Multiplex fluorescence immunomagnetic bead separation reagent card

Abstract

The utility model discloses a multiplex fluorescence immunomagnetic bead separation reagent card, and relates to the technical field of biological detection. The reagent card comprises an upper cover and a shell, wherein the upper cover is provided with a sample adding hole; the lower part of the sample adding hole is connected with a first microfluidic pipeline arranged in the shell; a reaction bin, a detection bin, a quality control bin, a first capturing bin and a second capturing bin are also arranged in the shell; the first microfluidic pipeline is communicated with the reaction bin; the reaction bin connects the quality control bin and the first capture bin in series through a second microfluidic pipeline; the reaction bin connects the detection bin and the second capture bin in series through a third microfluidic pipeline; each detection bin is communicated with the corresponding second capture bin through an independent third microfluidic pipeline; and magnetic strips are arranged in the first capturing bin and the second capturing bin. According to the scheme, a to-be-detected sample only needs to be dropwise added into the sample adding hole, filtering treatment, mixing, reaction and multi-item detection of the sample can be achieved, different target objects are shunted through the independent third microfluidic pipeline, and the detection sensitivity is improved.

Description

Technical Field

[0001] This utility model relates to the field of in vitro diagnostic technology, and in particular to a multi-fluorescent immunomagnetic bead separation reagent card. Background Technology

[0002] Immunomagnetic bead separation technology is a biological separation method based on the principle of antigen-antibody specific binding, which achieves efficient enrichment of target analytes through magnetic carriers. Traditional monochromatic immunomagnetic beads, by coupling a single antibody to their surface, complete target capture and separation under the action of an external magnetic field. Its technical limitations are reflected in: 1) the single-target recognition mechanism is difficult to cope with the need for simultaneous detection of multiple targets in complex biological samples; 2) the spatial distribution of magnetic beads and the efficiency of magnetic field action are limited by the non-directional magnetic field characteristics of traditional magnetic separation devices, resulting in insufficient binding kinetics; 3) for the detection of multiple indicators, existing technologies mostly adopt simple superimposed monochromatic magnetic bead systems, but have not solved the spectral crosstalk problem caused by multicolor fluorescent labeling, resulting in low detection resolution.

[0003] While commercially available multiplex fluorescent immunomagnetic bead reagent cards can achieve multi-target detection through magnetic beads encoded with different fluorescent groups, key technical bottlenecks remain: First, signal cross-interference caused by overlapping emission peaks of broadband fluorescent dyes necessitates a wide wavelength interval between detection channels, severely limiting the ability to detect multiple indicators in parallel; second, defects in the magnetic bead surface modification process and the adsorption of heterologous proteins in complex sample matrices result in non-specific binding rates as high as 15%-30%, directly affecting detection specificity. These problems severely limit the application of this technology in scenarios such as precision clinical diagnosis. Utility Model Content

[0004] The technical problem to be solved by this utility model is to provide a multi-fluorescent immunomagnetic bead separation reagent card based on the deficiencies and defects mentioned in the background art.

[0005] To solve the above problems, the present invention proposes the following technical solution:

[0006] A multi-fluorescent immunomagnetic bead separation reagent card includes a top cover and a shell, the top cover and the shell being assembled to form a closed structure:

[0007] The upper cover is provided with a sample feeding hole, and a first microfluidic channel disposed inside the housing is connected below the sample feeding hole;

[0008] The shell also includes a reaction chamber, at least one detection chamber, a quality control chamber, a first capture chamber, and at least one second capture chamber; the first microfluidic channel is connected to the reaction chamber; the reaction chamber connects the quality control chamber and the first capture chamber in series via a second microfluidic channel; the reaction chamber connects the detection chamber and the second capture chamber in series via a third microfluidic channel.

[0009] Each detection chamber is connected to the corresponding second capture chamber via an independent third microfluidic conduit;

[0010] Both the first and second capture chambers are equipped with magnetic strips; there is no communication between the detection chambers and the quality control chambers.

[0011] A further technical solution is that the shell is provided with at least one filter chamber; the filter chamber is located between the sample dispensing port and the reaction chamber, and the filter chamber is connected to the sample dispensing port through a filter pipe.

[0012] A further technical solution is that the aperture of the filter pipe is larger than that of the first microfluidic pipe.

[0013] A further technical solution is that the pore size of the first microfluidic channel is greater than or equal to 1 μm.

[0014] A further technical solution is that the total volume of the filter chamber is smaller than the volume of the reaction chamber.

[0015] A further technical solution is that the apertures of each of the third microfluidic channels are different.

[0016] A further technical solution is that the aperture of each of the third microfluidic channels is between 5 and 20 nm.

[0017] A further technical solution is that the shell is also provided with a waste liquid chamber, which is connected to the first capture chamber and the second capture chamber.

[0018] A further technical solution is that the magnetic strip is one of ferrite magnetic materials, rare earth permanent magnet materials, and metal alloy magnetic materials.

[0019] A further technical solution is that the upper cover is provided with observation windows at positions corresponding to the first capture chamber and the second capture chamber.

[0020] Compared with the prior art, the technical effects achieved by this utility model include:

[0021] The multi-fluorescent immunomagnetic bead separation reagent card provided by this utility model includes a top cover and a shell: the top cover is provided with a sample application hole, and a first microfluidic channel disposed inside the shell is connected below the sample application hole; the shell is further provided with a reaction chamber, at least one detection chamber, a quality control chamber, a first capture chamber, and at least one second capture chamber; the first microfluidic channel is connected to the reaction chamber; the reaction chamber is connected in series with the quality control chamber and the first capture chamber through a second microfluidic channel; the reaction chamber is connected in series with the detection chamber and the second capture chamber through a third microfluidic channel; each detection chamber is connected to the corresponding second capture chamber through an independent third microfluidic channel; a magnetic strip is disposed in both the first capture chamber and the second capture chamber; there is no communication between the detection chamber and the quality control chamber. This invention provides a multi-fluorescent immunomagnetic bead separation reagent card. Simply add the sample to the sample well to achieve filtration, mixing, reaction, and detection. Different target analytes are separated using independent third microfluidic channels. Simultaneously, a stable and controllable magnetic field generated by the magnetic strip within the capture chamber allows the target analytes to move along a preset path and be adsorbed onto the magnetic strip. Magnetic strip enrichment efficiently concentrates the target analytes, reduces background interference, and improves detection sensitivity. The technical solution provided by this invention allows for the simultaneous parallel detection and analysis of multiple samples or multiple target analytes, greatly improving detection efficiency. It can achieve efficient separation and enrichment of magnetic beads with different particle sizes or magnetic intensities, meeting diverse detection needs. The reagent card of this invention features a closed-loop flow from sample addition to detection, avoiding contamination and simplifying the operation. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of this utility model, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this utility model. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram of the structure of the multi-fluorescent immunomagnetic bead separation reagent card provided by this utility model.

[0024] Figure Labels

[0025] 1-Shell; 2-Sample dispensing port; 3-First microfluidic channel; 4-Filter chamber; 5-Reaction chamber; 6-Second microfluidic channel; 7-Quality control chamber; 8-First capture chamber; 9-Third microfluidic channel; 10-Detection chamber; 11-Second capture chamber; 12-Waste liquid chamber; 13-Magnetic strip; 14-Filter channel. Detailed Implementation

[0026] The technical solutions in the embodiments of this utility model will be clearly and completely described below with reference to the accompanying drawings. Similar component reference numerals in the drawings represent similar components. Obviously, the embodiments described below are only some embodiments of this utility model, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this utility model without creative effort are within the scope of protection of this utility model.

[0027] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0028] It should also be understood that the terminology used in this specification of embodiments of the present invention is for the purpose of describing particular embodiments only and is not intended to limit the embodiments of the present invention. As used in this specification of embodiments of the present invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0029] Immunomagnetic bead separation technology combines immunological reactions with magnetic bead enrichment to achieve specific capture and separation of target analytes. Traditional reagent cards mostly rely on monochromatic fluorescent labeling, which is prone to detection errors due to spectral overlap, and the problem of non-specific adsorption of magnetic beads is difficult to solve. To address this, this invention provides a multi-fluorescent immunomagnetic bead separation reagent card, which achieves automated sample processing and simultaneous detection of multiple target analytes through multi-stage microfluidic channel design, differentiated pore size configuration, and controllable magnetic strip enrichment technology.

[0030] See Figure 1 In specific implementation, the multi-fluorescent immunomagnetic bead separation reagent card of this utility model includes a top cover (not shown) and a shell 1, which are assembled to form a closed structure:

[0031] The upper cover is provided with a sample feeding hole 2, and the sample feeding hole 2 is connected to a first microfluidic channel 3 disposed inside the housing 1 below it;

[0032] The shell 1 is further provided with a reaction chamber 5, at least one detection chamber 10, a quality control chamber 7, a first capture chamber 8, and at least one second capture chamber 11; the first microfluidic pipe 3 is connected to the reaction chamber 5; the reaction chamber 5 is connected in series with the quality control chamber 7 and the first capture chamber 8 through the second microfluidic pipe 6; the reaction chamber 5 is connected in series with the detection chamber 10 and the second capture chamber 11 through the third microfluidic pipe 9;

[0033] Each detection chamber 10 is connected to the corresponding second capture chamber 11 via an independent third microfluidic conduit 9;

[0034] Both the first capture chamber 8 and the second capture chamber 11 are equipped with magnetic strips 13; there is no communication between the detection chambers 10 and the quality control chamber 7.

[0035] Understandably, the apertures of the second microfluidic channel 6 and the third microfluidic channel 9 can be the same or different, as long as some of the target material enters the quality control chamber 7, the effectiveness of the reaction can be monitored.

[0036] In a specific implementation, at least one filter chamber 4 is provided inside the shell; the filter chamber 4 is located between the sample dispensing port 2 and the reaction chamber 5, and the filter chamber 4 is connected to the sample dispensing port 2 through the filter pipe 14.

[0037] like Figure 1 As shown, the number of filter chambers 4 in this embodiment is 2.

[0038] In a specific implementation, the pore size of the filter channel 14 is larger than that of the first microfluidic channel 3, so that macromolecules or cell debris can be filtered out through the filter channel 14.

[0039] In specific implementation, the aperture of the first microfluidic channel 3 is greater than or equal to 1 μm.

[0040] It should be noted that, in some embodiments, for the detection of high-viscosity fluids (such as blood), the first microfluidic channel 3 needs to use a larger aperture (≥10μm) to reduce resistance and avoid excessively slow flow rate.

[0041] In some embodiments, for the detection of low-viscosity reagents (such as buffer solutions), the first microfluidic channel 3 may use a smaller pore size (1–5 μm) to improve separation accuracy.

[0042] In practice, the total volume of the filter chamber 4 is smaller than the volume of the reaction chamber 5.

[0043] In practice, the apertures of the third microfluidic channels 9 are different. This embodiment utilizes third microfluidic channels 9 with different apertures to achieve parallel detection of multiple items and supports simultaneous analysis of multiple detection items.

[0044] In specific implementations, the pore size of each of the third microfluidic channels 9 is between 5 and 20 nm. For example, in a multiplex fluorescent immunomagnetic bead separation reagent card, the pore size of one third microfluidic channel 9 is 5 nm, the pore size of one third microfluidic channel 9 is 10 nm, and the pore size of one third microfluidic channel 9 is 15 nm.

[0045] In a specific implementation, the shell 1 is also provided with a waste liquid chamber 12, which is connected to the first capture chamber 8 and the second capture chamber 11.

[0046] In specific implementation, the magnetic strip 13 is one of ferrite magnetic material, rare earth permanent magnet material, and metal alloy magnetic material.

[0047] In this embodiment, the magnetic strip 13 is a ferrite magnetic material.

[0048] In other embodiments, the magnetic field intensity of each second capture chamber 11 can be adjusted according to the different detection items to ensure efficient adsorption of the target and improve detection sensitivity.

[0049] In specific implementation, the top cover is provided with observation windows (not shown in the figure) at the positions corresponding to the first capture chamber 8 and the second capture chamber 11, which facilitates real-time monitoring of the magnetic bead enrichment status.

[0050] The multiplex fluorescent immunomagnetic bead separation reagent card provided in this embodiment only requires adding the sample to the sample well to achieve sample filtration, mixing, reaction, and detection. Different target analytes are separated using independent third microfluidic channels. Simultaneously, a stable and controllable magnetic field generated by the magnetic strip within the capture chamber allows the target analytes to move along a preset path and be adsorbed onto the magnetic strip. Magnetic strip enrichment efficiently concentrates the target analytes, reduces background interference, and improves detection sensitivity. The technical solution provided by this invention can simultaneously detect and analyze multiple samples or multiple target analytes in parallel, greatly improving detection efficiency. It can achieve efficient separation and enrichment of magnetic beads with different particle sizes or magnetic intensities to meet diverse detection needs. The reagent card of this invention features a closed-loop flow from sample addition to detection, avoiding contamination and simplifying the operation steps.

[0051] Example 1

[0052] The multiplex fluorescent immunomagnetic bead separation reagent card provided in this embodiment is a dual-target analyte detection reagent card. See further details. Figure 1 The reagent card includes:

[0053] The housing 1 has an elliptical sample feeding port 2, and a first microfluidic channel 3 with a pore diameter of 1μm is connected below the sample feeding port 2 and disposed inside the housing 1.

[0054] The housing 1 is also equipped with two filter chambers 4 (with a volume of 0.5 mL), which are connected to the sample feeding port 2 through filter pipes 14 with a pore size of 2 μm, to remove macromolecular impurities and improve separation accuracy.

[0055] The housing 1 also includes a reaction chamber 5 for receiving filtered samples. The reaction chamber 5 is connected in series with the quality control chamber 7 and the first capture chamber 8 via a second microfluidic channel 6 (15nm aperture). At the same time, the reaction chamber 5 is connected to two detection chambers 10 and the corresponding second capture chamber 11 via two independent third microfluidic channels 9 (5nm and 10nm apertures, respectively).

[0056] Both the first capture chamber 8 and the second capture chamber 11 are embedded with neodymium iron boron magnetic strips 13.

[0057] Waste liquid tank 12 is located downstream of the capture tank and is connected to the first capture tank 8 and the second capture tank 11 respectively, and is used to receive waste liquid.

[0058] The shell 1 has a transparent observation window at the capture chamber position to facilitate monitoring of the magnetic bead enrichment status.

[0059] The operation procedure for the multiplex fluorescent immunomagnetic bead separation reagent card provided in this embodiment is as follows:

[0060] The sample is added to the sample well 2, and after preliminary filtration through the filter pipe 14, it enters the reaction chamber 5 through the first microfluidic pipe 3. Larger cell debris and other impurities enter the filter chamber 4.

[0061] The filtered sample is mixed with fluorescently labeled magnetic beads in reaction chamber 5;

[0062] The mixed liquid flows in two separate streams:

[0063] The first path enters the quality control chamber 7 via the second microfluidic channel 6 to verify the effectiveness of the reaction, and then the target substance is adsorbed by the first capture chamber 8;

[0064] The second path enters the detection chamber 10 through the parallel third microfluidic channel 9. The target objects of different particle sizes are sorted by the aperture of the third microfluidic channel 9 and finally enriched in the corresponding second capture chamber 11.

[0065] Waste liquid flows into waste liquid tank 12, and fluorescence detection is performed after enrichment is confirmed through observation window 14.

[0066] In this embodiment, the volume of the filter chamber 4 is 1 / 3 of the volume of the reaction chamber 5, which can effectively accommodate impurities and avoid interference with the sample; the filter pipe 14 has a pore size of 2μm, which ensures that cell debris is trapped while the target substance passes through smoothly.

[0067] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0068] The above description describes specific embodiments of this utility model, but the scope of protection of this utility model is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this utility model, and these modifications or substitutions should all be covered within the scope of protection of this utility model. Therefore, the scope of protection of this utility model should be determined by the scope of the claims.

Claims

1. A multiplexed fluorescent immunomagnetic bead separation assay card, characterized in that, The upper cover and the shell are assembled to form a closed structure: The upper cover is provided with a sample adding hole, and the sample adding hole is connected with a first micro-fluidic channel arranged in the shell below; The shell is further provided with a reaction chamber, at least one detection chamber, a quality control chamber, a first capture chamber and at least one second capture chamber; the first micro-fluidic channel is in communication with the reaction chamber; the reaction chamber is connected with the quality control chamber and the first capture chamber in series through a second micro-fluidic channel; the reaction chamber is connected with the detection chamber and the second capture chamber in series through a third micro-fluidic channel; Each detection chamber is in communication with a corresponding second capture chamber through an independent third micro-fluidic channel; The first capture chamber and the second capture chamber are both provided with a magnetic strip; and the detection chambers and the quality control chamber are not in communication with each other.

2. The multiplexed fluorescent immunomagnetic bead separation test kit of claim 1, wherein, The shell is further provided with at least one filter chamber; the filter chamber is located between the sample adding hole and the reaction chamber, and is in communication with the sample adding hole through a filter channel.

3. The multiplexed fluorescent immunomagnetic bead separation test kit of claim 2, wherein, The pore size of the filter channel is larger than that of the first micro-fluidic channel.

4. The multiplexed fluorescent immunomagnetic bead separation test kit of claim 3, wherein, The pore size of the first micro-fluidic channel is greater than or equal to 1 μm.

5. The multiplexed fluorescent immunomagnetic bead separation test kit of claim 3, wherein the first and second fluorescently labeled antibodies are different. The total volume of the filter chamber is smaller than the volume of the reaction chamber.

6. The multiplexed fluorescent immunomagnetic bead separation test kit of claim 1, wherein, The pore sizes of the third micro-fluidic channels are different.

7. The multiplexed fluorescent immunomagnetic bead separation test kit of claim 6, wherein, The pore sizes of the third micro-fluidic channels are between 5 and 20 nm.

8. The multiplexed fluorescent immunomagnetic bead separation test kit of claim 1, wherein, The shell is further provided with a waste liquid chamber, which is in communication with the first capture chamber and the second capture chamber.

9. The multiplexed fluorescent immunomagnetic bead separation test kit of claim 1, wherein, The magnetic strip is one of ferrite magnetic material, rare earth permanent magnetic material and metal alloy magnetic material.

10. The multiplexed fluorescent immunomagnetic bead separation test kit of claim 1, wherein, The upper cover is provided with an observation window at positions corresponding to the first capture chamber and the second capture chamber.

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