Integrated chip card box system based on microfluidic technology and detection method and application thereof

Abstract

The invention relates to an integrated chip card box system based on a microfluidic technology. A pump valve driving module injects a whole blood sample to be detected into a plasma separation module; the plasma separation module is provided with a snakelike micro-nano array groove structure and a capillary tube, the snakelike micro-nano array groove structure realizes plasma separation based on cell size difference, and separated plasma is guided into the reaction detection module through the capillary tube under the driving and capillary action of the pump valve driving module; the reaction detection module comprises a sample treatment area and a reaction detection area, tumor markers in plasma are combined with magnetic beads fixed with antibodies in advance in the sample treatment area to form a compound, and the compound is cleaned in the sample treatment area; the cleaned compound enters a reaction detection area through dielectric wetting for chemiluminescence immunodetection of the tumor marker. The invention further relates to a detection method and application of the integrated chip card box system. According to the method, the full-process automation from'sample entering 'to'result outputting' can be realized.

Description

Technical Field

[0001] This invention relates to microfluidic chips, and more specifically to an integrated chip cartridge system based on microfluidic technology, its detection method, and its application. Background Technology

[0002] Cancer is a serious threat to human health, with high incidence and mortality rates. Taking pancreatic cancer as an example, the 5-year survival rate after diagnosis is only about 10%, with approximately 120,000 patients in China and a staggering 470,000 worldwide. Although pancreatic cancer can be treated surgically, many patients still face the risk of cancer recurrence after surgery, posing a significant challenge to both patients and healthcare professionals. CA19-9, a key biomarker for pancreatic cancer, can effectively indicate postoperative recurrence in blood levels. Therefore, early detection of tumor markers is of paramount importance for predicting disease progression.

[0003] Currently, medical testing methods typically involve obtaining plasma or serum from whole blood using a centrifuge, followed by the detection of tumor markers using a large, high-throughput chemiluminescence immunoassay analyzer. However, these traditional devices are bulky, energy-intensive, and unsuitable for portable operation or use in resource-constrained environments. Furthermore, their complex construction and high cost, as seen in products from companies like Roche, Abbott, and Mindray, limit their application in a wider range of scenarios.

[0004] Microfluidic chip technology, with its broad application prospects, enables high-sensitivity, high-throughput, and rapid detection and analysis through flexible chip structure design, making it particularly suitable for efficient tumor analysis. In recent years, this technology has been widely applied to the detection of tumor markers and has achieved significant progress. However, in existing microfluidic chip applications, traditional methods such as centrifugation and settling are still typically required to separate plasma from whole blood, followed by the use of pipetting devices to load the sample into the detection cartridge. This process is not only cumbersome but also requires human intervention during sample transport, increasing the risk of environmental contamination and affecting the automation level and reliability of the results. Summary of the Invention

[0005] To address the problems of existing technologies requiring human intervention and posing risks of contamination to the experimental environment, this invention aims to provide an integrated chip cartridge system based on microfluidic technology, along with its detection method and applications.

[0006] According to the integrated chip cartridge system based on microfluidic technology of the present invention, it includes a pump valve driving module, a plasma separation module, and a reaction detection module. The pump valve driving module injects a whole blood sample to be tested into the plasma separation module. The plasma separation module has a serpentine micro-nano array trench structure and a capillary. The serpentine micro-nano array trench structure achieves plasma separation based on cell size differences. The separated plasma is introduced into the reaction detection module through the capillary under the drive of the pump valve driving module and capillary action. The reaction detection module includes a sample processing area and a reaction detection area. Tumor markers in the plasma bind to magnetic beads pre-immobilized with antibodies in the sample processing area to form a complex. The complex is washed in the sample processing area, and the washed complex enters the reaction detection area through dielectric wetting for chemiluminescent immunoassay of the tumor markers.

[0007] In a preferred embodiment, the pump valve drive module includes a syringe and a stepper motor. The syringe is pre-loaded with a whole blood sample, and the stepper motor drives the piston of the syringe to quantitatively inject the whole blood sample into the plasma separation module.

[0008] In a preferred embodiment, the plasma separation module and the reaction detection module are cardboard structures encapsulated with multi-layered plastic materials.

[0009] In a preferred embodiment, the plasma separation module includes a base plate, a lid, and a microfluidic channel plate sandwiched therebetween. The base plate is made of photosensitive resin, the serpentine micro-nano array groove structure is formed on the microfluidic channel plate, and the microfluidic channel plate and the lid are made of PC material.

[0010] In a preferred embodiment, the reaction detection module includes a support plate, a reagent injection cover plate, and a chip sandwiched therebetween, wherein the support plate is made of PC material, the chip is made of PET material, and the reagent injection cover plate is made of PMMA material.

[0011] In a preferred embodiment, the chip surface is constructed with an electrode array and a hydrophobic layer, which, in conjunction with a magnetic field, enables the adsorption, cleaning, transfer, and reaction of the complex.

[0012] In a preferred embodiment, the integrated chip cartridge system further includes a PMT signal capture device, which works in conjunction with the reaction detection module to capture the light signal generated by the reaction and output the detection result.

[0013] According to the above-described integrated chip cartridge system based on microfluidic technology of the present invention, a method for in vitro detection of tumor markers includes injecting a whole blood sample into the plasma separation module through the pump valve drive module; the plasma separation module separating plasma from the whole blood sample using the serpentine micro-nano array groove structure; introducing the separated plasma into the reaction detection module through the capillary under the drive of the pump valve drive module and capillary action; the tumor markers in the plasma binding with magnetic beads pre-immobilized with antibodies in the sample processing area to form a complex; the complex being washed in the sample processing area; and the washed complex entering the reaction detection area through dielectric wetting. The obtained concentration information of the tumor markers is not directly used for disease diagnosis or treatment.

[0014] The application of the tumor markers of the present invention in the above-described integrated chip cartridge system is characterized in that the application includes: injecting a whole blood sample into the plasma separation module via the pump valve drive module; the plasma separation module separating plasma from the whole blood sample using the serpentine micro-nano array groove structure; introducing the separated plasma into the reaction detection module via the capillary under the drive of the pump valve drive module and capillary action; the tumor markers in the plasma binding with magnetic beads pre-immobilized with antibodies in the sample processing area to form a complex; the complex being washed in the sample processing area; and the washed complex entering the reaction detection area for chemiluminescent immunoassay of the tumor markers via dielectric wetting.

[0015] In a preferred embodiment, the tumor marker is CA19-9.

[0016] The integrated microfluidic chip cartridge system based on microfluidic technology according to the present invention includes a fully automated microfluidic chip cartridge for integrated plasma separation and tumor marker detection, integrating blood sample preparation and tumor marker detection into one unit, suitable for rapid detection of tumor markers. This system achieves a fully enclosed, high-precision detection process through the synergistic design of a pump-valve drive module (such as a stepper motor lead screw driving an injector) and dielectric wetting technology.

[0017] This invention integrates microfluidics, electrowetting control, magnetic bead separation, and chemiluminescence enzyme-linked immunosorbent assay (ELISA) technologies. Employing a modular design, it provides a highly efficient, accurate, and convenient solution for tumor marker detection, demonstrating significant practical value. Its innovation lies not only in the technical details, such as the combination of a micron-level channel array structure and dielectric wetting control technology, but also in effectively solving the problems of long detection times, complex chip structures, high costs, large equipment size, and risks of environmental contamination in existing tumor marker detection technologies.

[0018] According to the detection method of the present invention, the operation process requires only simple human intervention, such as injecting samples and reagents, to automatically send the cartridge into the detector to complete the detection of the target sample, greatly reducing the reliance on the professional skills of operators and auxiliary equipment. At the same time, the system has the advantages of precise droplet control, compact and portable structure, high sensitivity, and low cost, making it particularly suitable for the rapid on-site detection of tumor markers and showing broad application prospects in pathogen diagnosis and personalized medicine. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of a chip cartridge system according to a preferred embodiment of the present invention.

[0020] Figure 2 yes Figure 1 An exploded view of the card holder structure of the chip card holder system.

[0021] Figure 3 yes Figure 2 A side view of the card box structure.

[0022] Figure 4 yes Figure 1 A schematic diagram of the plasma separation module.

[0023] Figure 5 yes Figure 1 A 3D view of the reaction detection module.

[0024] Figure 6 yes Figure 1 A top view of the reaction detection module.

[0025] Figure 7 yes Figure 1 The operation flowchart of the reaction detection module.

[0026] Figure 8 yes Figure 1 The calibration flowchart for the reaction detection module.

[0027] Figure 9 The CA199 detection result is shown according to an example of the present invention. Detailed Implementation

[0028] The preferred embodiments of the present invention are given below with reference to the accompanying drawings and described in detail.

[0029] like Figures 1-2As shown, an integrated chip cartridge system according to a preferred embodiment of the present invention includes a pump valve drive module 1 and a cartridge structure 10, wherein the cartridge structure 10 comprises a plasma separation module 2 and a reaction detection module 3. Furthermore, the integrated chip cartridge system is further equipped with a PMT signal capture device 4 for detecting the light signal generated by the reaction and achieving quantitative analysis.

[0030] The pump valve drive module 1, located on the left side of the system, is used to inject the whole blood sample to be tested into the plasma separation module 2 connected below it. Figure 1 As shown, the pump valve drive module 1 includes a sealing plug 11, a syringe 12, a push plate 13, a push rod 14, and a stepper motor 15. The sealing plug 11 is installed at the inlet of the plasma separation module 2. The syringe 12 is pre-filled with a whole blood sample, and its needle passes through the sealing plug 11 to communicate with the internal channel of the plasma separation module 2. The stepper motor 15 drives the piston 121 of the syringe 12 through the push rod 14 and the push plate 13, accurately injecting the whole blood sample into the plasma separation module 2.

[0031] like Figure 2 and Figure 3 As shown, the cartridge structure 10 is encapsulated with multi-layered plastic materials to integrate functional modules. The plasma separation module 2 includes a base plate 2a, a microfluidic channel plate 2b, and a lid 2c, wherein the base plate 2a is made of photosensitive resin, and the microfluidic channel plate 2b and lid 2c are made of polycarbonate (PC). The reaction detection module 3 includes a support plate 3a, a chip 3b, and a reagent injection cover plate 3c, wherein the support plate 3a is made of PC, the chip 3b is made of polyethylene terephthalate (PET), and the reagent injection cover plate 3c is made of polymethyl methacrylate (PMMA).

[0032] Plasma separation module 2 uses microfluidic technology to separate plasma from whole blood samples. For example... Figure 4 As shown, the plasma separation module 2 internally includes a serpentine micro / nano array groove structure 21, a plasma separation pool 22, a capillary tube 23, and an exhaust port 24. When the whole blood sample flows in the groove structure 21, it is subjected to shear force, and plasma and blood cells are separated based on their size difference. The separated plasma flows into the plasma separation pool 22 and is then introduced into the reaction detection module 3 through the capillary tube 23 under capillary action. The exhaust port 24 is used to balance the internal pressure of the cartridge, ensuring fluid stability.

[0033] The reaction detection module 3 is connected to the plasma separation module 2 via capillary tube 23. For example... Figure 5 and Figure 6As shown, the reaction detection module 3 includes a reagent storage area 31, a sample processing area 32, and a reaction detection area 33. The reagent storage area 31 stores washing solutions (W1, W2), substrates, primary antibodies, secondary antibodies, standards, and calibrators. The sample processing area 32 utilizes the antigen-antibody binding properties of magnetic beads to capture and wash the antigen-primary antibody-secondary antibody complex under the influence of a magnetic field. The reaction detection area 33 is equipped with a sample reaction chamber R1, a negative control chamber R2, and positive control chambers R3 and R4 for chemiluminescent immunoassay.

[0034] To avoid errors and contamination caused by human operation, the reaction detection module 3 employs dielectric wetting technology, precisely controlling droplet movement through an electric field. An electrode array and a hydrophobic layer are constructed on the surface of chip 3b, which, in conjunction with a magnetic field, enables the adsorption, washing, transfer, and reaction of the magnetic bead-antigen-primary antibody-secondary antibody complex. The substrate generates photons under the enzyme catalysis of the secondary antibody; the PMT signal capture device 4 converts the light signal into an electrical signal, achieving quantitative detection.

[0035] The following is a brief introduction to the workflow of reaction detection module 3.

[0036] like Figure 7 As shown, the accuracy and reliability of the reaction detection zone 33 were first verified using a known concentration of CA19-9 standard. Specifically, this was done by introducing the standard into the negative control chamber R2 of the reaction detection zone 33. Next, whole blood samples were sequentially moved to the sample processing zone 32 with primary and secondary antibodies for thorough mixing, forming an antigen-primary antibody-secondary antibody complex. Since the primary antibody was pre-fixed on magnetic beads, the permanent magnet captured the antigen-primary antibody-secondary antibody complex as it rose. The mixed waste liquid left the sample processing zone 32 for waste recovery. Washing solution entered the sample processing zone 32 to perform the first wash on the magnetic bead-antigen-primary antibody-secondary antibody complex. The permanent magnet descended, releasing the magnetic beads, and the washing droplets reciprocated, ensuring full contact between the magnetic bead-antigen-primary antibody-secondary antibody complex and the washing solution. The permanent magnet rose again, capturing the magnetic beads, and the washing waste liquid left the sample processing zone 32. The above steps were repeated to complete four washes using the washing solution. Finally, the magnetic bead-antigen-primary antibody-secondary antibody complex enters the sample reaction chamber R1 of the reaction detection area 33 under the action of a magnetic field for luminescence detection, thereby quantitatively analyzing the level of CA19-9 in the whole blood sample.

[0037] like Figure 8 As shown, CA19-9 containing a known concentration level is guided into the positive control chambers R3 and R4 of the reaction detection zone 33. The measurement signal establishes a calibration curve, thereby converting the relative luminescence value (RLU) of the sample into a specific quantitative analyte concentration.

[0038] like Figure 9As shown, relevant data were obtained by actually measuring the CA199 concentration in relapsed and non-relapsed patients. The straight line y = 2.5867x + 32.937 shows R0. 2 =0.8836 is the calibration curve. The CA199 concentration of relapsed patients calculated by the calibration curve is consistent with the actual measured value, thus verifying the accuracy of the test results.

[0039] The following is a brief description of the specific usage method of the integrated chip card holder system according to the present invention.

[0040] In the sample preparation stage, approximately 5 ml of whole blood sample (venous blood) to be tested is first drawn using syringe 12, and syringe 12 is then installed onto the plasma separation module 2. Next, the various reagents are injected into the respective wells of the reaction detection module 3.

[0041] During the sample processing and preparation stage, the whole blood sample first enters the serpentine micro-nano array groove structure 21 of the plasma separation module 2 under the drive of the pump valve drive module 1, and the rapidly separated plasma enters the plasma separation pool 22. Next, driven by the pump valve drive module 1 and assisted by capillary force, about 10-20µL of plasma is injected from the plasma separation pool 22 into the reaction detection module 3 above through the capillary tube 23.

[0042] In reaction detection module 3, dielectric wetting technology combined with magnetic beads enables microfluidic manipulation. Through the synergistic effect of electric field force and permanent magnet force, droplet movement is controlled to complete adsorption, washing, and purification actions. This, combined with substrate and PMT optical detection, enables the detection of tumor markers (e.g., CA19-9). Specifically, an electrode array and a hydrophobic layer are constructed on the surface of chip 3b. Sequential activation of the electrodes generates electrostatic force, causing the droplets to move in a directional manner. Simultaneously, magnetic beads pre-bound with antibodies are added to the droplets. A movable permanent magnet or electromagnet above chip 3b applies a force to the magnetic beads using a magnetic field, which, combined with the electric field force, achieves the binding and purification of magnetic beads, antigen, and antibody. This process includes precise droplet positioning control → mixing of sample antigen, antibody-coated magnetic beads, and labeled antibodies → magnetic field-assisted washing → addition of substrate → driving droplet transfer to reaction detection area 33. After sufficient reaction, the instrument's internal optical detection and analysis program outputs a detection result report upon completion of the reaction.

[0043] Thus, this invention integrates multiple technologies, including pump valves, micro / nano array trench structures, dielectric wetting microfluidics, magnetic bead separation, and chemiluminescence immunoassay, to construct an integrated, automated, and highly sensitive tumor marker detection system. Based on plastic encapsulation, the necessary reagents for the reaction are sealed within the reagent storage chamber; the pump valve and micro / nano array trench design ensure the smooth introduction of blood samples into the plasma separation pool during the separation process; antigen-antibody molecules are adsorbed, enriched, and washed by magnetic beads adsorbed by permanent magnets; and the PMT optical module captures the signals of the tumor marker reaction, ultimately achieving tumor marker detection from whole blood sample to result analysis. This fully automated process from "sample in" to "result out" offers advantages such as compact structure, simple operation, rapid detection, and low cost, making it particularly suitable for rapid on-site detection of tumor markers such as pancreatic cancer, and has broad clinical application prospects.

[0044] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. That is, all simple and equivalent changes and modifications made based on the claims and description of this invention fall within the protection scope of the claims. All aspects not described in detail in this invention are conventional technical content.

Claims

1. An integrated chip cartridge system based on microfluidic technology, characterized in that, This integrated chip cartridge system includes a pump-valve drive module, a plasma separation module, and a reaction detection module. The pump-valve drive module injects the whole blood sample to be tested into the plasma separation module. The plasma separation module has a serpentine micro-nano array trench structure and a capillary. The serpentine micro-nano array trench structure achieves plasma separation based on cell size differences. The separated plasma is introduced into the reaction detection module through the capillary under the drive of the pump-valve drive module and capillary action. The reaction detection module includes a sample processing area and a reaction detection area. Tumor markers in the plasma bind to magnetic beads pre-immobilized with antibodies in the sample processing area to form a complex. The complex is washed in the sample processing area, and the washed complex enters the reaction detection area through dielectric wetting for chemiluminescent immunoassay of the tumor markers.

2. The integrated chip card holder system according to claim 1, characterized in that, The pump valve drive module includes a syringe and a stepper motor. The syringe is pre-loaded with a whole blood sample, and the stepper motor drives the piston of the syringe to quantitatively inject the whole blood sample into the plasma separation module.

3. The integrated chip card holder system according to claim 1, characterized in that, The plasma separation module and reaction detection module are encapsulated in a cartridge structure using multi-layered plastic materials.

4. The integrated chip card holder system according to claim 3, characterized in that, The plasma separation module includes a base plate, a lid, and a microfluidic channel plate sandwiched therebetween. The base plate is made of photosensitive resin, and the serpentine micro-nano array groove structure is formed on the microfluidic channel plate. The microfluidic channel plate and the lid are made of PC material.

5. The integrated chip card holder system according to claim 3, characterized in that, The reaction detection module includes a support plate, a reagent injection cover plate, and a chip sandwiched therebetween. The support plate is made of PC material, the chip is made of PET material, and the reagent injection cover plate is made of PMMA material.

6. The integrated chip card holder system according to claim 5, characterized in that, The chip surface is constructed with an electrode array and a hydrophobic layer, which, in conjunction with a magnetic field, enables the adsorption, cleaning, transfer, and reaction of the complex.

7. The integrated chip card holder system according to claim 1, characterized in that, The integrated chip cartridge system also includes a PMT signal capture device, which works in conjunction with the reaction detection module to capture the light signal generated by the reaction and output the detection result.

8. A method for in vitro detection of tumor markers using an integrated chip cartridge system according to any one of claims 1-7, characterized in that, The method includes: The whole blood sample is injected into the plasma separation module through the pump valve drive module; The plasma separation module utilizes the serpentine micro-nano array trench structure to separate plasma from the whole blood sample; The separated material is introduced into the reaction detection module through the capillary under the drive of the pump valve drive module and capillary action; the tumor marker in the plasma binds with magnetic beads pre-fixed with antibodies in the sample processing area to form a complex, the complex is washed in the sample processing area, and the washed complex enters the reaction detection area through dielectric wetting. The obtained concentration information of the tumor marker is not directly used for the diagnosis or treatment of the disease.

9. The application of a tumor marker in an integrated chip cartridge system according to any one of claims 1-7, characterized in that, The application includes: The whole blood sample is injected into the plasma separation module through the pump valve drive module; The plasma separation module utilizes the serpentine micro-nano array trench structure to separate plasma from the whole blood sample; The separated material is introduced into the reaction detection module through the capillary under the drive of the pump valve drive module and capillary action; the tumor marker in the plasma binds with magnetic beads pre-immobilized with antibodies in the sample processing area to form a complex, the complex is washed in the sample processing area, and the washed complex enters the reaction detection area through dielectric wetting for chemiluminescent immunoassay of the tumor marker.

10. The application according to claim 8 or 9, characterized in that, The tumor marker is CA19-9.

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