Quantum dot-based FRET (Fluorescence Resonance Energy Transfer) fluorescent probe, preparation method thereof and AchR-Ab detection method

Abstract

The invention relates to a fluorescent probe based on a quantum dot fluorescence resonance energy transfer effect, a preparation method of the fluorescent probe and a method for detecting an acetylcholine receptor antibody by using the probe. The method comprises the following steps: preparing particle size monodisperse carrier microspheres through a template method, then carrying out surface modification on the carrier microspheres and quantum dots, so that the quantum dots are attached to the surfaces of the carrier microspheres through electrostatic self-assembly or hydrophobic driving assembly to form quantum dot microspheres; the preparation method comprises the following steps: preparing a quantum dot microsphere, performing carboxyl modification on the surface of the quantum dot microsphere, activating, coupling an acetylcholine receptor antigen to the surface of the quantum dot microsphere, and sealing a non-specific binding site by using a sealing agent to obtain a quantum dot microsphere labeled AchR antigen as a fluorescent probe. The immobilization of the cross-linking agent enhances the binding strength between the quantum dots and the carrier microspheres, and closes non-specific binding sites on the surfaces of the microspheres, thereby ensuring the structural stability of the probe. In addition, an AchR-Ab detection method using the quantum dot FRET fluorescent probe provided by the invention can realize quantitative detection of AchR-Ab concentration.

Description

Technical Field

[0001] This application relates to the field of biological detection technology, and particularly to a quantum dot FRET fluorescence probe, a preparation method thereof, and an AchR-Ab detection method. Background Art

[0002] As a core biomarker for autoimmune diseases such as myasthenia gravis (MG), the pathogenic mechanism of acetylcholine receptor antibody (AchR-Ab) has been deeply elucidated. This antibody is mainly of IgG1 and IgG3 subtypes, which cause damage to the postsynaptic membrane by activating the complement system, leading to the degradation and conformational change of acetylcholine receptor (AchR), and ultimately resulting in the disorder of neuromuscular junction signal transmission. Clinical studies have shown that the quantitative detection of AchR-Ab is of great value for the early diagnosis, disease stratification, and efficacy evaluation of MG.

[0003] Currently, the detection of AchR-Ab mainly relies on radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and electrochemiluminescence (ECL) technology. However, these methods all have limitations: RIA brings environmental pollution and operation risks due to radioactive labels; ELISA is inexpensive but has insufficient sensitivity and requires multiple incubation steps; although ECL improves the detection sensitivity, it relies on expensive instruments and reagents, and the detection cycle is relatively long. Therefore, it is necessary to further develop a detection scheme with high sensitivity, rapid response, low cost, and environmental friendliness. Summary of the Invention

[0004] The purpose of this application is to provide a quantum dot FRET fluorescence probe. By optimizing the specific preparation method and composition of the probe, high-sensitivity, rapid, reliable, and stable detection of acetylcholine receptor antibody (AchR-Ab) can be achieved. In addition, by improving the detection method, the concentration of AchR-Ab can be quantitatively analyzed. The kit with this fluorescence probe has the advantages of simple operation, short detection time, and no need for radioactive substances, and can be widely applied to clinical diagnosis and scientific research. The purpose of this application is achieved through the following technical solutions. A preparation method of a quantum dot FRET fluorescence probe of this application includes the following steps: Prepare carrier microspheres and quantum dots respectively; Perform surface modification on the carrier microspheres and / or the quantum dots; After the modification is completed, mix the carrier microspheres and the quantum dots in a solution so that the quantum dots adhere to the surface of the carrier microspheres to obtain quantum dot microspheres; Perform carboxyl modification on the surface of the quantum dot microspheres, activate the carboxyl groups, and couple the quantum dot microspheres with AchR antigen through the carboxyl activation method; Block the non-specific binding sites on the surface of the closed microspheres to obtain quantum dot microsphere-labeled AchR antigen as a fluorescent probe.

[0005] In one embodiment, the attachment of quantum dots on the surface of the microspheres is achieved by electrostatic interaction, and after the attachment is completed, a cross-linking agent is also used to cross-link and fix the quantum dots.

[0006] In one embodiment, before blocking the non-specific binding sites on the surface of the closed microspheres, it further includes a step of washing to remove the unbound AchR antigen.

[0007] In one embodiment, 3-mercaptopropionic acid is used to modify the carboxyl group of the quantum dots or succinic anhydride is used to modify the surface of the quantum dot microspheres, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are used to activate the carboxyl group.

[0008] The present application further provides a quantum dot FRET fluorescent probe, including a carrier microsphere, quantum dots, and AchR antigen; The quantum dots are attached to the surface of the carrier microspheres to form quantum dot microspheres, and the quantum dots are cross-linked and fixed by a cross-linking agent; The AchR antigen is conjugated to the surface of the quantum dot microspheres; Among them, the non-specific binding sites on the surface of the quantum dot microspheres are blocked.

[0009] In one embodiment, the carrier microsphere is a PS microsphere or a SiO2 microsphere, and the quantum dot material is selected from CdSe / ZnS or InP / ZnS.

[0010] In one embodiment, the carrier microspheres include magnetic nanoparticles.

[0011] In one embodiment, the fluorescent probe is suspended in a PBS buffer solution.

[0012] The present application also provides an AchR-Ab detection method, including: Using the aforementioned quantum dot FRET fluorescent probe to prepare an AchR antigen working solution, preparing a Cy5-labeled anti-human IgG working solution, and preparing a series of diluted gradient concentration AchR-Ab standard solutions; Adding the AchR antigen working solution to the test sample and the reference sample, mixing well and performing the first incubation; Adding the anti-human IgG working solution to the solution after the first incubation, mixing well and reacting, and then performing the second incubation; Washing to remove the unbound components; Measuring the fluorescence signals and FRET efficiency of the test sample and the reference sample; The FRET efficiency of the test sample is compared with that of the reference sample to obtain the concentration of AchR-Ab in the test sample.

[0013] In one embodiment, a microplate or microfluidic chip is used to measure the fluorescence signal.

[0014] Compared with the prior art, the present application has the following beneficial effects: In the present application, a quantum dot microsphere structure is first constructed in the fluorescent probe. The carrier microspheres are used as the matrix to uniformly load quantum dots and crosslink and fix them, which can improve the quantum dot loading density and the stability of the fluorescent probe. Through the high affinity of the Cy5-labeled secondary antibody (IgG) and the AchR-Ab antibody, a more efficient FRET energy transfer system is constructed to improve the FRET efficiency.

[0015] When using the fluorescent probe of the present application for detection, the homogeneous FRET detection method can be selected. This method does not require immobilization operation and is carried out in a homogeneous solution throughout the process, which can eliminate the interference of non-specific adsorption on the solid phase surface, is beneficial to reducing the background signal, and thus can realize the "mix-incubate-detect" one-step operation. The dynamic FRET signal change can be monitored in real time, shortening the detection time and cycle.

[0016] In addition, magnetic nanoparticles can be integrated into the carrier microspheres, and rapid separation of the complex can be achieved by applying an external magnetic field, reducing the number of washing times and the non-specific adsorption rate. Further, the fluorescent probe of the present application can be compatible with different platforms and is suitable for rapid, low-sample-volume, high-throughput, and automated detection.

[0017] Through the improvement of materials science and surface chemistry technology, the quantum dot FRET probe of the present application can couple quantum dots with specific antigens and combine the immune recognition mechanism to improve the signal-to-noise ratio during the detection process and achieve highly sensitive detection of AchR-Ab. Description of the Drawings

[0018] Figure 1 is a schematic structural diagram of a quantum dot FRET fluorescent probe in an embodiment of the present application; Figure 2 is a schematic diagram of a detection method of a quantum dot FRET fluorescent probe in an embodiment of the present application.

[0019] Description of the reference numerals: 100, quantum dot microsphere; 200, quantum dot; 300, AchR antigen. Detailed Description of the Embodiments

[0020] To make the above objects, features, and advantages of the present application more apparent and understandable, the following describes the specific embodiments of the present application in detail with reference to the accompanying drawings. It can be understood that the specific embodiments described herein are only used to explain the present application, rather than limiting the present application. Additionally, it should be noted that for the convenience of description, only the parts related to the present application rather than all the structures are shown in the drawings. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present application.

[0021] The terms "including" and "having" and any variations thereof in the present application are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or device that includes a series of steps or units is not limited to the listed steps or units, but optionally further includes steps or units not listed, or optionally further includes other steps or units inherent to these processes, methods, products, or devices.

[0022] Referring to "embodiments" herein means that the specific features, structures, or characteristics described in connection with the embodiments can be included in at least one embodiment of the present application. The phrase appears at various positions in the specification and does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. Those skilled in the art explicitly and implicitly understand that the embodiments described herein can be combined with other embodiments.

[0023] Quantum dot fluorescence resonance energy transfer (FRET) probes, as a new generation of biosensing technologies, are based on the unique optical properties of quantum dots (QDs), such as continuous excitation with broad-spectrum emission, photobleaching resistance, and size-dependent fluorescence tunability. Combining with the distance-sensitive energy transfer effect of the donor-acceptor pair in the FRET mechanism, such probes have achieved the monitoring of dynamic interactions of biomolecules. Their preparation process usually involves quantum dot structure optimization, surface functionalization modification, and precise assembly of FRET pairs. For example, a donor-acceptor complex is constructed through molecular beacons or biotin-avidin systems. In the detection of myasthenia gravis-related antibody AchR-Ab, the quantum dot FRET probe can specifically recognize the target antibody, triggering fluorescence signal conversion through conformational changes or spatial distance changes induced by antibody binding, thereby achieving highly sensitive and highly selective quantitative analysis in complex biological samples. The design principle, controllable synthesis strategy of the probe and its application in the detection of neuroimmune disease markers will be systematically described below. The preparation method of the quantum dot FRET fluorescence probe in a preferred embodiment of this application includes the following steps: preparing carrier microspheres and quantum dots 200 respectively; performing surface modification on the carrier microspheres and / or the quantum dots 200; after the modification is completed, mixing the carrier microspheres with the quantum dots 200 in a solution to make the quantum dots 200 adhere to the surface of the carrier microspheres, obtaining quantum dot microspheres 100; performing carboxyl modification on the surface of the quantum dot microspheres 100, activating the carboxyl groups, and coupling the quantum dot microspheres 100 with AchR antigen 300 through a carboxyl activation method; blocking non-specific binding sites on the surface of the microspheres to obtain the AchR antigen 300 labeled with quantum dots 200 as a fluorescence probe.

[0024] The preparation method of the present application first uses the template method to prepare monodisperse carrier microspheres, with the specific particle size selected in the range of 200 - 500 nm. The microspheres can include PS microspheres and SiO2 microspheres. Synthesis of PS microspheres: Through the emulsion polymerization of styrene (St) under nitrogen protection (80 °C, 6 h), high specific surface area polymer microspheres are obtained by centrifugal purification. Synthesis of SiO2 microspheres: Using ammonia water to catalyze the sol - gel reaction of TEOS (6 h), silica microspheres with surface hydroxylation are obtained by ethanol washing. Modify the surface of quantum dots 200, including hydrophilic transformation. Specifically, for example, use 3 - mercaptopropionic acid (MPA) or polyvinylpyrrolidone (PVP) to perform carboxyl / amino modification on CdSe / ZnS (or InP / ZnS) quantum dots 200. Through ultrasonic treatment, the monodispersity of quantum dots 200 in deionized water is achieved (particle size < 10 nm), and the absolute value of Zeta potential > 30 mV. Surface charge regulation ensures the colloidal stability of quantum dots 200 at physiological pH, and the carboxyl / amino functional groups provide active sites for subsequent coupling reactions. Next, controllable assembly of quantum dots 200 - microspheres can be selected, either electrostatic self - assembly or hydrophobic - driven assembly. Under optimized ratios (5 mg / 10 mL of PS microspheres, 1 mg / mL of quantum dots 200), in electrostatic self - assembly, negatively charged PS microspheres (-40 mV) are combined with amino - modified quantum dots 200 (+35 mV) through electrostatic adsorption, and in hydrophobic - driven assembly, SiO2 microspheres are modified with APTES to introduce amino groups (+45 mV), and combined with hydrophobic ligand quantum dots 200 through hydrophobic interaction. Next, carboxyl functionalization and activation are carried out. Surface carboxylation is to introduce carboxyl groups on the surface of quantum dot microspheres 100 through succinic anhydride (SAA), and EDC / NHS activation is to activate the carboxyl groups in the MES buffer system (pH 6.0) to form highly reactive ester groups. The activated carboxyl groups using EDC / NHS react with the amino groups (-NH2) of AchR antigen 300 to form amide bonds, and non - specific blocking is carried out using 1% BSA for blocking treatment. The nanoscale carrier effect of quantum dot microspheres 100 enhances the fluorescence signal intensity, the FRET efficiency is optimized, and cross - linker immobilization (glutaraldehyde / EDC - NHS) keeps the structure of quantum dot microspheres 100 stable in the buffer solution.

[0025] Specifically, the attachment of quantum dots 200 on the surface of the microspheres is achieved through electrostatic interaction. After the attachment is completed, a cross - linker is also used to cross - link and fix quantum dots 200, blocking the non - specific binding sites on the surface of the microspheres and reducing non - specific adsorption. Before blocking the non - specific binding sites on the surface of the microspheres, it also includes the step of washing to remove the unbound AchR antigen 300.

[0026] In a specific embodiment of the present application, succinic anhydride is used to modify the surface of the quantum dot microsphere 100 with carboxyl groups, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are used to activate the carboxyl groups. Through experiments, it is found that the quantum dot FRET fluorescence probe formed by the above two materials is the most stable and the quantum dots 200 are more evenly distributed.

[0027] Please refer to Figure 1 , the present application further provides a quantum dot FRET fluorescence probe, including a carrier microsphere, quantum dots 200, and AchR antigen 300. The quantum dots 200 are attached to the surface of the carrier microsphere to form a quantum dot microsphere 100, and the quantum dots 200 are crosslinked and fixed through a crosslinking agent. The AchR antigen 300 is coupled to the surface of the quantum dot microsphere 100. Among them, the non-specific binding sites on the surface of the quantum dot microsphere 100 are blocked.

[0028] The structural composition of the quantum dot FRET fluorescent probe includes a carrier microsphere, quantum dots 200, and acetylcholine receptor (AchR) antigen. Specifically, the quantum dots 200 are attached to the surface of the carrier microsphere by physical adsorption or chemical bonding to form a microsphere structure modified with quantum dots 200 (abbreviated as "quantum dot microsphere 100"). To ensure the stability and uniform distribution of the quantum dots 200 on the microsphere surface, a crosslinking agent is used to crosslink and fix the quantum dots 200 to enhance their binding strength and resistance to environmental interference. Further, the AchR antigen 300 is precisely fixed on the surface of the quantum dot microsphere 100 through a specific coupling reaction to form a probe structure with specific biological recognition function. In addition, to reduce the influence of non-specific adsorption on the probe performance, a blocking agent is used to block the sites on the surface of the quantum dot microsphere 100 that are not occupied by the quantum dots 200 and the AchR antigen 300, thereby effectively reducing the background signal and improving the specificity and sensitivity of the probe. By crosslinking and fixing the quantum dots 200 attached to the surface of the carrier microsphere with a crosslinking agent, the binding strength between the quantum dots 200 and the carrier microsphere can be enhanced, preventing the detachment or aggregation of the quantum dots 200 during subsequent processing or application. At the same time, crosslinking and fixing also helps to maintain the uniform distribution of the quantum dots 200 and ensure the stable and consistent fluorescence performance of the probe. The crosslinking agent can react with the functional groups on the surface of the quantum dots 200 to form stable chemical bonds, thereby firmly fixing the quantum dots 200 on the surface of the carrier microsphere. Precisely coupling the AchR antigen 300 on the surface of the quantum dot microsphere 100 enables the probe to specifically recognize acetylcholine receptors. The AchR antigen 300 reacts with specific functional groups (such as carboxyl groups, amino groups, etc.) on the surface of the quantum dot microsphere 100 to form stable covalent bonds or ionic bonds, thereby achieving the coupling of the antigen. By blocking the sites on the surface of the quantum dot microsphere 100 that are not occupied by the quantum dots 200 and the AchR antigen 300 with a blocking agent, the non-specific adsorption of the probe to other non-target molecules in the biological sample can be effectively reduced, helping to reduce the background signal and improve the specificity and sensitivity of the probe. The blocking agent usually has chemical properties or functional groups similar to those on the surface of the carrier microsphere and can bind to the unoccupied sites, thereby occupying these sites and preventing the non-specific adsorption of other molecules.

[0029] In the fluorescence probe based on the quantum dot fluorescence resonance energy transfer (FRET) effect proposed in this application, the carrier microspheres are selected from polystyrene (PS) microspheres or silica (SiO2) microspheres. Both of these microsphere materials have good biocompatibility, chemical stability, and are easy to surface modify, providing an ideal platform for the immobilization of quantum dots 200 and subsequent bioconjugation reactions. At the same time, the quantum dot 200 material is selected from cadmium selenide / zinc sulfide (CdSe / ZnS) or indium phosphide / zinc sulfide (InP / ZnS) core-shell structure quantum dots 200. Both of these quantum dot 200 materials have excellent optical properties, including high fluorescence quantum yield, narrow emission spectral bandwidth, and good photostability, which can meet the requirements of high-sensitivity and high-specificity biological detection.

[0030] Furthermore, this application also incorporates magnetic nanoparticles inside or on the surface of the microspheres, so that the carrier microspheres not only retain the excellent properties of the original materials, such as good biocompatibility, chemical stability, and easy surface modification, but also endow them with magnetic responsiveness, providing great convenience for the manipulation, separation, and enrichment of the probe. Enhanced magnetic responsiveness: The introduction of magnetic nanoparticles enables the carrier microspheres to have magnetic responsiveness, and can achieve rapid and efficient manipulation and separation under the action of an external magnetic field. This property has broad application prospects in the fields of biological detection, drug screening, etc., such as rapidly enriching target molecules through magnetic separation technology to improve the detection sensitivity and efficiency. The addition of magnetic nanoparticles provides an additional functional dimension for the fluorescence probe, enabling it to be integrated with other magnetic materials or devices to achieve more complex biological detection and analysis tasks. By reasonably selecting the type, size, and surface modification method of magnetic nanoparticles, it is possible to ensure their good compatibility with the biological system and avoid damage to biomolecules and cells. At the same time, the introduction of magnetic nanoparticles does not significantly affect the original optical properties and stability of the carrier microspheres.

[0031] After the assembly and functional modification of the carrier microspheres (including magnetic nanoparticles), quantum dots 200, and acetylcholine receptor (AchR) antigen, they are uniformly and stably suspended in phosphate buffered saline (PBS). PBS buffer, as a widely used biological buffer system, has an appropriate pH value range (usually 7.2 - 7.6), moderate ionic strength, and good biocompatibility.

[0032] Please refer to Figure 2 , this application further provides a method for detecting acetylcholine receptor antibody (AchR-Ab) with high sensitivity and high specificity. This method integrates the fluorescence probe technology based on the quantum dot fluorescence resonance energy transfer (FRET) effect, and the specific operation steps are as follows: First, using the aforementioned quantum dot FRET fluorescence probe technology, the acetylcholine receptor (AchR) antigen is specifically immobilized on the surface of the fluorescence probe to form the AchR antigen 300 working solution. The fluorescence probe in this working solution can specifically bind to AchR-Ab under specific conditions and generate detectable fluorescence signal changes through the FRET effect. Next, prepare the Cy5-labeled anti-human IgG working solution. At the same time, use the Cy5 fluorescent dye to label the antibody against human IgG to prepare the Cy5-labeled anti-human IgG working solution. This working solution is used to recognize and bind to AchR-Ab after it binds to the AchR antigen 300, further amplifying the fluorescence signal and improving the detection sensitivity.

[0033] To establish a quantitative detection standard curve, prepare a series of AchR-Ab standard solutions with known concentrations that cover the expected detection range. Add an equal amount of the AchR antigen 300 working solution to the test samples to be measured and the reference samples with known concentrations. After thorough mixing, perform the first incubation. The incubation conditions (such as temperature and time) are optimized to ensure that the AchR antigen 300 binds fully to AchR-Ab in the samples. After completing the first incubation, add the Cy5-labeled anti-human IgG working solution to each sample solution, mix again, and react for a period of time before performing the second incubation. This incubation promotes the specific binding of the Cy5-labeled anti-human IgG to AchR-Ab bound to the AchR antigen 300, forming a fluorescent complex and further amplifying the fluorescence signal.

[0034] Washing and removal of unbound components: After incubation, remove unbound components such as fluorescence probes, antigens, and antibodies through the washing step to reduce background signal interference and improve detection accuracy. Use a highly sensitive fluorescence detection device to measure the fluorescence signal intensity and FRET efficiency of each sample solution. The FRET efficiency, as an indicator of the energy transfer efficiency between fluorescence probes, can reflect the binding degree of AchR-Ab to the AchR antigen 300. Based on the concentration of the AchR-Ab standard solution and the corresponding FRET efficiency values, establish a quantitative detection standard curve. By comparing the FRET efficiency of the test sample with that of the reference sample, calculate the concentration of AchR-Ab in the test sample using the standard curve.

[0035] The specific binding between the AchR antigen 300 and AchR-Ab, as well as the specific recognition between the Cy5-labeled anti-human IgG and AchR-Ab, ensure the high specificity of the detection method and reduce the interference of non-specific binding. The specific interaction between antigen and antibody is based on the complementarity of molecular structures. This interaction has high selectivity and affinity. By optimizing the selection of antigens and antibodies and the labeling conditions, the specificity of the detection method can be further improved. By establishing a quantitative relationship between the AchR-Ab standard solution and the FRET efficiency, the quantitative detection of AchR-Ab concentration is achieved, providing an accurate basis for clinical diagnosis and treatment. Under specific conditions, the binding degree between AchR-Ab and the AchR antigen 300 is proportional to the FRET efficiency. By measuring the FRET efficiency values of AchR-Ab standard solutions at different concentrations, a quantitative detection standard curve can be established, and then the concentration of AchR-Ab in unknown samples can be calculated.

[0036] To more conveniently use the AchR-Ab detection method of this application, this application directly provides a kit in specific applications, which includes the working solution of the AchR antigen 300 labeled with quantum dot microspheres 100, the working solution of anti-human IgG labeled with cyanine 5 (Cy5), the AchR-Ab standard product and the quality control product working solution, and the washing solution (PBS buffer solution with 0.05% Tween-20), providing all the working solutions required by the method of this application in one kit.

[0037] Specifically, the fluorescence signal is measured by using a microplate or a microfluidic chip. In the detection process of acetylcholine receptor antibody (AchR-Ab), in order to achieve efficient and accurate measurement of the fluorescence signal, the present invention innovatively uses a microplate or a microfluidic chip as the detection platform. After completing the pretreatment steps such as incubation and washing of the sample and the reagent, the treated solution is transferred to the microplate wells pre-coated with detection conditions adapted, or is distributed and guided through the precise channel network of the microfluidic chip, so that the sample solution fully contacts the detection area and a fluorescence signal reaction occurs. Subsequently, a highly sensitive fluorescence detection instrument, such as a fluorescence microplate reader (for microplates) or a laser-induced fluorescence detector (for microfluidic chips), is used to quickly and accurately collect and analyze the fluorescence signal in the microplate wells or specific areas of the microfluidic chip.

[0038] Both the microplate and the microfluidic chip possess excellent optical properties, which can reduce background noise interference, improve the collection efficiency of fluorescence signals, and thus enhance the sensitivity and accuracy of detection. The design of the smoothness and transparency of the microplate well walls, as well as the precise processing of the internal channels of the microfluidic chip, ensure low loss of fluorescence signals during transmission. Meanwhile, the chemical modification on the chip surface can effectively reduce non-specific adsorption and further lower the background signal. With the advantage of multi-well parallel detection, the microplate can process a large number of samples simultaneously, improving the detection throughput; while the microfluidic chip, through an integrated design, integrates multiple detection steps on a single chip to achieve continuous and rapid processing of samples, also supporting high-throughput detection requirements. The multi-well structure of the microplate allows independent detection of multiple samples simultaneously, reducing operation time and cost; the microfluidic chip realizes automatic distribution, mixing, and reaction of samples through a microchannel network, greatly shortening the detection cycle and improving the detection efficiency. Specific embodiments Some specific implementation manners will be further introduced below to further elaborate on the technical solutions of this application. Specific Embodiment 1 (1) Preparation of quantum dot microspheres: The PS microsphere template method is adopted: First, prepare a 10% mass fraction styrene (St) monomer solution, add 1% initiator azobisisobutyronitrile (AIBN), and perform emulsion polymerization at 80 °C for 6 h under nitrogen protection to obtain PS microspheres with a diameter of 200 - 500 nm. Remove the unreacted monomer by centrifugation and water washing, and dry in vacuum at 60 °C for 12 h. Immediately afterwards, carboxyl or amino modification is performed on the surface of CdSe / ZnS (or InP / ZnS) quantum dots with 3-mercaptopropionic acid (MPA) or polyvinylpyrrolidone (PVP) to improve water solubility and electrostatic stability, and ultrasonic treatment is used to ensure uniform dispersion of the quantum dots. Then, disperse 5 mg of PS microspheres in 10 mL of deionized water and sonicate for 10 min. Under stirring conditions, slowly add 10 mL of the modified quantum dot solution (1 mg / mL), and incubate at room temperature for 2 - 4 h to allow the quantum dots to attach to the microsphere surface through electrostatic adsorption or hydrophobic interaction. Separate the microspheres by centrifugation (6000 rpm, 10 min), and wash 3 times with deionized water to remove the unbound quantum dots to achieve the loading of quantum dots. To enhance the stability of the quantum dots, subsequently treat with a cross-linking agent (0.1% glutaraldehyde or 0.05% EDC / NHS) for 30 min, and then centrifuge and wash. The final product is dried in vacuum at 60 °C for 12 h and stored in a light-protected environment.

[0041] (2)Preparation of quantum dot microsphere-labeled AchR antigen: Quantum dot microspheres with an emission wavelength of 550 - 600 nm were selected and coupled with AchR antigen (purchased from a biological reagent company) through a carboxyl activation method. ① First, equimolar amounts of (3-mercaptopropyl)trimethoxysilane (MPTS) and succinic anhydride (SAA) were dissolved in N,N-dimethylformamide (DMF) and stirred for 4 h. Then, a SiO2@QDs quantum dot microsphere solution uniformly dispersed in DMF was added to the reaction system, and stirring was continued for 5 h under the same conditions. After the reaction was completed, the microspheres were collected by centrifugation (8000 rpm, 10 min) and washed 3 times with deionized water to finally obtain carboxyl-modified SiO2@QDs quantum dot microspheres. ② Then, 5 mg of carboxylated quantum dot microspheres were added to 5 mL of MES buffer (2-(N-morpholino)ethanesulfonic acid buffer, 0.1 M, pH 6.0); 2 mg of EDC and 5 mg of NHS were added, and the mixture was stirred at room temperature for 30 min to activate the carboxyl groups; after the reaction was completed, the free EDC / NHS was removed by centrifugation (8000 rpm, 10 min), and the carboxyl groups were activated after washing 2 times with PBS. ③ Finally, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were used to activate the carboxyl groups on the surface of the quantum dots and covalently couple them with the amino groups of the AchR antigen: 1 mg of AchR antigen was added to 5 mL of PBS buffer (pH 7.4), and it was added to the quantum dot microsphere solution treated with EDC / NHS, and the mixture was stirred at room temperature for 2 - 4 h to covalently couple the AchR antigen to the surface of the microspheres; the microspheres were collected by centrifugation (8000 rpm, 10 min) and washed 3 times with PBS to remove the unbound AchR antigen; the non-specific binding sites on the surface of the microspheres were blocked by gently stirring with a 1% BSA (bovine serum albumin) solution at 4 °C for 2 h to reduce non-specific adsorption; then, after washing 2 times by centrifugation (6000 rpm, 10 min), the microspheres were resuspended in PBS buffer to obtain the quantum dot microsphere-labeled AchR antigen.

[0042] (3)Preparation of Cy5-labeled anti-human IgG: ① Dissolve 1 mg of anti-human IgG in 1 mL of PBS at pH 7.4, and then place it in a dialysis bag with a MWCO of 10 kDa to dialyze the antibody (at 4 °C, dialysis is carried out 3 times, 2 h each time); after dialysis, adjust the antibody concentration to 1 mg / mL for subsequent labeling reactions. ② React Cy5-NHS ester (concentration 2 mg / mL) with anti-human IgG (1 mg / mL): Slowly add Cy5-NHS ester to the PBS buffer (10 mM) at pH 8.5 containing anti-human IgG (1 mg / mL) and react for 1 - 2 hours (at 4 °C); then add 0.1 M glycine to terminate the reaction and incubate at room temperature for 10 min to block non-specific binding sites; subsequently, remove free Cy5 through Sephadex G-25 gel filtration; after purification, the fluorescently labeled antibody is stored in PBS solution at pH 7.4 and can be stored short-term in the dark at 4 °C (-80 °C in the dark for longer storage), and repeated freezing and thawing should be avoided during use.

[0043] (4)AchR-Ab detection: ① Establishment of standard curve. Prepare a series of dilution gradients of AchR-Ab standards (such as 0, 10, 50, 100, 250, 500, 1000 ng / mL); dilute AchR-Ab standards to the required concentration using PBS (pH 7.4, containing 0.05% Tween-20); add 50 μL of AchR-Ab standard solutions with different concentrations to the designated wells of a black 96-well plate respectively. Finally, follow the detection process described below and draw the standard curve (FRET efficiency vs AchR-Ab concentration). ② Detection of samples. In a black 96-well microplate, add 50 μL of the sample to be tested (serum or cerebrospinal fluid) to each well. Use 50 μL of PBS to replace the sample in the blank control group, and add the AchR-Ab standard solution with a known concentration to the positive control group; then add 50 μL of the working solution of AchR antigen labeled with quantum dot microspheres to each well, mix well and incubate at 37 °C for 30 min; after incubation, add 50 μL of the working solution of anti-human IgG labeled with Cy5 to each well, make it react fully on an oscillating mixer, and then continue to incubate in a 37 °C incubator for 30 min to ensure that the secondary antibody binds to AchR-Ab. After the reaction, wash three times with PBS buffer containing 0.05% Tween-20, 200 μL each time, to remove unbound components; measure the respective fluorescence signals on a fluorescence detector (microspheres@QDs: λex = 550 nm, λem = 600 - 650 nm, Cy5: λex = 650 nm, λem = 670 nm); observe the FRET effect, specifically, the higher the AchR-Ab concentration, the greater the fluorescence quenching degree of the quantum dots and the higher the FRET efficiency. The FRET efficiency can be calculated by the fluorescence enhancement of the receptor: 𝐸=(FA - FA0) / FA, where FA represents the fluorescence intensity of the receptor (Cy5) in the presence of the donor (QDs), and FA0 represents the fluorescence intensity without the donor. For example: by comparing the FRET efficiencies (E, E'), qualitative and even quantitative analysis of the AchR-Ab concentration can be achieved. Here, E is the FRET efficiency without AchR-Ab, and E' is the FRET efficiency with the sample added. By drawing the standard curve of FRET efficiency or Cy5 fluorescence intensity (𝐹𝐴) vs antibody concentration, a quantitative relationship between antibody concentration and FRET signal can be established. Finally, calculate the AchR-Ab concentration in the unknown sample by standard curve regression. Specific Example 2 In Specific Embodiment 2, the preparation method of the quantum dot microspheres is as follows: or the SiO2 microsphere template method is adopted: Add 2 mL of ammonia water (NH3·H2O) to 50 mL of ethanol solution and stir for 30 min; Slowly dropwise add 2 mL of TEOS (tetraethyl orthosilicate) and continue stirring for 6 h to obtain SiO2 microspheres; Centrifuge (8000 rpm, 10 min), wash with ethanol 3 times, and dry in vacuum to complete the preparation of the microspheres; Modify the SiO2 microspheres with (3-aminopropyl)triethoxysilane (APTES) to make the surface positively charged; Disperse the modified SiO2 microspheres in 10 mL of quantum dot solution and stir at room temperature for 4 h to enable the quantum dots to bind through electrostatic interaction, thereby achieving the modification of the quantum dots on the surface of the microspheres; Crosslink and fix with 0.1% glutaraldehyde or chitosan to improve stability; Finally, after washing and drying, SiO2@CdSe / ZnS (or SiO2@InP / ZnS) microspheres can be obtained. The remaining methods and steps are the same as those in Specific Embodiment 1. Specific Embodiment 3 The purpose of Specific Embodiment 3 is to change or optimize the separation and purification means, and form a contrast with the separation effect in Specific Embodiment 1. For example, replace the ordinary nanoparticles with magnetic nanoparticles. Detect the AchR-Ab enriched by the magnetic nanoparticles in PBS buffer (pH 7.4). Activate Fe3O4@SiO2 magnetic nanoparticles (0.5 mg / mL) with EDC / NHS and incubate for 30 min; Add anti-AchR-Ab antibody (concentration 1 mg / mL) and incubate at room temperature for 2 hours to form functionalized magnetic nanoparticles; Add the AchR antigen labeled with quantum dot microspheres (final concentration 10 nM) and incubate at 37 °C for 1 hour; Enrich the target complex with an external magnetic field and wash with PBS (3 times, 200 μL each time); After resuspension, perform fluorescence detection. The remaining methods and steps are the same as those in Specific Embodiment 1. Specific Embodiment 4 The purpose of Specific Embodiment 4 is to form a contrast with the test results obtained by using the 96-well plate platform in (4) of Specific Embodiment 1 by changing the platform for the final-stage detection. Detection of AchR-Ab based on nano-microfluidic technology: Coat biotin-BSA (1 mg / mL) on the Y-shaped or T-shaped channels of the microfluidic chip (polydimethylsiloxane: PDMS material), incubate at room temperature for 1 hour, and wash with PBS; then add streptavidin (0.5 mg / mL), incubate at room temperature for 30 min; then add biotinylated microsphere@QDs-labeled AchR antigen (final concentration 10 nM) by using a pressure-driven mode, after incubating at room temperature for 30 - 60 min, wash with PBS to remove unbound substances (such as unbound biotinylated microsphere@QDs-labeled AchR antigen or other impurities), ensure that only those specifically bound antigens remain on the surface, and prevent non-specific adsorption; the test sample (10 μL) flows through the reaction area at a rate of 1 μL / min through the microfluidic system (pressure-driven mode, temperature controlled at 37 °C) and specifically binds to the immobilized antigen; then add Cy5-labeled anti-human IgG in the same sample addition manner, and incubate for 30 - 60 min; combine with a high-resolution CCD camera for real-time fluorescence detection of the reaction area, and evaluate the concentration of AchR-Ab through the FRET efficiency. The remaining methods and steps are the same as those in Specific Embodiment 1.

[0047] In the technical solutions of the above specific embodiments, in Example 1 and Example 2, conventional detection using a black 96-well plate was performed. In Example 3, magnetic enrichment using a magnetic nanoparticle enrichment system was carried out. In Example 4, a microfluidic platform with a PDMS microfluidic chip was adopted. The comparison of relevant detection indicators is as follows: In terms of the detection limit, the conventional detection scheme reached ≤0.1 ng / mL, which is more than 10 times higher than that of the traditional ELISA; the magnetic enrichment scheme was ≤0.05 ng / mL, with higher sensitivity, also at least 10 times higher than ELISA; the microfluidic platform scheme also reached ≤0.1 ng / mL, was compatible with trace samples, and was 10 times higher than ELISA. Among the three detection schemes, the linear ranges were different. The conventional detection was 10 - 1000 ng / mL, the magnetic enrichment was 5 - 1000 ng / mL, and the microfluidic platform was 1 - 1000 ng / mL. In terms of the detection time, the conventional detection required 1 - 2 hours, the magnetic enrichment scheme was slightly faster, at 1 - 1.5 hours, while the microfluidic platform required 2 - 3.5 hours due to chip pretreatment. In terms of the fluorescence quantum yield of quantum dot microspheres, the conventional detection was 80 - 90%, the magnetic enrichment decreased to 80% due to the coupling loss of magnetic particles, and the microfluidic platform remained at 85% - 90%. In terms of photostability, the signal attenuation of the conventional detection was ≤1% after 300 hours, the magnetic enrichment scheme was ≤3% (interfered by magnetic particles), and the attenuation of the optimized microfluidic platform was also ≤1%. In terms of the sample volume requirement, both the conventional detection and the magnetic enrichment scheme required 50 μL of sample, while the microfluidic platform only required 10 μL due to low consumption. In terms of non-specific adsorption control, the conventional detection was optimized by BSA blocking to reach ≤5%, the magnetic enrichment scheme was slightly higher, at ≤8%, while the microfluidic platform was controlled at ≤3% through directional binding. In terms of equipment dependence, the conventional detection relied on a multifunctional microplate reader, the magnetic enrichment scheme also required a magnetic separation device, and the microfluidic platform required a microfluidic pump and a high-resolution CCD camera. In terms of throughput, both the conventional detection and the magnetic enrichment scheme were medium throughput, while the microfluidic platform could achieve high-throughput detection. Under different schemes of this application, the R² values of the standard curves were all relatively high. Both the conventional detection and the microfluidic platform were >0.99, and the magnetic enrichment scheme also reached >0.98. It can be seen from this that the fluorescence probe provided by this application can be used in different detection platforms, and each method has its own characteristics and can be selected according to requirements in specific applications.

[0048] As described above, the present application proposes a fluorescence probe based on the fluorescence resonance energy transfer (FRET) effect of quantum dots, its preparation method, and a method for detecting acetylcholine receptor antibody (AchR-Ab) using the probe. The fluorescence probe is composed of carrier microspheres, quantum dots, and acetylcholine receptor (AchR) antigen. Through a series of fine preparation steps and functionalization modifications, high-sensitivity and high-specificity biological detection are achieved. The combination of the high fluorescence quantum yield of quantum dots and the FRET effect improves the sensitivity and specificity of the detection method. The fluorescence probe and detection method of the present application are not only applicable to the detection of AchR-Ab, but also can be applied to the detection of other biological molecules by adjusting the selection of antigens and antibodies and the labeling conditions, with wide applicability. In summary, the preparation method of the quantum dot FRET fluorescence probe proposed in the present application and its application in AchR-Ab detection provide an efficient, accurate, and sensitive detection means for the field of biological detection.

[0049] The above is only a specific embodiment of the present application, and any improvement made on the premise of the present application concept is regarded as the protection scope of the present application.

Claims

1. A preparation method of a quantum dot FRET fluorescence probe, characterized in that, It includes the following steps: Prepare carrier microspheres and quantum dots respectively; Perform surface modification on the carrier microspheres and / or the quantum dots; After the modification is completed, mix the carrier microspheres with the quantum dots in a solution to make the quantum dots attach to the surface of the carrier microspheres, obtaining quantum dot microspheres; Perform carboxyl modification on the surface of the quantum dot microspheres, activate the carboxyl groups, and couple the quantum dot microspheres with AchR antigen through a carboxyl activation method; Block the non-specific binding sites on the surface of the microspheres to obtain the AchR antigen labeled with quantum dot microspheres as a fluorescent probe.

2. The preparation method of the quantum dot FRET fluorescent probe according to claim 1, wherein The attachment of quantum dots on the surface of the microspheres is achieved through electrostatic interaction, and after the attachment is completed, a cross-linking agent is also used to cross-link and fix the quantum dots.

3. The preparation method of the quantum dot FRET fluorescence probe according to claim 1, characterized in that, Before blocking the non-specific binding sites on the surface of the microspheres, it also includes the step of washing to remove the unbound AchR antigen.

4. The preparation method of the quantum dot FRET fluorescent probe according to claim 1, wherein Use 3-mercaptopropionic acid to perform carboxyl modification on the quantum dots or use succinic anhydride to perform carboxyl modification on the surface of the quantum dot microspheres, and use 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to activate the carboxyl groups.

5. A quantum dot FRET fluorescence probe, characterized in that, It includes carrier microspheres, quantum dots, and AchR antigen; The quantum dots are attached to the surface of the carrier microspheres to form quantum dot microspheres, and the quantum dots are cross-linked and fixed through a cross-linking agent; The AchR antigen is coupled to the surface of the quantum dot microspheres; Among them, the non-specific binding sites on the surface of the quantum dot microspheres are blocked.

6. The quantum dot FRET fluorescence probe according to claim 5, characterized in that, The carrier microspheres are PS microspheres or SiO2 microspheres, and the quantum dot materials are selected from CdSe / ZnS or InP / ZnS.

7. The quantum dot FRET fluorescence probe according to claim 6, wherein The carrier microspheres include magnetic nanoparticles.

8. The quantum dot FRET fluorescence probe according to claim 5, wherein The fluorescent probe is suspended in PBS buffer.

9. A method for detecting AchR-Ab, characterized in that, It includes: Use the quantum dot FRET fluorescent probe described in any one of claims 5-8 to prepare an AchR antigen working solution, prepare a Cy5-labeled anti-human IgG working solution, and prepare a series of diluted gradient concentration AchR-Ab standard solutions; Add the AchR antigen working solution to the test samples and reference samples, mix well and perform the first incubation; Add the anti-human IgG working solution to the solution after the first incubation, mix well and react, and then perform the second incubation; Wash to remove the unbound components; Measure the fluorescence signals and FRET efficiencies of the test samples and reference samples; Compare the FRET efficiencies of the test samples and reference samples to obtain the AchR-Ab concentration in the test samples.

10. The AchR-Ab detection method according to claim 9, characterized in that, Use a microplate or microfluidic chip method to measure the fluorescence signals.

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