A flow fluorescence microsphere detection method and kit based on TSA signal amplification

Abstract

This invention discloses a flow cytometry method for detecting fluorescent microspheres based on TSA signal amplification. The method includes: incubating fluorescently encoded microspheres coated with a capture antibody with a sample; adding a biotinylated detection antibody to form a sandwich complex; subsequently introducing two high-density signal amplification polymers sequentially: SA-PolyHRP400 as the first signal amplification polymer, and a second signal amplification polymer with a polysaccharide backbone and covalently coupled with multiple tyrosine and luciferin molecules. Under HRP catalysis, the tyrosine is activated and covalently deposited in the vicinity of the microspheres, achieving exponential amplification of the fluorescence signal. This method is simple to operate, more efficient than traditional methods, and more suitable for high-throughput, high-timeliness platforms like flow cytometry. It also exhibits high sensitivity, high specificity, and good repeatability for detecting low-abundance proteins such as IL-6 based on the sandwich method, and is compatible with non-magnetic / magnetic fluorescently encoded microspheres of various sizes. It is suitable for multi-indicator detection and can be widely applied to early clinical diagnosis and trace biomarker analysis.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, specifically to a flow cytometry-based fluorescent microsphere detection method and kit based on TSA signal amplification. It is suitable for femtogram-level detection of low-abundance antigens and can be widely applied in clinical diagnostics and research scenarios such as early diagnosis of malignant diseases and biomarker screening. Background Technology

[0002] With the rapid development of biomedical technology, early detection and precision treatment of diseases have become core development directions in the medical field. Detection and diagnostic technologies, as key supports for achieving this goal, directly impact the effectiveness of disease diagnosis and treatment due to their sensitivity and practicality. Flow cytometry and flow fluorescence platforms, with their unique advantages of high-speed detection and high-throughput analysis, have been widely used in biomarker detection, immunoassay, and other fields, becoming important tools in clinical testing and scientific research. Flow cytometry microsphere technology (also known as liquid-phase chip technology) is a high-throughput, multi-index simultaneous detection platform. Its core utilizes microspheres encoded with different fluorescence intensities as a solid-phase carrier, capturing target analytes through a double-antibody sandwich immunoassay, and achieving quantitative analysis using the signal intensity of reporter molecules (such as fluorescent dyes). This technology has been widely applied in clinical diagnosis, drug screening, and basic research.

[0003] However, the detection sensitivity of existing flow cytometry and flow fluorescence platforms is generally only at the picogram level, which presents significant limitations when dealing with low-abundance antigens in samples such as blood (e.g., trace biomarkers released in the early stages of malignant diseases). When the antigen content in the reaction system is as low as the femtogram level, the number of antigen molecules is extremely small, and the average amount of antigen distributed to the microspheres in the flow cytometry system is negligible. At this point, even if a "fluorescence-secondary antibody-antigen-antibody" complex is formed, the number of fluorescent molecules bound to the surface of the microspheres is far below the detection limit of the flow cytometer, making it impossible to effectively distinguish the target signal from background noise. Ultimately, this prevents the achievement of ultrasensitive detection of extremely low concentrations of antigens, severely restricting the efficiency of early diagnosis of malignant diseases.

[0004] To overcome the bottleneck of detection sensitivity, various signal amplification techniques have been developed in related fields. Among them, tyramide signal amplification (TSA) is a highly sensitive in-situ detection technique based on enzyme-catalyzed reactions. Its core mechanism utilizes the highly efficient catalytic activity of horseradish peroxidase (HRP) to specifically and densely label target proteins or nucleic acid molecules in samples. Through its unique signal amplification effect, TSA technology can significantly enhance the intensity of target-related fluorescence signals in applications such as immunofluorescence cytochemistry (ICC), immunohistochemistry (IHC), and in situ hybridization (FISH), becoming a key means to overcome the sensitivity limitations of traditional detection techniques. However, to date, TSA technology is mainly applied to the aforementioned in-situ detection scenarios and has not yet been effectively integrated and optimized for use in flow cytometry and flow cytometry platforms. Therefore, it cannot leverage the high-throughput and rapid detection advantages of flow cytometry platforms to achieve ultrasensitive detection of low-abundance antigens.

[0005] Furthermore, existing detection methods for low-abundance biomolecules suffer from drawbacks such as operational complexity, time-consuming processes, and high costs. Some methods require the purchase of specialized auxiliary equipment, which not only increases detection costs but also limits their widespread application in routine clinical diagnostic scenarios. With the increasing demand for early disease diagnosis in the medical industry and the support of relevant policies and regulations for precision medicine technologies, developing a novel detection method that achieves femtogram-level ultrasensitive detection on flow cytometry and flow fluorescence platforms, while being easy to operate and cost-effective, has become an urgent technical challenge in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a detection method and kit for flow cytometry fluorescent microspheres based on TSA signal amplification. By optimizing tyrosine signal amplification (TSA) technology and combining it with a flow cytometry / flow cytometry fluorescence platform, this invention solves the problems of existing technologies being unable to achieve femtogram-level low-abundance antigen detection, being complex to operate, and being costly. It enables efficient and low-cost ultrasensitive detection of single or multiple indicators, meeting the clinical and research needs such as early diagnosis of malignant diseases.

[0007] To achieve the above-mentioned objectives, this application adopts the following technical solution: A flow cytometry method for detecting fluorescent microspheres based on TSA signal amplification, the method comprising the following steps: (a) Incubate fluorescently encoded microspheres coated with capture antibodies with the sample to be tested to allow the target analyte to specifically bind to the surface of the microspheres; (b) Add biotinylated detection antibody to form a microsphere-capture antibody-target analyte-biotinylated detection antibody complex; (c) Add a first signal amplification polymer, wherein the first signal amplification polymer is SA-PolyHRP400; (d) Add a second signal amplification polymer, which is a polymer with multiple tyrosine groups and multiple luciferin molecules covalently coupled on a polysaccharide backbone. Under HRP catalysis, the tyrosine groups are activated and covalently deposited in the vicinity of the microspheres to achieve fluorescence signal amplification. (e) Detect the fluorescence signal intensity of the fluorescently encoded microspheres and quantify the target analyte accordingly.

[0008] Furthermore, the polysaccharide is a dextran, and the molecular weight of the dextran is 30,000–500,000 Da.

[0009] Furthermore, the dextran molecule is coupled with an average of ≥20 tyrosine groups and ≥20 fluorescein molecules.

[0010] Furthermore, the microspheres include non-magnetic functionalized microspheres, non-magnetic functionalized fluorescently encoded microspheres, magnetic functionalized microspheres, or magnetic functionalized fluorescently encoded microspheres, and the particle size distribution of the microspheres is 0.5-20 micrometers.

[0011] Furthermore, the fluorescent dye molecule includes at least one of fluorescent small molecules such as fluorescein, AF488, rhodamine, Cy3, Cy5, PE or APC, and nanofluorescent materials such as quantum dots.

[0012] Furthermore, the target analytes include interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), or interferon-γ (IFN-γ) or other protein detection items based on sandwich assays.

[0013] Furthermore, the preparation method of the second signal amplification polymer includes the following steps: dissolving polysaccharide in buffer solution, adding sodium periodate for oxidation reaction to generate aldehyde-containing polysaccharide; then adding tyramine hydrazide to react with aldehyde to form tyramine polymer; then coupling the tyramine polymer with N-hydroxysuccinimide-activated fluorescein under alkaline conditions, and purifying it to obtain a polymer rich in multiple tyramine fluorescein molecules.

[0014] A detection kit for flow cytometry fluorescent microspheres based on TSA signal amplification, the kit comprising: (i) The second signal amplification reagent is a polymer covalently coupled with multiple tyrosine groups and multiple fluorescent dye molecules on a dextran backbone; (ii) A first signal amplification reagent, wherein the first signal amplification polymer is SA-PolyHRP400; (iii) Fluorescently encoded microspheres coated with capture antibodies; (iv) Biotin-labeled detection antibodies; and (v) Suitable reaction buffer for HRP catalysis.

[0015] This invention integrates a multivalent tyrosine-fluorescein polymer with SA-PolyHRP400 and a dextran backbone into a flow cytometry fluorescent microsphere detection system, successfully achieving efficient adaptation of TSA signal amplification technology to a liquid-phase microsphere platform. This method significantly improves detection sensitivity, achieving detection limits for low-abundance proteins such as interleukin-6 (IL-6) at the femtogram per milliliter (fg / mL) level, a substantial improvement over traditional flow cytometry microsphere methods. Simultaneously, it maintains good specificity, repeatability, and compatibility with multiple detection methods, and is applicable to various types and sizes of fluorescently encoded microspheres, providing a reliable technical means for early clinical diagnosis and trace biomarker analysis. Compared with existing technologies, the technical solution of this invention has the following significant advantages: 1. Achieving femtogram-level ultrasensitive detection: By preparing a polymer rich in multiple tyrosine fluorescein molecules, multiple fluorescein molecules are aggregated onto a single chain, achieving both fluorescence signal enrichment and phenolic hydroxyl enrichment. A high concentration of phenolic hydroxyl groups is formed around HRP, which increases catalytic efficiency and improves the binding probability to tyrosine residues of antigen-antibody complexes. Simultaneously, the polymer rich in multiple biotin and HRP molecules increases the enzyme loading on a limited antigen-antibody complex, accelerating the enzyme catalytic reaction. Under this dual effect, the signal is efficiently amplified, enabling flow cytometry and flow fluorescence platforms to detect femtogram-level low-abundance antigens, breaking through the sensitivity bottleneck of existing technologies.

[0016] 2. Simplified Operation and Reduced Detection Costs: This invention organically combines tyrosine signal amplification (TSA) technology with flow cytometry / fluid cytometry fluorescence platforms. No additional specialized auxiliary equipment is required. Leveraging the high speed and high throughput advantages of existing flow cytometry platforms, the detection operation is simple and time-saving, significantly reducing detection costs and facilitating widespread application in clinical diagnostic scenarios. This method is simple to operate, achieving secondary signal amplification through multiple conjugations of tyrosine and fluorescein, while also directly emitting fluorescence. Compared to the traditional tyrosine-biotin approach with the addition of SA-fluorescein molecules, this method is more efficient and better suited to high-throughput, high-time-sensitivity platforms like flow cytometry.

[0017] 3. Supports multi-indicator joint detection: With the help of the unique coded microsphere recognition technology of the flow cytometry platform, this invention can achieve femtogram-level ultrasensitive analysis of multiple indicators, further expanding the application scenarios of the detection method and meeting the needs of joint screening of multiple biomarkers.

[0018] 4. Excellent reagent stability and compatibility: The two polymers prepared through a specific coupling process (high reactivity and high selectivity of hydrazide and aldehyde groups) have high loading efficiency and stable structure. They have strong binding specificity with microspheres / magnetic beads and antigen-antibody complexes, which can effectively avoid signal interference caused by non-specific reactions and ensure the accuracy of detection results. Detailed Implementation

[0019] To make the technical solution of the present invention clearer and to verify its technical effects, a detailed description is provided below with reference to specific embodiments and experimental examples. It should be understood that these embodiments and experimental examples are only for explaining the present invention and not for limiting its scope of protection. Without departing from the spirit of the present invention, those skilled in the art can make conventional adjustments to parameters such as raw materials, concentration, reaction time, and microsphere type, all of which should fall within the scope of protection of the present invention.

[0020] Example 1: Preparation of a second signal amplification polymer (a polymer rich in multiple tyrosine fluorescein molecules) 10 mg of dextran with a molecular weight of approximately 40,000 Da was dissolved in 1 mL of phosphate-buffered saline (PBS) at pH 6.0. 100 μL of 5 mM sodium periodate (NaIO4) solution was added, and the reaction was carried out at room temperature in the dark for 30 minutes to selectively oxidize the vicinal diol structure in the dextran molecule, generating an aldehyde group. After the reaction was complete, an equal volume of ethylene glycol was added to quench any unreacted sodium periodate. The reaction solution was then placed in a dialysis bag with a molecular weight cutoff of 3,500 Da and dialyzed against deionized water for 24 hours to remove small molecule byproducts. The resulting lyophilized dextran was obtained.

[0021] The aldehyde-modified dextran was redissolved in 0.1M sodium acetate buffer (pH 5.5), and tyrosine hydrazide was added to a final concentration of 5 mM. The reaction was carried out at 37°C for 4 hours. Under these conditions, the hydrazide group of tyrosine hydrazide undergoes a condensation reaction with the aldehyde group on the dextran to form a stable hydrazone bond, thereby covalently linking multiple tyramine groups to the dextran backbone to obtain a tyramine polymer.

[0022] Subsequently, the pH of the reaction system was adjusted to 8.5 with 1M sodium bicarbonate solution, and N-hydroxysuccinimide (NHS) activated AF488 (AF488-NHS) was added to a final concentration of 10 mM. The reaction was continued for 12 hours at 4°C in the dark. At this time, the residual amino groups on the tyramine polymer underwent an amidation reaction with FITC-NHS, coupling multiple fluorescein molecules to the polymer. After the reaction was completed, the reaction solution was purified using 10 kDa ultrafiltration centrifuge tubes to remove unreacted small molecule dyes, and the polymer products were further separated by Sephadex G-50 gel filtration chromatography. The main peak component was collected to obtain the target product—a dextran polymer rich in multiple tyramides and AF488. Detection and calculation showed that each dextran molecule was coupled with an average of approximately 20 tyramide groups and 30 fluorescein molecules.

[0023] Example 2: Construction and Validation of a TSA-based Flow Cytometry Fluorescent Microsphere Ultrasensitive Detection Kit for IL-6 Detection The kit contains the following components: (i) Second signal amplification reagent: namely, the dextran polymer (TSA-AF488-Dextran) rich in multiple tyrosine and fluorescein obtained in Example 1, dissolved in Tris-HCl buffer (pH 8.5) containing 0.1% BSA, at a concentration of 1 mg / mL; (ii) First signal amplification reagent: SA-PolyHRP400 dissolved in PBS (pH 7.4) (iii) Fluorescently encoded microspheres coated with capture antibodies: Commercially available anti-IL-6 monoclonal antibodies were covalently coupled to 5.6 μm non-magnetic fluorescently encoded microspheres and resuspended in PBS containing 0.05% sodium azide at a concentration of 1250 microspheres / μL. (iv) Biotin-labeled detection antibody: Anti-IL-6 monoclonal antibody was labeled with NHS-Biotin, purified and dissolved in PBS containing 1% BSA at a concentration of 10 μg / mL. (v) HRP reaction buffer: 50 mM Tris-HCl buffer (pH 8.5) containing 0.003% H2O2, prepared immediately before use.

[0024] The above components are packaged in individual containers and stored at 4°C away from light. The shelf life is 6 months.

[0025] II. Testing Operation Procedure Mix 50 μL of microsphere suspension (approximately 2500 microspheres) with 50 μL of the test sample (containing serially diluted IL-6 standard) and incubate at 37°C with shaking for 30 minutes. After washing, add 50 μL of biotin-labeled detection antibody and incubate at 37°C for 30 minutes. After washing again, add 50 μL of the first signal amplification reagent (SA-PolyHRP400) and incubate at 37°C for 20 minutes. Then add 50 μL of the second signal amplification reagent and an equal volume of HRP reaction buffer and react at room temperature in the dark for 5 minutes. After stopping the reaction and washing thoroughly, resuspend in the detection buffer and read the MFI value using an EasyCell instrument.

[0026] Example 3: Multi-detection and microsphere type compatibility verification Capture antibodies against TNF-α and IFN-γ were coated onto microspheres with different fluorescent coding properties, including non-magnetic functionalized fluorescent coding microspheres (3.2 μm in diameter) and magnetic functionalized fluorescent coding microspheres (6.8 μm in diameter). Following the procedure in Example 3, three cytokines, IL-6, TNF-α, and IFN-γ, were simultaneously detected. The results showed that all three targets could be accurately detected at the fg / mL level, with no cross-interference between the detection channels, demonstrating that this invention is applicable to various microsphere types (0.5–20 μm in diameter) and multi-indicator simultaneous detection scenarios.

[0027] Example 4: Replacement of other fluorescent dyes with the polysaccharide backbone Replacing AF488 in Example 1 with Cy5-NHS yielded a tyrosine-Cy5 polymer, which was used for IL-6 detection, achieving fg-level sensitivity. Furthermore, functional second signal amplification polymers were successfully prepared by replacing dextran (molecular weight 70,000 Da) or hydroxyethyl starch as the backbone, verifying the applicability of the polysaccharide backbone in the 30,000–500,000 Da range.

[0028] Experiment 1: Sensitivity Comparison Test Objective: To verify the sensitivity improvement effect of the method of the present invention compared with the conventional TSA method.

[0029] method: Sample preparation: The recombinant human IL-6 standard was serially diluted with PBS containing 1% BSA at concentration gradients of 0.001 fg / mL, 0.01 fg / mL, 0.1 fg / mL, 1 fg / mL, 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, and 100 pg / mL.

[0030] Detection group (in this invention): The detection was performed using the kit described in Example 3 and the operating procedure.

[0031] Control group (conventional method): SA-PolyHRP400 was used as the first signal amplification reagent, and FITC-labeled single-molecule tyramide (FITC-tyramide, 1μM) was used instead of the second signal amplification reagent. The remaining steps were the same as those of the detection group.

[0032] Detection conditions: All reactions were performed in 96-well filter plates. Washing was performed using a vacuum filtration device (3 times, 300 μL PBS-Tween 20 each time). Signal readings were performed using a Luminex 200 flow cytometer. 50 microspheres were collected per sample, and the median fluorescence intensity (MFI) was reported.

[0033] Blank control: The background signal was measured using a buffer solution without IL-6 as a blank (mean MFI = 100 ± 8).

[0034] result: In the detection group, when the IL-6 concentration was ≥0.1 fg / mL, the MFI was significantly higher than that of the blank (P<0.01), and the LOD (calculated based on 3×SD background) was 0.1 fg / mL; The control group showed a significantly higher signal than the background only at ≥100 fg / mL, with a LOD of 100 fg / mL. At a concentration of 1 fg / mL, the MFI in the detection group was 850 ± 42, and the MFI in the control group was 120 ± 15.

[0035] Conclusion: The sensitivity of the method of the present invention is significantly improved compared with that of conventional TSA.

[0036] Experiment 2: Specificity test Objective: To evaluate the cross-reactivity of the present invention with non-target proteins.

[0037] method: The IL-6 concentration was fixed at 1 fg / mL (low positive level); High concentrations of interfering agents were added to the samples: TNF-α, IFN-γ, CRP, IgG, and BSA, with a final concentration of 100 ng / mL for each. Simultaneously set up: (a) a positive control containing only IL-6; (b) a negative control containing only the interfering substance; and (c) a blank buffer.

[0038] Parallel testing was performed using the kit from Example 3, with n=3 per group.

[0039] result: The MFI values ​​of all interfering samples (842–865) were not significantly different from those of the positive control (850±28) (P>0.05). The negative control MFI≈100, consistent with the blank control.

[0040] Conclusion: This invention shows no cross-reactivity with common cytokines and serum proteins, and has good specificity.

[0041] Experimental Example 3: Precision Test Objective: To evaluate the repeatability and reproducibility of the method.

[0042] method: Intra-assay precision: The same batch of kits was used to test IL-6 samples at three concentrations (1 fg / mL, 100 fg / mL, and 10 pg / mL) 10 times each; Inter-batch precision: For three consecutive days, tests were performed independently each day using freshly prepared reagents (but from the same batch of polymer raw materials), with each concentration tested three times per day; All operations are performed by the same operator on the same instrument.

[0043] result: The intra-assay CVs were 7.8% (1 fg / mL), 5.3% (100 fg / mL), and 4.2% (10 pg / mL), respectively. The inter-batch CVs were 11.5%, 9.6%, and 8.1%, respectively.

[0044] Conclusion: The method has good precision and meets the requirements of clinical testing (usually CV < 15%).

[0045] Test Example 4: Multi-connection Detection Compatibility Test Objective: To verify the performance of the present invention in simultaneous detection of multiple indicators.

[0046] method: Three fluorescently encoded microspheres were prepared: microsphere A (encoding #23, coated with anti-IL-6 antibody), microsphere B (encoding #45, coated with anti-TNF-α antibody), and microsphere C (encoding #67, coated with anti-IFN-γ antibody). Prepare a mixed sample containing IL-6 (1 fg / mL), TNF-α (5 fg / mL), and IFN-γ (10 fg / mL). The kit from Example 3 (in which equal amounts of the three types of microspheres were mixed and added to the same reaction well) was used to detect the virus according to the standard procedure. The signals from each channel are automatically distinguished and quantified using Luminex software.

[0047] result: IL-6 recovery rate = 98%, TNF-α = 105%, IFN-γ = 92%; The proportion of TNF-α / IFN-γ signal in channel A of the microspheres was <0.5%, and the same was true for other channels; Total signal crosstalk <1%.

[0048] Conclusion: This invention supports high-fidelity multi-connection detection and is suitable for multiplex application scenarios.

[0049] Experimental Example 5: Microsphere Type Compatibility Test Objective: To verify the compatibility of the present invention with microspheres of different physical properties.

[0050] method: Three types of commercially available microspheres were selected: (a) Non-magnetic fluorescently encoded microspheres (5.6 μm, Luminex standard); (b) Magnetic fluorescently encoded microspheres (6.8 μm, for use in magnetic separation platforms); (c) Ordinary non-coded functionalized microspheres (3.2 μm, requiring external fluorescent tags for identification); All samples were coated with the same anti-IL-6 antibody, and IL-6 was detected in samples with a concentration of 10 fg / mL. The washing method is adjusted according to the characteristics of the microspheres (magnetic microspheres are washed using a magnetic frame, while the rest are filtered by vacuum).

[0051] result: The three MFI values ​​were: 892±35, 876±41, and 868±38, respectively. ANOVA analysis showed no significant differences between groups (P=0.62).

[0052] Conclusion: This invention is applicable to non-magnetic or magnetic, coded or non-coded functionalized microspheres in the particle size range of 0.5–20 μm, and has strong platform adaptability.

[0053] All experiments were performed at room temperature (20–25°C). The pH of the buffer solution was calibrated. Microsphere counting was performed using a hemocytometer or concentrations provided by the manufacturer. Data are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 26.0 software.

[0054] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit it. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A flow cytometry method for detecting fluorescent microspheres based on TSA signal amplification, characterized in that, The method includes the following steps: (a) Incubate fluorescently encoded microspheres coated with capture antibodies with the sample to be tested to allow the target analyte to specifically bind to the surface of the microspheres; (b) Add biotinylated detection antibody to form a microsphere-capture antibody-target analyte-biotinylated detection antibody complex; (c) Add a first signal amplification polymer, wherein the first signal amplification polymer is SA-PolyHRP400; (d) Add a second signal amplification polymer, which is a polymer with multiple tyrosine groups and multiple luciferin molecules covalently coupled on a polysaccharide backbone. Under HRP catalysis, the tyrosine groups are activated and covalently deposited in the vicinity of the microspheres to achieve fluorescence signal amplification. (e) Detect the fluorescence signal intensity of the fluorescently encoded microspheres and quantify the target analyte accordingly.

2. The flow cytometry method for detecting fluorescent microspheres based on TSA signal amplification according to claim 1, characterized in that, The polysaccharide is a dextran, and the molecular weight of the dextran is 30,000–500,000 Da.

3. The flow cytometry method for detecting fluorescent microspheres based on TSA signal amplification according to claim 2, characterized in that, The dextran molecule is coupled with an average of ≥20 tyrosine groups and ≥20 fluorescein molecules.

4. The flow cytometry method for detecting fluorescent microspheres based on TSA signal amplification according to claim 1, characterized in that, The microspheres include non-magnetic functionalized microspheres, non-magnetic functionalized fluorescently encoded microspheres, magnetic functionalized microspheres, or magnetic functionalized fluorescently encoded microspheres, and the particle size distribution of the microspheres is 0.5-20 micrometers.

5. The flow cytometry method for detecting fluorescent microspheres based on TSA signal amplification according to claim 1, characterized in that, The fluorescent dye molecules include at least one of fluorescent small molecules such as fluorescein, AF488, rhodamine, Cy3, Cy5, PE or APC, and nanofluorescent materials such as quantum dots.

6. The flow cytometry method for detecting fluorescent microspheres based on TSA signal amplification according to claim 1, characterized in that, The target analytes include interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), or interferon-γ (IFN-γ), or other protein detection items based on the sandwich method.

7. The flow cytometry method for detecting fluorescent microspheres based on TSA signal amplification according to claim 1, characterized in that, The preparation method of the second signal amplification polymer includes the following steps: dissolving polysaccharide in buffer solution, adding sodium periodate for oxidation reaction to generate polysaccharide containing aldehyde group; then adding tyramine hydrazide to react with aldehyde group to form tyramine polymer; then coupling the tyramine polymer with N-hydroxysuccinimide activated fluorescein under alkaline conditions, and purifying it to obtain a polymer rich in multiple tyramine fluorescein molecules.

8. A test kit for implementing the method according to any one of claims 1–6, characterized in that, The kit contains: (i) The second signal amplification reagent is a polymer covalently coupled with multiple tyrosine groups and multiple fluorescent dye molecules on a dextran backbone; (ii) A first signal amplification reagent, wherein the first signal amplification polymer is SA-PolyHRP400; (iii) Fluorescently encoded microspheres coated with capture antibodies; (iv) Biotin-labeled detection antibodies; and (v) Suitable reaction buffer for HRP catalysis.