Luminescence immunodetection kit based on dynamic coupling and signal enhancement and application

Abstract

The invention provides a chemiluminescence immunoassay kit based on dynamic coupling and signal enhancement, which comprises a pH response dynamic coupling assembly, a multi-mode anti-interference assembly and an FRET (Fluorescence Resonance Energy Transfer) delayed luminescence probe which have a synergistic effect and form a closed-loop detection system through'coupling-reaction-signal 'time sequence adaptation; the kit also comprises an antigen standard substance, a diluent and an excitation liquid. Reversible fixation and cyclic utilization of an antibody are achieved through the pH response dynamic coupling assembly, the problem of complex matrix interference is solved through the multi-mode anti-interference assembly, the signal detection capacity is improved through the FRET delayed light-emitting probe, the immunodetection performance is remarkably improved through the synergistic effect of the pH response dynamic coupling assembly, the multi-mode anti-interference assembly and the FRET delayed light-emitting probe, and the characteristics of high sensitivity, high specificity and interference resistance are achieved.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic technology, specifically relating to a chemiluminescent immunoassay kit based on dynamic coupling and signal enhancement and its application. Background Technology

[0002] Chemiluminescence immunoassay (CLIA) technology, due to its advantages of high sensitivity, high specificity, and fast detection speed, has been widely used in clinical disease diagnosis (such as the detection of Alzheimer's disease biomarkers Aβ1-42 and p-Tau181), drug concentration monitoring, and food safety testing. The core principle of CLIA technology is to correlate the chemiluminescent signal with the target concentration through the specific binding of antigen and antibody, achieving quantitative analysis. The "capture efficiency, anti-interference ability, and signal stability" of the detection system are key factors determining its detection performance. However, traditional CLIA technology still has several bottlenecks that restrict its application in complex sample detection and precise diagnosis: 1. Antibody fixation is irreversible, costly, and difficult to recycle. Traditional CLIA typically uses EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride / N-hydroxysuccinimide) covalently coupled antibodies with magnetic beads / solid-phase carriers. This method has the following drawbacks: (1) Covalent bonds (such as amide bonds) are highly stable at physiological pH (7.4), resulting in irreversible binding between the antibody and the carrier. This makes it impossible to separate and recover the antibody through simple acid-base treatment, leading to the inability to reuse the antibody and significantly increasing the detection cost (especially for high-value antibodies). (2) During long-term storage, the antibody is easily inactivated due to steric hindrance or chemical degradation, affecting the detection stability.

[0003] 2. Weak anti-interference ability and low accuracy in detecting complex samples. Clinical samples (such as serum and plasma) often contain high abundance of interfering substances (such as hemoglobin, lipids, and immunoglobulins). These interfering substances can interfere with detection by quenching chemiluminescence signals, occupying antibody binding sites, or altering the pH of the reaction system. Traditional CLIA relies on simple sample pretreatment (such as centrifugation and dilution) or a single blocking agent (such as bovine serum albumin BSA), which is insufficient to effectively eliminate interference from complex samples such as lipemia (hemoglobin > 5 g / L) and hemolysis (hemoglobin > 2 g / L), resulting in low detection accuracy (usually < 80%) and a high risk of missed or false positives.

[0004] 3. Rapid signal attenuation limits sensitivity and detection window. Traditional CLIA often uses free acridine esters (such as NSP-SA) as chemiluminescent probes. The luminescence half-life of free acridine esters is extremely short (<1 second), and the signal decay rate far exceeds the instrument reading speed, resulting in: (1) a narrow detection time window: detection needs to be completed near the signal peak (usually within 0-1 second), which places extremely high demands on the instrument response speed; (2) insufficient sensitivity: the total signal amount in a short time is low (integrated signal intensity <5×10). 5 RLU), making it difficult to detect low-abundance biomarkers (such as the concentration of Aβ1-42 in the cerebrospinal fluid of early Alzheimer's patients being only 1-2 pg / mL); (3) low signal-to-noise ratio: the signal decays rapidly and is easily interfered with by background noise (such as autofluorescence of the sample), further reducing the reliability of detection.

[0005] Although researchers have attempted to improve CLIA performance in recent years by optimizing coupling methods (such as using reversible covalent bonds), adding blocking agents (such as casein), or improving probes (such as lanthanide chelates), existing reversible coupling technologies (such as disulfide-based coupling) are susceptible to reducing environments and lack stability. Existing single blocking agents struggle to simultaneously address multiple interferences (such as hemoglobin, lipids, and non-specific binding sites). Furthermore, existing coupling technologies between long-lived probes (such as quantum dots) and chemiluminescent probes are immature, exhibiting low energy transfer efficiency and failing to balance delayed luminescence with high sensitivity. Therefore, it is urgent to overcome the bottlenecks in traditional CLIA technology and promote the widespread application of immunoassay in precision medicine. Summary of the Invention

[0006] This invention addresses the shortcomings of existing technologies by providing a chemiluminescent immunoassay kit and its application based on dynamic coupling and signal enhancement. The kit integrates three functional components: dynamic reversible coupling, multimodal anti-interference, and FRET delayed luminescence. The synergistic effect of these three components can significantly improve immunoassay performance.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution: This invention provides a chemiluminescent immunoassay kit based on dynamic coupling and signal enhancement, characterized in that it includes a synergistic pH-responsive dynamic coupling component, a multimodal anti-interference component, and a FRET delayed luminescence probe, which form a closed-loop detection system through temporal adaptation of "coupling-reaction-signal"; it also includes antigen standards, diluent, and excitation solution; The pH-responsive dynamic coupling component includes the following reagents: a reagent containing PEG-CHO-coupled magnetic beads-antibody at pH 7.4, as well as spare PEG-CHO-coupled magnetic beads, detection antibody, neutral buffer at pH 7.4±0.2, and acidic buffer at pH 4.0±0.2; The PEG-CHO-coupled magnetic bead-antibody is formed by linking the aldehyde group of the PEG-CHO-coupled magnetic bead with the amino group of the detection antibody via hydrazone bonds. The hydrazone bonds have a half-life of >30 days in a neutral buffer solution at pH 7.4±0.2 and are hydrolyzed in an acidic buffer solution at pH 4.0±0.2. The PEG-CHO-coupled magnetic beads are Fe3O4@SiO2 nanomagnetic beads with a surface-modified PEG-CHO composite coating, a particle size of 100-300 nm, and a PEG-CHO modification density of 50-200 μmol / g magnetic beads. The multimodal anti-interference component is a composite anti-interference buffer system comprising a biomimetic adsorbent mesoporous SiO2@polydopamine, a pH-responsive hydrogel polymethacrylic acid-acrylamide copolymer [P(MAA-co-AM)], and a competitive blocking agent salmon sperm DNA+casein; the mesoporous SiO2@polydopamine has a particle size of 50-100 nm, a mesoporous pore size of 10-20 nm, and a polydopamine coating thickness of 5-10 nm; The FRET delayed-emission probe is a CdSe / ZnS quantum dot-acrididine ester FRET probe. The particle size of the CdSe / ZnS quantum dots is 10±2 nm, and the grafting density of acridine ester on the quantum dot surface is 4-8 quantum dots / quantum dot. The probe emission window delay is 0.1-5 s. The probe emission wavelength includes the emission peak of the quantum dots at 620±10 nm and the emission peak of the acridine ester at 540±10 nm. The Stokes shift of both is greater than 80 nm, which can avoid interference from the autofluorescence of biological samples. The antigen standard consists of 11 standard solutions with antigen concentrations of 0 pg / mL, 0.05 pg / mL, 0.1 pg / mL, 0.5 pg / mL, 1 pg / mL, 5 pg / mL, 10 pg / mL, 50 pg / mL, 100 pg / mL, and 500 pg / mL.

[0008] Furthermore, the present invention provides the application of the above-mentioned chemiluminescent immunoassay kit in the chemiluminescent immunoassay of Alzheimer's disease biomarkers.

[0009] The chemiluminescence immunoassay kit provided herein is characterized in that the kit is used for the chemiluminescence immunoassay to detect the content of Alzheimer's disease biomarker Aβ1-42 protein in samples, comprising a pH-responsive dynamic coupling component, a multimodal anti-interference component, and further comprising a FRET delayed luminescence probe, antigen standard, diluent, and excitation solution; the pH-responsive dynamic coupling component comprises the following reagents: a pH 7.4 reagent containing PEG-CHO-conjugated magnetic beads-Aβ1-42 antibody, and spare PEG-CHO-conjugated magnetic beads, Aβ1-42 antibody, pH 7.4±0.2 neutral buffer, pH... 4.0±0.2% acidic buffer solution; the multimodal anti-interference component comprises the following reagents: biomimetic adsorbent mesoporous SiO2@polydopamine, pH-responsive hydrogel polymethacrylic acid-acrylamide copolymer, competitive blocking agents salmon sperm DNA and casein; the antigen standard consists of 11 standard solutions with Aβ1-42 protein concentrations of 0 pg / mL, 0.05 pg / mL, 0.1 pg / mL, 0.5 pg / mL, 1 pg / mL, 5 pg / mL, 10 pg / mL, 50 pg / mL, 100 pg / mL, and 500 pg / mL; the excitation solution contains 0.2 mM H2O2, 0.02 mM DMAP, 0.095 M ethanolamine, and 0.015 mM vitamin C.

[0010] Furthermore, the chemiluminescent immunoassay kit is characterized in that, in the pH-responsive dynamic coupling component, the magnetic saturation intensity of the Fe3O4@SiO2 nanobeads is 50-80 emu / g, and the surface of the magnetic beads is first pretreated with an aminosilane coupling agent, and then grafted with PEG-CHO through an ethylene oxide ring-opening reaction to ensure the uniformity of the PEG-CHO modification density.

[0011] Furthermore, the chemiluminescent immunoassay kit is characterized in that, when used to detect the target content in a sample, the preferred amounts of the reagents included in the multimodal anti-interference component in the reaction system are as follows: The biomimetic adsorbent mesoporous SiO2@polydopamine is present at a concentration of 0.1-5 mg / mL in the reaction system, adsorbing hemoglobin in the sample; the pH-responsive hydrogel polymethacrylic acid-acrylamide copolymer is present at a mass concentration of 0.1-0.5 mg / mL in the reaction system, maintaining pH stability and controlling pH fluctuations to be less than 0.1; the competitive blocking agent is present at a mass concentration of 0.08-0.12 mg / mL salmon sperm DNA and 0.8-1.2 mg / mL casein in the reaction system, with salmon sperm DNA and casein synergistically blocking non-specific adsorption sites on the magnetic bead surface in the reaction system.

[0012] Furthermore, the chemiluminescent immunoassay kit is characterized in that, when used to detect the target content in a sample, the preferred amount of the FRET delayed luminescence probe in the reaction system is as follows: the concentration of the FRET delayed luminescence probe in the reaction system is 10–50 nmol / L.

[0013] Furthermore, the chemiluminescent immunoassay kit is characterized in that, when the pH-responsive dynamic coupling component, the multimodal anti-interference component, and the FRET delayed luminescence probe component work synergistically to detect the target content in the sample, the detection limit LOD is <1 pg / mL, which can detect biomarkers with extremely low abundance in the sample and is suitable for early disease screening and micro-sample detection.

[0014] Furthermore, the chemiluminescent immunoassay kit is characterized in that the FRET delayed luminescence probe has a surface ligand of mercaptoacetic acid or mercaptopropionic acid on the CdSe / ZnS quantum dots, and the surface ligand is modified with amino groups to ensure the dispersibility of the quantum dots in the aqueous buffer (particle size distribution PDI < 0.2) and the quantum yield of the quantum dots, which can improve the stability of FRET efficiency; the probe FRET efficiency is > 80%, and the quantum yield of the quantum dots is > 80%.

[0015] The FRET delayed emission probe achieves a luminescence window delay of 0.1–5 s through fluorescence resonance energy transfer between the long-lived excited state (fluorescence lifetime 10–50 ns) of CdSe / ZnS quantum dots and acridine ester, with an FRET efficiency >80%.

[0016] Furthermore, the chemiluminescent immunoassay kit is characterized in that the FRET delayed luminescence probe is prepared as follows: The carboxyl group of acridine ester is covalently coupled to the amino group modified on the surface of CdSe / ZnS quantum dots using the EDC / NHS activation method. The coupling reaction is carried out in MES buffer at pH 5.0-5.5, at a reaction temperature of 25±2℃, and for a reaction time of 1-2 h. After coupling, the acridine ester is purified by dialysis using a 10 kDa molecular weight cutoff bag to remove free acridine ester.

[0017] Furthermore, the chemiluminescent immunoassay kit is characterized in that the operating steps for detecting the target content in a sample are as follows: (1) Coupling and capture: PEG-CHO-conjugated magnetic beads and detection antibodies are mixed in a certain proportion in a neutral buffer solution at pH 7.4±0.2. The antibody concentration is 10-50 μg / mL and the magnetic bead concentration is 0.1-0.5 mg / mL. The mixture is incubated at 25°C for 1-2 h to generate PEG-CHO-conjugated magnetic beads-antibody. This coupling reaction is an optional step. Mix the PEG-CHO-conjugated magnetic bead-antibody generated in the above steps or the reagent in the kit containing PEG-CHO-conjugated magnetic bead-antibody at pH 7.4 with the sample to be tested, and incubate at 37°C for 15-30 min to allow the target in the sample to specifically bind with the PEG-CHO-conjugated magnetic bead-antibody to form a "magnetic bead-antibody-target" complex. (2) Anti-interference treatment: A multimodal anti-interference component was added to the "magnetic bead-antibody-target" complex, wherein the concentration of the biomimetic adsorbent mesoporous SiO2@polydopamine in the reaction system was 0.1-5 mg / mL, the mass concentration of the pH-responsive hydrogel polymethacrylic acid-acrylamide copolymer in the reaction system was 0.1-0.5 mg / mL, and the mass concentration of the competitive blocking agent in the reaction system was 0.08-0.12 mg / mL for salmon sperm DNA and 0.8-1.2 mg / mL for casein; incubation was carried out at 25℃ for 5-10 min; the mesoporous SiO2@polydopamine in the anti-interference component adsorbed hemoglobin in the sample, and the polymethacrylic acid-acrylamide copolymer maintained the pH stability of the system, which could control the pH fluctuation of the reaction system to <0.1; salmon sperm DNA and casein blocked the non-specific sites that the magnetic beads did not bind to; (3) Signal probe binding: Add FRET delayed luminescence probe, the concentration of the probe in the reaction system is 10-50 nmol / L, incubate at 37℃ for 10-15 min to form a complex of "magnetic bead-antibody-target-FRET probe"; separate this complex by magnetic field and discard the unbound free probe. (4) Signal detection and antibody recovery: Excitation solution was added to the magnetically separated "magnetic bead-antibody-target-FRET probe" complex, and the integrated luminescence signal was collected from 0 to 5 s using a chemiluminescence analyzer, with the detection wavelength covering 540 nm and 620 nm; after detection, acidic buffer pH 4.0±0.2 was added, and the mixture was incubated at 25℃ for 10 to 15 min to hydrolyze the hydrazone bonds, and the antibody was recovered by centrifugation; (5) Repeated antibody conjugation: The recovered antibody can be repeatedly conjugated with magnetic beads after being purified by dialysis with PBS buffer at pH 7.4.

[0018] The above steps (2) and (3) anti-interference and signal detection can be integrated together: the anti-interference buffer and FRET probe can be pre-mixed and added to the reaction system to make the detection suitable for high-throughput processing (>96 samples / batch) of automated equipment (such as Opentrons OT-2).

[0019] Experiments have shown that in step (1) above, the coupling reaction conditions for the antibody and PEG-CHO-conjugated magnetic beads are as follows: in Tris-HCl buffer (pH 7.4), the antibody concentration is 10-50 μg / mL, the PEG-CHO-conjugated magnetic bead concentration is 0.1-0.5 mg / mL, and the mixture is incubated at 25°C for 1-2 h. When the PEG-CHO-conjugated magnetic bead-antibody complex is stored at 4°C in the dark, the antibody binding activity retention rate is >90% within 6 months.

[0020] Experiments have shown that after repeated coupling of the antibody with PEG-CHO magnetic beads three times, the antibody conjugation activity retention rate is greater than 80%, the detection sensitivity does not decrease significantly, and the detection limit (LOD) fluctuation is <10%.

[0021] Beneficial effects of the present invention The detection kit provided by this invention integrates three functional components: a pH-responsive dynamic coupling component, a multimodal anti-interference component, and a FRET delayed luminescence probe. The pH-responsive dynamic coupling component enables reversible antibody immobilization and recycling; the multimodal anti-interference component addresses interference from complex matrices; and the FRET delayed luminescence probe enhances signal detection capabilities. The synergistic effect of these three components significantly improves immunoassay performance, as detailed below: Significantly improved detection sensitivity: The FRET delayed-emission probe in this kit utilizes the long-lived excited state (nanosecond to microsecond level) of CdSe / ZnS quantum dots to efficiently transfer energy to acridine ester via FRET, extending the luminescence time of acridine ester from a transient state (<1 second) to provide more sufficient signal accumulation time for instrument acquisition. This invention's FRET delayed-emission probe boasts high FRET efficiency; the high quantum yield of quantum dots synergistically enhances the strong luminescence properties of acridine ester, significantly improving the signal-to-noise ratio (SNR). The extended signal window and enhanced signal intensity work together to achieve a detection limit (LOD) <1 pg / mL, enabling the detection of extremely low abundance biomarkers in samples. It is suitable for early disease screening (such as AD, tumors) and trace sample detection (such as cerebrospinal fluid, aqueous humor), filling the gap in traditional detection methods for low concentration ranges. It solves the pain points of traditional detection methods, such as "rapid signal attenuation and high detection limit."

[0022] Significantly Enhanced Specificity: The multimodal anti-interference components in this kit achieve precise suppression of complex samples: Mesoporous SiO2@polydopamine (PDA): specifically adsorbs hemoglobin (adsorption rate > 90%), reducing the quenching effect of hemoglobin on chemiluminescence in lipemia / hemolysis samples. Polymethacrylic acid-acrylamide copolymer: pH-responsive hydrogel maintains system pH stability (fluctuation < 0.1) through proton absorption / release, avoiding antibody conformational changes or non-specific binding caused by pH fluctuations; Salmon sperm DNA + casein: competitively blocks non-specific sites on the magnetic bead surface, reducing the non-specific adsorption of impurities (such as IgG, fibrinogen) in the sample. High detection accuracy is achieved in complex samples with lipemia (hemoglobin > 5 g / L), hemolysis (hemoglobin > 2 g / L), and high-abundance protein interference, solving the problem of "numerous interferences and unstable results" in clinical samples.

[0023] Stability and repeatability optimization: The pH-responsive dynamic coupling component in this kit provides PEG-CHO-conjugated magnetic beads-antibody conjugates with reversible coupling and a long half-life. The PEG-CHO-conjugated magnetic beads are linked to the antibody via hydrazone bonds, which are stable at neutral pH 7.4 (half-life > 30 days), preventing antibody detachment during storage. After detection, hydrazone bond hydrolysis is triggered by acidic pH 4.0 buffer, resulting in antibody recovery > 80%, allowing for reuse in subsequent detections. Optimized performance parameters for PEG-CHO-conjugated magnetic beads: The PEG-CHO modification density on the Fe3O4@SiO2 magnetic beads (particle size 100-300 nm) is 50-200 μmol / g, balancing coupling efficiency (> 90%) and bead dispersibility (avoiding aggregation), ensuring reaction uniformity.

[0024] This invention provides a kit with an extended shelf life (half-life > 30 days), reduced antibody consumption due to reuse, and a batch-to-batch CV < 5% (compared to > 15% for traditional kits), meeting standardized testing requirements such as ISO 15189. It overcomes the shortcomings of traditional kits, which suffer from irreversible antibody conjugation (e.g., EDC / NHS covalent conjugation), leading to antibody shedding during long-term storage (half-life < 7 days), making antibody recovery and reuse impossible, resulting in high testing costs and significant batch-to-batch variations.

[0025] Simplified Detection Process: This kit achieves rapid detection through modular design: the conjugation step is highly efficient; magnetic beads and antibodies can be conjugated in 2 hours at neutral pH 7.4 (compared to 4 hours for traditional EDC / NHS), and no additional centrifugation / purification is required (magnetic separation is completed in one step). Integrated Anti-interference and Signal Detection: The anti-interference buffer and FRET probe can be pre-mixed, requiring only a three-step reaction of "sample + buffer + probe" (total time < 1 hour), suitable for high-throughput processing (>96 samples / batch) with automated equipment (such as Opentrons OT-2). Application Value: Suitable for scenarios requiring rapid results, such as emergency rooms and health check centers, promoting the popularization of immunoassay testing from "high-end laboratory testing" to "rapid screening at the grassroots level," overcoming the shortcomings of traditional testing which requires multiple manual steps (such as antibody coating, blocking, and washing), is time-consuming, and prone to errors. Attached Figure Description

[0026] Figure 1 The luminescence kinetic curves of the experimental and control groups in Example 4 are shown. Figure 2 The curve showing the correlation between Aβ1-42 concentration and RLU was established using the kit from Example 5 to detect Aβ1-42. Detailed Implementation

[0027] The chemiluminescent immunoassay kit of the present invention will be described in detail below with reference to embodiments, specifically using the application of the chemiluminescent immunoassay kit in the detection of the Alzheimer's disease biomarker Aβ1-42 protein content as an example. The chemiluminescent immunoassay kit of the present invention is also suitable for screening other early diseases and detecting trace samples (such as cerebrospinal fluid and aqueous humor), which will not be described in detail here. The embodiments provided by the present invention are implemented under the premise of the technical solution of the present invention, and detailed implementation methods and specific operating procedures are given, but the scope of protection of the present invention is not limited to the following embodiments. Example 1

[0028] The chemiluminescent immunoassay kit of the present invention is illustrated using a kit for detecting the content of Aβ1-42 protein, a biomarker of Alzheimer's disease, in a sample by chemiluminescent immunoassay.

[0029] This invention provides a chemiluminescent immunoassay kit based on dynamic coupling and signal enhancement, characterized in that it is used for detecting the content of the Alzheimer's disease marker Aβ1-42 protein in samples by chemiluminescent immunoassay. The kit includes three main functional components: a synergistic pH-responsive dynamic coupling component, a multimodal anti-interference component, and a FRET delayed luminescence probe. It also includes antigen standards, diluent, and activation solution. The following reagents constitute the pH-responsive dynamic coupling component: a reagent containing PEG-CHO-coupled magnetic beads-Aβ1-42 antibody at pH 7.4, and spare PEG-CHO-coupled magnetic beads, Aβ1-42 antibody, neutral buffer at pH 7.4±0.2, and acidic buffer at pH 4.0±0.2; The following reagents constitute the multimodal anti-interference component: biomimetic adsorbent mesoporous SiO2@polydopamine, pH-responsive hydrogel polymethacrylic acid-acrylamide copolymer, competitive blocking agents salmon sperm DNA and casein; It also includes FRET delayed luminescence probes, antigen standards, diluents, and activation solutions.

[0030] The antigen standard consists of 11 standard solutions with Aβ1-42 protein concentrations of 0 pg / mL, 0.05 pg / mL, 0.1 pg / mL, 0.5 pg / mL, 1 pg / mL, 5 pg / mL, 10 pg / mL, 50 pg / mL, 100 pg / mL, and 500 pg / mL.

[0031] The activating solution contains 0.2 mM H2O2, 0.02 mM DMAP, 0.095 M ethanolamine, and 0.015 mM vitamin C.

[0032] The PEG-CHO coupled magnetic beads are Fe3O4@SiO2 nanomagnetic beads with a surface modified PEG-CHO composite coating, with a particle size of 200 nm and a PEG-CHO modification density of 100 μmol / g magnetic beads on their surface.

[0033] The PEG-CHO-conjugated magnetic bead-Aβ1-42 antibody is formed by the reversible linkage of the aldehyde group of PEG-CHO and the amino group of the detection antibody Aβ1-42 via hydrazone bonds. The hydrazone bonds have a half-life of >30 days in neutral buffer solution at pH 7.4±0.2 and can be hydrolyzed in acidic buffer solution at pH 4.0±0.2, allowing for the recovery of more than 80% of the antibody.

[0034] The mesoporous SiO2@polydopamine has a particle size of 50–100 nm, a mesopore size of 10–20 nm, and a polydopamine coating thickness of 5–10 nm, and adsorbs hemoglobin in the sample.

[0035] The FRET delayed emission probe is a CdSe / ZnS quantum dot-acrididine ester FRET probe. The particle size of the CdSe / ZnS quantum dots is 10±2 nm, and the grafting density of acridine ester on the quantum dot surface is 4-8 per quantum dot. The probe emission window is delayed by 0.1-5 s. The emission wavelength of the probe includes the emission peak of the quantum dots at 620±10 nm and the emission peak of the acridine ester at 540±10 nm. Both have a Stokes shift greater than 80 nm, which can avoid interference from the autofluorescence of biological samples.

[0036] The FRET delayed emission probe has a CdSe / ZnS quantum dot yield of >80% and an FRET efficiency of >80%.

[0037] Furthermore, the operating steps of the chemiluminescent immunoassay kit of the present invention for detecting the target content in a sample are as follows: (1) Coupling and capture: PEG-CHO-conjugated magnetic beads and detection antibodies are mixed in a certain proportion in a neutral buffer solution at pH 7.4±0.2. The antibody concentration is 10-50 μg / mL and the magnetic bead concentration is 0.1-0.5 mg / mL. The mixture is incubated at 25°C for 1-2 h to generate PEG-CHO-conjugated magnetic beads-antibody. This coupling reaction is an optional step. Mix the PEG-CHO-conjugated magnetic bead-antibody generated in the above steps or the reagent in the kit containing PEG-CHO-conjugated magnetic bead-antibody at pH 7.4 with the sample to be tested, and incubate at 37°C for 15-30 min to allow the target in the sample to specifically bind with the PEG-CHO-conjugated magnetic bead-antibody to form a "magnetic bead-antibody-target" complex. (2) Anti-interference treatment: A multimodal anti-interference component was added to the "magnetic bead-antibody-target" complex, wherein the concentration of the biomimetic adsorbent mesoporous SiO2@polydopamine in the reaction system was 0.1-5 mg / mL, the mass concentration of the pH-responsive hydrogel polymethacrylic acid-acrylamide copolymer in the reaction system was 0.1-0.5 mg / mL, and the mass concentration of the competitive blocking agent in the reaction system was 0.08-0.12 mg / mL of salmon sperm DNA and 0.8-1.2 mg / mL of casein; incubated at 25℃ for 5-10 min; (3) Signal probe binding: Add FRET delayed luminescence probe, the concentration of the probe in the reaction system is 10-50 nmol / L, incubate at 37℃ for 10-15 min to form a complex of "magnetic bead-antibody-target-FRET probe"; separate this complex by magnetic field and discard the unbound free probe. (4) Signal detection and antibody recovery: Excitation solution was added to the magnetically separated "magnetic bead-antibody-target-FRET probe" complex, and the integrated luminescence signal was collected from 0 to 5 s using a chemiluminescence analyzer, with the detection wavelength covering 540 nm and 620 nm; after detection, acidic buffer pH 4.0±0.2 was added, and the mixture was incubated at 25℃ for 10 to 15 min to hydrolyze the hydrazone bonds, and the antibody was recovered by centrifugation; (5) Repeated antibody conjugation: The recovered antibody can be repeatedly conjugated with magnetic beads after being purified by dialysis with PBS buffer at pH 7.4.

[0038] Example 2 Example 2 mainly involves the preparation of "PEG-CHO-conjugated magnetic beads-Aβ1-42 antibody" and its performance testing.

[0039] 1. Main reagents: PEG-CHO coupled magnetic beads (Fe3O4@SiO2 nanomagnetic beads with surface modified PEG-CHO): The particle size is 200 nm, and the PEG-CHO modification density on the surface of the magnetic beads is 100 μmol / g magnetic beads.

[0040] Detection antibody: Aβ1-42 antibody; Comparison magnetic beads: EDC / NHS coupled magnetic beads; pH 7.4 PBS buffer; pH 4.0 citrate buffer; BCA kit; 2. Preparation of magnetic bead-antibody conjugates (1) Experimental group: Prepare PEG-CHO coupled magnetic beads-Aβ1-42 antibody.

[0041] Preparation method: PEG-CHO-conjugated magnetic beads and Aβ1-42 antibody are added to pH 7.4 PBS buffer in a certain ratio. The antibody concentration is 10 μg / mL and the amount of magnetic beads is 0.1-0.5 mg / mL. After incubation for 2 h, PEG-CHO-conjugated magnetic beads-Aβ1-42 antibody with hydrazone linkage is formed.

[0042] (2) Control group: EDC / NHS coupled magnetic beads-Aβ1-42 antibody.

[0043] The preparation method is as follows: the surface carboxyl groups of EDC / NHS-coupled magnetic beads are modified by adding 100 μmol / g of EDC / NHS-coupled magnetic beads and 1 μg / mL of Aβ1-42 antibody to PBS buffer at pH 6.5 with EDC / NHS concentration of 10 mM for 2 h to obtain EDC / NHS-coupled magnetic beads-Aβ1-42 antibody.

[0044] 3. Stability Test Experimental group: PEG-CHO conjugated magnetic beads-Aβ1-42 antibody; Control group: EDC / NHS conjugated magnetic beads-Aβ1-42 antibody.

[0045] The magnetic bead + antibody conjugates of the experimental and control groups were placed in pH 7.4 PBS (neutral) buffer, and the amount of free antibody in the supernatant was measured every 7 days (BCA method), and the half-life (time for 50% antibody dissociation) was calculated. The stability comparison data of the conjugates of the experimental and control groups are shown in Table 2-1 below.

[0046] Table 2-1: Comparison of stability data of conjugates between experimental and control groups Time (days) Free antibody level in the experimental group (μg / mL) Half-life (days) Free antibody level in the control group (μg / mL) Half-life (days) 0 0.1 (Initial dissociation) — 0.5 (Initial dissociation) — 7 0.08 (down 20%) — 2.5 (down 80%) 3.2 14 0.06 (a decrease of 40%) — 4.0 (down 90%) — 30 0.05 (a decrease of 50%) ＞30 >5.0 (a decrease of 95%) ＜7 The stability comparison data of the conjugates in the experimental and control groups show that: Experimental group: In pH 7.4 neutral buffer, the half-life of "PEG-CHO coupled magnetic beads-Aβ1-42 antibody" is >30 days, indicating that the hydrazone bond is highly stable in a neutral environment, meeting the requirements for long-term storage of the reagent.

[0047] Control group: In pH 7.4 neutral buffer, the half-life of "EDC / NHS coupled magnetic beads-Aβ1-42 antibody" is <7 days (EDC / NHS covalent bonds are easily hydrolyzed), indicating poor stability and failing to meet the requirements for long-term reagent storage.

[0048] 4. Antibody recovery rate test Experimental group: PEG-CHO conjugated magnetic beads-Aβ1-42 antibody; Control group: EDC / NHS conjugated magnetic beads-Aβ1-42 antibody.

[0049] The recovery rate was tested as follows: The magnetic bead-antibody conjugates from the experimental and control groups were magnetically separated in pH 4.0 citrate buffer (0.1 M, containing 0.1% BSA). The supernatant (containing dissociated antibody) was collected, and the amount of free antibody in the supernatant was detected using the BCA method. The supernatant was then re-incubated with fresh magnetic beads (unmodified antibody) at a ratio of 1:8 (w / w) for 2 h (pH 7.4). The number of cycles was 5 (first conjugation → separation → re-conjugation → separation → … → fifth conjugation). The test results are shown in Table 2-2 below.

[0050] The antibody recovery rate test results of the experimental group showed that the antibody recovery rate was ≥75% after 5 cycles, indicating that the stability of the magnetic bead-antibody hydrazone bond connection can support the reuse of antibodies.

[0051] The antibody recovery rate test of the control group showed that traditional EDC conjugation could only be repeated 1-2 times, with an antibody recovery rate of <50%.

[0052] Table 2-2: Comparison of antibody recovery rates between experimental and control groups Cycle number experimental group experimental group control group control group Free antibody level in the experimental group (μg / mL) Recovery rate (%) Free antibody level in the control group (μg / mL) Recovery rate (%) 1 time 0.085 85 0.05 50 2 times 0.082 82 0.04 40 3 times 0.08 80 0.03 30 4 times 0.078 78 0.01 10 5 times 0.075 75 0.01 10 5. Antibody activity retention rate test Experimental group: PEG-CHO conjugated magnetic beads-Aβ1-42 antibody; Control group: Free Aβ1-42 antibody; Control group 2: EDC / NHS conjugated magnetic beads-Aβ1-42 antibody.

[0053] The testing method is as follows: The experimental group, control group 1, and control group 2 were incubated with the Aβ1-42 antigen standard, and the binding ability was detected by ELISA (detection wavelength 450 nm, EC50 calculated by standard curve fitting). The results of the activity retention rate test are shown in Table 2-3 below.

[0054] Table 2-3. Results of Activity Retention Rate Test Group EC50 (pg / mL) Antibody-conjugated activity retention rate (%) experimental group 15.5 87.7 Control group 1 13.6 — Control group 2 45.2 30.1 The results of the above activity retention rate test show that the EC50 values ​​of the experimental group and control group 1 are similar, while the EC50 values ​​of the experimental group and control group 2 are significantly different, indicating that the hydrazone coupling process did not change the conformation of the antibody's antigen-binding site. Antibody-coupled activity retention rate (%) = (EC50 free antibody ÷ EC50 coupled antibody) × 100%), the antibody binding ability of the experimental group is close to that of the free antibody. Table 3 shows that the antibody-coupled activity retention rate of the experimental group is >85%, while the traditional EDC coupling activity retention rate is <70%, indicating that pH-responsive hydrazone coupling does not cause steric hindrance or chemical damage.

[0055] Example 3 Example 3 is a test of the effect of using the multimodal anti-interference component in the reaction system.

[0056] Sample types: lipemic sample (hemoglobin 5 g / L), hemolyzed sample (hemoglobin 2 g / L), normal sample (no hemoglobin).

[0057] (1) Background signal detection (when there is no antigen) Experimental group: Mesoporous SiO2@polydopamine 0.5 mg / mL, pH-responsive hydrogel polymethacrylic acid-acrylamide copolymer [P(MAA-co-AM)] 0.1 mg / mL, and competitive blocking agent (salmon sperm DNA 0.1 mg / mL + casein 0.9 mg / mL) were added to the reaction system.

[0058] Control group: PBS + 0.1% BSA (traditional anti-interference system) was added to the reaction system.

[0059] Detection method: The luminescence intensity of the supernatant was detected by a chemiluminescence analyzer (RLU, integration time 10 s), and the detection results are shown in Table 3-1 below.

[0060] Table 3-1: Comparison of background signal detection data between the experimental group and the control group Sample type Experimental group background RLU Control group background RLU Background suppression rate (%) Lipids (5 g / L) 320 2100 84.8 Hemolysis (2 g / L) 180 1800 89.9 normal samples 120 150 92 As shown in Table 4 above, the background RLU of the experimental group in the lipemia sample was 320, while that of the control group was 2100. The background inhibition rate (%) was calculated as (control group - experimental group) / control group × 100%, resulting in a background inhibition rate (%) of 84.8% for the experimental group in the lipemia sample. The background RLU of the hemolytic sample was 180, while that of the control group was 1800. The background inhibition rate (%) of the experimental group in the hemolytic sample was 89.9%, indicating that the specific adsorption of hemoglobin by the biomimetic adsorbent (mesoporous SiO2@polydopamine) is the core mechanism for reducing the background (the background of lipemia / hemolytic samples mainly comes from hemoglobin quenching).

[0061] The background RLU of the normal sample was 120, while that of the control group was 150. The background of the normal sample came from the blank sites on the surface of the magnetic beads that were not occupied by the antibody, thus demonstrating the effectiveness of the competitive blocking agent (salmon sperm DNA + casein) in blocking non-specific sites.

[0062] (2) Signal-to-noise ratio (S / N) verification Experimental group: Mesoporous SiO2@polydopamine 0.5 mg / mL, pH-responsive hydrogel polymethacrylic acid-acrylamide copolymer [P(MAA-co-AM)] 0.1 mg / mL, and competitive blocking agent (salmon sperm DNA 0.1 mg / mL + casein 0.9 mg / mL) were added to the reaction system.

[0063] Control group: PBS + 0.1% BSA (traditional anti-interference system) was added to the reaction system. Detection target: Low concentration of Aβ1-42 antigen (10 pg / mL, close to the detection limit).

[0064] Signal definition: Luminescence intensity after antigen binding (RLU); Background signal: Luminescence intensity (RLU) without antigen. Calculation: S / N = Signal RLU / Background RLU. Detection results are shown in Table 3-1 below.

[0065] Table 3-2: Signal-to-noise ratio data for the experimental and control groups Sample type Experimental group S / N Control group S / N S / N enhancement factor Lipids (5 g / L) 15.6 2.1 7.4 Hemolysis (2 g / L) 12.8 1.8 7.1 normal samples 10.5 1.5 7 The experimental results show that the S / N ratio of the experimental group is greater than 10:1, and the S / N ratio of the lipemia sample is 15.6:1, which is significantly higher than that of the control group (S / N < 5:1). This indicates that the multimodal anti-interference system (adsorption + buffering + blocking) of the present invention effectively improves the signal authenticity (signal / background ratio). The S / N ratio is improved by more than 7 times, which means that the instrument can more accurately distinguish between antigen signals and background noise (traditional methods are prone to false negatives due to background interference).

[0066] Example 4: Preparation and Verification of FRET Delay Probe (1) Preparation of quantum dot-acridone ester FRET probe Quantum dot pretreatment (removal of unreacted ligands): CdSe / ZnS quantum dot surfaces are usually modified with excess thioglycolic acid (HS-AA). Unreacted HS-AA must be removed first to avoid interfering with the coupling reaction: Take 1 mL of quantum dot solution (concentration 1 mg / mL, MES buffer pH 6.0); add 10 μL of ethanolamine (1 M, prepared in MES buffer), stir at room temperature for 30 minutes (blocking unreacted thiol groups); centrifuge using an ultrafiltration tube (MWCO 30 kDa) (4000 rpm, 10 minutes, 4℃), discard the supernatant; wash 3 times with MES buffer (pH 6.0), resuspend to 1 mL, and obtain "activated quantum dots" (surface exposed -COOH).

[0067] EDC / NHS activation of quantum dot surface carboxyl groups: Add EDC (final concentration 50 mM) and NHS (final concentration 50 mM) to activated quantum dots (1 mg / mL), and adjust the volume to 1 mL with MES buffer (pH 6.0); react at room temperature with magnetic stirring (200 rpm) for 30 minutes (optimized activation time); centrifuge using an ultrafiltration tube (MWCO 30 kDa) (4000 rpm, 10 minutes, 4℃), discard the supernatant (to remove unreacted EDC / NHS); wash twice with MES buffer (pH 6.0), resuspend to 1 mL, and obtain "EDC / NHS activated quantum dots".

[0068] Acridinium ester coupling reaction: Acridinium ester (final concentration 20 μM, diluted with PBS buffer pH 7.4) was added to EDC / NHS activated quantum dots (1 mg / mL); the reaction was carried out at room temperature in the dark with stirring (200 rpm) for 2 hours (optimized coupling time); the mixture was centrifuged (4000 rpm, 10 min, 4℃) using an ultrafiltration tube (MWCO 30 kDa), and the supernatant was discarded (to remove unbound acridinium ester); the mixture was washed 3 times with PBS buffer (pH 7.4) and resuspended to 1 mL to obtain "quantum dot-acriminium ester conjugate (QD-Acr)".

[0069] (2) Experimental methods Experimental group: Quantum dot-acridone ester FRET probe; Control group: Free acridinium ester NSP-SA probe; Table 4-1: Detection configuration for experimental and control groups experimental group control group Remark Probe / Reagent Concentration 100 nmol / L (quantum dot concentration) 200 nmol / L - probe type CdSe / ZnS quantum dots (10 nm) - acridine ester Free acridine ester (NSP-SA) The experimental group was connected via EDC / NHS. Testing instruments BPCL Weak Emission Detector BPCL Weak Emission Detector Sampling interval 0.1 s, excitation wavelength 430 nm Referring to Table 4-1 above, the luminescence intensity of the experimental group and the control group was measured using a BPCL weak luminescence detector. The detection data are shown in Table 4-2 below.

[0070] Table 4-2: Luminescence kinetics detection data (luminescence intensity 0-10 s) Time (s) Luminous intensity (RLU) of the experimental group Control group luminescence intensity (RLU) Experimental group intensity / Control group intensity (times) 0 12000 15000 0.8 0.1 850000 920000 0.92 0.2 1020000 780000 1.31 0.3 980000 650000 1.51 0.4 920000 520000 1.77 0.5 850000 410000 2.07 1 680000 180000 3.78 2 450000 65000 6.92 3 280000 22000 12.73 4 150000 8000 18.75 5 85000 3500 24.29 6 42000 1800 23.33 7 21000 950 22.11 8 10500 500 21 9 5200 280 18.57 10 2600 150 17.33 As shown in Table 4-2, the peak luminescence intensity was significantly improved: the experimental group reached a peak luminescence intensity of 1,020,000 RLU, while the control group was 920,000 RLU, a difference of 1.11 times. A stronger peak intensity means a more prominent signal can be output at the moment of excitation, which can reduce recognition errors caused by weak signals during detection, especially for low-concentration target detection, and helps to accurately capture the initial luminescence signal. (Appendix) Figure 1 This is the luminescence dynamics curve.

[0071] Enhanced integrated signal intensity: The integrated signal intensity of the experimental group was 12,856,000 RLU·s⁻¹ from 0 to 10 s, while that of the control group was 4,893,300 RLU·s⁻¹, a difference of 2.63 times. The total luminescence intensity increased significantly, directly improving detection sensitivity and signal-to-noise ratio, making it particularly suitable for the detection of low-concentration samples (such as trace biomolecule analysis). The integrated signal reflects the "total signal amount." Even if the peak intensity only increased slightly (1.11 times), the significant extension of the duration allowed for a substantial accumulation of the total signal, compensating for the difference in peak intensity and even achieving a net performance advantage.

[0072] The decay time is extended: the decay time of the experimental group is 4.8 s, while that of the control group is only 0.6 s, a difference of 8 times. The emission duration is significantly extended, making it suitable for scenarios requiring long-term stable detection (such as dynamic process monitoring and long-term signal acquisition). It reduces the detection time limitation caused by rapid signal decay, improving the flexibility of experiments or applications (e.g., allowing for a more relaxed detection time window and reducing stringent real-time requirements).

[0073] The comparison data of peak emission intensity (RLU), decay time (s), and 0-10 s integrated signal intensity (RLU·s) between the experimental group and the control group are shown in Table 4-3 below.

[0074] Table 4-3: Comparison of Peak Emission Intensity, Decay Time, and Integral Signal Intensity index experimental group control group Difference (times) Peak luminous intensity (RLU) 1020000 920000 1.11 Decay time (s) 4.8 0.6 8 Integrated signal strength (RLU·s) from 0 to 10 s 12856000 4893300 2.63 Note: Attenuation time = the time required for the intensity to drop from the peak to 10% (experimental group: 102000 × 10% = 102000, corresponding to 4.8 s; control group: 920000 × 10% = 92000, corresponding to 0.6 s); Integral signal intensity = the sum of the intensities from 0 to 10 s calculated using the SUMPRODUCT function in Excel.

[0075] Example 5: Comparison of Integrated Performance Tests 1. Comparison of experimental parameters Experimental group: The kit of this invention (magnetic beads-antibody dynamic conjugation + anti-interference buffer + FRET probe); Control group: Traditional kit (EDC conjugation + traditional buffer + free acridinium ester probe); The experimental items and instructions are shown in Table 5-1 below: Table 5-1: Experiment Items and Instructions project illustrate Detection markers Aβ1-42 (0~1000 pg / mL) instrument Chemiluminescence analyzer Blank Sample 0 pg / mL Aβ1-42 (0.01M PBS, pH7.4) Clinical samples plasma Experimental methods See the detection steps of the kit of this invention. The luminescence intensity (RLU) detection data of the experimental group kit when detecting the biomarker Aβ1-42 are shown in Table 5-2 below. Figure 2 The curve showing the correlation between Aβ1-42 concentration and RLU was established using the kit from Example 5 to detect Aβ1-42.

[0076] Table 5-2: RLU data of Aβ1-42 detected by the experimental kit Aβ1-42 concentration (pg / mL) Batch 1 Batch 2 Batch 3 mean Standard deviation CV% (coefficient of variation) blank sample 1205 1198 1202 1201.67 3.51 0.29 5 2460 2495 2502 2485.67 21.50 0.86 10 12230 12590 12410 12410.00 182.37 1.47 50 59050 57980 58020 58350.00 562.49 0.96 100 115100 114950 115020 115023.33 75.06 0.07 200 228050 227950 228000 228000.00 50.00 0.02 500 535200 544900 545050 541716.67 5446.79 1.01 800 811800 821500 801650 811650.00 9954.89 1.23 1000 1074800 1094500 1074650 1081316.67 11853.73 1.10 The luminescence intensity (RLU) detection data of the control group kit when detecting biomarker Aβ1-42 are shown in Table 5-3 below.

[0077] Table 5-3: RLU data for Aβ1-42 detection using conventional kits Aβ1-42 concentration (pg / mL) Batch 1 Batch 2 Batch 3 Mean Standard deviation (SD) Coefficient of variation (CV%) blank sample 805 798 802 801.67 3.51 0.44 5 1605 1505 1500 1536.67 55.08 3.58 10 5900 6080 5790 5923.33 145.47 2.46 50 26050 27950 30000 28000.00 2054.81 7.34 100 58050 54950 55000 56000.00 1825.74 3.26 200 150050 159950 160000 156666.67 5773.50 3.69 300 170050 189950 190000 183333.33 11547.01 6.30 500 235050 264950 255000 251666.67 14433.76 5.74 1000 340050 319950 350000 336666.67 17018.51 5.06 The sensitivity (LOD) comparison data between the experimental group and the control group are shown in Table 5-4 below.

[0078] Table 5-4: Sensitivity (LOD) Comparison (LOD signal = blank mean + 3 * blank SD value) project Experimental group (LOD, pg / mL) Control group (LOD, pg / mL) Difference multiple Compliance status Blank mean 1202 802 - - Blank SD 3.5 3.5 - - LOD signal 1212.5 812.5 - - LOD concentration 0.82 5.2 6.3 times Experimental group <1, control group >5 The results of the comparison of linear range between the experimental group and the control group are shown in Table 5-5 below.

[0079] Table 5-5: Comparison Results of Linear Range project Linear range of the experimental group (pg / mL) Linear range for the control group (pg / mL) Linear R² Compliance status Aβ1-42 1-1000 1-200 0.998 The system covers the entire course of the disease. The repeatability (CV%) comparison results between the experimental group and the control group are shown in Table 5-6 below.

[0080] Table 5-6: Comparison of Repeatability (CV%) Aβ1-42 concentration (pg / mL) CV% of the experimental group CV% of control group difference Compliance status (system < 5%) 5 0.86 3.58 Reduced by 76.0% Meets standards 10 1.47 2.46 Reduced by 40.2% Meets standards 50 0.96 7.34 Reduced by 86.9% Meets standards From the above experimental data tables 5-1 to 5-6 and appendices Figure 2 As can be seen, when the three functional components of this invention—pH-responsive dynamic coupling component, multimodal anti-interference component, and FRET delayed luminescence probe—work synergistically, the LOD < 1 pg / mL, enabling the detection of very early AD samples (such as the MCI stage) and avoiding missed diagnoses. The cross-reactivity rate is < 0.3%, with no significant interference when detecting complex samples, ensuring diagnostic accuracy. The target concentration ranges from 1 to 1000 pg / mL, eliminating the need to dilute high-concentration samples, making it suitable for "full-course disease monitoring." The CV < 0.5%, making it suitable for large-scale screening. The three modules are conflict-free, requiring no pretreatment of clinical samples, and the detection can be completed within 30 minutes, improving laboratory turnover.

Claims

1. A chemiluminescent immunoassay kit based on dynamic coupling and signal enhancement, characterized in that, It includes a synergistic pH-responsive dynamic coupling component, a multimodal anti-interference component, and a FRET delayed luminescence probe, which form a closed-loop detection system through timing adaptation of "coupling-reaction-signal"; it also includes antigen standards, diluents, and excitation solutions; The pH-responsive dynamic coupling component includes the following reagents: a reagent containing PEG-CHO-coupled magnetic beads-antibody at pH 7.4, as well as spare PEG-CHO-coupled magnetic beads, detection antibody, neutral buffer at pH 7.4±0.2, and acidic buffer at pH 4.0±0.2; The PEG-CHO-coupled magnetic bead-antibody is formed by linking the aldehyde group of the PEG-CHO-coupled magnetic bead with the amino group of the detection antibody via hydrazone bonds. The hydrazone bonds have a half-life of >30 days in a neutral buffer solution at pH 7.4±0.2 and are hydrolyzed in an acidic buffer solution at pH 4.0±0.

2. The PEG-CHO-coupled magnetic beads are Fe3O4@SiO2 nanomagnetic beads with a surface-modified PEG-CHO composite coating, a particle size of 100-300 nm, and a PEG-CHO modification density of 50-200 μmol / g magnetic beads. The multimodal anti-interference component is a composite anti-interference buffer system comprising a biomimetic adsorbent mesoporous SiO2@polydopamine, a pH-responsive hydrogel polymethacrylic acid-acrylamide copolymer, and a competitive blocking agent salmon sperm DNA+casein; the mesoporous SiO2@polydopamine has a particle size of 50-100 nm, a mesoporous pore size of 10-20 nm, and a polydopamine coating thickness of 5-10 nm; The FRET delayed emission probe is a CdSe / ZnS quantum dot-acrididine ester FRET probe. The particle size of the CdSe / ZnS quantum dots is 10±2 nm, the grafting density of acridine ester on the quantum dot surface is 4 to 8 per quantum dot, and the probe emission window delay is 0.1 to 5 s. The probe emission wavelength includes the emission peak of the quantum dots at 620±10 nm and the emission peak of the acridine ester at 540±10 nm.

2. The chemiluminescent immunoassay kit according to claim 1, characterized in that, The kit is for detecting the content of Aβ1-42 protein, a biomarker of Alzheimer's disease, in samples using chemiluminescent immunoassay. It includes a pH-responsive dynamic coupling component, a multimodal anti-interference component, a FRET delayed luminescence probe, antigen standards, diluent, and excitation solution. The pH-responsive dynamic coupling component includes the following reagents: a reagent containing PEG-CHO-coupled magnetic beads-Aβ1-42 antibody at pH 7.4, and spare PEG-CHO-coupled magnetic beads, Aβ1-42 antibody, neutral buffer at pH 7.4±0.2, and acidic buffer at pH 4.0±0.2; The multimodal anti-interference component comprises the following reagents: biomimetic adsorbent mesoporous SiO2@polydopamine, pH-responsive hydrogel polymethacrylic acid-acrylamide copolymer, competitive blocking agents salmon sperm DNA and casein; The antigen standard consists of 11 standard solutions with Aβ1-42 protein concentrations of 0 pg / mL, 0.05 pg / mL, 0.1 pg / mL, 0.5 pg / mL, 1 pg / mL, 5 pg / mL, 10 pg / mL, 50 pg / mL, 100 pg / mL, and 500 pg / mL. The activating solution contains 0.2 mM H2O2, 0.02 mM DMAP, 0.095 M ethanolamine, and 0.015 mM vitamin C.

3. The chemiluminescent immunoassay kit according to claim 1 or 2, characterized in that, In the pH-responsive dynamic coupling assembly, the magnetic saturation intensity of the Fe3O4@SiO2 nanobeads is 50-80 emu / g. The surface of the magnetic beads is first pretreated with an aminosilane coupling agent, and then PEG-CHO is grafted through an ethylene oxide ring-opening reaction to ensure the uniformity of the PEG-CHO modification density.

4. The chemiluminescent immunoassay kit according to claim 1 or 2, characterized in that, When used to detect the target content in a sample, the preferred amounts of the reagents included in the multimodal anti-interference component in the reaction system are as follows: The biomimetic adsorbent mesoporous SiO2@polydopamine is present at a concentration of 0.1–5 mg / mL in the reaction system, adsorbing hemoglobin from the sample; the pH-responsive hydrogel polymethacrylic acid-acrylamide copolymer is present at a mass concentration of 0.1–0.5 mg / mL in the reaction system, maintaining pH stability; the competitive blocking agent is present at a mass concentration of 0.08–0.12 mg / mL salmon sperm DNA and 0.8–1.2 mg / mL casein in the reaction system, with salmon sperm DNA and casein synergistically blocking non-specific adsorption sites on the magnetic bead surface in the reaction system.

5. The chemiluminescent immunoassay kit according to claim 1 or 2, characterized in that, When used to detect the target content in a sample, the preferred amount of FRET delayed emission probe in the reaction system is as follows: the concentration of the FRET delayed emission probe in the reaction system is 10-50 nmol / L.

6. The chemiluminescent immunoassay kit according to claim 1 or 2, characterized in that, When the pH-responsive dynamic coupling component, multimodal anti-interference component, and FRET delayed luminescence probe component work together to detect the target content in a sample, the detection limit LOD is <1 pg / mL, which can detect biomarkers with extremely low abundance in the sample and is suitable for early disease screening and trace sample detection.

7. The chemiluminescent immunoassay kit according to claim 1 or 2, characterized in that, The FRET delayed emission probe uses mercaptoacetic acid or mercaptopropionic acid as the surface ligand for CdSe / ZnS quantum dots, and the surface ligands are modified with amino groups to ensure the dispersibility of quantum dots in aqueous buffer and the quantum yield of quantum dots.

8. The chemiluminescent immunoassay kit according to claim 1 or 2, characterized in that, The FRET delayed luminescence probe is prepared as follows: using the EDC / NHS activation method, the carboxyl group of acridine ester is covalently coupled to the amino group modified on the surface of CdSe / ZnS quantum dots. The coupling reaction is carried out in MES buffer at pH 5.0-5.5, the reaction temperature is 25±2℃, and the reaction time is 1-2 h. After coupling, the acridine ester is purified by dialysis with a molecular weight cutoff of 10 kDa to remove free acridine ester.

9. The chemiluminescent immunoassay kit according to claim 1 or 2, characterized in that, The operating steps for detecting the target content in a sample are as follows: (1) Coupling and capture: PEG-CHO-conjugated magnetic beads and detection antibodies are mixed in a certain proportion in a neutral buffer solution at pH 7.4±0.

2. The antibody concentration is 10-50 μg / mL and the magnetic bead concentration is 0.1-0.5 mg / mL. The mixture is incubated at 25℃ for 1-2 h to generate PEG-CHO-conjugated magnetic beads-antibody. This coupling reaction is an optional step. Mix the PEG-CHO-conjugated magnetic bead-antibody generated in the alternative step or the reagent containing PEG-CHO-conjugated magnetic bead-antibody at pH 7.4 in the kit with the sample to be tested, and incubate at 37°C for 15-30 min to allow the target in the sample to specifically bind to the PEG-CHO-conjugated magnetic bead-antibody to form a "magnetic bead-antibody-target" complex. (2) Anti-interference treatment: A multimodal anti-interference component was added to the "magnetic bead-antibody-target" complex, wherein the concentration of the biomimetic adsorbent mesoporous SiO2@polydopamine in the reaction system was 0.1-5 mg / mL, the mass concentration of the pH-responsive hydrogel polymethacrylic acid-acrylamide copolymer in the reaction system was 0.1-0.5 mg / mL, and the mass concentration of the competitive blocking agent in the reaction system was 0.08-0.12 mg / mL of salmon sperm DNA and 0.8-1.2 mg / mL of casein; incubated at 25℃ for 5-10 min; (3) Signal probe binding: Add FRET delayed luminescence probe, the concentration of the probe in the reaction system is 10-50 nmol / L, incubate at 37℃ for 10-15 min to form a complex of "magnetic bead-antibody-target-FRET probe"; separate this complex by magnetic field and discard the unbound free probe. (4) Signal detection and antibody recovery: Excitation solution was added to the magnetically separated "magnetic bead-antibody-target-FRET probe" complex, and the integrated luminescence signal was collected from 0 to 5 s using a chemiluminescence analyzer, with the detection wavelength covering 540 nm and 620 nm; after detection, acidic buffer pH 4.0±0.2 was added, and the mixture was incubated at 25℃ for 10 to 15 min to hydrolyze the hydrazone bonds, and the antibody was recovered by centrifugation; (5) Repeated antibody conjugation: The recovered antibody can be repeatedly conjugated with magnetic beads after being purified by dialysis with PBS buffer at pH 7.4.

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