of this application is to provide a FRET probe and its construction method that can be used for dynamic monitoring of HER2 levels, in order to solve the problems of existing detection methods such as complex operation, insufficient response sensitivity, and inability to achieve continuous monitoring. This application first provides a FRET fluorescent probe with a controllable configuration, which includes a first complex solution and a second complex solution; The first complex solution includes a first complex formed by coupling a nanoluminescent material, a first biotin and a multi-site avidin, wherein the multi-site avidin includes an empty site that is not coupled with biotin; The second complex solution includes a second complex formed by coupling a detection antibody, a receptor molecule dye and a second biotin, wherein the second biotin includes a group that can be bound to a vacant site.

[0007] In one embodiment, the multi-site avidin is streptavidin, and the first biotin occupies three sites of the streptavidin.

[0008] In one embodiment, the method further comprises a coated capture antibody, wherein the coated capture antibody captures the HER2 antigen.

[0009] In one embodiment, the first biotin is NHS-PEG4-Biotin, and the second biotin is Sulfo-NHS-Biotin.

[0010] The present application further provides a method for preparing a FRET fluorescent probe with a controllable configuration, comprising: Conjugating a first biotin on the surface of the nanoluminescent material to obtain a first conjugate; Mixing the first conjugate with multisite avidin and reacting them to obtain a first complex, wherein the number of sites of the multisite avidin is n, and the molar ratio of the first conjugate to the multisite avidin is (n-1):1; The detection antibody is coupled to an acceptor dye molecule to obtain a second conjugate, and the second conjugate is then mixed and reacted with a second biotin to obtain a second complex, wherein during the coupling process, the molar amount of the acceptor dye molecule is 5-10 times the molar amount of the detection antibody, and the molar amount of the second biotin is 3-5 times the molar amount of the second conjugate.

[0011] In one embodiment, a verification step is further included, wherein the mobility of the first complex is tested using electrophoretic mobility analysis, or the particle size of the first complex is characterized using dynamic light scattering to confirm that the first complex includes a vacant site.

[0012] In one embodiment, a purification step is included to remove unreacted substances in the solution after obtaining the first conjugate, the first complex, the second conjugate or the second complex.

[0013] In one embodiment, the method further comprises adding a stabilizer to the solution containing the first complex and the solution containing the second complex.

[0014] The present application also provides a method for detecting HER2 antigen concentration, comprising the following steps: Adding the second complex solution of the aforementioned FRET fluorescent probe with controllable configuration to a sample containing the HER2 antigen to be detected; Further adding the first complex solution of the aforementioned FRET fluorescent probe with controllable configuration to construct a FRET system; Perform fluorescence detection, record the donor and acceptor fluorescence emission intensities corresponding to the excitation wavelength, and calculate the energy transfer ratio or fluorescence lifetime change.

[0015] In one embodiment, the HER2 antigen to be detected is captured by a HER2-specific antibody coated in a well plate to form a solid-phase immunobinding platform.

[0016] Compared with the prior art, this application has the following beneficial effects: This application constructs a FRET probe system based on a sparse biotinylation and limited avidin coupling strategy. The system comprises QDs surface-modified with a first biotin, a limited amount of introduced avidin molecules, and a detection antibody simultaneously labeled with an acceptor dye molecule and a second biotin. Through the specific biotin (Biotin)-streptavidin (SA) interaction, a controllable self-assembly structure of "multi-quantum dot-antibody" is achieved. By limiting the coupling ratio and spatial configuration, the spacing between the donor and acceptor is made consistent, improving the uniformity of the probe structure and the efficiency of energy transfer, effectively reducing background fluorescence signals, and improving the predictability and reproducibility of detection signals. This provides a new approach to building a highly sensitive, low-background dynamic molecular detection and bioimaging platform.

[0017] The biotin-avidin system used in this application to construct the probe has good modularity and platform compatibility, and can be quickly adapted to different biomarker detections. By simply replacing the corresponding detection antibodies, different biomarker detections can be achieved, which has strong versatility. At the same time, it can be adapted to a variety of antibodies and fluorescent labels to meet different detection needs. In addition, the probe can be integrated into platforms such as POCT and microfluidics. The detection process can be completed by self-assembly in the liquid phase, eliminating the tedious elution steps and is suitable for multi-time point sampling and online continuous monitoring.

[0018] This application constructs a multi-donor-single acceptor FRET structure, in which multiple donors (QDs) simultaneously transfer energy to a single acceptor to achieve signal amplification. This structure is particularly suitable for high-sensitivity detection of low-abundance targets such as HER2. It can still produce a clearly detectable fluorescent signal in the presence of low concentrations of HER2 antigen, thereby improving the sensitivity of detection and facilitating accurate detection in cases where HER2 antigen expression levels are low, such as in the early stages of tumors.

[0019] In a specific manner, the present application can combine the four-site binding characteristics of streptavidin and adopt a strategy of limited coupling of biotinylated molecules. By controlling the coupling amount of biotinylated molecules, precise regulation of the spatial structure of the probe can be achieved, and the donor-acceptor distance can be effectively controlled to avoid the problem of low energy transfer efficiency or increased background signal due to excessive or small donor-acceptor distance. At the same time, the reasonable spatial structure makes the binding of the probe to the HER2 antigen more specific, reduces the interference signal generated by nonspecific binding, thereby improving the specificity of the detection and being able to more accurately distinguish HER2 positive and negative samples. Due to the reasonable design of the probe structure, the energy transfer process has good signal gradient and partial reversibility. When the HER2 expression level changes, the fluorescence signal of the probe will change accordingly, which can reflect the dynamic process of HER2 expression status in real time. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1Schematic diagram of the construction process of the first complex in the FRET fluorescent probe with controllable configuration of the present application; Figure 2 Schematic diagram of the construction process of the second complex in the FRET fluorescent probe with controllable configuration of the present application; Figure 3 It is a flow chart of the HER2 antigen concentration detection method of the present application.

[0021] Explanation of the accompanying drawings: 100, first complex; 110, nanoluminescent material; 120, first biotin; 130, multi-site avidin; 200, second complex; 210, detection antibody; 220, receptor molecule dye; 230, second biotin; 300, capture antibody; 400, antigen. DETAILED DESCRIPTION

[0022] In order to make the above-mentioned objects, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below in conjunction with the accompanying drawings. It will be understood that the specific embodiments described herein are only used to explain the present application, rather than to limit the present application. It should also be noted that, for ease of description, only some, rather than all, structures related to the present application are shown in the accompanying drawings. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0023] As used herein, the terms "comprise," "comprising," and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, system, product, or apparatus comprising a series of steps or elements is not limited to the listed steps or elements but may optionally include steps or elements not listed, or may optionally include other steps or elements inherent to the process, method, product, or apparatus.

[0024] References herein to "embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute an independent or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0025] In the field of cancer diagnosis and treatment, measuring tumor marker expression levels is crucial for disease assessment, treatment plan development, and efficacy monitoring. Therefore, ensuring the accuracy of tumor marker detection is crucial. For example, human epidermal growth factor receptor 2 (HER2) plays a crucial role in monitoring the development and progression of various malignancies, particularly breast, gastric, and ovarian cancers. HER2 gene amplification or protein overexpression is closely associated with high tumor invasiveness, rapid progression, increased risk of metastasis, and poor prognosis. For example, approximately 15%-20% of breast cancer patients are HER2-positive, and these patients often face serious challenges such as accelerated tumor growth, increased recurrence rates, and treatment resistance. Therefore, accurate, sensitive, and dynamic measurement of HER2 expression levels is crucial for achieving precise diagnosis and personalized treatment of cancer. Currently, commonly used HER2 detection methods in clinical practice rely on single-point sample collection and endpoint assessment, making it difficult to continuously monitor HER2 expression as the disease progresses or during treatment. This, in particular, fails to meet the clinical need for real-time, accurate information for early cancer screening, efficacy assessment, and personalized treatment.

[0026] This application focuses on a FRET fluorescent probe with a controllable configuration, its preparation method and application method, and provides a technical solution that can be used for dynamic monitoring of HER2 levels. The fluorescent probe designed in this application is based on a unique sparse biotinylation and limited avidin coupling strategy. By controlling the spatial configuration and donor-acceptor spacing of the probe, it achieves optimization in many aspects such as signal amplification, background reduction, and dynamic response. At the same time, in conjunction with the detection process, the fluorescent probe system can improve the sensitivity and specificity of HER2 detection, and has the ability of real-time and continuous monitoring, providing strong technical support for early diagnosis, precise treatment, and efficacy evaluation of tumors. The specific composition, preparation method, and HER2 antigen concentration detection method based on the fluorescent probe in this application will be introduced in detail below.

[0027] The present application first provides a FRET fluorescent probe with a controllable configuration, which includes a first complex solution and a second complex solution; The first complex solution includes a first complex 100 formed by coupling a nanoluminescent material 110, a first biotin 120, and a multi-site avidin 130, wherein the multi-site avidin 130 includes an empty site that is not coupled with biotin; The second complex solution includes a second complex 200 formed by coupling a detection antibody 210, a receptor molecule dye 220, and a second biotin 230, wherein the second biotin 230 includes a group that can bind to a vacant site.

[0028] The nanoluminescent material 110, the first biotin 120, and the multi-site avidin 130 form a first complex 100 through a specific chemical coupling reaction. A vacant site on the multi-site avidin 130, uncoupled from biotin, is intentionally left unattached. This provides a site for subsequent specific binding with other functional molecules, ensuring the directional and orderly assembly of the probe. The second biotin 230 possesses a specific group that can precisely bind to the vacant site on the multi-site avidin 130 in the first complex 100, enabling the second complex 200 to assemble with the first complex 100, thereby constructing the overall structure of the fluorescent probe.

[0029] There are many options for nanoluminescent materials 110, and quantum dots can be used as fluorescence resonance energy transfer (FRET) donors. Quantum dots have excellent optical properties such as narrow emission spectrum, high fluorescence intensity and good photostability, which can provide a stable and efficient energy supply for the FRET process. In addition, other nanoluminescent materials 110 with similar optical properties can also be used in this application, such as quantum rods, which have unique anisotropic optical properties and can provide stronger fluorescence emission in specific directions; upconversion nanoparticles, which can convert low-energy near-infrared light into high-energy visible light, bringing a new energy transfer mode to the FRET system. These nanoluminescent materials 110 can all achieve similar energy transfer processes, providing more options for the design and application of probes.

[0030] This application constructs a "multi-donor, one-acceptor" FRET structure, specifically using a single antibody coupled with multiple quantum dot donors. This allows for flexible adjustments based on actual application needs. For example, depending on different detection scenarios, target concentration ranges, and signal amplification requirements, a structure connecting two quantum dots can be used as an alternative, demonstrating the flexibility and versatility of this application's probe structure.

[0031] During the energy transfer process, multiple quantum dots act as donors, simultaneously transferring energy to a single acceptor molecule, dye 220, improving the efficiency of energy transfer. Compared to the traditional single-donor-single-acceptor structure, energy transfer is more concentrated and efficient, thereby enhancing the intensity of the fluorescence signal. This signal amplification effect is crucial for detecting low-abundance biomarkers (such as HER2). In the early stages of a tumor or under certain special physiological conditions, the expression level of the biomarker is often low, making it difficult to accurately detect with traditional detection methods. However, the "multi-donor, one-acceptor" structure of the present application can generate a sufficiently strong fluorescence signal, enabling sensitive detection of low-abundance biomarkers, thereby improving the sensitivity and accuracy of detection.

[0032] The present application uses avidin to limit the coupling of sparse biotinylated quantum dots and the acceptor dye-biotin double-labeled detection antibody 210. This spatial structure regulation method realizes the control of the spatial distance between the donor and the acceptor. In the FRET process, the spatial distance between the donor and the acceptor is one of the key factors affecting the energy transfer efficiency. By precisely controlling the spatial distance, the donor and the acceptor are within the optimal energy transfer range, thereby effectively optimizing the FRET efficiency. At the same time, this precise spatial structure regulation can also reduce the interference of nonspecific binding and background signals. Since the assembly of the donor and the acceptor is directional and orderly, the structural heterogeneity caused by random coupling is avoided, making the signal of the probe clearer and more accurate. Therefore, the spatial structure regulation method of the present application improves the specificity of detection and can more accurately distinguish target biomarkers and non-target substances.

[0033] This application uses HER2 as a primary detection target as a specific example. Accurate detection of HER2 expression levels is of great significance for tumor diagnosis, treatment, and prognosis assessment. However, the scope of application of this application is not limited to HER2 and can also be widely applied to the detection of other protein biomarkers, such as epidermal growth factor receptor (EGFR), prostate-specific antigen 400 (PSA), interleukin-6 (IL-6), etc. Specific detection of different biomarkers can be achieved by simply replacing the corresponding specific detection antibody 210.

[0034] The probe system constructed using this application has good gradual and partially reversible response characteristics. The FRET signal can respond continuously as the target concentration changes, enabling visual monitoring of the dynamic expression level of the target molecule. During the occurrence and development of tumors, the expression level of biomarkers will fluctuate continuously with the changes in the disease. Traditional detection methods can usually only provide static detection results at a certain point in time and are difficult to reflect the dynamic changes of biomarkers. The probe system of this application can monitor the changes in the expression level of biomarkers in real time and continuously.

[0035] See also Figures 1 to 2 Therefore, in one specific and representative embodiment, the multi-site avidin 130 is streptavidin (SA). In this embodiment, the first biotin 120 occupies three sites of streptavidin, which serves as the basis for subsequent probe assembly and functional realization.

[0036] This application specifically illustrates how to control the spatial structure by means of limited coupling of SA with sparse biotinylated molecules. In addition to SA, other proteins with multi-site binding capabilities can also be used, such as NeutrAvidin, which also has four sites that can bind to biotin and has low non-specific binding properties; there are also modified avidins, whose structures are modified by genetic engineering methods to give them multi-site binding capabilities and spatial conformations that are more suitable for the requirements of this application. In addition, chemical cross-linkers can also be used as an effective means to achieve similar spatial configuration regulation. Chemical cross-linkers can form stable covalent bonds between molecules, connecting different functional molecules together in a predetermined manner, thereby constructing a probe system with a specific spatial structure.

[0037] During probe construction, sparse biotinylation technology was used to regulate the density of the quantum dot (QD) surface and the functional sites of the detection antibody 210. This controlled the amount of biotin modification to achieve single-point coupling, ensuring that only a limited number of biotin molecules bind to each QD surface, thereby avoiding steric hindrance and signal interference caused by excessive modification. Furthermore, sparse biotinylation of the functional sites of the detection antibody 210 ensures that the binding activity of the antibody to the target (such as HER2) is not affected.

[0038] In terms of spatial structural design, three of the four sites of SA preferentially bind to the quantum dot-biotin complex. This is achieved by optimizing the reaction conditions and molecular ratios to ensure that all three sites are occupied, leaving only one site for binding to the biotinylated detection antibody 210. This gives the probe a clear spatial configuration, ensuring subsequent efficient energy transfer. The Fc tail of the detection antibody is also labeled with a fluorescent receptor. Labeling the fluorescent receptor on the Fc tail of the detection antibody can form an efficient donor-acceptor configuration with it. When the quantum dot is irradiated with excitation light as a donor, its energy can be efficiently transferred to the acceptor molecule, thereby realizing the construction of a FRET probe with a "3QDs+1 antibody" structure.

[0039] When the probe binds to HER2, it triggers spatial conformational changes and distance reconstruction. The specific binding of HER2 to the detection antibody 210 alters the spatial structure of the entire probe system, shifting the distance between the donor (QDs) and the acceptor (fluorescent receptor). When the distance between the donor and acceptor is appropriate, energy transfer efficiency is maximized and the fluorescence signal is strongest. Therefore, by detecting changes in the fluorescence signal, quantitative analysis of HER2 expression can be achieved. Furthermore, because the probe can monitor dynamic changes in HER2 expression levels in real time, signal enhancement and dynamic trend tracking can be achieved through improved FRET efficiency.

[0040] In addition to the aforementioned probe structure, a coated capture antibody 300 can also be introduced. This antibody possesses highly specific capture capabilities for the HER2 antigen 400. In the detection system, the coated capture antibody 300 is pre-immobilized on a specific solid support surface, such as a microplate, magnetic microspheres, or chip. When a sample containing the HER2 antigen 400 is added to the system, the coated capture antibody 300 recognizes and binds to the HER2 antigen 400, immobilizing it on the solid support and providing a stable anchor for subsequent detection steps. Antibodies are used as recognition and linking units to achieve precise targeting of the donor. Antibodies possess high specificity and affinity, enabling them to specifically recognize target molecules (such as HER2) and link them to donor molecules (such as quantum dots), thereby constructing a functional energy transfer structure. Specifically, one end of the antibody binds to the donor molecule through chemical coupling or a biotin-avidin system, while the other end specifically binds to the target molecule, enabling the donor to be positioned near the target molecule and creating conditions for efficient energy transfer.

[0041] However, the design of this application is not limited to the use of antibodies as recognition and binding units. Considering the needs of different application scenarios and the diversity of recognition elements, recognition elements such as nucleic acid aptamers can also be used to replace antibodies. Nucleic acid aptamers are short single-stranded DNA or RNA sequences screened through the systematic evolution of ligands by exponential enrichment (SELEX) method. They have similar specificity and affinity to antibodies and can specifically bind to various target molecules, including proteins, small molecules, and cells. Compared with antibodies, nucleic acid aptamers have several unique advantages, such as ease of synthesis and modification, high stability, and low immunogenicity. When using nucleic acid aptamers instead of antibodies, the nucleic acid aptamer can also be linked to the donor molecule through specific chemical modification or connection methods to achieve target binding and energy transfer structure construction. For example, biotin can be modified at one end of the nucleic acid aptamer and linked to the donor molecule through a biotin-avidin system; or the nucleic acid aptamer can be directly covalently linked to the donor molecule using a chemical crosslinker.

[0042] In one specific embodiment, based on the specific properties of biotin molecules required for the biotinylation process, the first biotin 120 is NHS-PEG4-Biotin, and the second biotin 230 is Sulfo-NHS-Biotin. The NHS (N-hydroxysuccinimide) group in NHS-PEG4-Biotin is a highly reactive ester group that can covalently react with molecules containing amino groups (-NH2), such as proteins and antibodies, to form stable amide bonds. The reaction conditions are relatively mild and proceed rapidly at near-physiological pH. The reaction is highly specific and effectively avoids unwanted side reactions with other functional groups. The PEG4 (polyethylene glycol 4 monomer units) chain segment has excellent biocompatibility and water solubility, increasing the solubility of the biotinylated molecules and ensuring more stable dispersion in solution. Furthermore, the PEG chain offers a certain degree of flexibility and steric hindrance, preventing aggregation of biotinylated molecules and reducing the possibility of nonspecific binding, thereby improving detection accuracy and sensitivity. In addition, the length of PEG4 is moderate, which can provide sufficient space and will not affect the binding affinity between biotin and avidin due to excessive chain length.

[0043] Sulfo-NHS-Biotin incorporates a sulfonic acid group (-SO3H) ortho-to the NHS group, making it more water-soluble and highly stable and active in aqueous solutions. During the biotinylation reaction, Sulfo-NHS-Biotin can also covalently react with amino-containing molecules to form stable amide bonds. Furthermore, the presence of the sulfonic acid group makes Sulfo-NHS-Biotin more adaptable to pH values ​​during the reaction and maintains high reactivity over a wide pH range (typically pH 6-9), providing greater flexibility in optimizing biotinylation reaction conditions. Furthermore, the sulfonic acid group can also reduce nonspecific adsorption between the biotinylated molecule and cell surfaces or other biomacromolecules, further reducing background signal and improving detection specificity.

[0044] In a specific probe system of the present application, biotin NHS-PEG4-Biotin and biotin Sulfo-NHS-Biotin are used in the biotinylation steps during the formation of the first and second complex solutions, respectively, based on their respective properties. When biotinylating quantum dots (QDs), considering the potential steric hindrance and aggregation issues on the QD surface, the choice of NHS-PEG4-Biotin can utilize the spatial spacing effect of its PEG chain to uniformly modify biotin on the QD surface, avoiding aggregation of the QDs while ensuring good binding ability between biotin and the subsequently bound avidin. When biotinylating the detection antibody 210, Sulfo-NHS-Biotin's good water solubility and wide pH adaptability enable it to efficiently bind to the amino groups on the antibody molecule under mild conditions, achieving stable biotinylation modification of the antibody without affecting the antibody's biological activity and specificity.

[0045] The present application further provides a method for preparing a FRET fluorescent probe with a controllable configuration, comprising: A first biotin 120 is coupled to the surface of the nanoluminescent material 110 to obtain a first conjugate; The first conjugate is mixed with the multisite avidin 130 and reacted to obtain a first complex 100, wherein the number of sites of the multisite avidin 130 is n, and the molar ratio of the first conjugate to the multisite avidin 130 is (n-1):1; The detection antibody 210 is coupled to an acceptor dye molecule to obtain a second conjugate, and then the second conjugate is mixed with a second biotin 230 and reacted to obtain a second complex 200, wherein, during the coupling process, the molar amount of the acceptor dye molecule is 5-10 times the molar amount of the detection antibody 210, and the molar amount of the second biotin 230 is 3-5 times the molar amount of the second conjugate.

[0046] First, a suitable nanoluminescent material 110 is selected as the basic building block. Nanoluminescent materials 110 are available in a variety of types, including quantum dots, quantum rods, upconversion nanoparticles, organic fluorescent dyes, carbon quantum dots, or metal nanoclusters. After selecting the nanoluminescent material 110, a first biotin 120 is coupled to its surface using a chemical coupling method. Reaction conditions, such as reaction temperature, pH value, reaction time, and reactant concentration, are controlled to ensure that the first biotin 120 is stably coupled to the surface of the nanoluminescent material 110. After coupling, to reduce interference from nonspecific binding on subsequent experiments, an appropriate blocking agent is used to block unreacted sites on the surface of the nanoluminescent material 110, thereby obtaining a first conjugate with a specific biotin modification.

[0047] Next, the prepared first conjugate is mixed and reacted with multisite avidin 130. Multisite avidin 130 is a protein molecule with multiple binding sites, assuming the number of sites is n. During the reaction, the molar ratio of the first conjugate to multisite avidin 130 is controlled to (n-1):1. This ratio ensures the presence of a vacant site on multisite avidin 130 and avoids insufficient binding or waste due to improper reactant ratios.

[0048] Then, the second conjugate and the second complex 200 are prepared. The detection antibody 210 is first coupled to the receptor dye molecule. During the coupling process, the molar amount of the receptor dye molecule is set to 5-10 times the molar amount of the detection antibody 210 to achieve sufficient labeling of the detection antibody 210 with the receptor dye molecule and ensure that a sufficient number of receptor dye molecules can bind to each detection antibody 210 molecule, thereby enhancing the fluorescence signal intensity. By optimizing the coupling reaction conditions, such as selecting a suitable coupling agent and controlling the reaction temperature and time, a stable covalent bond is formed between the receptor dye molecule and the detection antibody 210 molecule, thereby obtaining the second conjugate. Subsequently, the second conjugate is mixed and reacted with the second biotin 230. To enable the second biotin 230 to fully modify the second conjugate and enhance its interaction with subsequent binding molecules, the molar amount of the second biotin 230 is set to 3-5 times the molar amount of the second conjugate. By controlling the reaction conditions, the second biotin 230 is specifically bound to the second conjugate, ultimately obtaining the second complex 200.

[0049] The first biotin 120 is connected to the surface of the nanoluminescent material 110 by a chemical coupling method, providing a binding site for subsequent specific binding with the multi-site avidin 130. The stable coupling of the first biotin 120 and the nanoluminescent material 110 ensures that it will not fall off easily during the subsequent preparation and use process, thereby ensuring the stability and reliability of the fluorescent probe. At the same time, the unreacted sites on the surface of the nanoluminescent material 110 are blocked, which can effectively reduce the occurrence of non-specific binding. In biological detection, non-specific binding will lead to an increase in background signal and reduce the sensitivity and specificity of detection. By blocking the unreacted sites, the background noise can be reduced and the signal-to-noise ratio of the fluorescent probe can be improved, thereby more accurately detecting the target molecule. The method of precisely controlling the reaction ratio ensures that the binding sites of the multi-site avidin 130 are fully utilized, while avoiding insufficient binding or waste caused by improper reactant ratios, thereby improving the reaction efficiency and product purity.

[0050] After the preparation of the first complex 100 is complete, a verification step is further performed to ensure that the structure of the first complex 100 conforms to the intended design and to guarantee the accuracy and reliability of subsequent fluorescent probe assembly and functional realization. Specifically, two effective analytical methods can be used to characterize the properties of the first complex 100.

[0051] On the one hand, the mobility of the first complex 100 can be tested using an electrophoretic mobility assay. Electrophoretic mobility assays are based on the migration characteristics of molecules under the influence of an electric field. Molecules of different structures and sizes migrate at different rates in an electric field. Because the first complex 100 has a specific structural composition, including a key feature called a vacant site, the presence of the vacant site affects the overall charge distribution and molecular conformation of the first complex 100, thereby causing its mobility in an electric field to differ from that of a fully saturated complex. By precisely controlling electrophoresis experimental conditions, such as electric field strength, buffer composition and pH, and electrophoresis time, a sample of the first complex 100 is placed in an electrophoresis system for separation and analysis. Using appropriate staining methods or fluorescent labeling techniques, the first complex 100 is visualized, its migration distance in the electric field is measured, and compared with the mobility of known standard molecules to accurately determine the mobility of the first complex 100. By comparing the theoretically predicted value with the actual measured value, if the actual mobility falls within the range predicted based on the structural model containing the vacant site, the presence of a vacant site in the first complex 100 can be preliminarily inferred.

[0052] Alternatively, the particle size of the first complex 100 can be characterized using dynamic light scattering. Dynamic light scattering analyzes particle size and distribution by measuring the intensity fluctuations of light scattered by particles in a sample. The presence of vacant sites in the first complex 100 can affect its overall spatial structure and intermolecular interactions. In a dynamic light scattering experiment, a sample of the first complex 100 is dispersed in an appropriate solvent to ensure uniform dispersion and an appropriate concentration. A dynamic light scattering instrument then illuminates the sample with a laser, and the intensity of the scattered light is measured over time. Data analysis software processes the scattered light intensity fluctuation data to calculate the hydrodynamic radius of the first complex 100, i.e., the particle size. The measured particle size is then compared with the expected particle size of the first complex 100, calculated based on a theoretical model, for both the presence and absence of vacant sites. If the measured particle size matches the theoretical particle size for the presence of vacant sites, the presence of vacant sites in the first complex 100 is further confirmed.

[0053] In the preparation process of fluorescent probes, the purification step is a key step to ensure quality and performance. Specifically, after successfully obtaining the first conjugate, the first complex 100, the second conjugate, and the second complex 200, a purification operation is performed to remove residual unreacted substances in the solution.

[0054] Regarding the purification of the first conjugate, considering that it is the product obtained after the first biotin 120 is coupled to the surface of the nanoluminescent material 110, unconjugated first biotin 120, excess coupling reagent, and other impurities may be present in the solution. A method combining centrifugation and dialysis can be used. Due to the difference in particle size and density between the nanoluminescent material 110 and the first conjugate, high-speed centrifugation causes small molecules such as unreacted first biotin 120 and coupling reagent to precipitate or suspend in the upper layer of the solution, while the first conjugate forms a precipitate or aggregates in a specific area, thereby achieving preliminary separation. The first complex 100 is obtained by the reaction of the first conjugate with the multi-site avidin 130. In addition to the unreacted first conjugate and multi-site avidin 130, the solution may also contain byproducts produced during the reaction. In this case, gel filtration chromatography can be used for purification.

[0055] The second conjugate is the product of the coupling between the detection antibody 210 and the receptor dye molecule. Impurities such as unconjugated detection antibody 210, receptor dye molecules, and catalysts used in the coupling process may be present in the solution. Affinity chromatography can be used for purification. Since the detection antibody 210 typically has a specific binding site for the antigen 400, an antigen 400 or an anti-antibody that specifically binds to the detection antibody 210 can be selected as an affinity ligand and immobilized on a chromatography medium to prepare an affinity chromatography column. The second conjugate solution is loaded onto the affinity chromatography column. Unconjugated detection antibody 210, receptor dye molecules, and other impurities will flow directly through the column, while the second conjugate will specifically bind to the affinity ligand and be retained on the column. An appropriate elution buffer is then used to adjust the binding conditions and elute the second conjugate from the column, thereby obtaining a highly pure second conjugate. The second complex 200 is obtained by reacting the second conjugate with the second biotin 230. Unreacted second conjugate and second biotin 230 may be present in the solution. At this point, purification can be performed again by combining centrifugation with ultrafiltration.

[0056] In the preparation process of the fluorescent probe, to further improve its performance and stability and ensure that it maintains excellent properties and reliable detection results during subsequent storage, transportation, and actual detection applications, this application specifically adds a key step to the preparation process: precisely adding a stabilizer of appropriate type and concentration to the solution containing the first complex 100 and the solution containing the second complex 200. Specifically, for the solution containing the first complex 100, since the first complex 100 is a complex structure system formed by coupling the first biotin 120 to the surface of the nanoluminescent material 110 and then reacting with the multi-site avidin 130, the nanoluminescent material 110 in this system may be sensitive to environmental factors such as light, temperature, and solution pH. At the same time, the binding of the multi-site avidin 130 to the first conjugate may also exist in a certain dynamic equilibrium and be easily dissociated by external interference. Therefore, it is necessary to select a stabilizer that can work against these potential destabilizing factors. When adding, the amount of the carbohydrate stabilizer must be precisely controlled according to the volume and concentration of the first complex solution.

[0057] The solution containing the second complex 200 is formed by coupling the detection antibody 210 with an acceptor dye molecule, followed by a reaction with the second biotin 230. As a biomacromolecule, the activity of the detection antibody 210 is easily affected by factors such as temperature and solution ionic strength, which can reduce the activity. The acceptor dye molecule may also be susceptible to photofading, resulting in a decrease in fluorescence signal intensity. To address these issues, a composite stabilizer system consisting of a protein stabilizer (such as bovine serum albumin (BSA)) and an antioxidant (such as vitamin C or glutathione) can be added.

[0058] See also Figure 3 The present application also provides a method for detecting HER2 antigen 400 concentration, comprising the following steps: Adding the second complex solution of the aforementioned FRET fluorescent probe with controllable configuration to a sample containing the HER2 antigen 400 to be detected; Further adding the first complex solution of the aforementioned FRET fluorescent probe with controllable configuration to construct a FRET system; Perform fluorescence detection, record the donor and acceptor fluorescence emission intensities corresponding to the excitation wavelength, and calculate the energy transfer ratio or fluorescence lifetime change.

[0059] First, a certain volume of sample containing the HER2 antigen 400 to be detected is measured and transferred to a container. Then, the second complex solution in the fluorescent probe is slowly and evenly added to the sample containing the HER2 antigen 400 to be detected using a pipette. During the addition process, the container should be gently shaken to thoroughly mix the second complex solution with the sample, ensuring that the detection antibody 210 in the second complex 200 can quickly and effectively bind specifically to the HER2 antigen 400 in the sample.

[0060] After the second complex 200 and the HER2 antigen 400 have fully bound and reached dynamic equilibrium, the first complex solution of the fluorescent probe is slowly added to the mixed system. During this addition process, the container should be gently shaken to promote further interaction between the first complex 100 and the second complex 200 already bound to the HER2 antigen 400. The multisite avidin 130 in the first complex 100 specifically recognizes and binds to the second biotin 230 in the second complex 200, thereby bringing the first complex 100 (donor) and the second complex 200 (acceptor) within a suitable distance, successfully establishing a FRET system. When establishing a FRET system, it is important to control reaction conditions, such as temperature, pH, and reaction time.

[0061] After the FRET system is established, the mixed system is transferred to a fluorescence detection instrument for detection. Based on the characteristics of the fluorescent probe used, an appropriate excitation wavelength is selected to excite the sample. Under the influence of the excitation light, the donor (nanoluminescent material 110 in the first complex 100) is excited to produce fluorescence. Simultaneously, a portion of the energy is transferred to the acceptor (acceptor dye molecule in the second complex 200) through the FRET process, causing the acceptor to fluoresce. The fluorescence emission intensities of the donor and acceptor corresponding to the excitation wavelength are recorded using the fluorescence detection instrument. The energy transfer ratio (E) or fluorescence lifetime change is calculated based on the recorded donor and acceptor fluorescence emission intensities. The energy transfer ratio (E) can be calculated using a formula, and the fluorescence lifetime change can be measured and analyzed using time-resolved fluorescence detection. The FRET efficiency is assessed by comparing the donor fluorescence lifetime in the presence and absence of the acceptor. By establishing a quantitative relationship curve between the energy transfer ratio or fluorescence lifetime change and the HER2 antigen 400 concentration, the HER2 antigen 400 concentration in the sample can be accurately calculated based on the energy transfer ratio or fluorescence lifetime change values ​​obtained.

[0062] In the HER2 antigen 400 concentration detection process, a solid-phase immunoassay strategy can be further employed to achieve efficient, accurate, and stable capture of the HER2 antigen 400, thereby establishing a reliable detection foundation. Specifically, a well plate with excellent biocompatibility and chemical stability is first selected as the solid phase carrier. A common example is a 96-well polystyrene plate. Its surface has been specially treated to possess certain hydrophobic and charge properties, which facilitates subsequent antibody coating.

[0063] Next, the screened and purified HER2-specific antibody is appropriately diluted, typically using a buffer (such as phosphate-buffered saline (PBS)) containing a certain concentration of protein (such as bovine serum albumin (BSA)) to prevent nonspecific adsorption and aggregation of the antibody during the dilution process. The diluted HER2-specific antibody is then slowly and evenly added to each well of the plate in a precisely controlled volume (usually 50-100 μL per well). During the addition process, ensure that the antibody solution completely covers the bottom surface of the plate to avoid bubbles or uneven distribution.

[0064] Subsequently, the well plate with the antibody solution added is placed in a suitable environment for incubation. During the incubation process, the HER2-specific antibody is firmly bound to the well plate by physical adsorption (such as hydrophobic interaction, electrostatic interaction, etc.) and chemical coupling (such as covalent bond binding, which can be achieved by the reaction of active groups pre-treated on the well plate surface with amino or carboxyl groups on the antibody) through its antigen 400 binding site, thereby forming a uniform antibody coating layer inside the well plate.

[0065] Finally, to remove unbound antibodies and possible impurities, the plate is washed multiple times with a wash buffer (such as PBS containing 0.05% Tween-20, or PBST), typically 3-5 times. Each wash allows the wash buffer to remain in the wells for 1-2 minutes before discarding the wash buffer. After washing, the HER2 antigen 400 to be tested can be effectively captured on the plate through a specific binding reaction with the HER2-specific antibody coated on the plate, thereby forming a solid-phase immunobinding platform.

[0066] This application proposes a fluorescence resonance energy transfer (FRET) probe system based on a sparse biotinylation and limited streptavidin coupling strategy. The system consists of quantum dots (QDs) modified with only a small amount of biotin, a limited amount of introduced streptavidin molecules, and a detection antibody 210 labeled with both an acceptor dye molecule and a small amount of biotin. Through the high-affinity interaction between specific biotin and streptavidin (SA), the system can precisely construct a controllable self-assembly structure of "3QDs + 1 antibody."

[0067] The quantum dots are sparsely biotinylated to ensure a fixed number of donors (quantum dots) per assembly unit; limited amounts of streptavidin are introduced to construct an ordered core structure. Because the four streptavidin sites are arranged in a fixed pattern with a well-defined spacing (approximately 2.5-3 nm), the overall structural space is predictable. The specific biotin-streptavidin linkage replaces conventional random reactions such as N-hydroxysuccinimide (NHS)-based reactive coupling, resulting in a more uniform and controllable probe conformation and effectively avoiding the structural heterogeneity that can result from random coupling. Labeling the acceptor dye on the Fc region of the detection antibody 210, distal to the antigen 400 binding site, does not affect the specific binding of the detection antibody 210 to the antigen 400, while also facilitating the proximity of the acceptor dye to the streptavidin-bound quantum dots, further optimizing the spatial relationship between the donor and acceptor. This probe system effectively reduces background fluorescence and improves the predictability and reproducibility of detection signals, offering a new approach for highly sensitive, low-background dynamic molecular detection and bioimaging platforms.

[0068] Multiple quantum dots act as donors to simultaneously transfer energy to a single acceptor dye molecule, achieving signal amplification and thus improving detection sensitivity. This is particularly suitable for highly sensitive detection of low-abundance targets such as HER2, and can accurately capture signals even when biomarker expression levels are low. Combined with the four-site binding characteristics of streptavidin, this application adopts a strategy of limited coupling of biotinylated molecules to achieve regulation of the spatial structure of the probe. By controlling the spacing between the donor and acceptor, the interference of the background signal is effectively reduced, the specificity of the detection is improved, and the target biomarker and non-target substances can be more accurately distinguished. The FRET fluorescence signal of the present application has excellent dynamic response characteristics, which can change continuously with the dynamic changes in the HER2 expression level, showing good signal gradient and partial reversibility, so that the probe system can monitor the target expression status in real time and continuously. In addition, the present application uses a standardized biotin-streptavidin system to construct a probe with good modularity and platform compatibility. This design facilitates the rapid expansion of the technology to the detection of other biomarkers, and can be adapted to a variety of antibodies and fluorescent labels to meet different detection needs. The probe system can also be integrated into point-of-care (POCT) and microfluidics platforms, enabling more convenient and efficient testing. Furthermore, the detection process can be self-assembled in liquid phase, eliminating the need for cumbersome elution steps, greatly simplifying the operational workflow and making it suitable for multi-time point sampling and online continuous monitoring.

[0069] Some specific implementation methods will be further introduced below to further explain the technical solution of this application in detail.

[0070] QDs particles modified with surface carboxyl or amino groups were selected and reacted with NHS-PEG4-Biotin after EDC / NHS activation to achieve sparse biotinylation. During this process, the reaction ratio was controlled to an average of <2 biotins per quantum dot to prevent cross-linking and aggregation.

[0071] The specific implementation steps are as follows: First, the carboxylated QDs and EDC / NHS solution are placed in MES buffer (pH 6.0) and reacted for 30 minutes (room temperature, gentle shaking, and in the dark). Subsequently, the prepared NHS-PEG4-Biotin solution is immediately added to a molar concentration ratio of QDs:Biotin = 1:20. After gentle mixing, the mixture is incubated at room temperature in the dark for 1 hour. Then, Tris (pH 7.4) buffer is added to a final concentration of 20 mM and the reaction is continued at room temperature for 15 minutes to block unreacted sites. Next, unbound small molecules are removed by ultrafiltration, and the purified conjugate is washed multiple times with PBS or ultrapure water. The resulting purified conjugate is then resuspended in an appropriate volume of aqueous solution to obtain a QDs-Biotin complex stock solution of appropriate concentration. Finally, the density of conjugated biotin can be determined by changes in the complex's particle size, precipitation, turbidity, or sudden changes in fluorescence intensity.

[0072] When the quantum dots used are amino-group QDs, they can be directly coupled with NHS-PEG4-Biotin without the activation catalysis of EDC / NHS.

[0073] For storage, follow the following procedure: Add 0.01% Tween-20 to maintain good colloidal dispersion and 0.02% NaN₃ as a preservative. Mix thoroughly, aliquot into dark-protected brown centrifuge tubes, and store in the dark. It is recommended to use within two weeks. For long-term storage, resuspend the complex in PBS buffer containing 50% glycerol (v / v) (optionally, add 5% trehalose to enhance cryopreservation stability), aliquot in the dark, and store at -20°C or -80°C. Avoid repeated freeze-thaw cycles. During storage, observe the dispersion regularly. If necessary, restore homogeneity by gentle shaking or brief low-power sonication. Avoid aggregation or fluorescence intensity loss caused by vigorous shaking. Quantum dot material systems include, but are not limited to, CdSe / ZnS, CdTe / ZnS, CuInS₂ / ZnS, and InP / ZnS.

[0074] Next, SA pre-coupling is performed, and the reaction is mixed at a molar ratio of SA:QDs-biotin complex = 1:3, so that the three sites of streptavidin are respectively bound to three QDs-biotin complexes, leaving only one site unbound. The specific implementation steps are as follows: First, take the required amount of biotinylated QDs and place them in a low-adsorption EP tube. Then, add 1 / 3 molar equivalent of freshly prepared SA, immediately rotate slowly or gently mix, and incubate at room temperature and in the dark for 30 minutes; after the reaction is complete, ultrafiltration centrifugation is used to remove and purify residual unconjugated free SA, polymers, and small molecule biotin substances; finally, the mobility of the QDs-biotin complex before and after SA modification is tested using electrophoretic mobility shift analysis to confirm the formation of the "3QDs-1SA" complex.

[0075] Next, the fluorescent energy acceptor molecule is coupled based on the labeling order of "first coupling the acceptor dye molecule - then biotin". Since both mainly react with the exposed lysine residues in the Fc segment of the antibody, and the dye molecules have higher labeling efficiency and optical sensitivity, the order of dye first and biotin later can not only prevent the biotin label from "occupying" the dye site and affecting the dye uniformity, but also make it easier to control the low coupling degree of biotin, realizing the double modification of the detection antibody. The specific implementation steps are as follows: First, the concentration of the purchased commercial HER2 detection antibody is adjusted to 1-2 mg / mL with PBS buffer (pH 7.4), and then a 5-10-fold molar amount of the receptor dye molecule A-dyes is added. After reacting at room temperature in the dark for 30 minutes, free dye molecules are removed with Sephadex G-25. Finally, the purified A-dyes-antibody complex is tested for absorbance or OD at 280 nm and 650 nm using a UV-visible spectrophotometer to preliminarily verify the dye labeling. Secondly, the A-dyes-antibody complex (concentration adjusted to >1 mg / mL) is placed in PBS buffer (pH 7.4), and an appropriate amount of Sulfo-NHS-Biotin (prepared in DMSO or DMF as solvent, used immediately) is added to the reaction system at a molar ratio of 1:5 between the antibody and Sulfo-NHS-Biotin. After gently mixing, the reaction is allowed to react at room temperature in the dark for 30 minutes. After the reaction is complete, Tris buffer (pH 7.4) is added to the system to a final concentration of 20 mM and the reaction is continued for 10 minutes to block residual NHS active esters. Three buffer exchanges are then performed using ultrafiltration centrifuge tubes (50 kDa cutoff), followed by washing with PBS to completely remove free, unreacted biotin, yielding the dually modified antibody. Finally, the resulting dually modified antibody, A-dyes / Biotin-antibody, is resuspended in PBS buffer (pH 7.4) and supplemented with sodium azide (NaN3) at a final concentration of 0.02% as a preservative, along with 0.1% bovine serum albumin (BSA) by volume to prevent low-concentration antibody adsorption to the tube walls during storage. For long-term storage or freezing at -20°C, 50% v / v glycerol can be added to the above base buffer to enhance protein stability at low temperatures, prevent freeze-thaw damage, and avoid repeated freeze-thaw cycles. All storage operations should be carried out in the dark, and the solution should be dispensed into low-adsorption EP tubes and refrigerated (4°C) or frozen (-20°C or -80°C) as needed.

[0076] A-dyes / Biotin-detection antibody recognizes HER2. The specific binding of HER2 to the antibody determines the spatial position of A-dyes, ensuring the optimal distance and orientation of the FRET donor-acceptor, and providing a structural foundation for constructing an efficient and controllable spatially ordered FRET energy transfer system. The specific implementation steps are as follows: In PBS buffer (containing 0.1% BSA to reduce nonspecific adsorption), the A-dyes / Biotin-detection antibody complex is added to a sample containing the HER2 antigen to be detected (such as serum, cell lysate, or standard buffer). The sample involved in this process has been captured with a HER2-specific primary antibody previously coated in an ELISA plate to form a solid-phase immunobinding platform. Incubate in the dark for 60 minutes at 4°C or room temperature. Finally, wash at least three times with PBS buffer containing 0.1% Tween-20 (containing 0.5% BSA). Add 200 μL of wash solution to each well, let it stand for 2 minutes to fully remove unbound components, and then aspirate dry to remove unbound or non-specifically adsorbed A-dyes / Biotin-detection antibody complexes.

[0077] Next, the FRET system is constructed, utilizing the remaining site on the SA to bind to the A-dyes / Biotin-antibody, forming a "3QDs + 1Ab" structure and completing the QDs / A-dyes FRET system. The specific binding of SA to Biotin brings the A-dyes on the detection antibody closer to the QD donor surface, shortening the distance between the donor and acceptor and enhancing the FRET effect. This is further manifested as a typical fluorescence energy transfer phenomenon characterized by quenching of the donor-QD fluorescence signal (Fd) and enhancement of the A-dyes fluorescence (Fa). The specific steps are as follows: In PBS buffer (pH 7.4) containing 0.1% BSA, the pre-assembled SA-QD complex is slowly added to the aforementioned 96-well plate immobilization system at an appropriate molar ratio. Gently mix while adding, avoiding vigorous vortexing. A 20% excess of SA-QD complex is sufficient. Ensure that the amount added to each experimental well is consistent, ensuring that all A-dyes / Biotin-antibodies can bind to the QD-SA and maximize the FRET signal. In addition, titration experiments are required to verify the addition amount: 1. For a fixed HER2 concentration, QD-SA was serially diluted to 200% of the theoretically calculated amount; 2. The lowest QD-SA concentration was achieved at which the Fa / Fd signal ratio reached a plateau, indicating saturated binding sites. After mixing, the mixture was incubated at 4°C in the dark for 30 minutes. To remove unbound SA-QD complexes, effectively reduce background interference from SA-QDs, and ensure the specificity and accuracy of subsequent FRET signals, after the FRET reaction is complete, the system was gently washed with PBS buffer (pH 7.4) containing 0.1% Tween-20 and 0.1% BSA, avoiding vigorous shaking and pipetting to reduce nonspecific adsorption and preserve the formed "3QDs + 1Ab" structure. During the wash process, 200 μL of wash buffer was added to each well, incubated at room temperature for 2 minutes, and then aspirated to dryness. This wash was repeated three times, followed by a final rinse with PBS to remove residual surfactant.

[0078] Signal reading: Analyze the dynamic changes in HER2 concentration over time using fluorescence intensity ratios or lifetime changes. Higher target analyte concentrations lead to more frequent binding events and a more pronounced FRET effect, with changes in fluorescence readings showing a good correlation with HER2 concentration. The specific steps are as follows: After all reactions are completed, fluorescence detection can be performed directly in a 96-well black plate using a microplate reader. The donor and acceptor fluorescence emission intensities (Fd and Fa) corresponding to the excitation wavelength (e.g., QDs-λex = 488 nm) are recorded, and the energy transfer ratio (FRET ratio = Fd / Fa) or fluorescence lifetime change (via TCSPC) is calculated. Multiple controls should also be included during the experiment, including a negative control (buffer without HER2 to confirm nonspecific binding levels) and a positive control (a HER2 standard with a known concentration to verify detection sensitivity and linear range). In theory, in the absence of HER2, donor QDs cannot be incorporated, and no signal is observed after washing (theoretically, no QD residue remains, but in practice, very low background noise may still be observed due to nonspecific adsorption to the plate, denoted as Fd0). At low HER2 concentrations, due to the low amount of solid-phase complex formed (i.e., the amount of biotin present), only a small amount of QD-SA can subsequently bind to the complex. The fluorescence of the very few bound QDs is quenched by A-dyes, but the signal intensities of both are extremely low (close to background, denoted as Fd). At high HER2 concentrations, the amount of solid-phase complex formed increases (i.e., the amount of biotin present increases), resulting in a large number of QDs binding to the complex and undergoing FRET with A-dyes. Compared to the negative control, the donor QD fluorescence decreases (denoted as Fd), while the acceptor A-dyes fluorescence signal increases. The signal change can be quantitatively described using the QD fluorescence quenching rate: quenching rate = (Fd0 - Fd) / Fd0 × 100%. The higher the HER2 concentration, the greater the quenching rate. Therefore, based on the above theoretical foundation, if the same sample or cell population is tested at multiple time points, a trend curve of HER2 level changes can be obtained, thereby realizing continuous signal acquisition and dynamic monitoring of the target object, accurately reflecting the changing trend of HER2 expression levels during tumor development and therapeutic intervention, and providing reliable technical support for early screening, efficacy evaluation and personalized treatment.

[0079] As can be seen from the foregoing, this application focuses on fluorescent probes, their preparation methods, and HER2 antigen concentration detection methods, and provides a technical solution for dynamic monitoring of HER2 levels. The fluorescent probe is based on a sparse biotinylation and limited avidin coupling strategy. By controlling the spatial configuration and donor-acceptor spacing, it achieves signal amplification, background reduction, and dynamic response optimization. It can improve the sensitivity and specificity of HER2 detection in conjunction with the detection process, and has real-time, continuous monitoring capabilities, providing strong support for tumor diagnosis, treatment, and efficacy evaluation. Multi-site avidin retains an empty site that is not coupled to biotin, providing a site for subsequent assembly to ensure directionality and orderliness. The second biotin has a group that binds to the empty site, so that the second complex and the first complex are assembled into the overall structure of the fluorescent probe.

[0080] This application constructs a "multi-donor, one-acceptor" FRET structure. By combining a single antibody with multiple quantum dot donors, it can be flexibly adjusted according to actual needs, achieving effective signal amplification and being of great significance for the detection of low-abundance biomarkers. At the same time, avidin is used to achieve limited coupling of sparse biotinylated quantum dots with an acceptor dye-biotin dual-labeled detection antibody, controlling the spatial distance between the donor and acceptor, optimizing FRET efficiency, reducing nonspecific binding and background signal interference, and improving detection specificity.

[0081] During probe preparation, sparse biotinylation technology is used to regulate the density of quantum dot and detection antibody functional sites, control the amount of biotin modification, avoid steric hindrance and signal interference, and ensure antibody binding activity. In terms of spatial structural design, three of the four SA sites preferentially bind to the quantum dot-biotin complex, ensuring that one site binds to the biotinylated detection antibody. The Fc tail of the detection antibody is labeled with a fluorescent receptor, forming a highly efficient donor-acceptor configuration. Alternatively, a coated capture antibody can be introduced and pre-fixed on the surface of a solid support. Alternatively, alternative antibodies such as nucleic acid aptamers can be used as recognition and binding units.

[0082] The HER2 antigen concentration detection method involves adding the second complex solution to a sample containing the HER2 antigen to be detected, then adding the first complex solution to establish a FRET system. Fluorescence detection is performed and the energy transfer ratio or fluorescence lifetime change is calculated to calculate the HER2 antigen concentration in the sample. The detection process can utilize a solid-phase immunobinding strategy to improve the capture efficiency and stability of the HER2 antigen.

[0083] The above is only a specific implementation of the present application. Any other improvements made based on the concept of the present application are considered to be within the scope of protection of the present application.

Claims

1. A FRET fluorescent probe with a controllable configuration, characterized in that: including a first complex solution and a second complex solution; The first complex solution includes a first complex formed by coupling a nanoluminescent material, a first biotin and a multi-site avidin, wherein the multi-site avidin includes an empty site that is not coupled with biotin; The second complex solution includes a second complex formed by coupling a detection antibody, a receptor molecule dye and a second biotin, wherein the second biotin includes a group that can be bound to a vacant site.

2. The FRET fluorescent probe with controllable configuration according to claim 1, wherein The multi-site avidin is streptavidin, and the first biotin occupies three sites of streptavidin.

3. The FRET fluorescent probe with controllable configuration according to claim 1, wherein The method further comprises a coated capture antibody, which captures the HER2 antigen.

4. The FRET fluorescent probe with controllable configuration according to claim 3, wherein The first biotin is NHS-PEG4-Biotin, and the second biotin is Sulfo-NHS-Biotin.

5. A method for preparing a FRET fluorescent probe with a controllable configuration, characterized in that: include: Conjugating a first biotin on the surface of the nanoluminescent material to obtain a first conjugate; Mixing the first conjugate with multisite avidin and reacting them to obtain a first complex, wherein the number of sites of the multisite avidin is n, and the molar ratio of the first conjugate to the multisite avidin is (n-1):1; The detection antibody is coupled to an acceptor dye molecule to obtain a second conjugate, and the second conjugate is then mixed and reacted with a second biotin to obtain a second complex, wherein during the coupling process, the molar amount of the acceptor dye molecule is 5-10 times the molar amount of the detection antibody, and the molar amount of the second biotin is 3-5 times the molar amount of the second conjugate.

6. The method for preparing a FRET fluorescent probe with a controllable configuration according to claim 5, wherein: The method further includes a verification step of testing the mobility of the first complex by electrophoretic mobility analysis or characterizing the particle size of the first complex by dynamic light scattering to confirm that the first complex includes a vacant site.

7. The method for preparing a FRET fluorescent probe with a controllable configuration according to claim 5, wherein: The method comprises a purification step, wherein after obtaining the first conjugate, the first complex, the second conjugate or the second complex, unreacted substances in the solution are removed.

8. The method for preparing a FRET fluorescent probe with a controllable configuration according to claim 5, wherein: The method further comprises adding a stabilizer to the solution containing the first complex and the solution containing the second complex.

9. A method for detecting HER2 antigen concentration, characterized in that: The steps include: adding the second complex solution of the FRET fluorescent probe with controllable configuration according to any one of claims 1 to 4 to a sample containing the HER2 antigen to be detected; further adding the first complex solution of the FRET fluorescent probe with controllable configuration according to any one of claims 1 to 4 to construct a FRET system; Perform fluorescence detection, record the donor and acceptor fluorescence emission intensities corresponding to the excitation wavelength, and calculate the energy transfer ratio or fluorescence lifetime change.

10. The method for detecting HER2 antigen concentration according to claim 9, wherein: The HER2 antigen to be tested is captured by the HER2-specific antibody coated in the well plate to form a solid-phase immune binding platform.

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