Flow fluorescence analyzer and flow fluorescence analysis method

Abstract

The invention relates to the technical field of biomedical detection, in particular to a flow fluorescence analyzer and a flow fluorescence analysis method. The flow type fluorescence analyzer comprises a pretreatment module, a magnetic bead enrichment and resuspension module and a flow type detection module, the pretreatment module comprises a plurality of independent reaction container stations which are used for respectively bearing independent samples to be detected and corresponding single-index fluorescence coding magnetic beads and carrying out independent immunoreactions so as to obtain a reaction product suspension containing the fluorescence coding magnetic beads carrying objects to be detected; the magnetic bead enrichment and resuspension module comprises at least one enrichment container and a magnetic field generating device, the same enrichment container is used for receiving multiple reaction product suspension liquid from the pretreatment module, and the magnetic field generating device is used for generating or removing a magnetic field at the enrichment container; enriching the fluorescent coded magnetic beads carrying the to-be-detected substances in the suspension liquid of the reaction products, and obtaining a suspension liquid of the to-be-detected magnetic beads; and the flow type detection module is used for detecting fluorescence signals of the fluorescence coding magnetic beads.

Description

Technical Field

[0001] This application relates to the field of biomedical detection technology, and in particular to a flow cytometry analyzer and a flow cytometry analysis method. Background Technology

[0002] In clinical diagnosis, to comprehensively assess a patient's physiological or pathological state, it is often necessary to simultaneously detect multiple biomarkers in blood or body fluid samples, such as a group of cytokines, autoimmune antibodies, or allergens. This combined multi-marker detection has significant clinical value for early disease screening, diagnosis, disease monitoring, and prognosis.

[0003] Currently, there are two main technical approaches to achieving multi-indicator joint detection. The first is single-indicator detection technology, represented by chemiluminescence immunoassay. Its advantages lie in the use of independent reagent kits and reaction procedures for each detection item, specific reaction conditions, accurate and reliable results, and the ability to flexibly charge for each detection item according to medical orders. However, when a sample needs to be tested for multiple indicators, multiple repeated tests are required, severely consuming instrument resources, resulting in low overall throughput and significantly extended sample turnaround time (TAT), making it difficult to meet the high-efficiency requirements of modern laboratories. The other approach is liquid-phase chip (or flow cytometry multiplex immunoassay) technology based on flow cytometry. This technology uses microspheres with different fluorescent codes as carriers to simultaneously obtain results for dozens of indicators in a single reaction and detection of a single sample, offering significant advantages such as high throughput, low cost, and sample conservation. However, existing conventional flow cytometry multiplex detection methods typically pre-mix the capture microspheres for all items into a fixed detection kit. This model fails to meet the flexibility required for on-demand testing and billing in clinical settings. Developing various possible combination packages to achieve this flexibility would place a significant burden on reagent manufacturers in terms of R&D, registration, and inventory management. More importantly, incubating multiple antibodies and antigens targeting different analytes in the same reaction system easily leads to non-specific interferences such as cross-reactions and steric hindrance, potentially affecting the sensitivity and accuracy of the tests. It also makes it difficult to strictly adhere to the independent, regulatory-approved optimized reaction conditions for each individual test. How to solve these problems is a question that those skilled in the art need to consider. Summary of the Invention

[0004] To address the problems in the prior art, this application provides a flow cytometry fluorescence analyzer and a flow cytometry fluorescence analysis method.

[0005] This application provides a flow cytometry fluorescence analyzer, including a pretreatment module, a magnetic bead enrichment and resuspension module, and a flow cytometry detection module. The pretreatment module includes multiple independent reaction vessel stations for carrying independent test samples and corresponding single-index fluorescently coded magnetic beads, and performing independent immunoreactions to obtain multiple reaction product suspensions containing fluorescently coded magnetic beads carrying analytes. The magnetic bead enrichment and resuspension module includes at least one enrichment container and a magnetic field generator. The same enrichment container receives multiple reaction product suspensions from the pretreatment module. The magnetic field generator is located outside the enrichment container and generates or removes a magnetic field at the enrichment container to enrich the multiple reaction product suspensions containing fluorescently coded magnetic beads carrying analytes. The same enrichment container also contains test magnetic bead suspensions containing different fluorescently coded magnetic beads carrying analytes. The flow cytometry detection module is fluidly connected to the magnetic bead enrichment and resuspension module. The flow cytometry detection module includes a detection channel, a laser, and a photodetector. The detection channel is used for multiple fluorescently coded magnetic beads carrying different analytes in the magnetic bead suspension to pass sequentially through the laser irradiation area. The laser is used to irradiate the fluorescently coded magnetic beads carrying the analytes that have passed through the laser irradiation area to excite fluorescence signals. The photodetector is used to collect the fluorescence signals emitted by the fluorescently coded magnetic beads carrying different analytes and to identify at least the classification fluorescence signal used to identify the magnetic bead code and the report fluorescence signal used to reflect the concentration of the analyte.

[0006] Understandably, the multiple independent reaction vessel stations of the pretreatment module support parallel and independent immunoreaction of different tests on the same sample. This provides each test with an independent reaction environment free from interference from reagents from other tests, helping to ensure the accuracy and sensitivity of individual tests. It also provides a basis for flexibly combining tests according to clinical needs. The magnetic bead enrichment and resuspension module receives multiple reaction product suspensions from the pretreatment module through a single enrichment container. A magnetic field generator then gathers the fluorescently coded magnetic beads in these suspensions together, draining excess liquid to form a mixed test bead suspension. In other words, multiple independent reaction products are integrated into a single test sample for subsequent centralized detection. Finally, the flow cytometry module performs a single sample loading and detection on the mixed test bead suspension. A photodetector identifies the classification fluorescence signal and the report fluorescence signal, allowing for the simultaneous acquisition of results for multiple tests. Therefore, the flow cytometry fluorescence analyzer of this application retains the specificity of single-index detection reaction conditions through the independent reaction of the pretreatment module; at the same time, it utilizes the high-throughput characteristics of flow cytometry by collecting data through the magnetic bead enrichment and resuspension module and realizing single detection through the flow cytometry detection module; thus, while ensuring the accuracy of detection results, it improves the efficiency of multi-index joint detection and enhances the combination flexibility of detection items.

[0007] In one embodiment, the magnetic bead enrichment and resuspension module further includes a liquid collection assembly, which includes a liquid collection pipe, a switching valve, a sample loading tube assembly, and a drain pipe assembly. The liquid collection pipe includes a suction end and a connecting end. The suction end extends into the enrichment container, and the connecting end is optionally connected to the sample loading tube assembly or the drain pipe assembly via the switching valve, for introducing different reaction product suspensions into the enrichment container or extracting liquid from the enrichment container.

[0008] Understandably, by controlling the connection between the liquid collection pipe and the sample loading or drainage pipe assembly via a switching valve, the same liquid collection pipe extending into the enrichment container can perform different tasks. When it is necessary to introduce the reaction product suspension, the switching valve can connect to the corresponding inlet flow path; when it is necessary to remove waste liquid, the switching valve connects to the drainage pipe assembly; when it is necessary to send the final test magnetic bead suspension to the flow cytometry detection module, the switching valve connects to the sample loading pipe assembly. By reusing the liquid collection pipe, the flow path structure around the enrichment container is simplified, and the accuracy of liquid transfer is improved.

[0009] In one embodiment, the enrichment container is made of titanium alloy; the enrichment container includes a bottom wall and a side wall, which cooperate to form an enrichment cavity; the magnetic field generating device includes a magnetic component and a driving component, which are connected in a transmission manner; the driving component is used to drive the magnetic component to move closer to or further away from the side wall; the magnetic component is used to attract fluorescent coded magnetic beads carrying the analyte to enrich the inner surface of the side wall by magnetic force; the suction end extends into the enrichment cavity, and the opening of the suction end faces the bottom wall.

[0010] Understandably, on the one hand, the enrichment container is made of titanium alloy. Since titanium alloy is paramagnetic or nonmagnetic, it is less likely to generate residual magnetism after the external magnetic field is removed. This prevents the fluorescently encoded magnetic beads from being attracted by the residual magnetism of the container wall during the resuspension step, thus helping to improve the uniformity of resuspension and the recovery rate of the magnetic beads. On the other hand, the magnetic components are positioned close to or far from the sidewalls, allowing the magnetic beads to be enriched on the inner surface of the sidewalls. Simultaneously, the opening of the suction end faces the bottom wall, physically separating the area where the magnetic beads are enriched from the area where the liquid is extracted. This arrangement ensures that when extracting the liquid, the suction port is far from the magnetic beads bound to the sidewalls by magnetic force, thereby reducing the risk of accidentally removing the magnetic beads during the drainage process.

[0011] In one embodiment, the magnetic bead enrichment and resuspension module further includes an ultrasonic generator component connected to the bottom wall. The ultrasonic generator component is used to generate ultrasonic waves to drive different fluorescently coded magnetic beads carrying the analyte in the magnetic bead suspension to mix and resuspend.

[0012] Understandably, under the influence of a magnetic field, fluorescently coded magnetic beads carrying the analyte will be tightly adsorbed together, forming aggregates. The ultrasonic generator, connected to the bottom wall, transmits ultrasonic energy to the liquid within the enrichment container. The cavitation effect and high-frequency vibrations generated by the ultrasound in the liquid break up the aggregates, allowing the fluorescently coded magnetic beads carrying the analyte from different reaction containers to be uniformly suspended in the liquid, which helps improve the stability and accuracy of subsequent detection results.

[0013] In one embodiment, the side of the bottom wall away from the magnetic field generating device is configured to be inclined downward along the direction of gravity and cooperate with the side wall to form a suction area, with the suction end extending into the suction area.

[0014] Understandably, by designing the bottom wall as a sloping structure, gravity allows the liquid in the enrichment container to naturally converge into the lower-lying suction zone. Placing the suction end within this zone effectively reduces dead zones at the bottom of the container, resulting in less liquid residue upon extraction. Reduced liquid residue means more thorough removal of the supernatant or washing solution after the reaction, thus reducing background interference and providing a cleaner environment for subsequent resuspension and detection.

[0015] In one embodiment, the driving component includes a driving member, a slide block, a slide rail, and a fixed plate. The driving member and the enrichment container are respectively connected to opposite sides of the fixed plate. The slide rail is fixedly connected to the fixed plate, the slide block is slidably connected to the slide rail, the magnetic component is fixedly connected to the slide block, and the driving member is drivenly connected to the slide block and used to drive the magnetic component to move closer to or away from the opposite sidewall. The enrichment container also includes a cantilever, one end of which is connected to the sidewall and the other end is connected to the fixed plate.

[0016] Understandably, the cooperation between the slide rail and the slide block provides precise guidance for the movement of the magnetic components, ensuring good consistency in their path and final position each time they approach or move away from the sidewall. The slide block, driven by a drive unit, allows for automated and precise control of the application and removal of the magnetic field. The enrichment container is connected to the fixed plate via a cantilever structure. This suspended installation method reduces direct contact between the enrichment container and other components, helps isolate unnecessary mechanical vibration, facilitates the effective transfer of ultrasonic energy, and makes the installation and maintenance of the enrichment container more convenient.

[0017] This application also provides a flow cytometry fluorescence analysis method using a flow cytometer as described above. The flow cytometry fluorescence analysis method includes the following steps: S1. Independent reaction steps: In multiple independent reaction vessel stations of the pretreatment module, the test sample and the corresponding single-index fluorescent coded magnetic bead are subjected to independent immune reactions to obtain multiple reaction product suspensions containing fluorescent coded magnetic beads carrying the test analyte. S2. Enrichment and Resuspension Steps: Multiple sample suspensions of reaction products are introduced into the same enrichment container of the magnetic bead enrichment and resuspension module. A magnetic field is generated by a magnetic field generator to enrich the fluorescently encoded magnetic beads in the multiple sample suspensions of reaction products and resuspend them to form a test magnetic bead suspension containing different fluorescently encoded magnetic beads in the same enrichment container. S3. Flow cytometry detection step: The magnetic bead suspension to be tested is delivered to the flow cytometry detection module, so that multiple different fluorescently coded magnetic beads in the magnetic bead suspension pass through the laser irradiation area in sequence. The laser excites the beads and the photodetector collects the fluorescence signal, and identifies the classification fluorescence signal used to identify the magnetic bead code and the report fluorescence signal used to reflect the concentration of the analyte.

[0018] Understandably, the independent reaction steps allow the immune response of each detection indicator to proceed in its own optimized and independent environment, avoiding potential cross-interference issues caused by simultaneous reactions of multiple indicators. The enrichment and resuspension steps, through magnetic enrichment, combine the products (fluorescently encoded magnetic beads) from multiple independent reactions into a single suspension of test beads in a unified medium, achieving sample integration and concentration. The flow cytometry detection step utilizes the single-injection and multi-parameter analysis capabilities of flow cytometry technology to efficiently perform simultaneous detection of all indicators. The flow cytometry fluorescence analysis method provided in this application combines the accuracy of the reaction process with the high throughput advantages of the detection process.

[0019] In one embodiment, the enrichment and resuspension steps further include: Multiple reaction product suspensions are introduced into the same enrichment container once or multiple times using a single liquid collection assembly, and a magnetic field is generated by a magnetic field generator to enrich fluorescent coded magnetic beads carrying the analyte. After the magnetic field generator generates a magnetic field and constrains the position of the fluorescent coded magnetic beads carrying the analyte, the liquid in the enrichment container is extracted using the liquid collection assembly. Add resuspension liquid to the enrichment container or retain at least a portion of the liquid in the last added reaction product suspension, remove the magnetic field, and mix and resuspend the enriched fluorescently encoded magnetic beads to form the test magnetic bead suspension.

[0020] Understandably, the collection component introduces multiple reaction product suspensions and enriches them under a magnetic field, thus achieving the aggregation of fluorescently encoded magnetic beads. Simultaneously, while the magnetic field continuously confines the position of the magnetic beads, the reaction supernatant is removed, effectively eliminating unbound markers, sample matrix, and other potential interfering substances. Furthermore, the option to add resuspension liquid or retain the last added reaction product suspension provides two strategies: the former allows for the replacement with a sheath solution or buffer solution more favorable for flow cytometry, optimizing detection conditions; the latter simplifies the process and reduces reagent consumption in specific situations.

[0021] In one embodiment, during the enrichment and resuspension steps, fluorescently coded magnetic beads carrying the analyte are magnetically attracted and enriched on the inner surface of the sidewall of the enrichment container; ultrasonic waves are generated by an ultrasonic generator to drive the fluorescently coded magnetic beads to be uniformly suspended to achieve mixing and resuspension.

[0022] Understandably, enriching fluorescently coded magnetic beads on the inner surface of the sidewalls effectively prevents them from agglomerating near the bottom drain, reducing the risk of them being sucked away during drainage. After removing the magnetic field, since the magnetic beads may aggregate, ultrasonic resuspension is used to disperse the particles using high-frequency vibration energy. This helps ensure that the fluorescently coded magnetic beads in the final suspension are mostly dispersed as individual beads, facilitating flow cytometry analysis of each bead and obtaining accurate signals.

[0023] In one embodiment, the method for introducing multiple reaction product suspensions in the enrichment and resuspension steps is as follows: Multiple portions of the reaction product suspension were sequentially introduced into the enrichment container, with magnetic field enrichment and liquid extraction performed after each portion was introduced; or After collecting all the reaction product suspensions into the enrichment container, a one-time magnetic field enrichment and liquid extraction operation is performed.

[0024] Understandably, on the one hand, sequentially introducing multiple product suspensions into the enrichment container, processing only one suspension at a time, means the enrichment container's volume does not need to exceed the total volume of all suspensions. This offers excellent scalability for handling large numbers of samples or large volumes. On the other hand, collecting all multiple product suspensions into the enrichment container, provided the container volume is large enough, simplifies the control process and shortens the total time spent on the enrichment and resuspension steps by reducing repeated magnetic field loading / removal and liquid extraction. The system can flexibly select the optimal processing flow based on the number of analytes and sample volume, improving the applicability and efficiency of flow cytometry fluorescence analysis.

[0025] In one embodiment, the flow cytometry fluorescence analysis method further includes a data analysis step: S4. Data Analysis Steps: Identify the test items corresponding to each fluorescent coded magnetic bead based on the classified fluorescence signal, and call the independent calibration equation corresponding to each test item based on the reported fluorescence signal to convert the reported fluorescence signal value into a concentration value, so as to output a concentration result report for multiple test items.

[0026] Understandably, by interpreting the categorized fluorescence signals, the system can accurately distinguish which test item each fluorescently coded magnetic bead in the test bead suspension corresponds to. Subsequently, for each identified item, the system calls its corresponding independent calibration equation to analyze the reported fluorescence signal intensity of the magnetic bead and convert it into a specific concentration value. Even when all magnetic beads are tested together, the calculation of the results maintains their independence. This ensures that even temporarily and flexibly combined test items can be accurately quantified based on their respective independent standards. Attached Figure Description

[0027] Figure 1 This is a general schematic diagram of the flow cytometry analyzer provided in the embodiments of this application.

[0028] Figure 2 This is a schematic diagram of the pretreatment module of the flow cytometry fluorescence analyzer provided in the embodiments of this application.

[0029] Figure 3 This is a schematic diagram of the liquid collection component of the magnetic bead enrichment and resuspension module of the flow cytometry fluorescence analyzer provided in the embodiments of this application.

[0030] Figure 4 This is a schematic diagram showing the combination of the enrichment container, magnetic field generating device, and ultrasonic generating component of the magnetic bead enrichment and resuspension module of a flow cytometer provided in an embodiment of this application.

[0031] Figure 5 This is a three-dimensional schematic diagram of the enrichment container of the magnetic bead enrichment and resuspension module of a flow cytometer provided in an embodiment of this application.

[0032] Figure 6 This is a cross-sectional schematic diagram of the enrichment container of the magnetic bead enrichment and resuspension module of a flow cytometer provided in an embodiment of this application.

[0033] Figure 7 This is a schematic diagram of the enrichment container and magnetic field generating device of the magnetic bead enrichment and resuspension module of a flow cytometer provided in another embodiment of this application.

[0034] Figure 8 This is a schematic diagram of the enrichment container and magnetic field generating device of the magnetic bead enrichment and resuspension module of a flow cytometer provided in another embodiment of this application.

[0035] Figure 9 This is a schematic diagram of the enrichment container and magnetic field generating device of the magnetic bead enrichment and resuspension module of the flow cytometer provided in another embodiment of this application.

[0036] Figure 10 This is a schematic diagram of the flow cytometry detection module of the flow cytometry fluorescence analyzer provided in the embodiments of this application.

[0037] Figure 11 This is a schematic flowchart of the flow cytometry fluorescence analysis method provided in the embodiments of this application.

[0038] Figure 12 This is a flowchart illustrating a specific implementation of the flow cytometry fluorescence analysis method provided in this application.

[0039] Explanation of reference numerals in the attached drawings: 11. Pretreatment module; 111. Reaction vessel station; 1111. Reaction tray; 1112. Reaction vessel; 12. Magnetic bead enrichment and resuspension module; 121. Enrichment vessel; 1211. Bottom wall; 1212. Side wall; 1213. Enrichment cavity; 1214. Suction area; 1215. Liquid inlet port; 1216. Liquid outlet port; 1217. Cantilever; 122. Magnetic field generator; 1221. Magnetic component; 1222. Drive unit Components; 1223, Drive component; 1224, Slide seat; 1225, Slide rail; 1226, Fixing plate; 123, Liquid collection assembly; 1231, Liquid collection pipe; 12311, Suction end; 12312, Connecting end; 1232, Switching valve; 1233, Sample loading tube assembly; 1234, Drainage tube assembly; 124, Ultrasonic generating assembly; 125, Support frame; 13, Flow cytometry module; 131, Detection channel; 132, Laser; 133, Photodetector. Detailed Implementation

[0040] The following is in conjunction with the appendix Figures 1 to 12 This application will be described in further detail below.

[0041] The technical solutions in the embodiments of this application will be further described in detail below with reference to the accompanying drawings. The described embodiments are only possible technical implementations of this application, but are not limited thereto. Other embodiments obtained by those skilled in the art in conjunction with the embodiments of this application without creative effort are also within the protection scope of this application.

[0042] Further integration Figures 1 to 10As shown, this application provides a flow cytometry fluorescence analyzer, including a pretreatment module 11, a magnetic bead enrichment and resuspension module 12, and a flow cytometry detection module 13. The pretreatment module 11 includes multiple independent reaction vessel stations 111, each used to hold an independent test sample and a corresponding single-index fluorescently coded magnetic bead, and to perform independent immunoreactions to obtain multiple reaction product suspensions containing fluorescently coded magnetic beads carrying the analyte. The magnetic bead enrichment and resuspension module 12 includes at least one enrichment container 121 and a magnetic field generator 122. The same enrichment container 121 receives multiple reaction product suspensions from the pretreatment module 11. The magnetic field generator 122 is located outside the enrichment container 121 and is used to generate or remove a magnetic field at the enrichment container 121 to enrich the fluorescently coded magnetic beads carrying the analyte in the multiple reaction product suspensions. The same enrichment container 121 also contains test magnetic bead suspensions containing different fluorescently coded magnetic beads carrying the analyte. The flow cytometry detection module 13 is in fluid communication with the magnetic bead enrichment and resuspension module 12. The flow cytometry detection module 13 includes a detection channel 131, a laser 132, and a photodetector 133. The detection channel 131 is used for multiple fluorescently coded magnetic beads carrying different analytes in the magnetic bead suspension to pass sequentially through the laser irradiation area. The laser 132 is used to irradiate the fluorescently coded magnetic beads carrying analytes that have passed through the laser irradiation area to excite fluorescence signals. The photodetector 133 is used to collect the fluorescence signals emitted by the fluorescently coded magnetic beads carrying different analytes and to identify at least the classification fluorescence signal used to identify the magnetic bead code and the reporting fluorescence signal used to reflect the concentration of the analyte.

[0043] Understandably, the multiple independent reaction vessel stations 111 of the pretreatment module 11 support parallel and independent immunoreaction of different test items for the same sample, providing an independent reaction environment for each test item, free from interference from reagents of other items. This helps ensure the accuracy and sensitivity of individual tests and also provides a basis for flexibly combining test items according to clinical needs. The magnetic bead enrichment and resuspension module 12 receives multiple reaction product suspensions from the pretreatment module 11 through the same enrichment container 121, and uses a magnetic field generator 122 to gather the fluorescently encoded magnetic beads in these suspensions together, draining excess liquid to form a mixed test magnetic bead suspension; that is, multiple independent reaction products are integrated into a single test sample for subsequent centralized detection. Finally, the flow cytometry module 13 performs a single sample loading and detection on the mixed test magnetic bead suspension, and uses a photodetector 133 to identify the classification fluorescence signal and the report fluorescence signal, thus obtaining the results of multiple test items simultaneously. Therefore, the flow cytometry fluorescence analyzer of this application retains the specificity of the single-index detection reaction conditions through the independent reaction of the pretreatment module 11; at the same time, it utilizes the high-throughput characteristics of flow cytometry by collecting the magnetic bead enrichment and resuspension modules 12 and realizing single detection through the flow cytometry detection module 13; thus, while ensuring the accuracy of the detection results, it improves the efficiency of multi-index joint detection and enhances the combination flexibility of detection items.

[0044] Further integration Figure 2 As shown, in this embodiment, the multiple independent reaction vessel stations 111 of the pretreatment module 11 can be configured as a rotatable reaction disk 1111 supporting multiple reaction vessels 1112. The reaction vessels 1112 can be, for example, independent reaction cups. The magnetic bead enrichment and resuspension module 12 and the pretreatment module 11 are fluidly connected via tubing or a pipetting robotic arm. The outlet of the enrichment vessel 121 within the magnetic bead enrichment and resuspension module 12 is connected to the inlet of the flow cytometry detection module 13 via tubing, forming a complete fluid pathway. The pretreatment module 11 can utilize a known and feasible structure in the field of chemical fluorescence detection; the rotation method of the reaction disk 1111 and the arrangement of the reaction vessels 1112 are not detailed here.

[0045] Further integration Figure 3 As shown, in one embodiment, the magnetic bead enrichment and resuspension module 12 further includes a liquid collection assembly 123, which includes a liquid collection pipe 1231, a switching valve 1232, a sample loading tube assembly 1233, and a drain pipe assembly 1234. The liquid collection pipe 1231 includes a suction end 12311 and a connecting end 12312. The suction end 12311 extends into the enrichment container 121, and the connecting end 12312 is optionally connected to the sample loading tube assembly 1233 or the drain pipe assembly 1234 via the switching valve 1232, for introducing different reaction product suspensions into the enrichment container 121 or extracting liquid from the enrichment container 121.

[0046] In this embodiment, the liquid collection pipe 1231 is a liftable aspiration needle, with its suction end 12311 located at the end of the aspiration needle and its connecting end 12312 located at the upper part of the aspiration needle. The switching valve 1232 is a multi-port valve, with its multiple ports connected to the connecting end 12312, the pipeline of the sample loading tube group 1233, the pipeline of the drain pipe group 1234, and the cleaning liquid line, etc. The sample loading tube group 1233 is connected to the flow cytometry detection module 13, and the drain pipe group 1234 is connected to the waste liquid collection device. By controlling the rotation of the valve core of the switching valve 1232, the flow path of the liquid collection pipe 1231 can be switched to aspirate the liquid in the enrichment container 121 to the waste liquid collection device, or to aspirate the resuspended magnetic bead suspension to the flow cytometry detection module 13.

[0047] Understandably, by controlling the connection between the connecting end 12312 of the collection pipe 1231 and the sample loading tube assembly 1233 or the drain assembly 1234 via the switching valve 1232, the same collection pipe 1231 extending into the enrichment container 121 can perform different tasks. When it is necessary to introduce the reaction product suspension, the switching valve 1232 can connect to the corresponding inlet flow path; when it is necessary to remove waste liquid, the switching valve 1232 connects to the drain assembly 1234; when it is necessary to send the final test magnetic bead suspension to the flow cytometry detection module 13, the switching valve 1232 can connect to the sample loading tube assembly 1233. By reusing the collection pipe 1231, the flow path structure around the enrichment container 121 is simplified, and the accuracy of liquid transfer is improved.

[0048] Further integration Figures 4 to 6 As shown, in one embodiment, the enrichment container 121 is made of titanium alloy. The enrichment container 121 includes a bottom wall 1211 and a side wall 1212, which cooperate to form an enrichment cavity 1213. The magnetic field generating device 122 includes a magnetic component 1221 and a driving component 1222, which are connected to each other. The driving component 1222 is used to drive the magnetic component 1221 to move closer to or further away from the side wall 1212. The magnetic component 1221 is used to attract fluorescent coded magnetic beads carrying the analyte to enrich the inner surface of the side wall 1212 by magnetic force. The suction end 12311 extends into the enrichment cavity 1213, and the opening of the suction end 12311 faces the bottom wall 1211.

[0049] In this embodiment, the enrichment container 121 is generally cup-shaped. The magnetic component 1221 is a permanent magnet, which is mounted on the moving end of the driving component 1222. The driving component 1222 drives the magnetic component 1221 to reciprocate in the horizontal direction. When the magnetic component 1221 moves to the position closest to the enrichment container 121, its working surface is in close contact with or maintains a small gap with the outer surface of the side wall 1212 of the enrichment container 121. At this time, the magnetic field passes through the side wall 1212 and acts on the enrichment cavity 1213. When the magnetic component 1221 moves away from the enrichment container 121, the magnetic field effect basically disappears. The opening of the suction end 12311 is lower than the area where the magnetic component 1221 generates magnetic attraction.

[0050] Understandably, on the one hand, the enrichment container 121 is made of titanium alloy. Since titanium alloy is a paramagnetic or nonmagnetic material, it is not prone to residual magnetism after the external magnetic field is removed. This avoids the fluorescently encoded magnetic beads being attracted by the residual magnetic force of the container wall during the resuspension step, thereby helping to improve the uniformity of resuspension and the recovery rate of magnetic beads. On the other hand, the magnetic component 1221 is positioned close to or far from the sidewall 1212, so that the magnetic beads are enriched on the inner surface of the sidewall 1212; at the same time, the opening of the suction end 12311 faces the bottom wall 1211, physically separating the area where the magnetic beads are enriched from the area where the liquid is extracted. This arrangement ensures that when extracting the liquid, the suction port is far away from the magnetic beads that are magnetically bound to the sidewall 1212, thereby reducing the risk of accidentally extracting the magnetic beads during the drainage process.

[0051] In one embodiment, the magnetic bead enrichment and resuspension module 12 further includes an ultrasonic generator component 124, which is connected to the bottom wall 1211. The ultrasonic generator component 124 is used to generate ultrasonic waves to drive different fluorescently coded magnetic beads carrying the analyte in the magnetic bead suspension to mix and resuspend.

[0052] In this embodiment, the ultrasonic generating component 124 may include a piezoelectric transducer, which is fixedly connected to the outer surface of the bottom wall 1211 of the enrichment container 121 by means of acoustic coupling agent or direct welding. When the magnetic component 1221 of the magnetic field generating device 122 moves away from the enrichment container 121 to remove the magnetic field, the control system activates the ultrasonic generating component 124 to generate high-frequency vibration, and the energy is transmitted to the liquid in the enrichment cavity 1213 through the bottom wall 1211.

[0053] Understandably, under the influence of a magnetic field, fluorescently coded magnetic beads carrying the analyte will be tightly adsorbed together, forming bead agglomerates. The ultrasonic generator 124, connected to the bottom wall 1211, transmits ultrasonic energy to the liquid within the enrichment container 121. The cavitation effect and high-frequency vibration generated by the ultrasound in the liquid break up the bead agglomerates, allowing the fluorescently coded magnetic beads carrying the analyte from different reaction containers 1112 to be uniformly suspended in the liquid, which helps improve the stability and accuracy of subsequent detection results.

[0054] In one embodiment, the side of the bottom wall 1211 away from the magnetic field generating device 122 is configured to be inclined downward along the direction of gravity, and cooperates with the side wall 1212 to form a suction area 1214, with the suction end 12311 extending into the suction area 1214.

[0055] In this embodiment, the bottom wall 1211 of the enrichment container 121 is an inclined plane, with its lowest point located on the side away from the movement path of the magnetic component 1221. This lowest point and the adjacent side wall 1212 enclose a sharp-angled region, namely the suction region 1214. The suction end 12311 of the liquid collection assembly 123 is positioned during operation so that its opening is very close to the bottom of the suction region 1214, thereby effectively suctioning the liquid collected therein.

[0056] Understandably, by designing the bottom wall 1211 as a sloping structure, gravity allows the liquid in the enrichment container 121 to naturally converge into the lower-lying suction zone 1214. Placing the suction end 12311 within the suction zone 1214 effectively reduces dead zones at the bottom of the container, resulting in less residue when the liquid is removed. Reduced liquid residue means more thorough removal of the supernatant or washing solution after the reaction, thereby reducing background interference and providing a cleaner environment for subsequent resuspension and detection.

[0057] In one embodiment, the driving component 1222 includes a driving member 1223, a slide block 1224, a slide rail 1225, and a fixing plate 1226. The driving member 1223 and the enrichment container 121 are respectively connected to opposite sides of the fixing plate 1226. The slide rail 1225 is fixedly connected to the fixing plate 1226, the slide block 1224 is slidably connected to the slide rail 1225, and the magnetic component 1221 is fixedly connected to the slide block 1224. The driving member 1223 is drivenly connected to the slide block 1224 and is used to drive the magnetic component 1221 to move closer to or away from the opposite sidewall 1212. The enrichment container 121 also includes a cantilever 1217, one end of which is connected to the sidewall 1212 and the other end is connected to the fixing plate 1226.

[0058] In this embodiment, the fixing plate 1226 is generally rectangular, with its thickness and length directions corresponding to different horizontal directions, and its width direction corresponding to the vertical direction. The slide rail 1225 extends approximately along the length of the fixing plate 1226 and is disposed on one side of the fixing plate 1226 (which can be the side with the largest area of ​​the fixing plate 1226). The slide block 1224 is slidably connected to the slide rail 1225 and located on the corresponding side. The enrichment container 121 is spaced apart from this side along the thickness direction of the fixing plate 1226. The enrichment container 121 and the driving member 1223 are located at opposite ends of the fixing plate 1226, approximately along its length. The driving member 1223 is a stepper motor, connected to the slide block 1224 via a lead screw and nut mechanism to control the position of the slide block 1224. The slide rail 1225 is a linear guide rail, ensuring the smoothness and straightness of the slide block 1224's movement. The enrichment container 121 is fixed to the fixing plate 1226 in a suspended state by the cantilever 1217 structure, with space left below and to the side to facilitate the installation of the ultrasonic generator assembly 124 and avoid mechanical interference with other components.

[0059] Understandably, the cooperation between the slide rail 1225 and the slide block 1224 provides precise guidance for the movement of the magnetic component 1221, ensuring good consistency in its path and final position each time it approaches or moves away from the side wall 1212. The slide block 1224, driven by the drive component 1223, enables automated and precise control of the application and removal of the magnetic field. The enrichment container 121 is connected to the fixed plate 1226 via a cantilever structure 1217. This suspended installation method reduces direct contact between the enrichment container 121 and other components, helps isolate unnecessary mechanical vibration, facilitates the effective transfer of ultrasonic energy, and makes the installation and maintenance of the enrichment container 121 more convenient.

[0060] Further integration Figure 7 As shown, in another embodiment, the enrichment container 121 has an enrichment cavity 1213, an inlet port 1215, and an outlet port 1216. The inlet port 1215 and the outlet port 1216 are respectively connected to the enrichment cavity 1213. Multiple reaction product suspensions from the pretreatment module 11 are introduced into the enrichment cavity 1213 through the inlet port 1215, and the magnetic bead suspension to be tested is discharged from the enrichment cavity 1213 through the outlet port 1216.

[0061] Understandably, by setting up independent inlet ports 1215 and outlet ports 1216 to construct a flow-through enrichment chamber 1213 structure, multiple reaction product suspensions can be continuously or batch-wise introduced into the chamber through the inlet port 1215. Under the influence of a magnetic field, fluorescently coded magnetic beads carrying the analyte are trapped within the enrichment chamber 1213, while the liquid can flow out through the outlet port 1216. After processing, the suspension of the magnetic beads is then discharged through the outlet port 1216. This method is suitable for scenarios requiring the processing of large volumes of liquid or continuous operation, improving liquid processing efficiency compared to using a single opening for inlet and outlet.

[0062] In one embodiment, the enrichment container 121 is configured to be horizontally positioned, and the magnetic component 1221 is suspended above the enrichment container 121.

[0063] In this embodiment, the enrichment container 121 is a flat tubular or box-shaped structure placed horizontally. A magnetic component 1221 is located directly above it and can move up and down to apply or remove a magnetic field. An inlet port 1215 and an outlet port 1216 are located at opposite ends of the container. When a magnetic field is applied, fluorescently coded magnetic beads are attracted to the upper inner wall of the container, while the liquid flows out from the outlet port 1216 under gravity.

[0064] In this embodiment, the liquid inlet port 1215 can be connected to the liquid collection assembly 123 and / or the cleaning liquid path, and the liquid outlet port 1216 can be connected to the waste liquid collection device and / or the flow cytometry detection module 13.

[0065] Understandably, suspending the magnetic component 1221 above for magnetic adsorption allows the fluorescently coded magnetic beads to accumulate on the upper wall of the container, while the liquid naturally remains at the bottom due to gravity. This gravity-assisted solid-liquid separation layout facilitates smoother drainage and further reduces the likelihood of the magnetic beads being carried away during drainage.

[0066] Further integration Figure 8 As shown, in another embodiment, the enrichment container 121 is configured to be vertically arranged, at least a portion of the enrichment container 121 is funnel-shaped, and the cross-sectional diameter of the inlet port 1215 is larger than the cross-sectional diameter of the outlet port 1216; the magnetic component 1221 is configured to be able to move up and down relative to the enrichment container 121.

[0067] In this embodiment, the inlet port 1215 is located at the upper part of the enrichment container 121, and the outlet port 1216 is located at the lower constriction. The magnetic component 1221 can be designed as a ring magnet or a magnetic sleeve composed of multiple bar magnets, which is fitted over the enrichment container 121 and can move up and down along its vertical axis. By reciprocating the raising and lowering of the magnetic component 1221, the capture efficiency of fluorescently coded magnetic beads can be improved, and more fluorescently coded magnetic beads can be acquired as much as possible.

[0068] Understandably, the vertically positioned enrichment container 121, with its funnel-shaped structure wider at the top and narrower at the bottom, facilitates the rapid convergence of liquid towards the outlet port 1216 under gravity, reducing liquid residue adhering to the inner wall. The magnetic component 1221 is configured to move up and down relative to the enrichment container 121. This allows it not only to control the application and removal of the magnetic field but also to control the specific location of the magnetic beads being enriched by adjusting its dwell height, or to assist the cleaning process through reciprocating motion, thus enabling more efficient and thorough enrichment and cleaning operations.

[0069] Further integration Figure 9 As shown, in another embodiment, the magnetic bead enrichment and resuspension module 12 further includes a support frame 125, and the enrichment container 121 is detachably connected to the support frame 125.

[0070] In this embodiment, the support frame 125 is provided with a buckle or positioning structure that matches the bottom of the enrichment container 121. The enrichment container 121 can be quickly installed and removed from the support frame 125 through simple plugging, unplugging or rotating actions.

[0071] Understandably, designing the enrichment container 121 to be detachably connected to the support frame 125 allows the enrichment container 121 to be used as a replaceable component, such as a disposable consumable. Replacing the enrichment container 121 with a new one each time a different sample is tested reduces cross-contamination between samples due to incomplete container cleaning, making it more suitable for applications requiring high sensitivity or higher biosafety standards.

[0072] Further integration Figure 10 As shown, in this embodiment, the detection channel 131 allows multiple fluorescently coded magnetic beads carrying different analytes in the test bead suspension to sequentially pass through the laser irradiation area. The laser 132 irradiates the corresponding laser beam towards the laser irradiation area to excite the fluorescently coded magnetic beads carrying the analytes to emit fluorescence signals. The photodetector 133 collects the fluorescence signals emitted by the different fluorescently coded magnetic beads carrying the analytes and identifies at least the classification fluorescence signal used to identify the bead coding and the report fluorescence signal used to reflect the concentration of the analyte.

[0073] It is understandable that the detection channel 131, laser 132 and photodetector 133 can be selected from known and feasible structures in the field of flow cytometry detection. The configuration of the channel, the model and operation mode of laser 132 and photodetector 133 are not described in detail.

[0074] Further integration Figure 11 As shown in the embodiments of this application, a flow cytometry fluorescence analysis method is also provided, using a flow cytometer as described in any of the above claims.

[0075] In one embodiment, the flow cytometry fluorescence analysis method uses a flow cytometer as described in any of the foregoing embodiments, and the flow cytometry fluorescence analysis method includes the following steps: S0. Work Order Creation Steps: Based on all the items that need to be tested for the sample, the system searches the instrument's internal reagent library and calibration curve library, and can create an optimized testing work order for the sample according to a preset combination algorithm. This work order can be a combination of single-index tests, a combination of multi-index reagents and single-index tests, or a combination of multiple different multi-index reagents.

[0076] Understandably, the reagent library configuration allows for flexible combinations of testing items with maximum freedom, and by optimizing the combination, the number of work orders can be reduced as much as possible, thereby reducing the number of subsequent sample preparations and improving the overall testing throughput.

[0077] S1. Independent reaction steps: In multiple independent reaction vessel stations 111 of the pretreatment module 11, the test sample is independently immunoreacted with the corresponding single-index fluorescently encoded magnetic beads to obtain multiple reaction product suspensions containing fluorescently encoded magnetic beads carrying the test analyte.

[0078] In this embodiment, the system allocates a separate reaction container 1112 for each target test item (or combination of test items) according to the work order. Following the instructions of the regulatory-approved test reagents for each test item, the system adds the sample, corresponding fluorescently coded magnetic beads, and other necessary reagents to each reaction container 1112, and performs independent incubation and washing reaction steps. This process ensures that the detection environment for each indicator is independent and optimized, thereby minimizing cross-interference between items and improving the detection limit and sensitivity of the items. After the reaction is complete, each reaction container 1112 contains the prepared sample, i.e., the magnetic beads that have bound the analyte and the reporter fluorescent molecule. At this point, the reaction has terminated, and the reaction product is stable.

[0079] S2. Enrichment and Resuspension Steps: Multiple portions of reaction product suspensions are introduced into the same enrichment container 121 of the magnetic bead enrichment and resuspension module 12. A magnetic field is generated by the magnetic field generator 122 to enrich the fluorescently encoded magnetic beads in the multiple portions of reaction product suspensions and resuspend them to form a test magnetic bead suspension containing different fluorescently encoded magnetic beads in the same enrichment container 121.

[0080] Understandably, since the reaction products are in a stable state after each independent reaction step is completed, they can be mixed together without cross-interference between the individual products.

[0081] S3. Flow cytometry detection step: The magnetic bead suspension to be tested is delivered to the flow cytometry detection module 13, so that multiple different fluorescently coded magnetic beads in the magnetic bead suspension to be tested pass through the laser irradiation area in sequence. The laser 132 excites the beads and the photodetector 133 collects the fluorescence signal and identifies the classification fluorescence signal used to identify the magnetic bead code and the report fluorescence signal used to reflect the concentration of the analyte.

[0082] Understandably, the independent reaction steps allow the immune response of each detection indicator to proceed in its own optimized and independent environment, avoiding potential cross-interference issues caused by simultaneous reactions of multiple indicators. The enrichment and resuspension steps, through magnetic enrichment, combine the products (fluorescently encoded magnetic beads) from multiple independent reactions into a single suspension of test beads in a unified medium, achieving sample integration and concentration. The flow cytometry detection step utilizes the single-injection and multi-parameter analysis capabilities of flow cytometry technology to efficiently perform simultaneous detection of all indicators. The flow cytometry fluorescence analysis method provided in this application combines the accuracy of the reaction process with the high throughput advantages of the detection process.

[0083] In one embodiment, the enrichment and resuspension steps further include: using a liquid collection component 123 to introduce multiple portions of reaction product suspensions into the same enrichment container 121 once or multiple times, causing the magnetic field generating device 122 to generate a magnetic field, enriching fluorescently coded magnetic beads carrying the analyte; after the magnetic field generating device 122 generates a magnetic field and constrains the position of the fluorescently coded magnetic beads carrying the analyte, using the liquid collection component 123 to extract the liquid from the enrichment container 121; adding resuspension liquid to the enrichment container 121 or retaining at least a portion of the liquid in the last added reaction product suspension, removing the magnetic field, and mixing and resuspending the enriched fluorescently coded magnetic beads to form a suspension of magnetic beads to be tested.

[0084] In other embodiments, one of the reaction containers 1112 in the pretreatment module 11 can be used as the enrichment container 121. For example, the first reaction container 1112 is moved to the station of the magnetic bead enrichment and resuspension module 12. After applying a magnetic field, the reaction product suspensions in the subsequent reaction containers 1112 are sequentially transferred to the reaction container 1112 using a pipette. Magnetic field enrichment and supernatant extraction are performed after each transfer. When the reaction product suspension is added to the last reaction container 1112, the liquid is no longer extracted; instead, the magnetic field is directly removed and the mixture is mixed and resuspended. At this point, the liquid in the last reaction container 1112 is used as the resuspension liquid.

[0085] Understandably, the collection component 123 introduces multiple reaction product suspensions and enriches them using a magnetic field, thus achieving the aggregation of fluorescently encoded magnetic beads. Simultaneously, while the magnetic field continuously confines the position of the magnetic beads, the reaction supernatant is removed, effectively eliminating unbound markers, sample matrix, and other potential interfering substances. Furthermore, the option to add resuspension liquid or retain the last added reaction product suspension provides two strategies: the former allows for the replacement with a sheath solution or buffer solution more favorable for flow cytometry, optimizing detection conditions; the latter simplifies the process and reduces reagent consumption in specific situations.

[0086] In one embodiment, during the enrichment and resuspension steps, fluorescently coded magnetic beads carrying the analyte are attracted by magnetic force and enriched on the inner surface of the sidewall 1212 of the enrichment container 121; ultrasonic waves are generated by an ultrasonic generator 124 to drive the fluorescently coded magnetic beads to be uniformly suspended to achieve mixing and resuspension.

[0087] In other embodiments, the mixing and resuspension method can also be implemented by mechanical vibration or gas-liquid blowing.

[0088] Understandably, enriching the fluorescently coded magnetic beads on the inner surface of the sidewall 1212 effectively prevents them from agglomerating near the bottom drain, reducing the risk of them being sucked away during drainage. After the magnetic field is removed, since the magnetic beads may agglomerate, ultrasonic mixing and resuspension are used. This utilizes the high-frequency vibration energy to disperse the magnetic bead particles, helping to ensure that the fluorescently coded magnetic beads are mostly dispersed as individual beads in the final test suspension. This facilitates flow cytometry analysis of each fluorescently coded magnetic bead and the acquisition of accurate signals.

[0089] In one embodiment, in the enrichment and resuspension steps, the multiple portions of reaction product suspension are introduced in the following ways: multiple portions of reaction product suspension are introduced into the enrichment container 121 in sequence, and a magnetic field enrichment and liquid extraction operation is performed after each portion of reaction product suspension is introduced; or multiple portions of reaction product suspension are collected into the enrichment container 121 and then a one-time magnetic field enrichment and liquid extraction operation is performed.

[0090] In this embodiment, when the volume of the enrichment container 121 is small, for example, less than the total volume of all reaction product suspensions, the first method of sequential introduction and operation is used. When the volume of the enrichment container 121 is large enough, for example, to accommodate the total volume of all reaction product suspensions, the second method of one-time collection operation can be used.

[0091] Understandably, on the one hand, the method of sequentially introducing multiple product suspensions into the enrichment container 121 processes only one volume of product suspension at a time, meaning the volume of the enrichment container 121 does not need to exceed the total volume of all product suspensions. This provides excellent scalability for processing a large number of items or large samples. On the other hand, the method of collecting all multiple product suspensions into the enrichment container 121, provided the volume of the enrichment container 121 is large enough, simplifies the control process and shortens the total time spent on the entire enrichment and resuspension steps by reducing repeated magnetic field loading / removal and liquid extraction. The system can flexibly select a better processing flow based on the number of analytes and the sample volume, improving the applicability and efficiency of the flow cytometry fluorescence analysis method.

[0092] In one embodiment, the flow cytometry fluorescence analysis method further includes a data analysis step: S4. Data Analysis Steps: Identify the test items corresponding to each fluorescent coded magnetic bead based on the classified fluorescence signal, and call the independent calibration equation corresponding to each test item based on the reported fluorescence signal to convert the reported fluorescence signal value into a concentration value, so as to output a concentration result report for multiple test items.

[0093] In this embodiment, the data analysis step is automatically completed by the data processing module built into the flow cytometer. This module pre-stores the coding rules for each test item and its corresponding fluorescently coded magnetic beads, as well as the independent calibration equations or standard curves obtained after calibration for each test item. Upon receiving signal data collected by the photodetector 133, the software first categorizes each magnetic bead event to its corresponding test item based on the intensity or color combination of the classified fluorescence signal. Then, it extracts the reported fluorescence signal value of that magnetic bead event, substitutes it into the corresponding calibration equation for that item, and calculates the concentration result. Finally, the concentration results of all items are integrated into a single report. It is particularly important to note that all items in the reagent library must have independent codes; codes cannot be mixed or shared to avoid calculation confusion.

[0094] Understandably, by interpreting the categorized fluorescence signals, the system can accurately distinguish which test item each fluorescently coded magnetic bead in the test bead suspension corresponds to. Subsequently, for each identified item, the system calls its corresponding independent calibration equation to analyze the reported fluorescence signal intensity of the magnetic bead and convert it into a specific concentration value. Even when all magnetic beads are tested together, the calculation of the results maintains their independence. This ensures that even temporarily and flexibly combined test items can be accurately quantified based on their respective independent standards.

[0095] Further integration Figure 12 As shown, the method provided in this application will be described below through a specific embodiment: In this embodiment, we take the detection of four cytokines, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), and interleukin-10 (IL-10), in a patient sample as an example.

[0096] First, the analyzer receives the sample information and test item request, obtains the patient information of the blood sample ID through LIS / HIS query, and decodes the items to be tested (IL-2, IL-4, IL-6, IL-10).

[0097] Subsequently, based on the required test items (IL-2, IL-4, IL-6, IL-10), the system searches the current reagent library for reagents for all items, the current calibration curve library for calibration curves for all items, and the quality control library for quality control results for all required tests, as well as whether these quality control results are under control. A work order for the tests is then created, and the system establishes four independent lists of items (IL-2, IL-4, IL-6, IL-10).

[0098] Next, independent reaction steps are performed. The system automatically allocates four independent reaction containers in the pretreatment module. The sample and fluorescently coded magnetic beads from the IL-2 single-index detection kit (let's assume the code is 01) are added to the first reaction container; the sample and fluorescently coded magnetic beads from the IL-4 single-index detection kit (code 02) are added to the second reaction container; the sample and fluorescently coded magnetic beads from the IL-6 single-index detection kit (code 03) are added to the third reaction container; and the sample and fluorescently coded magnetic beads from the IL-10 single-index detection kit (code 04) are added to the fourth reaction container. Subsequently, each of the four reaction containers independently completes incubation, washing, and other steps according to its respective reagent instructions, yielding four reaction product suspensions.

[0099] Next, the enrichment and resuspension steps are performed. The system sequentially or simultaneously transfers the reaction product suspensions from the four reaction vessels to the same enrichment vessel of the magnetic bead enrichment and resuspension module. A magnetic field is generated by activating the magnetic field generator, adsorbing and enriching all fluorescently coded magnetic beads (coded 01, 02, 03, 04) onto the inner wall of the enrichment vessel. The supernatant is then removed. Afterward, an appropriate amount of resuspension buffer (e.g., 200 μL) is added to the enrichment vessel, the magnetic field is removed, and ultrasound is generated by the ultrasonic generator to thoroughly mix all the magnetic beads, forming a mixed suspension of the test magnetic beads.

[0100] Subsequently, a flow cytometry detection step is performed. The suspension of magnetic beads to be tested is delivered to the flow cytometry module. Under the constraint of the sheath fluid, the mixed fluorescently coded magnetic beads pass sequentially through the laser irradiation area. A photodetector collects the fluorescence signal emitted by each magnetic bead after it is excited.

[0101] Finally, the data analysis step is executed. After receiving the fluorescence signal data, the data processing module analyzes it: when the system identifies the categorical fluorescence signal corresponding to code 01, it calls the independent calibration equation for IL-2 to convert the reported fluorescence signal intensity of the magnetic bead into an IL-2 concentration value; when code 02 is identified, it calls the calibration equation for IL-4 to calculate its concentration value; and so on. Ultimately, the system outputs a complete test report containing the concentration values ​​of four items: IL-2, IL-4, IL-6, and IL-10.

[0102] The embodiments described herein are preferred embodiments and are not intended to limit the scope of protection of this application. Identical components are represented by the same reference numerals. All equivalent changes made to the structure, shape, and principle of this application should be included within the scope of protection of this application.

Claims

1. A flow cytometer, characterized in that, include: The pretreatment module (11) includes multiple independent reaction vessel stations (111) for carrying independent test samples and corresponding single-index fluorescently encoded magnetic beads, and performing independent immune reactions to obtain multiple reaction product suspensions containing fluorescently encoded magnetic beads carrying the test analytes. The magnetic bead enrichment and resuspension module (12) includes at least one enrichment container (121) and a magnetic field generating device (122). The same enrichment container (121) is used to receive multiple portions of the reaction product suspension from the pretreatment module (11). The magnetic field generating device (122) is located outside the enrichment container (121) and is used to generate or remove a magnetic field at the enrichment container (121) to enrich the fluorescently coded magnetic beads carrying the analyte in the multiple portions of the reaction product suspension. The same enrichment container (121) is also used to contain test magnetic bead suspensions containing different fluorescently coded magnetic beads carrying the analyte. The flow cytometry detection module (13) is in fluid communication with the magnetic bead enrichment and resuspension module (12). The flow cytometry detection module (13) includes a detection channel (131), a laser (132), and a photodetector (133). The detection channel (131) is used to allow multiple fluorescently coded magnetic beads carrying the analyte in the magnetic bead suspension to pass sequentially through the laser irradiation area. The laser (132) is used to irradiate the fluorescently coded magnetic beads carrying the analyte through the laser irradiation area to excite a fluorescence signal. The photodetector (133) is used to collect the fluorescence signals emitted by the different fluorescently coded magnetic beads carrying the analyte, and to identify at least the classification fluorescence signal used to identify the magnetic bead code and the report fluorescence signal used to reflect the concentration of the analyte in the fluorescence signal.

2. The flow cytometer according to claim 1, characterized in that: The magnetic bead enrichment and resuspension module (12) further includes a liquid collection assembly (123), which includes a liquid collection pipe (1231), a switching valve (1232), a sample loading tube assembly (1233), and a drain assembly (1234). The liquid collection pipe (1231) includes a suction end (12311) and a connecting end (12312). The suction end (12311) extends into the enrichment container (121), and the connecting end (12312) is optionally connected to the sample loading tube assembly (1233) or the drain assembly (1234) via the switching valve (1232) for introducing different reaction product suspensions into the enrichment container (121) or extracting liquid from the enrichment container (121).

3. The flow cytometer according to claim 2, characterized in that: The enrichment container (121) is made of titanium alloy; the enrichment container (121) includes a bottom wall (1211) and a side wall (1212), the bottom wall (1211) and the side wall (1212) cooperate to form an enrichment cavity (1213); the magnetic field generating device (122) includes a magnetic component (1221) and a driving component (1222), the magnetic component (1221) and the driving component (1222) are connected in a transmission manner, the driving component (1222) is used to drive the magnetic component (1221) to move closer to or further away from the side wall (1212), the magnetic component (1221) is used to attract the fluorescent coded magnetic beads carrying the analyte to enrich the inner surface of the side wall (1212) by magnetic force; the suction end (12311) extends into the interior of the enrichment cavity (1213), the opening of the suction end (12311) faces the bottom wall (1211).

4. The flow cytometer according to claim 3, characterized in that: The magnetic bead enrichment and resuspension module (12) further includes an ultrasonic generator component (124), which is connected to the bottom wall (1211). The ultrasonic generator component (124) is used to generate ultrasonic waves to drive the different fluorescently coded magnetic beads carrying the analyte in the magnetic bead suspension to be tested to mix and resuspend.

5. The flow cytometer according to claim 3, characterized in that: The side of the bottom wall (1211) away from the magnetic field generating device (122) is configured to be inclined downward along the direction of gravity and cooperates with the side wall (1212) to form a suction area (1214), and the suction end (12311) extends into the suction area (1214).

6. The flow cytometer according to claim 3, characterized in that: The driving component (1222) includes a driving element (1223), a slide block (1224), a slide rail (1225), and a fixing plate (1226). The driving element (1223) and the enrichment container (121) are respectively connected to opposite sides of the fixing plate (1226). The slide rail (1225) is fixedly connected to the fixing plate (1226). The slide block (1224) is slidably connected to the slide rail (1225). The magnetic component (1221) is fixedly connected to the slide block (1224). The driving element (1223) is drivenly connected to the slide block (1224) and is used to drive the magnetic component (1221) to move closer to or away from the side wall (1212). The enrichment container (121) also includes a cantilever (1217). One end of the cantilever (1217) is connected to the side wall (1212), and the other end is connected to the fixing plate (1226).

7. A flow cytometry fluorescence analysis method, characterized in that, Using the flow cytometer as described in any one of claims 1 to 6, the flow cytometer fluorescence analysis method comprises the following steps: S1. Independent reaction steps: In the multiple independent reaction container stations (111) of the pretreatment module (11), the test sample and the corresponding single-index fluorescent coded magnetic beads are subjected to independent immune reactions to obtain multiple reaction product suspensions containing fluorescent coded magnetic beads carrying the test analytes. S2, Enrichment and Resuspension Step: Multiple portions of the reaction product suspension are introduced into the same enrichment container (121) of the magnetic bead enrichment and resuspension module (12). A magnetic field is generated by the magnetic field generator (122) to enrich the fluorescently encoded magnetic beads in the multiple portions of the reaction product suspension and resuspend them to form a test magnetic bead suspension containing different fluorescently encoded magnetic beads in the same enrichment container (121). S3, Flow cytometry detection step: The test magnetic bead suspension is transported to the flow cytometry detection module (13), so that multiple different fluorescently coded magnetic beads in the test magnetic bead suspension pass through the laser irradiation area in sequence, are excited by the laser (132) and the fluorescence signal is collected by the photodetector (133), and the classification fluorescence signal used to identify the magnetic bead code and the report fluorescence signal used to reflect the concentration of the test substance are identified in the fluorescence signal.

8. The flow cytometry fluorescence analysis method according to claim 7, characterized in that, The enrichment and resuspension steps further include: Multiple portions of the reaction product suspension are introduced into the same enrichment container (121) once or multiple times using a liquid collection assembly (123), so that the magnetic field generating device (122) generates a magnetic field to enrich the fluorescent coded magnetic beads carrying the analyte. After the magnetic field generating device (122) generates a magnetic field and constrains the position of the fluorescent coded magnetic bead carrying the analyte, the liquid in the enrichment container (121) is extracted using the liquid collection assembly (123). Add resuspension liquid to the enrichment container (121) or retain at least a portion of the liquid in the last added reaction product suspension, remove the magnetic field, and mix and resuspend the enriched fluorescently encoded magnetic beads to form the test magnetic bead suspension.

9. The flow cytometry fluorescence analysis method according to claim 8, characterized in that, In the enrichment and resuspension steps, the fluorescently coded magnetic beads carrying the analyte are attracted by magnetic force and enriched on the inner surface of the side wall (1212) of the enrichment container (121); ultrasonic waves are generated by an ultrasonic generator (124) to drive the fluorescently coded magnetic beads to be uniformly suspended to achieve mixing and resuspension.

10. The flow cytometry fluorescence analysis method according to claim 7, characterized in that, In the enrichment and resuspension steps, the multiple reaction product suspensions are introduced in the following manner: Multiple portions of the reaction product suspension are sequentially introduced into the enrichment container (121), with magnetic field enrichment and liquid extraction performed after each portion of the reaction product suspension is introduced; or After all the multiple portions of the reaction product suspension are collected into the enrichment container (121), a one-time magnetic field enrichment and liquid extraction operation is performed.

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