EB (Epstein-Barr) virus and echinococcosis antibody joint detection magnetic immunofluorescence detection micro-fluidic chip

Abstract

The invention relates to the technical field of in-vitro diagnosis, and discloses an EB virus and echinococcosis antibody joint detection magnetic immunofluorescence detection micro-fluidic chip which comprises a chip body, the chip body is provided with a sample adding chamber, a liquid separation pool and a detection cavity which are communicated in sequence, and the chip body is further provided with a detergent chamber and a waste liquid pool; the detergent chamber is connected with the detection cavity through a washing valve; a hydrophobic modified layer is arranged on the surface of the inner wall of the washing valve; the opening rotating speed threshold value of the washing valve is higher than the centrifugal rotating speed for realizing plasma separation in the liquid separation pool; and one-step immunoreaction freeze-dried microspheres are preset in the detection cavity. According to the invention, a fluid control switch with a high opening threshold value for a liquid containing a surfactant is constructed by arranging a hydrophobic coating with a specific geometric size at the washing valve, so that the problem that the flow is out of control is solved, and the time sequence accuracy of a multi-step reaction is ensured.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic technology, specifically to a microfluidic chip for the combined detection of EB virus and echinococcosis antibodies using magnetic immunofluorescence assay. Background Technology

[0002] Both Epstein-Barr virus (EBV) and echinococcosis are widespread infectious diseases. Serological antibody detection, especially the differentiation between IgM and IgG antibodies, is of great value for early diagnosis, infection staging, and epidemiological investigation. Traditional laboratory testing methods, such as enzyme-linked immunosorbent assay (ELISA), while accurate, have lengthy procedures, rely on large equipment and specialized personnel, and are difficult to meet the needs of point-of-care testing (POCT).

[0003] To automate and miniaturize detection processes, centrifugal microfluidics has emerged. This technology integrates microchannels and reaction chambers onto a single, disc-shaped chip. By programmably controlling the chip's rotation, centrifugal force acts as a liquid-driven virtual pump, enabling precise sample measurement, dispensing, and sequential transfer to different reaction zones. This design requires only a simple rotary actuator to automate the entire process from sample addition to the basic reaction, demonstrating its potential for integration and ease of operation in fields such as biochemical analysis and cell separation.

[0004] However, when attempting to port multi-step immunoassays, which include standard washing steps, to this platform, inherent technical contradictions become apparent. To ensure effective washing in immunoassays, surfactants must be added to the washing solution. This directly conflicts with the working principle of passive valves commonly used in centrifugal microfluidics. These valves, which rely on liquid surface tension, experience a significant decrease in their fluid resistance upon contact with surfactants, leading to premature valve opening and leakage of the washing solution into the reaction chamber before the reaction is complete. Furthermore, to achieve multi-indicator detection, capture reagents and detection probes often need to be added step-by-step. This step-by-step reaction mode places higher demands on the flow path design and valve control reliability within the chip, making automated processes more difficult to implement. Therefore, existing technology faces a dilemma: either simplify or omit critical washing steps to avoid valve failure, which directly affects the sensitivity and accuracy of detection; or introduce complex and costly active valve systems, which deviates from the original intention of centrifugal microfluidics to pursue low cost and ease of operation. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a magnetic immunofluorescence detection microfluidic chip for the combined detection of EB virus and echinococcosis antibodies. This chip solves the technical problem that the passive valves of existing centrifugal microfluidic chips are prone to failure due to incompatibility with washing solutions containing surfactants, making it difficult to integrate automated multi-step immunoassays on a simple centrifugation platform.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a magnetic immunofluorescence detection microfluidic chip for the combined detection of EB virus and echinococcosis antibodies. The chip includes a chip body on which a sample application chamber, a dispensing reservoir, and a detection chamber are integrated in sequence, as well as a separate detergent chamber and a waste liquid reservoir. The detergent chamber is connected to the detection chamber via a washing valve. The inner wall surface of the washing valve is provided with a hydrophobic modified layer, and its opening speed threshold is higher than the centrifugation speed used to achieve plasma separation in the dispensing reservoir. One-step immunoreaction lyophilized microspheres are pre-placed in the detection chamber.

[0007] By adopting the above technical solution, this invention establishes a fluid timing control mechanism. Its principle is as follows: 1. Graded Threshold Control: Due to its hydrophobic modified layer and specific geometry, the washing valve exhibits high fluid resistance to liquid flow. This necessitates a high centrifugal speed for its opening, i.e., a high opening speed threshold. This threshold is designed to be higher than the low-speed centrifugation used for plasma sample separation.

[0008] 2. Process Sequence Guarantee: During the low-speed centrifugation stage, the chip completes the separation of the blood sample. The plasma automatically flows into the detection chamber to react with the lyophilized microspheres, while the washing solution in the detergent chamber is blocked because the centrifugal force is insufficient to exceed the threshold of the washing valve. This prevents the washing solution from entering the detection chamber before the immune reaction is completed, ensuring the independence and integrity of the reaction.

[0009] 3. Washing triggered on demand: After the immune reaction is completed, the centrifugal speed is increased to a level higher than the opening threshold of the washing valve. The centrifugal force overcomes the valve resistance, allowing the washing liquid to flow into the detection chamber for washing.

[0010] 4. Integrated reaction: The pre-placed one-step immunoreaction lyophilized microspheres in the detection chamber integrate magnetic microparticles and fluorescent probes, simplifying the complex immunoreaction process into a one-step operation.

[0011] Therefore, this technical solution solves the technical problem of valve failure caused by surfactants in centrifugal microfluidic chips through the synergistic design of structure and reagents, and realizes automated, high-throughput and high-sensitivity immunoassay from sample addition to signal detection.

[0012] Preferably, the one-step immunoreaction lyophilized microspheres comprise magnetically captured microparticles, a fluorescent probe, and a lyophilization protective matrix composed of D-mannitol, D(+)-trehalose dihydrate, bovine serum albumin, and polyoxyethylene (20) sorbitan monolaurate.

[0013] By employing the above technical solution, the components of this freeze-drying protective matrix work synergistically: D-mannitol acts as a molding framework, giving the microspheres a stable physical morphology; D(+)-trehalose dihydrate and bovine serum albumin act as bioactive protectants, maintaining the spatial conformation and bioactivity of antigens and antibodies during freeze-drying and long-term storage by forming a glassy structure and replacing water molecules; polyoxyethylene (20) sorbitan monolaurate, as a nonionic surfactant, helps the microspheres resolubilize after contact with plasma and reduces nonspecific adsorption in the reaction system. The specific ratio of each component ensures the physical stability and integrity of the biological functions of the freeze-dried microspheres.

[0014] Preferably, the lyophilized protective matrix is ​​prepared from a lyophilized matrix solution containing the following components at concentrations: D-mannitol 20 mg / mL to 40 mg / mL; D(+)-trehalose dihydrate 40 mg / mL to 60 mg / mL; bovine serum albumin 10 mg / mL to 20 mg / mL; and polyoxyethylene (20) sorbitan monolaurate 0.5 mg / mL to 1.0 mg / mL.

[0015] By employing the above technical solution, within this concentration range, each component can achieve suitable protective effects and physical properties. Too low a concentration may result in insufficient protection or poor spheroidization, while too high a concentration may affect the reconstitution rate or introduce background interference. This preferred concentration range is an optimized choice that achieves a balance between microsphere stability and reactivity.

[0016] Preferably, the capturing magnetic microparticles are magnetic microspheres with recombinant EB virus capsid antigen and / or recombinant Echinococcus multilocularis Em18 antigen covalently coupled to their surface; the fluorescent probe is FITC-labeled goat anti-human IgM polyclonal antibody or Cy3-labeled goat anti-human IgG polyclonal antibody.

[0017] By employing the above technical solution, multi-indicator joint detection and infection staging diagnosis were achieved. Two specific recombinant antigens were covalently coupled to the surface of magnetic microparticles, enabling the capture of EB virus and echinococcosis-specific antibodies in the test sample. Simultaneously, two different fluorescently labeled (FITC and Cy3) goat anti-human IgM and IgG polyclonal antibodies were used as probes, allowing them to pass through different fluorescence channels in a single test, simultaneously distinguishing and quantifying IgM antibodies (often indicating acute or recent infection) and IgG antibodies (often indicating past infection or chronic phase) in the sample, thus improving the information dimension and clinical diagnostic value of the detection.

[0018] Preferably, the hydrophobic modified layer is a coating formed by curing 1H,1H,2H,2H-perfluorooctyltrichlorosilane (FDTS).

[0019] By employing the above technical solution, FDTS molecules can chemically react with the hydroxyl groups on the surface of the chip substrate through their terminal silane groups, forming a stable covalent bond. This allows them to self-assemble on the substrate surface into a dense, low-surface-energy perfluoroalkyl chain monolayer. This coating exhibits hydrophobicity and chemical stability, enhancing the washing valve's barrier ability against surfactant-containing liquids and ensuring the stability and reliability of the valve's opening threshold.

[0020] Preferably, the washing valve has the following geometric dimensions: a width of 80μm to 120μm and a depth of 40μm to 60μm.

[0021] By employing the above technical solution, the specific microchannel size is the result of fluid dynamics optimization. Within this size range, the capillary effect of the channel and the surface effect provided by the FDTS hydrophobic coating achieve a synergistic balance, enabling the establishment of the valve's opening pressure threshold. Simultaneously, this size also ensures smooth liquid flow during high-speed centrifugation, achieving control and optimization of the valve's performance in both open and closed states.

[0022] Secondly, this invention provides a detection method using a microfluidic chip for the combined detection of EB virus and echinococcosis antibodies via magnetic immunofluorescence assay. The method includes the following steps: (a) Add the blood sample to be tested into the sample addition chamber; (b) The chip is centrifuged at a first rotation speed to separate the sample into plasma in the separation pool and flow into the detection chamber, where it is reconstituted with the one-step immunoreaction lyophilized microspheres and subjected to an immunoreaction. The first rotation speed is lower than the opening speed threshold of the washing valve. (c) Centrifuge the chip at a second speed higher than the first speed, so that the washing liquid in the detergent chamber opens the washing valve and enters the detection chamber for washing; (d) Detect the fluorescence signal in the detection cavity.

[0023] By adopting the above technical solution, this invention utilizes graded programmed control of centrifugal force to automate a multi-step immunoassay process. Its working mechanism can be broken down as follows: 1. Automated Sample Separation and Reaction Stage: In step (b), a low initial centrifugal force drives the blood cells in the whole blood sample to settle at the bottom of the separator, while the lower-density supernatant plasma overflows into the detection chamber. Simultaneously, this centrifugal force is insufficient to overcome the fluid resistance of the washing valve, ensuring that the washing solution is contained within the washing chamber. The plasma entering the detection chamber rehydrates upon contact with pre-placed lyophilized microspheres. The captured magnetic microparticles and fluorescent probes specifically bind to the target antibodies (EBV and echinococcosis IgM / IgG) in the plasma, forming an immune complex of "magnetic microparticles-antigen-antibody-fluorescent probe".

[0024] 2. Automatic Washing Stage: In step (c), the centrifuge speed is increased to a higher second speed. At this time, the centrifugal force applied to the washing liquid exceeds the opening threshold of the washing valve, and the washing liquid breaks through the valve and enters the detection chamber. This flowing washing liquid can flush the detection chamber and the surface of the immune complex, carrying away impurities such as unbound plasma components and free fluorescent probes, and discharging them into the waste liquid pool.

[0025] This method integrates multiple processes, such as plasma separation, immune reaction, and washing, into a single continuous centrifugation program by controlling two different centrifugation speeds and utilizing the on / off characteristics of a specially designed washing valve. It eliminates the need for external pumps, valves, or complex fluid control components, simplifying operation, shortening detection time, and reducing errors introduced by human operation.

[0026] Preferably, between step (b) and step (c), the chip is further controlled to oscillate and incubate at a frequency of 5Hz within a range of ±180° for 10 to 12 minutes.

[0027] By employing the above-described technical solution, the reciprocating oscillation provides active mixing for the immune reaction. The oscillation causes the magnetic immune complexes suspended in the plasma to move continuously, breaking down diffusion limitations near the reaction interface and increasing the effective collision frequency between antigen, antibody, and probe molecules. This accelerates the immune binding reaction process, helping the reaction reach equilibrium in a shorter time, thereby improving detection sensitivity and result uniformity.

[0028] Preferably, the first rotational speed is 1000 rpm to 2000 rpm, and the second rotational speed is 2000 rpm to 4000 rpm.

[0029] By employing the above technical solution, these two rotational speed ranges are optimized parameters that match the chip structure (especially the separatory tank structure and the washing valve threshold). The first rotational speed range is used for effective sedimentation and separation of blood cells, while ensuring that the centrifugal force is below the washing valve's opening threshold. The second rotational speed range ensures that the washing valve can be opened and provides sufficient fluid shear force to achieve cleaning. This parameter range is the foundation for ensuring the stable operation of this automated method.

[0030] Preferably, before step (c), a magnetic separation step is included: the immune complex formed in step (b) is adsorbed by a magnetic field and the unbound sample matrix is ​​removed by centrifugation.

[0031] By adopting the above technical solution, a pre-washing step is added before the formal washing. An external magnetic field is used to immobilize the magnetic microparticle complex bound to the target antibody in a specific area of ​​the detection chamber. Subsequently, centrifugation or decanting removes most of the plasma matrix containing interfering substances. This step reduces the load on subsequent washing steps, making washing more thorough, thereby reducing the fluorescence background signal and improving the signal-to-noise ratio and sensitivity of the detection.

[0032] This invention provides a magnetic immunofluorescence detection microfluidic chip for the combined detection of EB virus and echinococcosis antibodies. It has the following beneficial effects: 1. This invention constructs a fluid control switch with a high opening threshold for surfactant-containing liquids by setting specific geometric dimensions and a hydrophobic coating at the washing valve. In the prior art, the surface tension barrier of conventional hydrophobic valves is prone to failure when in contact with surfactant-containing washing liquid, causing the washing liquid to enter the reaction zone prematurely. This invention solves this process control problem and ensures the timing accuracy of multi-step reactions.

[0033] 2. This invention pre-places the capture magnetic microparticles and fluorescent probes within a single lyophilized microsphere, enabling a one-step immunoreaction after sample addition. This changes the multi-step pipetting process in traditional immunoassay, which requires adding the sample, capture reagent, and detection probe in separate steps. The new approach not only simplifies the operation but also avoids volume errors and reagent activity loss that may result from multiple sample addition steps.

[0034] 3. This invention combines graded speed control, high-threshold hydrophobic valve, and integrated freeze-dried microsphere technology to construct a complete solution for automatic multi-step immunoassay on a single centrifuge platform. Compared with existing technologies, it can achieve precise reagent timing control and washing without the need for complex fluid drive components such as external pumps and valves, and solves the technical problem of integrating reliable multi-step biochemical reactions on a compact, low-cost centrifuge chip. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the structure of a microfluidic chip according to an embodiment of the present invention.

[0036] Figure 2 The bar chart shows the average burst speed of the chip washing valves in different groups in Test Example 1.

[0037] Figure 3 This is a bar chart comparing the intra-batch imprecision (CV%) of detection signals for different detection schemes in Test Example 2.

[0038] Figure 4 This is a graph showing the change in signal retention rate between Example 1 and Comparative Example 1 under accelerated aging conditions at 37°C in Test Example 3.

[0039] in: 1. Sample loading chamber; 2. Separator; 3. Detergent chamber; 4. Detection chamber; 5. Waste liquid tank. Detailed Implementation

[0040] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Inorganic salts, acid and alkali solutions, organic solvents and conventional biochemical reagents not specifically mentioned are all commercially available analytical grade or higher products.

[0042] Polymethyl methacrylate (PMMA) sheet, CAS No. 9011-14-7.

[0043] Polyethylene terephthalate-1,4-cyclohexanediol (PETG) film, CAS No. 25038-91-9.

[0044] Carboxylated superparamagnetic iron oxide nanospheres with an average particle size of 1.0 μm to 3.0 μm, a solid content of 10 mg / mL, and a surface carboxyl group density of 100 μmol / g to 150 μmol / g.

[0045] Recombinant EB virus capsid antigen (rVCA) and recombinant Echinococcus multilocularis Em18 antigen were obtained through genetic engineering expression and purification, with a purity greater than 95%.

[0046] Goat anti-human IgM polyclonal antibody and goat anti-human IgG polyclonal antibody, with affinity constant Ka greater than 1.0 × 10⁻⁶. 9 M -1 .

[0047] Fluorescein isothiocyanate (FITC), CAS No. 3326-32-7.

[0048] Anthocyanin Cy3-N-hydroxysuccinimide ester (Cy3-NHS), CAS No. 146368-16-3.

[0049] 1H,1H,2H,2H-Perfluorooctyltrichlorosilane (FDTS), CAS No. 78560-45-9.

[0050] D(+)-trehalose dihydrate, CAS No. 6138-23-4.

[0051] D-Mannitol, CAS No. 69-65-8.

[0052] Preparation Example 1: This preparation example provides a one-step immunoreaction lyophilized microsphere, the preparation process of which includes the following steps: 1. Preparation of immunomagnetic microparticles: Take 10 mg of carboxylated superparamagnetic iron oxide nanosphere suspension, magnetically remove the supernatant, and resuspend in 1 mL of 50 mM, pH 6.0 2-(N-morpholine) ethanesulfonic acid monohydrate (MES) buffer; add 0.5 mL of 10 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) solution and 0.5 mL of 10 mg / mL N-hydroxysuccinimide. (NHS) solution was activated at room temperature for 30 minutes; after magnetic washing, the particles were resuspended in PBS buffer (pH 7.4), and recombinant EB virus capsid antigen and recombinant Echinococcus multilocularis Em18 antigen were added respectively (50 μg of antigen was added per milligram of magnetic beads). The particles were incubated at 37°C with shaking for 3 hours; the supernatant was discarded by magnetic removal, and blocking solution containing 1% bovine serum albumin was added for 1 hour. After washing, the particles were resuspended in the preservation solution to obtain EB virus capturing magnetic particles and echinococcosis capturing magnetic particles.

[0053] 2. Preparation of fluorescent probes: Fluorescein isothiocyanate (FITC) was dissolved in DMSO and mixed with goat anti-human IgM polyclonal antibody at a dye to protein molar ratio of 20:1. The mixture was reacted in the dark for 12 hours and purified by Sephadex G-25 gel column chromatography to obtain FITC-labeled anti-human IgM antibody. Anthocyanin Cy3-N-hydroxysuccinimide ester was dissolved in anhydrous DMF and mixed with goat anti-human IgG polyclonal antibody at a molar ratio of 15:1. The mixture was reacted in the dark for 2 hours and purified by Sephadex G-25 gel column chromatography to obtain Cy3-labeled anti-human IgG antibody.

[0054] 3. Preparation of lyophilized matrix solution: Prepare 10mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer (pH 7.4) as solvent, add D-mannitol to make the concentration 30mg / mL, add D(+)-trehalose dihydrate to make the concentration 50mg / mL, add bovine serum albumin to make the concentration 15mg / mL, add polyoxyethylene (20) sorbitan monolaurate (Tween-20) to make the concentration 0.8mg / mL, mix well to obtain lyophilized matrix solution.

[0055] 4. Microsphere Formation and Lyophilization: In the lyophilization matrix solution obtained in step 3, add the two types of magnetic trapping microparticles prepared in step 1 (both with a final concentration of 0.8 mg / mL), the two types of fluorescent probes prepared in step 2 (both with a final concentration of 8 μg / mL), and the heterophile antibody blocking agent (HBR, with a final concentration of 80 μg / mL). After mixing, drop the mixture into liquid nitrogen through a precision dispensing system and freeze it into microspheres. Then, perform lyophilization in a vacuum freeze dryer (vacuum degree <10 Pa, drying for 24 hours) to obtain one-step immunoreaction lyophilized microspheres.

[0056] Preparation Example 2: This preparation example provides a one-step immunoreaction lyophilized microsphere, which differs from Preparation Example 1 only in the concentration of each component in the lyophilized matrix solution (corresponding to the lower end of the numerical range).

[0057] Its preparation process includes the following steps: Steps 1 and 2 are the same as in Preparation Example 1.

[0058] 3. Preparation of lyophilized matrix solution: Prepare 10mM Tris-HCl buffer (pH 7.4) as solvent, add D-mannitol to make the concentration 20mg / mL, add D(+)-trehalose dihydrate to make the concentration 40mg / mL, add bovine serum albumin to make the concentration 10mg / mL, add polyoxyethylene (20) sorbitan monolaurate to make the concentration 0.5mg / mL, mix well to obtain lyophilized matrix solution.

[0059] 4. Microsphere molding and freeze-drying: Add the same amount of bioactive components (capturing magnetic microparticles, fluorescent probes and blocking agents) as in Preparation Example 1 to the freeze-drying matrix solution obtained in step 3, mix well, drop into liquid nitrogen to freeze and freeze-dry, and obtain one-step immunoreaction freeze-dried microspheres.

[0060] Preparation Example 3: This preparation example provides a one-step immunoreaction lyophilized microsphere, which differs from Preparation Example 1 only in the concentration of each component in the lyophilized matrix solution (corresponding to the high end of the numerical range).

[0061] Its preparation process includes the following steps: Steps 1 and 2 are the same as in Preparation Example 1.

[0062] Step 3: Preparation of lyophilized matrix solution: Prepare 10mM Tris-HCl buffer (pH 7.4) as solvent, add D-mannitol to make the concentration 40mg / mL, add D(+)-trehalose dihydrate to make the concentration 60mg / mL, add bovine serum albumin to make the concentration 20mg / mL, add polyoxyethylene (20) sorbitan monolaurate to make the concentration 1.0mg / mL, mix well to obtain lyophilized matrix solution.

[0063] Step 4, Microsphere Forming and Freeze-drying: Add the same amount of bioactive components (capturing magnetic microparticles, fluorescent probes and blocking agents) as in Preparation Example 1 to the freeze-drying matrix solution obtained in Step 3, mix well, drop into liquid nitrogen to freeze and freeze-dry, and obtain one-step immunoreaction freeze-dried microspheres.

[0064] Preparation Example 4: This preparation example provides a method for hydrophobic modification of the surface of a valve in a microfluidic chip, including the following steps: 1H,1H,2H,2H-perfluorooctyltrichlorosilane was dissolved in n-hexane to prepare a hydrophobic modification solution with a volume percentage concentration of 1.0%. The hydrophobic modification solution was then coated onto the inner wall of the capillary valve channel at the outlet of the detergent chamber of the microfluidic chip using a micro-dispensing method. Subsequently, the chip was placed in a 60°C oven for heat treatment for 30 minutes to allow the solvent to evaporate and solidify to form a hydrophobic layer.

[0065] Preparation Example 5: This preparation example provides a method for hydrophobic modification of the surface of a microfluidic chip valve. The only difference between this method and Preparation Example 4 is the concentration of the hydrophobic modification solution (corresponding to the lower end of the numerical range). The method includes the following steps: 1H,1H,2H,2H-perfluorooctyltrichlorosilane was dissolved in n-hexane to prepare a hydrophobic modified solution with a volume percentage concentration of 0.5%; the subsequent coating and heat treatment steps were the same as those in Preparation Example 4.

[0066] Preparation Example 6: This preparation example provides a method for hydrophobic modification of the surface of a microfluidic chip valve. The only difference between this method and Preparation Example 4 is the concentration of the hydrophobic modification solution (corresponding to the higher end of the numerical range). The method includes the following steps: 1H,1H,2H,2H-perfluorooctyltrichlorosilane was dissolved in n-hexane to prepare a hydrophobic modified solution with a volume percentage concentration of 2.0%; the subsequent coating and heat treatment steps were the same as those in Preparation Example 4.

[0067] Preparation Example 7: This preparation example provides a flow channel structure for a magnetic immunofluorescence detection microfluidic chip for the combined detection of EB virus and echinococcosis antibodies. The flow channel layer is made of polymethyl methacrylate board through laser engraving.

[0068] The washing valve (high-speed valve) connecting the detergent chamber and the detection chamber has the following geometric dimensions: width 100 μm, depth 50 μm. The sample valve (low-speed valve) connecting the sample dispensing chamber and the dispensing tank has the following geometric dimensions: width 200 μm, depth 100 μm. These dimensions are designed to achieve a suitable washing liquid release speed threshold (e.g., around 2500 rpm).

[0069] Preparation Example 8: This preparation example provides a flow channel structure for a magnetic immunofluorescence detection microfluidic chip for the combined detection of EB virus and echinococcosis antibodies. The only difference between this chip and preparation example 7 is the geometry of the washing valve.

[0070] The washing valve (high-speed valve) connecting the detergent chamber and the detection chamber has the following geometric dimensions: width 120μm, depth 60μm. This size is designed to reduce the speed threshold for detergent release (e.g., around 2000rpm), making it suitable for low-speed centrifuges.

[0071] Preparation Example 9: This preparation example provides a flow channel structure for a magnetic immunofluorescence detection microfluidic chip for the combined detection of EB virus and echinococcosis antibodies. The only difference between this chip and preparation example 7 is the geometry of the washing valve.

[0072] The washing valve (high-speed valve) connecting the detergent chamber and the detection chamber has the following geometric dimensions: width 80μm and depth 40μm. This size is designed to increase the speed threshold for detergent release (e.g., around 3000rpm) to enhance fluid control stability and prevent premature leakage.

[0073] Example 1: This embodiment provides a magnetic immunofluorescence detection microfluidic chip for combined detection of EB virus and echinococcosis antibodies and its detection method. Its structure is referenced below. Figure 1 As shown, the optimized reagent formulation and medium rotation speed threshold structure are suitable for standard detection scenarios. This embodiment utilizes the one-step immunoreaction lyophilized microspheres prepared in Preparation Example 1, the hydrophobic modification process of Preparation Example 4, and the flow channel structure of Preparation Example 7.

[0074] This embodiment includes the following steps: Assembly of microfluidic chips: 1. Substrate preparation: Take the polymethyl methacrylate (PMMA) flow channel layer (with the washing valve size being 100 μm wide × 50 μm deep) obtained from Preparation Example 7, ultrasonically clean it with ethanol and dry it.

[0075] 2. Reagent preparation: Place one one-step immunoreaction lyophilized microsphere (containing preferred protective matrix, immunomagnetic microparticles, and fluorescent probe) prepared in Preparation Example 1 into the detection chamber 4.

[0076] 3. Washing buffer loading: Inject 300 μL of PBS washing buffer (pH 7.4) containing 0.05% (w / v) Tween-20 into the washing chamber 3.

[0077] 4. Chip bonding: The PETG valve layer with the hydrophobic coating (1.0% FDTS) prepared in Example 4 was selected and aligned with the flow channel layer so that the hydrophobic modified area accurately covered the washing valve flow channel; the chip packaging was completed by using a solvent-assisted hot pressing process at 70°C and 0.5MPa pressure for 5 minutes.

[0078] Detection methods and procedures: 1. Sample loading: Take 20 μL of whole blood sample from the subject and add it to sample loading chamber 1 of the chip. Place the chip in a centrifugal fluorescence detector.

[0079] 2. Plasma separation (first stage): Start the instrument and centrifuge at 1500 rpm for 2 minutes. At this speed, the whole blood sample breaks through the sample valve and enters the separatory chamber 2. Blood cells settle, and the supernatant plasma overflows into the detection chamber 4. At this time, the washing valve (designed burst threshold of about 2500 rpm) is kept closed by the hydrophobic layer and capillary force, and the washing solution does not leak.

[0080] 3. Immunological reaction: Stop centrifugation, switch the instrument to Shake Mode, and incubate the chip at a frequency of 5Hz within a range of ±180° for 10 minutes. The lyophilized microspheres in the detection chamber rapidly disintegrate and rehydrate in the plasma. The EBV-VCA-IgM antibody and echinococcosis antibody in the sample specifically bind to the released magnetic microparticles and fluorescent probes to form immune complexes.

[0081] 4. Magnetic separation and waste disposal: The internal magnet of the instrument rises and adheres to the bottom of the detection chamber 4 to adsorb and fix the immune complex; then the chip is centrifuged at 2000 rpm for 30 seconds to throw the unbound sample matrix and excess free probe into the waste liquid pool 5.

[0082] 5. Washing (Second Stage): Increase the rotation speed to 3000 rpm and maintain it for 10 seconds. The centrifugal pressure generated at this speed exceeds the threshold of the washing valve, and the washing liquid rushes open the valve and enters the detection chamber 4.

[0083] 6. Rinsing and secondary separation: Stop centrifugation and remove the magnet. Shake and wash for 60 seconds to resuspend the magnetic particles. Then the magnet rises again to attract the particles, and centrifuge at 3000 rpm to drain the washing waste liquid.

[0084] 7. Detection: Maintain magnetic field adsorption, and the optical module reads the fluorescence signal values ​​at 525nm and 570nm respectively.

[0085] Example 2: This embodiment provides a magnetic immunofluorescence detection microfluidic chip and detection method for the combined detection of EB virus and echinococcosis antibodies. It employs a low-concentration matrix formulation and a low-flow-resistance valve structure to verify its feasibility under low-speed equipment. This embodiment utilizes the one-step immunoreaction lyophilized microspheres prepared in Example 2, the hydrophobic modification process of Example 5, and the flow channel structure of Example 8.

[0086] This embodiment includes the following steps: Assembly of microfluidic chips: 1. Substrate preparation: Take the flow channel layer obtained from Preparation Example 8 (the washing valve is relatively wide, 120μm×60μm, and has low flow resistance).

[0087] 2. Reagent preparation: Place one lyophilized microsphere (low sugar / low protein matrix, dissolves very quickly but has a relatively loose structure) prepared in Preparation Example 2 into the detection chamber 4.

[0088] 3. Loading of washing liquid: Same as in Example 1.

[0089] 4. Chip bonding: The valve layer prepared in Example 5 (0.5% FDTS, slightly weaker hydrophobicity) was selected for bonding.

[0090] Detection methods and procedures: 1. Sample loading: Same as in Example 1.

[0091] 2. Plasma separation (first stage): Start the instrument and centrifuge at 1000 rpm for 3 minutes. Because the valve size of Preparation Example 8 is relatively wide, 1000 rpm is sufficient to drive sample separation, and this speed is below the burst threshold of the washing valve (about 1800 rpm), so the washing solution remains stable.

[0092] 3. Immune response: The reconstitution and shaking incubation steps are the same as in Example 1.

[0093] 4. Magnetic separation and waste discharge: After being attracted by a magnet, the waste liquid is discharged by centrifugation at 1200 rpm for 30 seconds.

[0094] 5. Washing (Second Stage): Increase the rotation speed to 2000 rpm and maintain it for 15 seconds. This speed is sufficient to exceed the washing valve threshold of the structure prepared in Example 8, releasing the washing liquid.

[0095] 6. Rinsing and secondary separation: After shaking and washing, the washing waste liquid is drained at 2000 rpm.

[0096] 7. Testing: Same as in Example 1.

[0097] Example 3: This embodiment provides a magnetic immunofluorescence detection microfluidic chip and detection method for the combined detection of EB virus and echinococcosis antibodies. It employs a high-concentration matrix formulation and a high-flow-resistance valve structure to verify the fluid control stability under high-speed centrifugation and its ability to prevent premature leakage. This embodiment utilizes the one-step immunoreaction lyophilized microspheres prepared in Example 3, the hydrophobic modification process of Example 6, and the flow channel structure of Example 9.

[0098] This embodiment includes the following steps: Assembly of microfluidic chips: 1. Substrate preparation: Take the flow channel layer obtained from Preparation Example 9 (the washing valve is narrow, 80μm×40μm, with a large flow resistance).

[0099] 2. Reagent pre-positioning: Place one lyophilized microsphere (high sugar / high protein matrix, dense structure) prepared in Preparation Example 3 into the detection chamber 4.

[0100] 3. Loading of washing liquid: Same as in Example 1.

[0101] 4. Chip bonding: The valve layer prepared in Example 6 (2.0% FDTS, strong hydrophobicity) was selected for bonding.

[0102] Detection methods and procedures: 1. Sample loading: Same as in Example 1.

[0103] 2. Plasma separation (first stage): Start the instrument and centrifuge at 2000 rpm for 2 minutes. Higher speed separation efficiency is higher, and because the washing valve threshold of Preparation Example 9 is relatively high (about 3500 rpm), the washing solution can withstand this speed without premature leakage.

[0104] 3. Immunological reaction: The reconstitution and shaking incubation steps are the same as in Example 1. Due to the denser microsphere matrix, the shaking time is extended to 12 minutes to ensure complete reaction.

[0105] 4. Magnetic separation and waste discharge: After being attracted by a magnet, the waste liquid is discharged by centrifugation at 2500 rpm for 30 seconds.

[0106] 5. Washing (Second Stage): Increase the rotation speed to 4000 rpm and maintain it for 5 seconds. Use the extremely high centrifugal force to break through the high threshold valve of Preparation Example 9 and release the washing liquid.

[0107] 6. Rinsing and secondary separation: After shaking and washing, drain the washing waste liquid at 4000 rpm.

[0108] 7. Testing: Same as in Example 1.

[0109] Comparative Example 1: The difference from Example 1 is that a one-step immunoreaction lyophilized microsphere method is not used. No reagents are pre-placed in detection chamber 4 during chip assembly. Before the detection operation begins, an equal volume of liquid magnetic microparticles and fluorescent probe mixture is manually added to detection chamber 4, followed immediately by sealing and detection. All other structures and steps are the same.

[0110] Comparative Example 2: Compared to Example 1, the difference lies in that the microvalve channel at the outlet of the detergent chamber 3 of the chip was not subjected to the hydrophobic modification treatment as in Preparation Example 4, and was directly bonded to the surface of the unmodified PETG / PMMA material. The rest of the structure and steps are the same.

[0111] Comparative Example 3: The difference from Example 1 lies in the formulation of the freeze-drying protective matrix for the one-step immunoreaction lyophilized microspheres. The matrix of Comparative Example 3 does not contain D(+)-trehalose dihydrate and polyoxyethylene (20) sorbitan monolaurate (Tween-20), but only contains 30 mg / mL of D-mannitol and 15 mg / mL of bovine serum albumin. The remaining preparation process is the same as in Example 1.

[0112] Comparative Example 4: Compared to Example 1, the difference lies in the altered geometry of the washing valve. In Comparative Example 4, the dimensions of the washing valve were adjusted to match those of the sample valve (i.e., 200 μm wide and 100 μm deep), thereby reducing the theoretical opening speed threshold of the washing valve to be close to the sample separation speed (1500 rpm). All other structures and procedures remained the same.

[0113] Test Example 1: Experimental objective: This test aims to quantitatively evaluate the fluid control performance of the hydrophobically modified washing valve in the microfluidic chip of this invention under different centrifugation conditions. Two core indicators are mainly examined: 1) the anti-leakage capability of the washing valve against washing liquid containing surfactants under low-speed centrifugation (simulating sample processing stage); 2) the speed threshold for the washing valve to open (i.e., burst) and its controllability and repeatability under high-speed centrifugation.

[0114] Experimental methods: Microfluidic chips prepared in Examples 1-3, Comparative Examples 2 and 4 were selected, with 5 replicates per group. 300 μL of PBS buffer containing 0.1% (w / v) red dye (e.g., amaranth red) and 0.05% (w / v) Tween-20 was injected into the detergent chamber 3 of each chip, while the sample loading chamber and other chambers remained blank. The chips were placed in a centrifuge equipped with a high-speed camera system.

[0115] Experimental steps: 1. Leakage prevention test: Set the centrifuge speed to 1000 rpm and rotate continuously for 5 minutes. Observe the test chamber 4 and waste liquid pool 5 for red dye using a high-speed camera system or visual inspection, and record whether liquid leakage occurs.

[0116] 2. Bursting Speed ​​Test: After completing the leak-proof test (or using a new chip), set the centrifuge program so that its speed increases from 0 rpm at a constant acceleration of 100 rpm / s. Monitor the washing valve outlet in real time using a high-speed camera system, accurately recording the instant the red liquid first breaks through the valve and enters the downstream flow channel; this speed is the bursting speed of the chip. Repeat the test 5 times for each chip group, calculating the average and standard deviation.

[0117] Experimental data: Table 1: Chip Fluid Control and Valve Burst Test Data The average blasting speed data for each group in Table 1 are plotted as a bar chart, such as... Figure 2 As shown.

[0118] Conclusion: Test results show that the unmodified Comparative Example 2 chip exhibited liquid leakage at 1000 rpm. This is because the surfactant in the washing liquid reduced the surface tension of the liquid, making the capillary resistance of the material itself insufficient to seal the liquid under centrifugal force. The washing valves in Examples 1, 2, and 3, after treatment with 1H,1H,2H,2H-perfluorooctyltrichlorosilane, formed a low-surface-energy hydrophobic interface, increasing the liquid contact angle. The resulting capillary resistance effectively prevented the washing liquid from flowing at low speeds.

[0119] like Figure 2 As shown, the burst speed data indicates that the valve's geometric parameters directly determine its opening threshold. The washing valves in Examples 1, 2, and 3 have different widths and depths, with average burst speeds of 2512 rpm, 1808 rpm, and 3518 rpm respectively, exhibiting a clear gradient distribution. Furthermore, the standard deviation of the data within each group is small, indicating that the control method has good repeatability.

[0120] The results of Comparative Example 4 illustrate the necessity of the staged valve design. Because the washing valve in this comparative example has similar geometry to the sample valve, its burst speed (1512 rpm) falls within the same range as the speed required for sample separation (e.g., 1500 rpm). This causes the washing solution and sample to enter the detection chamber at the same time, disrupting the required timing of the immunoassay and rendering the detection process ineffective.

[0121] This test confirms that by combining hydrophobic treatment of the valve surface with geometric design, a passive valve system with a preset burst threshold can be constructed, enabling sequential fluid control under centrifugal conditions. This method provides a foundation for automating multi-step biochemical reactions without human intervention.

[0122] Test Example 2: Experimental objective: This test aims to verify the detection performance of the microfluidic chip of the present invention for EB virus and echinococcosis antibodies, including accuracy (compared with the clinical gold standard) and precision (reproducibility), and to compare it with comparative samples using different reagent forms and formulations, in order to evaluate the technical advantages of the integrated freeze-dried microspheres and automated process.

[0123] Experimental methods: The microfluidic chips prepared in Example 1, Comparative Example 1, and Comparative Example 3 were selected, and a commercially available enzyme-linked immunosorbent assay (ELISA) kit was used as the reference method. The test samples were 30 clinical serum samples verified by the CDC, including 10 EBV-IgM positive samples, 10 echinococcosis IgG positive samples, and 10 double-negative samples.

[0124] Experimental steps: 1. Chip Detection: For the chips used in Example 1, Comparative Example 1, and Comparative Example 3, each sample was tested. 20 μL of serum sample was added to the chip's sample well, and the accompanying centrifugal fluorescence detector was started to execute the fully automated detection program. After the program ended, the relative fluorescence units (RFU) of the EBV channel and the echinococcosis channel output by the instrument were recorded. Three times the standard deviation (Mean + 3SD) of the mean RFU obtained from 10 negative samples in each group was used as the cut-off value for judging a positive result.

[0125] 2. Additional instructions for Comparative Example 1: For Comparative Example 1, since there are no pre-prepared reagents in its detection chamber, it is necessary to manually add an equal amount of liquid magnetic microparticles and fluorescent probe mixture to the detection chamber before proceeding with sample addition and instrument operation.

[0126] 3. ELISA reference method detection: All 30 serum samples were tested according to the instructions of the commercial ELISA kit. The absorbance (OD) value at 450 nm was read using an enzyme-linked immunosorbent assay (ELISA) reader, and the results were calculated and judged according to the standards provided in the instructions.

[0127] 4. Data Analysis: The test results of each group method on 30 samples were statistically analyzed, and the positive and negative concordance rates compared with the gold standard (clinical review results) were calculated. Simultaneously, a positive sample with a moderate titer was selected and tested 10 times each using the chips from Example 1, Comparative Example 1, and Comparative Example 3, and the intra-batch imprecision (CV%) of the detection signal RFU value was calculated.

[0128] Experimental data: Table 2: Comparison of Chip Detection Performance and Methodologies Plot the intra-batch imprecision (CV%) data of the detection signals for each group in Table 2 into a bar chart, as follows: Figure 3 As shown.

[0129] Conclusion: The detection results of Example 1 were consistent with the ELISA reference method, with a positive and negative concordance rate of 100%.

[0130] like Figure 3 As shown, Comparative Example 1 used liquid reagents, resulting in a lower detection accuracy but a higher CV value (12.5%-14.2%). This is related to the stability of liquid biological reagents and errors introduced by manual operation. Example 1 integrated the reaction components into a chip in the form of lyophilized microspheres, ensuring uniform reagent dosage and reducing operational steps, resulting in higher detection precision (CV value 4.8%-5.3%).

[0131] Comparative Example 3, using a lyophilized matrix lacking trehalose and Tween-20, exhibited the lowest detection concordance and precision among all groups. Trehalose forms a glassy matrix during lyophilization, maintaining the spatial conformation of proteins. Tween-20 facilitates microsphere resolubilization and reduces non-specific adsorption of biomolecules at the interface. The absence of these two components leads to reduced activity of the immunoassay reagent, thus affecting detection performance.

[0132] The automated process in Example 1 integrates sample processing, incubation, and washing steps within a closed flow channel, completed through programmed control. This avoids the errors introduced by the multiple manual steps of ELISA, thus achieving superior detection precision compared to the ELISA method. This test demonstrates that combining optimized lyophilization reagent technology with an automated centrifugation platform yields accurate and reproducible detection results.

[0133] Test Example 3: Experimental objective: This test aims to evaluate the storage stability of the integrated lyophilized microspheres in the microfluidic chip of this invention under accelerated aging conditions, and to compare it with that of conventional liquid reagents. Stability is a key indicator for evaluating the shelf life and reliability of diagnostic products.

[0134] Experimental methods: The finished chip prepared in Example 1 and the liquid immunoassay reagent used in Comparative Example 1 (magnetic microparticle suspension and fluorescent probe solution stored separately) were selected. Both forms of reagent (chip and liquid reagent tube) were placed in a 37°C incubator for accelerated aging. Three replicate chips or reagents were taken out on days 0, 7, 14, and 21. High- and low-value positive EBV-IgM standards and high- and low-value positive echinococcosis IgG standards from the same batch were used as test samples.

[0135] Experimental steps: At each test time point, the chip and liquid reagent corresponding to the storage time were removed. For the chip of Example 1, 20 μL of standard sample was added directly, and fully automated detection was performed according to the standard procedure. For the liquid reagent of Comparative Example 1, magnetic microparticles and fluorescent probes were manually mixed according to a predetermined ratio, then added to the detection chamber of the blank chip, followed by the addition of the sample for detection. The relative fluorescence units (RFU) of each channel were recorded, and the signal retention rate at each time point was calculated ([RFU at that time point / RFU on day 0] × 100%).

[0136] Experimental data: Table 3: Accelerated Aging Stability Test Data Plot the signal retention rate data in Table 3 as a line graph, such as... Figure 4 As shown.

[0137] Conclusion: The results of the accelerated aging test are as follows Figure 4 As shown, the chip of Example 1 retained over 94% of its detection signal after being stored at 37°C for 21 days. The liquid reagent of Comparative Example 1 showed decreased activity under the same conditions, with a signal retention rate below 20% after 21 days, and the detection signal of low-value positive samples was below the cutoff value.

[0138] This performance difference stems from the physical form of the reagent. Figure 4 The curves clearly show that in a liquid environment, protein molecules have a high degree of freedom of movement and are prone to irreversible denaturation under thermal stress. In Example 1, the freeze-drying process encapsulates bioactive components in a high-viscosity amorphous glassy matrix composed of trehalose, mannitol, etc. This glassy structure restricts the mobility of biomolecules and reduces their chemical reaction rates. Trehalose, in particular, maintains conformational stability by forming hydrogen bonds with proteins, replacing water molecules on the protein surface.

[0139] The test demonstrates that preparing immunoassay reagents into integrated lyophilized microspheres and pre-positioning them in a chip can improve the stability of the reagents under thermal stress conditions, which is of practical significance for enabling the transportation and storage of diagnostic products under non-cold chain conditions.

[0140] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A magnetic immunofluorescence detection microfluidic chip for combined detection of EB virus and echinococcosis antibodies, characterized in that, include: The chip body is provided with a sample application chamber, a liquid dispensing tank and a detection chamber connected in sequence, and is also provided with a detergent chamber and a waste liquid tank. The detergent chamber is connected to the detection chamber via a washing valve; The inner wall surface of the washing valve is provided with a hydrophobic modified layer; The opening speed threshold of the washing valve is higher than the centrifugation speed used to achieve plasma separation in the separatory tank. The detection chamber is pre-loaded with one-step immunoreaction lyophilized microspheres.

2. The microfluidic chip for combined detection of EB virus and echinococcosis antibodies according to claim 1, characterized in that, The one-step immunoreaction lyophilized microspheres comprise: Capture magnetic particles; Fluorescent probe; The freeze-dried protective matrix is ​​composed of D-mannitol, D(+)-trehalose dihydrate, bovine serum albumin and polyoxyethylene (20) sorbitan monolaurate.

3. The microfluidic chip for combined detection of EB virus and echinococcosis antibodies according to claim 2, characterized in that, The freeze-drying protective matrix is ​​prepared from a freeze-drying matrix solution containing the following components at the following concentrations: D-Mannitol: 20 mg / mL to 40 mg / mL; D(+)-trehalose dihydrate: 40 mg / mL to 60 mg / mL; Bovine serum albumin: 10 mg / mL to 20 mg / mL; Polyoxyethylene (20) sorbitan monolaurate: 0.5 mg / mL to 1.0 mg / mL.

4. The microfluidic chip for combined detection of EB virus and echinococcosis antibodies according to claim 2, characterized in that, The captured magnetic microparticles are magnetic microspheres with recombinant EB virus capsid antigen and / or recombinant Echinococcus multilocularis var. Em18 antigen covalently coupled to their surface. The fluorescent probe is either FITC-labeled goat anti-human IgM polyclonal antibody or Cy3-labeled goat anti-human IgG polyclonal antibody.

5. The microfluidic chip for combined detection of EB virus and echinococcosis antibodies according to claim 1, characterized in that, The hydrophobic modified layer is a coating formed by curing 1H,1H,2H,2H-perfluorooctyltrichlorosilane.

6. The microfluidic chip for combined detection of EB virus and echinococcosis antibodies according to claim 1, characterized in that, The washing valve has the following geometric dimensions: width 80μm to 120μm and depth 40μm to 60μm.

7. A testing method for a magnetic immunofluorescence detection microfluidic chip for combined detection of EB virus and echinococcosis antibodies as described in any one of claims 1-6, characterized in that, Includes the following steps: (a) Add the blood sample to be tested into the sample addition chamber; (b) The chip is centrifuged at a first rotation speed to separate the sample into plasma in the separation pool and flow into the detection chamber, where it is reconstituted with the one-step immunoreaction lyophilized microspheres and subjected to an immunoreaction. The first rotation speed is lower than the opening speed threshold of the washing valve. (c) Centrifuge the chip at a second speed higher than the first speed, so that the washing liquid in the detergent chamber opens the washing valve and enters the detection chamber for washing; (d) Detect the fluorescence signal in the detection cavity.

8. The testing method for a microfluidic chip for the combined detection of EB virus and echinococcosis antibodies according to claim 7, characterized in that, Between step (b) and step (c), the following is also included: The chip is controlled to oscillate back and forth at a frequency of 5Hz within a range of ±180° for 10 to 12 minutes.

9. The testing method for a magnetic immunofluorescence detection microfluidic chip for combined detection of EB virus and echinococcosis antibodies according to claim 7, characterized in that, The first speed is 1000 rpm to 2000 rpm, and the second speed is 2000 rpm to 4000 rpm.

10. The testing method for a magnetic immunofluorescence detection microfluidic chip for combined detection of EB virus and echinococcosis antibodies according to claim 7, characterized in that, Prior to step (c), a magnetic separation step is also included: The immune complex formed in step (b) is adsorbed by magnetic field and the unbound sample matrix is ​​removed by centrifugation.

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