Method for detecting CAR-T (Chimeric Antigen Receptor-T) cells by combining microfluidics and immunofluorescence

Abstract

The invention relates to the field of CAR-T cell concentration detection, in particular to a method for detecting CAR-T cells through combination of microfluidics and immunofluorescence. Through immunomagnetic bead enrichment and micro-fluidic chip partitioning, CAR-T cells are separated from a blood sample through immunomagnetic beads, then labeling is carried out or enhanced by adding a labeling antigen, anti-TCR-PE and DAPI, three-channel fluorescence labeling is achieved, quantitative analysis can be carried out on the number, the cell morphology and the function of the CAR-T cells through automatic immunofluorescence imaging, and the accuracy of CAR-T cell detection is improved. And the detection limit reaches 0.001%.

Description

Technical Field

[0001] This application relates to the field of CAR-T cell concentration detection, specifically to a method for detecting CAR-T cells using a combination of microfluidics and immunofluorescence. Background Technology

[0002] Chimeric antigen receptor T (CAR-T) cell therapy is an important tool in tumor immunotherapy. Since its approval in 2017-2018, more than 35,000 patients with malignant tumors of the lymphatic system worldwide have received treatment, significantly changing the treatment landscape for relapsed / refractory B-cell lymphoma (B-LNH), acute lymphoblastic leukemia (B-ALL), and multiple myeloma (MM).

[0003] Currently, the short- and medium-term management of CAR-T cell therapy patients relies heavily on clinical and imaging assessments, as well as some non-specific biological tests. However, the complexity, specificity, and reproducibility of biological monitoring remain inconclusive, and healthcare professionals use varying standards, ranging from simple biomarkers to cytokine measurements and in vivo CAR-T cell tracking.

[0004] The extent of CAR-T cell expansion in vivo is closely related to clinical response and can also affect the incidence and severity of early and late toxicities, such as cytokine release syndrome [CRS], immune effector cell-associated neurotoxicity syndrome [ICANS], and immune effector cell-associated hematologic toxicity [ICAHT].

[0005] In recent years, significant progress has been made in technologies for monitoring the in vivo concentration of CAR-T cells. Digital PCR technology (such as patent CN202411113482) achieves absolute quantification at the single-copy level by designing specific primer-probe combinations. It can accurately detect the copy number of CAR-T cells from products such as Acetaminophen injection and Qilu BCMACART without the need for a standard curve. It also exhibits high tolerance to PCR inhibitors and is suitable for analyzing complex clinical samples. Real-time PCR (patent CN202211627293) designs universal primers and probes for FMC63scFv and humanized variants, with a linear range of 1.1 × 10⁻⁶. 0 -1.1×10 7 The reference gene is PCBP2, with copies / μL, to improve detection stability and is suitable for quality control of CD19CAR-T products.

[0006] However, existing technologies still have significant limitations: digital PCR equipment is expensive and complex to operate; quantitative real-time PCR relies on standards, leading to poor comparability of results; while flow cytometry (patent CN202311621814) can simultaneously analyze cell phenotype and function, its detection limit is limited to 0.01% CAR+ cell proportion and requires fresh samples. Furthermore, research on the correlation between the dynamic changes of CAR-T cells in vivo and clinical efficacy and toxicity is insufficient, and there is a lack of unified monitoring standards and threshold settings. These technological bottlenecks limit the precision management and personalized treatment optimization of CAR-T cell therapy. Summary of the Invention

[0007] To address the technical challenges of insufficient sensitivity (flow cytometry detection limit of 0.1%) and complex operation (high cost of digital PCR equipment) in existing methods for detecting CAR-T cell concentration in vivo, this application utilizes immunomagnetic bead enrichment and microfluidic chip partitioning to separate CAR-T cells from blood samples. These cells are then further labeled with or enhanced with labeled antigens, anti-TCR-PE, and DAPI, achieving three-channel fluorescence labeling. This allows for automated immunofluorescence imaging to quantitatively analyze the number, morphology, and function of CAR-T cells, achieving a detection limit of 0.001%.

[0008] This application provides a method for detecting CAR-T cells using a microfluidic-immunofluorescence coupled approach, employing the following technical solution: A microfluidic-immunofluorescence coupled method for detecting CAR-T cells includes the following steps: S1: Mix the blood sample and immunomagnetic beads and incubate to obtain a mixed sample; S2: The mixed sample is added to a microfluidic chip for separation to obtain enriched CAR-T immunomagnetic beads; S3: Mix the enriched CAR-T immunomagnetic beads with a labeling solution to obtain initially labeled enriched CAR-T immunomagnetic beads; the labeling solution includes the following components: labeled antigen and anti-TCR-PE solution; the labeled antigen includes at least one of CD19, BCMA, CD5, CD7, CD30, Ep-CAM, CLDN18.2, GPC3, GUCY2C, MSLN, and ROR1-FTIC; S4: Mix the pre-labeled enriched CAR-T immunomagnetic beads with DAPI solution to obtain the enriched CAR-T immunomagnetic beads to be detected; S5: The number of CAR-T cells in the blood sample can be determined by performing immunofluorescence staining and scanning analysis on the enriched CAR-T immunomagnetic beads to be detected. The immunomagnetic beads are superparamagnetic Fe3O4@SiO2 nanoparticles with surface-modified antigens, and the adsorbed antigens include at least one of CD19, BCMA, CD5, CD7, CD30, Ep-CAM, CLDN18.2, GPC3, GUCY2C, MSLN, and ROR1-FTIC.

[0009] This application proposes a combined detection method integrating immunomagnetic bead enrichment, microfluidic chip partitioning, and immunofluorescence imaging, employing the aforementioned technical solution. The immunomagnetic beads are modified with adsorbed antigens, which bind well to CAR-T cells. After incubation with a blood sample, the immunomagnetic beads adsorb CAR-T cells from the blood sample onto their surface. The mixed sample is then precisely partitioned into single cells using a microfluidic chip, effectively separating CAR-T cells from other substances in the blood sample. The separated magnetic beads are then further enhanced with labeled antigens to strengthen the binding of CAR-T cells; anti-TCR-PE is added to bind to the "endogenous TCR" on the surface of CAR-T cells; and DAPI (4',6-diamidindo-2-phenylindole) is added to bind to the nuclei of CAR-T cells. Using three-channel fluorescent labeling, automated immunofluorescence imaging can be used to quantitatively analyze the number, morphology, and function of CAR-T cells, achieving a detection limit of 0.001% (1 CAR-T cell / 102). 6 Peripheral blood mononuclear cells).

[0010] This application utilizes immunomagnetic bead enrichment and microfluidic chip partitioning to separate CAR-T cells from blood samples via immunomagnetic beads. Then, by adding labeled antigens, anti-TCR-PE, and DAPI for labeling or enhanced labeling, three-channel fluorescence labeling is achieved. This allows for automated immunofluorescence imaging to quantitatively analyze the number, morphology, and function of CAR-T cells, achieving a detection limit of 0.001%.

[0011] Preferably, the method for preparing the immunomagnetic beads includes the following steps: Aminoation: Superparamagnetic Fe3O4@SiO2 nanoparticles were added to APTES ethanol solution, reacted under nitrogen protection, centrifuged, washed with water and dried to obtain aminated magnetic beads; Activation: Dissolve the adsorbed antigen in pre-cooled MES buffer, add EDC solution and NHS solution sequentially to form a mixed solution, and incubate on ice with shaking to obtain an activated antigen solution; Activation: The aminated magnetic beads are added to the activated antigen solution for reaction, and then glycine solution or Tris buffer is added to block the reaction. After incubation, the mixture is washed to obtain immunomagnetic beads.

[0012] By adopting the above technical solution, superparamagnetic Fe3O4@SiO2 nanoparticles refer to core-shell structured nanoparticles formed by coating a SiO2 shell around a Fe3O4 magnetic core. Amino groups are introduced from the silanol groups on the SiO2 shell surface through a silanization reaction with APTES (3-aminopropyltriethoxysilane).

[0013] EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) activates the carboxyl group on the adsorbed antigen, transforming it into an unstable intermediate (O-acylisourea). NHS (N-hydroxysuccinimide) is converted into a stable NHS ester. The NHS ester reacts with the amino groups of the aminated magnetic beads to form a strong amide bond, thereby stably binding the antigen to the superparamagnetic Fe3O4@SiO2 nanoparticles, thus preparing immunomagnetic beads.

[0014] Preferably, the mass ratio of the adsorbed antigen to the superparamagnetic Fe3O4@SiO2 nanoparticles is 1:5-10.

[0015] By adopting the above technical solution, when the content of superparamagnetic Fe3O4@SiO2 nanoparticles is too low, steric hindrance easily occurs between adsorbed antigens, leading to a decrease in binding efficiency; when the content of superparamagnetic Fe3O4@SiO2 nanoparticles is too high, the probability of adsorbed antigens binding to them decreases, also leading to a decrease in binding efficiency. Therefore, after extensive research and experimental verification, the applicant finally determined that the mass ratio of adsorbed antigens to superparamagnetic Fe3O4@SiO2 nanoparticles in this application is preferably as described above.

[0016] Preferably, the molar concentration of EDC in the mixed solution is 2-8 mM.

[0017] By adopting the above technical solution, when the molar concentration of EDC is too low, the activation degree of carboxyl groups on the adsorbed antigen is insufficient; when the molar concentration of EDC is too high, the activation degree of carboxyl groups on the adsorbed antigen is too high, and they aggregate with each other, which is not conducive to the subsequent adsorption of CAR-T cells. Therefore, after a lot of research and experimental verification, the applicant finally determined that the molar concentration of EDC in the mixed solution of this application should be as described above.

[0018] Preferably, the molar concentration of NHS in the mixed solution is 5-15 mM.

[0019] Preferably, the superparamagnetic Fe3O4@SiO2 nanoparticles are pretreated, including the following steps: The superparamagnetic Fe3O4@SiO2 nanoparticles were added to hydrochloric acid and ultrasonically mixed, then washed with water and dried to obtain pretreated magnetic beads.

[0020] By employing the above technical solution, the superparamagnetic Fe3O4@SiO2 nanoparticles are pre-acid-washed, which can further increase the silanol groups on the surface of the SiO2 shell, thereby better promoting the binding of the superparamagnetic Fe3O4@SiO2 nanoparticles with adsorbed antigens.

[0021] Preferably, the excitation wavelength of the adsorbed antigen is 488 nm and the emission wavelength is 575 nm.

[0022] Preferably, the excitation wavelength of the anti-TCR-PE solution is 494 nm and the emission wavelength is 517 nm.

[0023] Preferably, the excitation wavelength of DAPI in the DAPI solution is 405 nm, and the emission wavelength is 460 nm.

[0024] In summary, this application has the following beneficial effects: This application utilizes immunomagnetic bead enrichment and microfluidic chip partitioning to separate CAR-T cells from blood samples. Then, by adding or enhancing labeling with labeled antigens, anti-TCR-PE, and DAPI, three-channel fluorescence labeling is achieved. This allows for automated immunofluorescence imaging to quantitatively analyze the number, morphology, and function of CAR-T cells, achieving a detection limit of 0.001%. The immunomagnetic beads of this application are made by introducing amino groups through the silanization reaction of superparamagnetic Fe3O4@SiO2 nanoparticles with APTES (3-aminopropyltriethoxysilane), and activating and adsorbing antigens with EDC and NHS, thereby stably binding the antigens to the superparamagnetic Fe3O4@SiO2 nanoparticles. In this application, the superparamagnetic Fe3O4@SiO2 nanoparticles are pre-acid-washed, which can further increase the silanol groups on the surface of the SiO2 shell, thereby better promoting the binding of the superparamagnetic Fe3O4@SiO2 nanoparticles to the adsorbed antigen. Detailed Implementation

[0025] The raw materials in this application include the following: Superparamagnetic Fe3O4@SiO2 nanoparticles: diameters ranging from 200nm to 5μm are all acceptable; this application only uses 1μm as an example.

[0026] APTES: 3-aminopropyltriethoxysilane, using the commercially available product with CAS number 919-30-2; EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, using the commercially available product with CAS number 25952-53-8; NHS: N-hydroxysuccinimide, using the commercially available product with CAS number 6066-82-6; DAPI: 4',6-Diamidinyl-2-phenylindole, using the commercially available product with CAS number 28718-90-3; The present application will be further described in detail below with reference to embodiments and comparative examples.

[0027] Example 1 A method for preparing immunomagnetic beads includes the following steps: Aminoation: 8 mg of superparamagnetic Fe3O4@SiO2 nanoparticles were added to 4 mL of APTES ethanol solution (APTES volume fraction 1%), ultrasonically dispersed for 15 min to form a uniform suspension, reacted for 18 h under nitrogen protection, centrifuged, washed with water and dried to obtain aminated magnetic beads. Activation: Dissolve 1 mg of adsorbed antigen (CD19) in 80 μL of pre-cooled MES buffer, then add 10 μL of EDC (50 mM) solution and 10 μL of NHS (100 mM) solution to form a mixed solution. Incubate on ice with shaking for 30 min to obtain the activated antigen solution. The mixed solution is 100 μL, with EDC at a molar concentration of 5 mM and NHS at a molar concentration of 10 mM. Activation: Aminated magnetic beads were added to the activated antigen solution and reacted for 3 hours. Then, 100 μL of Tris buffer (100 mM molar concentration, glycine solution can also be used) was added to block unreacted sites. After incubation for 30 minutes, the mixture was washed to obtain immunomagnetic beads.

[0028] A microfluidic-immunofluorescence coupled method for detecting CAR-T cells includes the following steps: S1: Dilute 7.5 mL of blood sample 4 times and mix with 50 μL of immunomagnetic beads. Incubate at 10 rpm for 1.5 h to obtain a mixed sample. S2: Add the mixed sample to the microfluidic chip for separation to obtain enriched CAR-T immunomagnetic beads; S3: Mix the enriched CAR-T immunomagnetic beads with 40 μL of labeling solution and shake well. Let stand for 30 min, and shake well every 10 min to obtain the initially labeled enriched CAR-T immunomagnetic beads. The labeling solution consists of the following components: labeled antigen (CD19, 1 mg / mL), anti-TCR-PE (2 mg / mL), and sterile water. S4: Mix the initially labeled and enriched CAR-T immunomagnetic beads with 100 μL of DAPI solution (5 μg / mL), let stand for 5 min, and obtain the CAR-T immunomagnetic beads to be detected; S5: Immunofluorescence staining and scanning analysis of the CAR-T enriched immunomagnetic beads to be detected can determine the number of CAR-T cells in the blood sample. There are three sets of excitation and emission wavelengths for immunofluorescence staining and scanning analysis, targeting adsorbed or labeled antigens, anti-TCR-PE, and DAPI, respectively.

[0029] The excitation wavelength for adsorbed or labeled antigens is 488 nm, and the emission wavelength is 575 nm. The excitation wavelength for TCR-PE resistance is 494 nm, and the emission wavelength is 517 nm. The excitation wavelength of DAPI is 405nm, and the emission wavelength is 460nm.

[0030] Example 2-3 Examples 2-3 are based on Example 1, but the types of adsorbed antigens and labeled antigens are adjusted, as shown in Table 1.

[0031] Comparative Examples 1-2 Comparative Example 1 was based on Example 1, but the labeling solution was adjusted to have the following components: labeled antigen (CD19, 1 mg / mL) and sterile water.

[0032] Comparative Example 2, based on Example 1, involved directly performing immunofluorescence staining and scanning analysis on the initially labeled and enriched CAR-T immunomagnetic beads without mixing them with 100 μL of DAPI solution (5 μg / mL).

[0033] Performance testing Sensitivity Using serial dilutions, 300, 30, and 3 CAR-T cells were added to 30 mL of sample, respectively, resulting in a blood cell count of 3*10⁻⁶ cells per sample. 7 The samples were tested according to the microfluidic-immunofluorescence assays used in Examples 1-3 and Comparative Examples 1-2, wherein a 7.5 mL blood sample was diluted 4-fold and replaced with a 30 mL sample.

[0034] Clinical validation The clinical remission rate of 10 B-cell lymphoma patients treated with azithromycin was evaluated according to the lymphoma efficacy evaluation criteria, and the correlation coefficient between CAR-T cell concentration monitoring results and clinical remission rate was analyzed.

[0035] Based on the above detection method, the test results of Examples 1-3 and Comparative Examples 1-2 were obtained, as shown in Table 1 below: Table 1. Molar concentrations of EDC and NHS in the activation solution and performance test results for Examples 1-3 and Comparative Examples 1-2. project Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Types of adsorbed antigens CD19 BCMA Ep-CAM CD19 CD19 Types of antigens with price tags CD19 BCMA Ep-CAM CD19 CD19 Does it have anti-TCR-PE? yes yes yes no yes Is there DAPI solution? yes yes yes yes no Theoretical detection value 1 300 300 300 300 300 Actual measured value 1 299.93 299.88 299.83 294.35 296.72 Theoretical detection value 2 30 30 30 30 30 Actual measured value 2 29.88 29.74 29.69 26.81 27.33 Theoretical detection value 3 3 3 3 3 3 Actual measured value 3 0.86 0.81 0.79 0.14 0.16 Correlation coefficient 0.92 0.91 0.91 0.71 0.74 Referring to Table 1, and comparing Examples 1-3 and Comparative Examples 1-2, it can be seen that the detection method integrating immunomagnetic bead enrichment, microfluidic chip partitioning, and immunofluorescence imaging of this application can achieve a low detection limit (down to 1 mL / s). One CAR-T cell was detected in the sample. The reason is that CAR-T cells are separated from blood samples by immunomagnetic beads enrichment and microfluidic chip partitioning. Then, by adding labeled antigens, anti-TCR-PE and DAPI for enhanced labeling, three-channel fluorescence labeling is achieved. Automated immunofluorescence imaging can then be used to quantitatively analyze the number, morphology and function of CAR-T cells.

[0036] Effective detection requires the use of three-channel fluorescent labeling: one for the antigen, one for anti-TCR-PE, and one for DAPI. Otherwise, the absence of fluorescent labeling for anti-TCR-PE or DAPI will reduce the detection accuracy.

[0037] Examples 4-5 Examples 4-5 are based on Example 1, with adjustments made to the amount of superparamagnetic Fe3O4@SiO2 nanoparticles added. The specific adjustments are shown in Table 2.

[0038] The performance tests of Examples 4-5 were performed as described above, and the test results are shown in Table 2.

[0039] Table 2. Data on the addition amount and performance of superparamagnetic Fe3O4@SiO2 nanoparticles in Examples 1 and 4-5. project Example 1 Example 4 Example 5 Dosage added / mg 8 5 10 Correlation coefficient 0.92 0.85 0.88 Referring to Table 2, a comparison of Examples 1 and 4-5 shows that as the amount of superparamagnetic Fe3O4@SiO2 nanoparticles added increases, the correlation coefficient first rises and then falls. This may be because as the amount of superparamagnetic Fe3O4@SiO2 nanoparticles increases, the adsorbed antigens gradually overcome steric hindrance, leading to improved binding efficiency and thus better detection results. However, beyond a certain range, the probability of the adsorbed antigen binding to a single superparamagnetic Fe3O4@SiO2 nanoparticle decreases, also resulting in a decrease in binding efficiency and reduced detection performance.

[0040] Examples 6-7 Examples 6-7 are based on Example 1, but the molar concentrations of EDC and NHS in the mixed solution are adjusted as shown in Table 3.

[0041] The performance tests of Examples 6-7 were performed as described above, and the test results are shown in Table 3.

[0042] Table 3. Molar concentrations and performance test data of EDC and NHS in the mixed solutions of Examples 1 and 6-7. project Example 1 Example 6 Example 7 EDC / mM 5 2 8 NHS / mM 10 5 15 Correlation coefficient 0.92 0.84 0.89 Referring to Table 3, a comparison of Examples 1 and 6-7 shows that the correlation coefficient initially increases and then decreases as the molar concentrations of EDC and NHS in the mixed solution rise. This may be because the increasing molar concentrations of EDC and NHS in the mixed solution lead to a higher degree of activation of the adsorbed antigen, facilitating its binding to the superparamagnetic Fe3O4@SiO2 nanoparticles and improving detection efficiency. However, beyond a certain range, the carboxyl groups on the adsorbed antigen become excessively activated, causing them to aggregate and hindering subsequent adsorption of CAR-T cells, thus reducing detection efficiency.

[0043] Examples 8-9 In Example 8, based on Example 1, 8 mg of superparamagnetic Fe3O4@SiO2 nanoparticles were added to 1 mL of HCl solution (0.1 M), sonicated for 10 min, then washed with water and dried to obtain pretreated magnetic beads. The pretreated beads were then added to 5 mL of APTES ethanol solution (1% APTES by volume) for further processing.

[0044] Example 9: Based on Example 1, 8 mg of superparamagnetic Fe3O4@SiO2 nanoparticles were added to 1 mL of HCl solution (0.1 M), sonicated for 10 min, centrifuged and the supernatant was discarded. The nanoparticles were then added to another 1 mL of HCl solution (0.1 M), sonicated for 10 min, washed with water and dried to obtain pretreated magnetic beads. The pretreated beads were then added to 5 mL of APTES ethanol solution (1% APTES volume fraction) for further processing.

[0045] The performance tests of Examples 8-9 were performed as described above, and the test results are shown in Table 4.

[0046] Table 4 Performance test data for Examples 1 and 8-9 project Example 1 Example 8 Example 9 Correlation coefficient 0.92 0.94 0.95 Referring to Table 4, a comparison of Examples 1 and 8-9 shows that pre-acid washing of the superparamagnetic Fe3O4@SiO2 nanoparticles improves the detection effect. Two acid washes further enhance the detection performance. This is because acid washing increases the number of silanol groups on the SiO2 shell surface, thereby better promoting the binding of the superparamagnetic Fe3O4@SiO2 nanoparticles to the adsorbed antigen.

[0047] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for detecting CAR-T cells using microfluidics-immunofluorescence coupling, characterized in that, Includes the following steps: S1: Mix the blood sample and immunomagnetic beads and incubate to obtain a mixed sample; S2: The mixed sample is added to a microfluidic chip for separation to obtain enriched CAR-T immunomagnetic beads; S3: Mix the enriched CAR-T immunomagnetic beads with a labeling solution to obtain initially labeled enriched CAR-T immunomagnetic beads; the labeling solution includes the following components: labeled antigen and anti-TCR-PE solution; the labeled antigen includes at least one of CD19, BCMA, CD5, CD7, CD30, Ep-CAM, CLDN18.2, GPC3, GUCY2C, MSLN, and ROR1-FTIC; S4: Mix the pre-labeled enriched CAR-T immunomagnetic beads with DAPI solution to obtain the enriched CAR-T immunomagnetic beads to be detected; S5: The number of CAR-T cells in the blood sample can be determined by performing immunofluorescence staining and scanning analysis on the enriched CAR-T immunomagnetic beads to be detected. The immunomagnetic beads are superparamagnetic Fe3O4@SiO2 nanoparticles with surface-modified antigens, and the adsorbed antigens include at least one of CD19, BCMA, CD5, CD7, CD30, Ep-CAM, CLDN18.2, GPC3, GUCY2C, MSLN, and ROR1-FTIC.

2. The method for detecting CAR-T cells using microfluidic-immunofluorescence coupling according to claim 1, characterized in that: The method for preparing the immunomagnetic beads includes the following steps: Aminoation: Superparamagnetic Fe3O4@SiO2 nanoparticles were added to APTES ethanol solution, reacted under nitrogen protection, centrifuged, washed with water and dried to obtain aminated magnetic beads; Activation: Dissolve the adsorbed antigen in pre-cooled MES buffer, add EDC solution and NHS solution sequentially to form a mixed solution, and incubate on ice with shaking to obtain an activated antigen solution; Activation: The aminated magnetic beads are added to the activated antigen solution for reaction, and then glycine solution or Tris buffer is added to block the reaction. After incubation, the mixture is washed to obtain immunomagnetic beads.

3. The method for detecting CAR-T cells using microfluidic-immunofluorescence coupling according to claim 2, characterized in that: The mass ratio of the adsorbed antigen to the superparamagnetic Fe3O4@SiO2 nanoparticles is 1:5-10.

4. The method for detecting CAR-T cells using microfluidic-immunofluorescence coupling according to claim 2, characterized in that: The molar concentration of EDC in the mixed solution is 2-8 mM.

5. The method for detecting CAR-T cells using microfluidic-immunofluorescence coupling according to claim 4, characterized in that: The molar concentration of NHS in the mixed solution is 5-15 mM.

6. The method for detecting CAR-T cells using microfluidic-immunofluorescence coupling according to claim 2, characterized in that: The superparamagnetic Fe3O4@SiO2 nanoparticles undergo pretreatment, including the following steps: The superparamagnetic Fe3O4@SiO2 nanoparticles were added to hydrochloric acid and ultrasonically mixed, then washed with water and dried to obtain pretreated magnetic beads.

7. The method for detecting CAR-T cells using microfluidic-immunofluorescence coupling according to claim 1, characterized in that: The excitation wavelength of the adsorbed antigen is 488 nm, and the emission wavelength is 575 nm.

8. The method for detecting CAR-T cells using microfluidic-immunofluorescence coupling according to claim 1, characterized in that: The excitation wavelength of the anti-TCR-PE solution is 494 nm, and the emission wavelength is 517 nm.

9. The method for detecting CAR-T cells using microfluidic-immunofluorescence coupling according to claim 1, characterized in that: The excitation wavelength of DAPI in the DAPI solution is 405 nm, and the emission wavelength is 460 nm.