An ultrasonic-assisted integrated microfluidic ELISA rapid detection system

Abstract

This invention relates to the field of bioassay technology, and in particular to an ultrasound-assisted integrated microfluidic ELISA rapid detection system. The system includes an ultrasound device with a microfluidic chip mounted on it. The ultrasound device applies ultrasonic vibrations to the microfluidic chip during the detection process to assist in sample mixing and filtration. The microfluidic chip includes a chip body with a buffer injection chamber connected to a sample chamber via a primary herringbone burst valve. The sample chamber is connected to a microcolumn filter array via a serpentine micro-serrated mixing channel. The microcolumn filter array is connected to three parallel immunoreaction chambers via a Tesla one-way valve. These three parallel immunoreaction chambers are connected to a waste liquid chamber. The chip body also contains multiple calibration chambers. This invention, by combining a microfluidic chip with an ultrasound device, not only effectively simplifies the entire detection process and improves detection speed, but also makes it effective for home or bedside testing.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to an ultrasound-assisted integrated microfluidic ELISA rapid detection system. Background Technology

[0002] Traditional protein detection methods have many significant shortcomings, making it difficult to meet the high-efficiency and convenient requirements of practical testing scenarios. In the field of protein detection, traditional ELISA technology, as the most widely used mainstream method, has obvious limitations: the operation process is cumbersome, requiring a series of manual steps such as sample dilution, incubation, multiple washings, and color development. This not only results in a long detection cycle and is time-consuming, but also allows for significant errors due to variations in human operation, affecting the accuracy of the results. Furthermore, this technology has stringent requirements for the testing environment, relying on large laboratory equipment and professional operators, making rapid on-site testing impossible and greatly limiting its application in scenarios such as point-of-care diagnosis and on-site screening. Even when combining microfluidic chip technology with traditional ELISA to improve protein detection, existing traditional microfluidic protein detection methods still have several prominent drawbacks: poor homogeneity of protein sample and detection reagent mixing, easily leading to insufficient protein-antibody binding, further increasing detection errors; impurities such as other proteins and cell debris in the protein sample easily deposit in the microfluidic channels, causing blockages, and the lack of effective unblocking methods severely affects the continuity and stability of the detection. Furthermore, traditional microfluidic detection methods often suffer from poorly designed flow control structures, leading to issues such as liquid backflow and disordered flow sequence, which interfere with the accuracy of detection results. Simultaneously, they do not achieve truly integrated detection, still requiring additional auxiliary equipment for sample preprocessing or signal reading, resulting in insufficient operational convenience. Additionally, the antibody encapsulation methods of traditional detection methods are outdated, easily leading to reduced antibody activity and affecting the specific binding efficiency between proteins and antibodies, thereby reducing detection stability and sensitivity. Moreover, they exhibit poor target adaptability, failing to flexibly adjust to the detection needs of different types of target proteins, lacking versatility and struggling to adapt to diverse protein detection scenarios.

[0003] Therefore, there is an urgent need for an ultrasound-assisted integrated microfluidic ELISA rapid detection system. Summary of the Invention

[0004] The purpose of this invention is to provide an ultrasound-assisted integrated microfluidic ELISA rapid detection system to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution: an ultrasound-assisted integrated microfluidic ELISA rapid detection system, comprising an ultrasound device, wherein a microfluidic chip is disposed on the ultrasound device, the ultrasound device being used to apply ultrasonic vibration to the microfluidic chip during the detection process to assist sample mixing and filtration; the microfluidic chip comprising a chip body, wherein a buffer injection chamber is formed within the chip body, the buffer injection chamber being connected to a sample chamber via a primary herringbone burst valve, the sample chamber being connected to a microcolumn filter array via a serpentine micro-serrated mixing channel, the microcolumn filter array being connected to three parallel immunoreaction chambers via a Tesla one-way valve, the three parallel immunoreaction chambers being connected to a waste liquid chamber; and multiple calibration chambers are also formed within the chip body.

[0006] Preferably, the three parallel immune reaction chambers include three immune reaction chambers, and the three immune reaction chambers are respectively connected to the waste liquid chamber through a three-stage fishbone burst valve.

[0007] Preferably, the channel between the Tesla one-way valve and the three parallel immune reaction chambers is connected to a buffer chamber via a secondary fishbone burst valve, and the secondary fishbone burst valve is located close to the Tesla one-way valve.

[0008] Preferably, the microfluidic chip is prepared by replication molding of PDMS material. The PDMS prepolymer and curing agent are mixed at a mass ratio of 10:1 and fully degassed before being poured onto a microstructure mold and cured at 60-80°C to form a PDMS layer with a microchannel structure.

[0009] Preferably, the microstructure mold is formed by spin-coating SU-8 photoresist on a 4-inch silicon wafer and then performing an ultraviolet exposure and development process.

[0010] Preferably, the PDMS layer is bonded to the glass substrate after oxygen plasma treatment, thereby forming a closed microfluidic chip structure.

[0011] Preferably, the internal channels of the microfluidic chip are hydrophobic to form a hydrophobic surface.

[0012] Preferably, the ultrasonic device includes a base, a cover plate is installed on the top surface of the base, an aluminum alloy plate is installed on one end of the cover plate away from the base, a plurality of chip fixing holes are provided around the aluminum alloy plate, the plurality of chip fixing holes are opened on the cover plate, and the microfluidic chip is installed on the cover plate through the plurality of chip fixing holes.

[0013] Preferably, a piezoelectric ceramic drive board is installed inside the base, and the piezoelectric ceramic drive board is electrically connected to a program control board, an ultrasonic piezoelectric ceramic, and a two-prong power plug.

[0014] Preferably, the ultrasonic piezoelectric ceramic is installed through the cover plate on one end of the aluminum alloy plate facing the base.

[0015] The present invention discloses the following technical effects: This invention combines a microfluidic chip with an ultrasonic device, which not only effectively simplifies the entire detection process, reduces operational complexity, and increases detection speed, but also allows for operation without the need for professional technicians, and can be effectively applied to home or bedside detection. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a schematic diagram of the overall structure of the present invention; Figure 2 This is a schematic diagram of the microfluidic chip structure of the present invention; Figure 3 This is a schematic diagram of the ultrasonic device structure of the present invention; Figure 4 This is a schematic diagram of the internal structure of the ultrasonic device of the present invention; Figure 5 This is a schematic diagram illustrating the principle of the protein detection method of the present invention; Figure 6 The results of the competitive pairing experiment for the calprotectin-specific monoclonal antibody of this invention; Figure 7 This is a schematic diagram illustrating the dissolution of the present invention under different trehalose concentrations; Figure 8 This is a logical diagram of the data reading software of the present invention; Figure 9 This is the page of the data reading software of the present invention; Figure 10 This is a comparison table of recovery rate and repeatability results of the present invention applied to the detection of calprotectin-containing samples; Figure 11 These are the detection values ​​at different concentrations when the present invention is applied to the detection of calprotectin-containing samples; The components include: 1. Buffer injection chamber; 2. Primary fishbone burst valve; 3. Sample chamber; 4. Serpentine micro-serrated mixing channel; 5. Microcolumn filter array; 6. Tesla one-way valve; 7. Buffer chamber; 8. Secondary fishbone burst valve; 9. Three parallel immune reaction chambers; 10. Tertiary fishbone burst valve; 11. Waste liquid chamber; 12. Calibration chamber; 21. Base; 22. Cover plate; 23. Aluminum alloy plate; 24. Chip fixing hole; 31. Piezoelectric ceramic drive board; 32. Program control board; 33. Ultrasonic piezoelectric ceramic; 34. Two-pin power plug; 41. Polydopamine-modified antibody carrier; 42. Capture antibody; 43. HRP-labeled detection antibody. Detailed Implementation

[0018] The technical solutions of 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.

[0019] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] Reference Figures 1 to 11 This invention provides an ultrasound-assisted integrated microfluidic ELISA rapid detection system, including an ultrasound device with a microfluidic chip mounted on it. The ultrasound device applies ultrasonic vibration to the microfluidic chip during the detection process to assist in sample mixing and filtration. The microfluidic chip includes a chip body with a buffer injection chamber 1 inside. The buffer injection chamber 1 is connected to a sample chamber 3 via a first-stage herringbone burst valve 2. The sample chamber 3 is connected to a microcolumn filter array 5 via a serpentine micro-serrated mixing channel 4. The microcolumn filter array 5 is connected to a three-parallel immunoreaction chamber 9 via a Tesla one-way valve 6. The three-parallel immunoreaction chamber 9 is connected to a waste liquid chamber 11. The chip body also has multiple calibration chambers 12.

[0021] The buffer injection chamber 1 is connected to an external syringe via a tubing to provide buffer driving force to the chip system.

[0022] Sample chamber 3 is used to add the sample to be tested into the chip and to pre-mix the sample with the buffer solution before it enters the subsequent channels.

[0023] The serpentine micro-serrated mixing channel 4 is used to enhance the turbulence and lateral mixing efficiency of fluid in the microscale channel, so that the sample and buffer solution can be fully mixed during the flow process.

[0024] The micropillar filter array 5 is used to physically filter larger particulate impurities in the sample to reduce the interference of impurities entering the subsequent detection area and causing immune reactions.

[0025] The Tesla check valve 6 is used to achieve unidirectional flow of liquid and prevent backflow.

[0026] The buffer chamber 7 is used to connect the liquid storage device to buffer and store the sample and buffer solution mixture. It can dilute the sample according to the experimental requirements, so as to maximize the liquid storage capacity of the platform while ensuring that the microfluidic chip platform is small in size and simple in manufacturing process.

[0027] The waste liquid chamber 11 is used to collect the liquid after the reaction is completed inside the chip. The end of the waste liquid chamber 11 away from the three parallel immune reaction chambers 9 has an open structure to facilitate the discharge of waste liquid or connection to an external extraction device.

[0028] This invention combines a microfluidic chip with an ultrasonic device, which not only effectively simplifies the entire detection process, reduces operational complexity, and increases detection speed, but also allows for operation without the need for professional technicians, and can be effectively applied to home or bedside detection.

[0029] The scheme is further optimized. The three parallel immune reaction chambers 9 include three immune reaction chambers, which are connected to the waste liquid chamber 11 through three-stage fishbone burst valves 10.

[0030] The three parallel immunoreaction chambers 9 adopt a parallel three-chamber structure design, and each immunoreaction chamber can independently perform immunodetection reactions. Through the parallel structure design, multi-channel detection or parallel experiments can be performed simultaneously on the same chip, improving detection efficiency and experimental repeatability.

[0031] An immobilized antibody carrier is placed inside the immunoreaction chamber. The sample enters the immunoreaction chamber through a microchannel under external pressure, reacts with the immobilized capture antibody, and forms a double-antibody sandwich immune complex by HRP labeling. A colorimetric signal is generated under the action of an enzyme reaction. An ultrasonic device is used to apply ultrasonic vibration to the chip body during the detection process to assist in sample mixing and filtration. Images of the immunoreaction chamber and calibration chamber are acquired by a data reading device, and the true concentration is calculated using RGB values.

[0032] A calibration solution with known optical properties is pre-added to calibration chamber 12. During image analysis, the RGB values ​​of the immunoreaction chamber and calibration chamber 12 are extracted simultaneously. The color ratio of the two is calculated and normalized. The equivalent optical density value (OD value) is obtained through logarithmic transformation. The concentration of the target protein in the sample to be tested is then calculated by combining it with a pre-established standard curve model.

[0033] The immune reaction chamber contains a polydopamine-modified capture antibody carrier, which is a polycarbonate disc with a diameter of approximately 5 mm.

[0034] The polydopamine-modified layer is formed by the auto-oxidative polymerization of dopamine. Specifically, dopamine hydrochloride is dissolved in Tris-HCl buffer at pH 8.5 to a concentration of 2 mg / mL, and reacted at room temperature for about 2 hours to form a polydopamine coating on the carrier surface.

[0035] A capture antibody solution with a concentration of 20 μg / mL was dropped onto the surface of a polydopamine-modified carrier in a volume of approximately 10 μL, and the carrier was incubated at 37°C for approximately 1 hour to immobilize the capture antibody onto the carrier surface.

[0036] After the capture antibody is fixed, it is blocked at 37°C for about 1 hour using PBS buffer containing 1.5% BSA to reduce non-specific adsorption during the detection process.

[0037] The detection antibody was an HRP-labeled antibody, prepared using an HRP conjugation kit, and diluted to approximately 500-fold with PBST buffer as the working concentration for detection.

[0038] Add 3% trehalose as a protein protectant to the HRP-labeled antibody solution, and drop it onto the surface of the solid support in a sample volume of about 1 μL. After drying at room temperature, a stable enzyme-labeled antibody solid phase layer is formed.

[0039] The scheme is further optimized so that the channel between the Tesla one-way valve 6 and the three parallel immune reaction chambers 9 is connected to the buffer chamber 7 through the secondary fishbone burst valve 8, and the secondary fishbone burst valve 8 is set close to the Tesla one-way valve 6.

[0040] The primary fishbone burst valve 2 and the secondary fishbone burst valve 8 are used to rupture when the pressure in the channel reaches a set threshold, thereby realizing the sequential release and flow control of liquid inside the chip body.

[0041] Further optimization of the scheme: the microfluidic chip is prepared by replication molding of PDMS material. The PDMS prepolymer and curing agent are mixed at a mass ratio of 10:1 and fully degassed before being poured onto the microstructure mold. The mixture is then cured at 60-80℃ to form a PDMS layer with a microchannel structure.

[0042] Further optimization of the scheme: the microstructure mold is formed by spin-coating SU-8 photoresist on a 4-inch silicon wafer and then preparing it through a UV exposure and development process.

[0043] Further optimization involves bonding the PDMS layer to the glass substrate after oxygen plasma treatment, thereby forming a closed microfluidic chip structure.

[0044] Further optimization involves creating hydrophobic surfaces within the microfluidic chip's internal channels through a hydrophobication process. This hydrophobication is achieved by injecting a 10-1 concentration of hydrophobic material into the chip's internal structure. -2 A fluorosilane solution of M is prepared and allowed to stand for reaction, so that the fluorosilane forms a hydrophobic modification layer on the channel surface.

[0045] A colorimetric sample with a specific OD value is pre-stored in calibration chamber 12 for use in the color calibration step during the subsequent data reading stage.

[0046] The buffer chamber 7, the immune reaction chamber, and the waste liquid chamber 11 are all connected via capillary liquid channels. By rationally designing the size of the capillary liquid channels, capillary action can be used to achieve stable transport and distribution of liquids within the chip.

[0047] Regarding the liquid-driven method, the microfluidic chip of this invention adopts a pneumatic driving mode. To simplify the equipment, the three liquid storage devices connected to the buffer injection chamber 1, buffer chamber 7, and waste liquid chamber 11 all use precision syringes as liquid storage devices.

[0048] The driving process is as follows: First, connect each precision syringe to its corresponding chamber, ensuring no leakage and unobstructed flow. Once ready, first actuate the precision syringe connected to buffer injection chamber 1. The negative pressure (pneumatic drive) generated by the actuation drives the liquid flow, causing the liquid in buffer injection chamber 1 to flow out and sequentially through sample chamber 3, serpentine mixing channel 4, microcolumn filter array 5, and Tesla one-way valve 6, ultimately collecting the mixture of sample and buffer solution completely into the syringe.

[0049] After the mixture has been collected, stop pumping the syringe and instead gently push or squeeze it to slowly inject the mixture into the three parallel immune reaction chambers using capillary action and thrust.

[0050] Subsequent cleaning steps are also pneumatically driven: negative pressure is generated by pumping a precision syringe connected to the waste liquid chamber 11, and this negative pressure is used to draw out the buffer solution in the syringe connected to the buffer solution injection chamber 1, thereby flushing the immune reaction chamber and ensuring a smooth and efficient cleaning process.

[0051] In terms of structural dimensions, the contraction channel width of the primary herringbone burst valve 2 and the tertiary herringbone burst valve 10 is 120 μm, and the contraction channel width of the secondary herringbone burst valve 8 is 200 μm. The width of the capillary fluid passage is 100 μm to 200 μm, and the width of the remaining fluid passages is 1 mm to 1.5 mm.

[0052] The primary fishbone burst valve 2, secondary fishbone burst valve 8, and tertiary fishbone burst valve 10 adopt a four-stage structural design with a channel width-to-depth ratio of 1.5 to 2. By setting up a multi-layered fishbone structure, the fluid flow resistance is effectively increased, improving the stability and reliability of the capillary burst valve. When the liquid driving force is low, the capillary burst valve can prevent the liquid from continuing to flow; when the liquid pressure gradually increases and exceeds the threshold pressure, the capillary burst valve is breached, and the liquid begins to pass through the structure, thus achieving staged control of the liquid flow.

[0053] The chip body is fabricated using polydimethylsiloxane (PDMS) material via a casting process. First, a chip mold is prepared using photolithography, with a negative photoresist used to create microstructure patterns. After photolithography, the mold surface undergoes a fluorosilane vapor phase treatment to reduce the mold surface energy, thereby improving the PDMS release quality and minimizing microstructure damage.

[0054] In the preparation of PDMS materials, the mechanical properties of the material are closely related to the ratio of curing agent. Through experimental optimization, this invention controls the mass ratio of PDMS to curing agent to be 1:10. This ratio ensures good elasticity of the material while improving the chip's resistance to deformation under high liquid pressure conditions, thereby enhancing the chip's structural stability and increasing its reusability.

[0055] Specifically, the diameter of the waste liquid chamber 11 is 6 mm, the diameter of the sample chamber 3 is 5 mm, and the diameter of the calibration chamber 12 is 4 mm. The first and third stage fishbone burst valves have a contraction width of 120 μm and an expansion width of 1.5 mm; the second stage fishbone burst valve 8 has a contraction width of 200 μm and an expansion width of 1.6 mm. The serpentine micro-serrated mixing channel 4 has a bending angle of 90° and a channel width of 0.75-1.2 mm. The microcolumn filter array 5 consists of three layers: the first layer has a gap of 30 μm, the second layer has a gap of 15 μm, and the third layer has a gap of 5 μm, with a 2 mm spacing between each layer.

[0056] Silicon wafer pretreatment: A 4-inch monocrystalline silicon wafer was taken and cleaned sequentially under full ultrasonic conditions. First, the silicon wafer was placed in a piranha solution for ultrasonic cleaning for 20 minutes to thoroughly remove organic contaminants from the wafer surface. After discarding the piranha solution, ultrapure water was added for ultrasonic cleaning for 20 minutes. Then, the ultrapure water was discarded, and anhydrous ethanol was added for ultrasonic cleaning for another 20 minutes. After discarding the anhydrous ethanol, acetone was added for ultrasonic cleaning for 20 minutes. Finally, the acetone was discarded, and ultrapure water was added again for ultrasonic cleaning for 20 minutes. The amount of reagent added in each cleaning step was sufficient to completely immerse the silicon wafer. After cleaning, nitrogen gas was used to dry both sides of the silicon wafer, and it was placed on a 95°C heating plate for further drying to remove residual moisture from the surface. After drying, it was allowed to cool naturally to room temperature with the heating plate for later use.

[0057] Spin Coating: The cooled silicon wafer was placed in the center of the spin coater stage. Approximately 5 mL of SU-82075 photoresist was dropped into the center of the wafer, and the vacuum suction device was activated to fix the wafer. The spin coating process was performed in two steps: first, the photoresist was spread evenly on the silicon wafer surface at a lower speed; then, a fixed-thickness spin coating was performed at a higher speed to obtain the desired thickness of the SU-8 photoresist film. In this example, the spin speed was set to 1500 r / min, and the spin coating time was 2 min. After spin coating, the silicon wafer was allowed to stand for 3 min to allow the photoresist to spread evenly on the silicon wafer surface and release some internal stress. Under the conditions of this example, these spin coating parameters could obtain a photoresist structure layer with a thickness of approximately 80–90 μm.

[0058] Pre-baking: Place the silicon wafer, after it has been left to stand, in a 65°C oven for 5 minutes to remove some of the solvent from the photoresist; then transfer it to a 95°C heating plate and continue heating for 10 minutes to further stabilize and cure the photoresist. After heating, allow it to cool naturally to room temperature with the heating plate.

[0059] Exposure: The mask with the microfluidic channel pattern is precisely aligned and attached to the silicon wafer surface to ensure accurate pattern positioning and no significant offset; then it is exposed to a parallel ultraviolet light source with a wavelength of 365nm. Under the conditions of this example, the exposure time is set to 25s, corresponding to an ultraviolet exposure dose controlled at approximately 300mJ / cm², to ensure sufficient cross-linking of the photoresist.

[0060] Post-baking: Immediately after exposure, the silicon wafer is transferred to a 65°C oven for 5 minutes, then transferred to a 95°C heating plate for another 10 minutes to promote further cross-linking and curing of the photoresist in the exposed area. After heating, the wafer is allowed to cool naturally to room temperature along with the heating plate.

[0061] Development: Place the baked silicon wafer in propylene glycol methyl ether ethyl acetate (PGMEA) developer and gently agitate for 20 minutes to fully dissolve and remove the photoresist in the unexposed areas. After development, remove the silicon wafer and clean it in isopropanol solution, alternating between PGMEA and isopropanol solutions. If no white foam is observed in the isopropanol, and the photolithographic pattern on the silicon wafer surface is clear with no photoresist residue in the non-photolithographic areas, the development effect is good.

[0062] Hardening: The developed silicon wafer is baked at 150°C for 10 minutes to enhance the mechanical strength and adhesion stability of the SU-8 photolithography structure. It is then allowed to cool naturally to room temperature with the heating plate, thereby obtaining a structurally complete and stable SU-8 photolithography silicon wafer mold.

[0063] Mold silanization treatment: The prepared photolithographic silicon mold is placed in a vacuum oven, and a clean small container with approximately 10 μL of perfluorooctyltrichlorosilane (FOTS) reagent is added inside the oven. The oven is closed and a vacuum is created, allowing the silanizing reagent to evaporate in the vacuum phase and deposit uniformly on the surface of the silicon mold, thereby forming a low surface energy hydrophobic protective layer on the mold surface to reduce the adhesion between PDMS and the mold. The silanization treatment time is 30 min. In this example, the heating function of the vacuum oven can be appropriately turned on to accelerate the evaporation rate of the fluorosilane reagent, thereby speeding up the silanization reaction process.

[0064] PDMS prepolymer preparation: The PDMS prepolymer and curing agent were thoroughly mixed and stirred at a mass ratio of 10:1 to ensure uniform mixing. In this example, a total mass of approximately 20g of PDMS polymer mixture was prepared to ensure that the final PDMS chip structure has suitable thickness and mechanical strength. The stirred PDMS mixture was placed in a vacuum chamber for vacuum degassing treatment for approximately 30 minutes to remove air bubbles generated during stirring.

[0065] PDMS casting and secondary degassing: The degassed PDMS mixture is uniformly cast onto the surface of the silanized photolithography silicon wafer mold, ensuring complete coverage of the microstructure area on the mold. The silicon wafer mold, along with the PDMS mixture, is then placed in a vacuum oven for approximately 30 minutes for further vacuuming to remove any remaining air bubbles within the microstructure, ensuring a clear and intact microchannel structure.

[0066] PDMS curing: Place the cast silicon mold on a heating table and heat it at 70°C for 2 hours to allow the PDMS prepolymer to undergo a full cross-linking reaction and completely cure to form an elastomer structure.

[0067] PDMS peeling and cutting: After the PDMS has fully cured, allow the mold to cool naturally to room temperature, and slowly and steadily peel the PDMS substrate off along the edge of the silicon mold. Then, use a utility knife to cut the PDMS sheet to obtain the PDMS microfluidic chip substrate of the required size.

[0068] Cavity perforation: A dedicated perforator is used to precisely perforate the corresponding positions in the buffer injection chamber, sample chamber, buffer chamber, immune reaction chamber, waste liquid chamber, and calibration chamber to form the chip's sample inlet and functional cavity interface.

[0069] Chip cleaning: The PDMS chip with holes completed is placed in anhydrous ethanol and ultrapure water for ultrasonic cleaning for 10 minutes each to remove residual particles and impurities on the surface; after cleaning, the chip surface is dried with nitrogen gas for later use.

[0070] Plasma bonding: The dried PDMS chip and glass substrate are simultaneously placed in a plasma cleaner for surface activation treatment for 40 seconds. After treatment, they are quickly removed, and the microstructure surface of the PDMS chip is precisely aligned and bonded to the glass substrate. Residual air bubbles at the interface are removed by gentle pressure, thus completing the initial bonding.

[0071] Post-bake strengthening: The bonded chip is placed in a 65°C oven and baked for another 30 minutes to further enhance the bonding strength between PDMS and the glass. The chip is then removed and cooled to room temperature to obtain a complete integrated microfluidic chip body.

[0072] Hydrophobic treatment: After plasma treatment, a large number of silanol groups (–Si–OH) are generated on the surface of the PDMS material, changing the surface from its original hydrophobic state to a hydrophilic state. This hydrophilicity usually lasts for about 2–3 days. The change in surface wettability significantly affects the interfacial tension behavior of the liquid within the microchannel, which may cause the burst valve structure to lose its original liquid hindrance function. To further improve the stability and reliability of the burst valve structure, the internal channels of the chip are hydrophobized. In this study, liquid-phase fluorosilanes are used to modify the channel surface. A 10⁻²M fluorosilane solution is prepared using organic solvents such as anhydrous ethanol or acetone, and the solution is slowly injected into the chip's inlet using a perfusion method to ensure that the solution fully fills the entire microchannel system and makes full contact with the inner wall of the channel. Subsequently, the reaction is allowed to stand at room temperature for about 20–30 minutes, allowing the fluorosilane molecules to chemically react with the hydroxyl groups on the PDMS or glass surface, forming a low surface energy hydrophobic silane modification layer on the channel surface. After the reaction, anhydrous ethanol and deionized water were sequentially injected into the channel via a syringe to rinse it and remove unreacted fluorosilane molecules and byproducts. The channel was then dried using air or nitrogen, and the chip was placed in a 60°C oven for approximately 30 minutes to promote the stable formation of the surface modification layer. In this example, after hydrophobication treatment, the hydrophobic angle of the PDMS material increased from 15° to 105-110°.

[0073] The actual usage method is as follows: Connect the required syringe / reservoir to the buffer injection chamber 1, buffer chamber 7, and waste chamber 11. For simplification, this example uses precision syringes, named 1, 2, and 3 respectively. Syringe 1 is pre-stored with a specified volume of PBS buffer, while syringes 2 and 3 are empty. Place a certain amount of sample to be tested in the sample chamber. Create negative pressure by pumping syringe 2, using the negative pressure to drive the liquid forward. Slowly pump the syringe to allow the buffer to break through the first multi-stage fishbone burst valve. Continue pumping the syringe to allow the buffer to enter the sample chamber and come into contact with the sample. Depending on the sample characteristics, the ultrasonic device can be turned on or off. For easily soluble solid or liquid samples, the ultrasonic device can be left off. For viscous or impurity-rich solid samples, the ultrasonic device should be turned on. With ultrasonic assistance, continue to slowly pump the syringe to quickly and thoroughly mix the sample and buffer. The mixture is further mixed through the serpentine micro-serrated mixing channel 4. Combined with the ultrasonic action, this ensures that the sample is completely dissolved and mixed evenly before reaching the microcolumn filter array 5.

[0074] The syringe is then slowly withdrawn, forcing the mixture through the microcolumn filter array 5 under negative pressure. Ultrasonic vibration clears microparticles clogging the microcolumn gaps, preventing channel blockage and improving chip throughput. After passing through the Tesla valve, the syringe is continuously withdrawn to collect all the buffer solution containing the sample, filtered by the microcolumn filter array 5, completely into syringe 2. Syringe 2 serves as both a mixer and buffer, allowing the platform to be applied to detect different target proteins. Depending on the target protein concentration, the required dilution factor can be designed to match the developed colorimetric method. Depending on the detection requirements, other storage devices can be replaced with syringes; the sample dilution factor is determined by the total volume of buffer solution pre-stored in syringe 1.

[0075] Slowly push syringe No. 2, using thrust and capillary force to accurately retain the sample in three parallel immune reaction chambers. The second and third multi-stage fishbone burst valves on the liquid path can intercept the flow, ensuring that all the mixture enters the immune reaction chamber smoothly without leakage or backflow. The sample and the pre-fixed solid-phase capture antibody carrier in the chamber are incubated at room temperature for 1 hour to complete the immune capture process.

[0076] The sample and the pre-fixed solid-phase capture antibody carrier in the chamber were incubated at room temperature for 1 hour to complete the immunocapture. After incubation, the syringe was pushed to thoroughly wash away any uncaptured residual antigens with buffer solution. Then, the pre-prepared solid-phase enzyme-labeled antibody carrier was added to the three immunoreaction chambers. After the enzyme-labeled antibody preserved with trehalose was completely dissolved, the chamber was incubated at room temperature for 1 hour to allow the double antibody sandwich structure to form smoothly. After incubation, the syringe was pushed again to wash away any free enzyme-labeled antibody that had not formed the double antibody sandwich. Then, a certain volume of TMB chromogenic substrate was added to the immunoreaction chamber. After the substrate broke through the corresponding multi-stage fishbone burst valve, it was uniformly released into the three immunoreaction chambers under capillary action. The color development was carried out at room temperature for about 10 minutes. After the color development was completed, the stop solution was added to terminate the color development reaction. Finally, the data reading program provided with the chip was used to complete the reading and analysis of the color development signal.

[0077] The ultrasonic device is used to apply ultrasonic vibration to the microfluidic chip to achieve ultrasonic-assisted micro-mixing of the sample in the chip's microchannels and to clear the channels, thereby reducing microchannel blockage or local stagnation.

[0078] Further optimization of the scheme: the ultrasonic device includes a base 21, a cover plate 22 is installed on the top surface of the base 21, an aluminum alloy plate 23 is installed on the end of the cover plate 22 away from the base 21, and multiple chip fixing holes 24 are arranged around the aluminum alloy plate 23. The multiple chip fixing holes 24 are opened on the cover plate 22, and the microfluidic chip is installed on the cover plate 22 through the multiple chip fixing holes 24.

[0079] Both the base 21 and the cover plate 22 were designed using CAD software for 3D modeling and then manufactured using a 3D printer. The printing material is rigid ABS plastic to ensure the device has good mechanical strength and stability. The specific dimensions of each component are as follows: the base 21 has external dimensions of 93×90×38mm and internal cavity dimensions of 85×82×35mm; the cover plate 22 has dimensions of 93×90×4mm; and the aluminum alloy plate 23 has dimensions of 50×50×1mm. This structural design provides a stable mounting space for internal electronic components and the ultrasonic vibration module, while also reserving chip mounting holes for reliable fixation of the microfluidic chip during experiments.

[0080] The design is further optimized by installing a piezoelectric ceramic drive board 31 inside the base 21. The piezoelectric ceramic drive board 31 is electrically connected to a program control board 32, an ultrasonic piezoelectric ceramic 33, and a two-prong power plug 34.

[0081] The scheme was further optimized so that the ultrasonic piezoelectric ceramic 33 penetrated the cover plate 22 and was installed on the end of the aluminum alloy plate 23 facing the base 21.

[0082] The piezoelectric ceramic drive board 31 is embedded inside the base 21, while the program control board 32 is embedded in a pre-set square slot on the front side of the base 21 and connected to the drive board via a data cable. The power cable is introduced through a pre-drilled hole at the rear of the base 21 and connected to the drive board, providing power input to the entire device. The cover plate 22 is fixed to the base 21 using M1.4 round-head screws and matching nuts, ensuring the stability and sealing of the device structure. In the actual assembly process, the aluminum alloy plate 23 is first embedded into the pre-drilled mounting position on the cover plate 22, and the ultrasonic piezoelectric ceramic 33 is adhered to the lower surface of the aluminum alloy plate 23 using a special piezoelectric ceramic composite adhesive. After bonding, it needs to stand for approximately 24 hours to ensure a firm bond between the ultrasonic piezoelectric ceramic 33 and the aluminum alloy plate 23. Subsequently, the piezoelectric ceramic drive board 31 and the program control board 32 are installed in their corresponding positions inside the base 21, and the cable connections are completed. Finally, the cover plate 22 and the base 21 are locked and fixed with screws, thus completing the overall assembly and construction of the ultrasonic equipment.

[0083] The ultrasonic piezoelectric ceramic 33 generates ultrasonic vibration through a driving circuit.

[0084] The ultrasonic piezoelectric ceramic 33 is a circular structure made of PZT-5 material with a diameter of about 50 mm, an operating resonant frequency of about 40 kHz, and a driving power in the range of 10-50 W.

[0085] The resonant frequency of the piezoelectric ceramic ultrasonic transducer is as described in the formula: in, It is the radial resonant frequency; is the radial frequency constant of the material (Hz·m); The diameter of the piezoelectric ceramic disc is given. Considering that the vibration frequency provides the optimal working conditions for the piezoelectric ceramic at the resonant frequency, and based on the piezoelectric ceramic having a diameter of 50mm and the material being PZT-5, the resonant frequency is calculated to be 40kHz.

[0086] The radial linear elongation of a piezoelectric ceramic disc under the influence of an electric field is as described in the formula: in, Change in diameter of the piezoelectric ceramic disc Transverse piezoelectric constant (mm / V) Apply voltage (V). : Piezoelectric ceramic thickness (mm), D: Piezoelectric ceramic diameter (mm). Considering that higher voltage will bring greater deformation, the highest voltage that the equipment can support, 400V, is selected as the driving voltage.

[0087] The detection principle of this integrated microfluidic chip is as follows: Figure 5As shown, the detection principle is based on the double-antibody sandwich method, with HRP used for colorimetric development. The raw materials required for this detection method are as follows: polydopamine-modified antibody carrier 41, capture antibody 42, and HRP-labeled detection antibody 43. In this embodiment, the target protein being detected is calprotectin, and the antibody pairs used are specific antibody pairs that have undergone competitive antibody pairing experiments. The screening results are shown below. Figure 6 As shown. The experimental procedure for this method is as follows: 1) Preparation of enzyme-labeled antibody: HRP-labeled antibody was prepared using an HRP conjugation kit. Following the kit instructions, the detection antibody was conjugated with HRP to obtain the HRP-labeled detection antibody. The prepared enzyme-labeled antibody was then diluted 50,000 times with PBST (0.1% Tween-20) buffer to obtain a working concentration suitable for the detection system.

[0088] 2) Antibody Competitive Binding Assay: Antibody pairing screening was evaluated using a competitive binding assay. In the experiment, calprotectin antigen was first coated onto the wells of an ELISA plate (200 ng / well) and incubated at 37°C for 1 h to allow the antigen to stably adsorb onto the bottom surface of the plate. Then, the plate was blocked with 200 μL of PBS buffer containing 1.5% BSA for 1 h. After blocking, an antibody competition assay was performed: HRP-labeled antibody 1 was added to well 1, HRP-labeled antibody 2 to well 2, and both HRP-labeled antibody 1 and HRP-labeled antibody 2 were added to well 3. The plates were incubated at 37°C for 1 h to allow the antibodies to bind to the coated antigen. After incubation, the plates were washed to remove unbound antibody molecules, and then a chromogenic substrate was added for enzymatic colorimetric reaction. The absorbance (OD450) of each well was measured at 450 nm using an ELISA reader. Based on the OD450 values ​​measured in each experiment, the antibody competition index AI (%) was calculated using the following formula: Where A1 and A2 represent the OD450 values ​​measured when labeled antibody 1 and labeled antibody 2 are added individually, respectively, and A(1+2) represents the OD450 value measured when both labeled antibodies are added simultaneously. When the calculated AI value is greater than 45%, it indicates that there is no significant competition between the binding sites of the two antibodies, and they can simultaneously recognize different epitopes of the antigen, making them effective antibody pairs in sandwich immunoassay systems. If the AI ​​value is low, it indicates that the two antibodies may recognize the same or adjacent epitopes, making them unsuitable for sandwich assay systems. This method was used to screen multiple candidate antibodies, ultimately obtaining the optimal antibody combination suitable for the subsequent construction of the assay system. Monoclonal antibody 2 and monoclonal antibody 4 were the preferred antibody pair. Figure 6Using monoclonal antibody 2 as the capture antibody and monoclonal antibody 4 as the labeling antibody, the standard curve of this method is shown below, with R2=0.9999 and LOD=2.96pg / mL.

[0089] Preparation of polydopamine-modified antibody carrier 41: First, a dopamine hydrochloride solution was prepared by dissolving an appropriate amount of dopamine hydrochloride in Tris-HCl buffer at pH 8.5 to a final concentration of 2 mg / mL. Under weakly alkaline conditions, dopamine can undergo auto-oxidative polymerization, thereby forming a uniform polydopamine (PDA) coating on the surface of the carrier material. A suitably sized PC plastic sheet was selected and immersed in the dopamine hydrochloride solution. The reaction was carried out with gentle shaking at room temperature for 2 hours, allowing dopamine to self-polymerize and deposit on the carrier surface, forming a stable polydopamine-modified layer. The material surface gradually turned brownish-red. After the reaction was complete, the carrier material was removed and repeatedly washed with ultrapure water to remove unreacted dopamine monomers and weakly adsorbed polymer residues. It was then dried at room temperature. The dried material was processed into small discs with a diameter of 5 mm using a punch and subjected to plasma treatment for 1 minute in a plasma cleaner to improve the hydrophilicity of the material surface. Subsequently, a capture antibody solution with a concentration of 20 μg / mL was prepared using PBS buffer. 10 μL of the antibody solution was then added dropwise to the surface of the treated discs, and the mixture was incubated at 37°C for 1 hour. This allowed the capture antibody 42 to be immobilized on the polydopamine surface via covalent binding or physical adsorption, thus forming a polydopamine-modified antibody carrier. After incubation, the material was washed multiple times with PBS buffer to remove unbound antibody molecules. The carrier surface was then blocked for 1 hour at 37°C using PBS buffer containing 1.5% BSA blocking agent to reduce non-specific adsorption during subsequent detection. A stable, immobilized antibody carrier was finally obtained for subsequent immunoassay reactions.

[0090] Preparation of Trehalose-Protected Immobilized Antibody Carriers: The carrier sheets used were the same PC sheets used in the preparation of the polydopamine-modified antibody carriers described above. First, the PC plastic sheets were cleaned and dried at room temperature. Then, they were punched into small discs with a diameter of 5 mm and subjected to plasma treatment for 1 min in a plasma cleaner to improve the hydrophilicity of the material surface, thus facilitating the uniform spreading of the subsequent solution. Trehalose was added as a protein protectant to a 500-fold diluted enzyme-labeled antibody solution, achieving a mass fraction of 3%, which was experimentally verified as the optimal protection condition. Trehalose forms a stable glassy protective layer during drying, effectively maintaining the spatial structure and enzyme activity of the HRP-labeled antibody. After thoroughly mixing the prepared antibody-trehalose mixture, it was spotted onto the surface of the plasma-treated PC discs. The single spotting volume for each carrier was 1 μL, ensuring uniform distribution of the solution on the carrier surface. The carrier was then allowed to air dry at room temperature, allowing the trehalose-protected HRP-labeled detection antibody to form a stable solid-phase layer on the carrier surface, thus obtaining a trehalose-protected labeled antibody solid-phase carrier for subsequent microfluidic chip detection reactions. After drying, the prepared solid-phase labeled antibody carrier was stored under dry, light-protected, and low-temperature conditions to maintain the activity of the antibody and enzyme labeling. In actual detection, when the sample solution enters the microfluidic chip and comes into contact with the carrier, the trehalose layer rapidly dissolves and releases the HRP-labeled detection antibody, enabling it to participate in the subsequent immune reaction. (Refer to...) Figure 7 The figure shows the dissolution of enzyme-labeled antibodies under different concentrations of trehalose as a protective agent.

[0091] Reference Figures 8 to 9 This is a schematic diagram of the page and logic of the data reading mini-program described in this invention. The program is developed using WeChat Developer Tools (version: 2.01.2510280win32-x64), and its functions are implemented in the following ways: The detection image acquisition function is achieved by utilizing the mobile terminal's camera component. Multiple preset sampling areas are set in the photo-taking interface to correspond to the positions of the standard wells and the wells of the sample to be tested in the detection plate, ensuring that each detection area is within a fixed position range during each shot, thereby improving the stability of the detection results. During detection, the user places the detection plate containing the standard sample and the sample to be tested within the shooting area and completes a photo-taking operation according to the interface prompts. The mini-program then acquires image data containing information on all detection well positions. Subsequently, the mini-program extracts pixel information from each preset area in the image, reading the RGB color information of all pixels within the area and calculating its average value to reduce the impact of local noise on the detection results. Since the OD450 colorimetric reaction in ELISA detection typically manifests as a yellow solution, and yellow is most sensitive to changes in the blue channel (B value) in the RGB color space, this invention primarily extracts the blue channel value in the RGB color space as the detection signal parameter.

[0092] In the data processing, this invention first converts the blue channel values ​​into relative signal values ​​related to transmittance, and then performs a conversion based on the logarithmic relationship between absorbance and transmittance. According to the Beer-Lambert law, absorbance (OD) and transmittance (T) satisfy the following relationship: Where A is the transmittance and T is the transmittance. Based on this formula, the conversion formula between OD and B values ​​is obtained: in, The B value of the blank solution. The B value of the solution after color development. In this invention, the relative transmittance is obtained by normalizing the average value B of the blue channel in the detection well area with the signal of the reference area. Then, the relative transmittance is converted into a pseudo-OD value using the aforementioned logarithmic relationship, i.e.: Where B_sample is the average value of the blue channel in the test well area, and B_reference is the average value of the blue channel in the reference or standard well area. Through this logarithmic transformation process, the color information in the image can be converted into pseudo-OD values ​​that correspond to the detection results of traditional microplate readers.

[0093] This invention sets three standard sample wells with known concentrations in the calibration wells of a microfluidic chip. The corresponding true OD values ​​are pre-determined and fixed using an ELISA reader as calibration benchmarks. During each mobile terminal test, the app first reads the average blue channel value of the three standard well regions and obtains the corresponding pseudo-OD value through the aforementioned logarithmic transformation. Then, it establishes a mapping relationship between this pseudo-OD value and the true OD value, constructing a real-time calibration curve for the current detection environment through three-point linear fitting. Subsequently, the blue channel signal of the sample well to be tested is substituted into this calibration relationship for calculation, thereby obtaining the OD value or equivalent OD value of the sample. Using the pre-stored standard curve parameters in the program, the concentration of the target substance in the sample is calculated based on the obtained OD value and displayed on the results page, thus realizing rapid reading and quantitative analysis of ELISA detection data based on mobile terminal image acquisition.

[0094] To evaluate the accuracy of the established detection method for the target substance, this experiment used a spike recovery experiment to verify the performance of the detection system. By adding a known concentration of the target substance standard to the sample system and performing detection according to the established method, the detection results were compared with the theoretical amount added, thereby assessing the accuracy and reliability of the method.

[0095] The standards used in the experiment were target protein standard solutions of known concentrations. Before the experiment, standard solutions of different concentration gradients were prepared, and an appropriate sample matrix was selected as the detection system. This experiment used a buffer solution or simulated sample without the target analyte as the matrix solution. Based on this, spiked samples at three levels—low, medium, and high—were prepared by adding a certain volume of standard solution to the sample matrix. Each spiked sample was thoroughly mixed after adding the standard solution to ensure uniform distribution of the target analyte in the sample system.

[0096] After sample preparation, each spiked sample was tested according to the established detection procedure. The detection process strictly followed a unified operating procedure, including sample addition, reaction incubation, washing, and colorimetric reaction. At least three parallel experiments were set up for each concentration level to reduce random errors and improve the reliability of the experimental results. After the colorimetric reaction, the detection signal value was acquired using a predetermined data reading method, and the signal value was converted into the corresponding target concentration according to a pre-established standard curve. The recovery rate and standard deviation of each experimental group were calculated to evaluate the accuracy of the detection method described in this invention. The recovery rate was calculated using the formula: Recovery rate (%) = Measured concentration / Theoretical concentration × 100%. Simultaneously, the standard deviation of each parallel result group was calculated to evaluate the repeatability of the detection method described in this invention. Experiments showed that the recovery rate of this method was approximately 90%, within the ideal range of 80%-120%, indicating good accuracy. The standard deviation of approximately 5% in each group of ten measurements indicated good repeatability. The recovery rate, standard deviation, and distribution of results from multiple tests for this detection method are referenced. Figures 10 to 11 As shown.

[0097] The data reading device is a mobile terminal-based mini-program image analysis system. It captures images of the detection area by calling the mobile terminal's camera and extracts the RGB color values ​​of the detection area for analysis.

[0098] The ultrasonic device is used to apply ultrasonic vibration to the microfluidic chip to achieve ultrasonic-assisted micro-mixing of the sample in the chip's microchannels and to clear the channels, thereby reducing microchannel blockage or local stagnation.

[0099] The ultrasonic device mainly comprises an ultrasonic piezoelectric ceramic 33, a boost drive module, a control module, and a base structure. The ultrasonic piezoelectric ceramic 33 is a circular structure with a diameter of 50 mm and a thickness of 2 mm, made of PZT-8 piezoelectric ceramic material. PZT-8 material has a high electromechanical coupling coefficient and strong mechanical stability, making it suitable for long-term stable operation. The ultrasonic piezoelectric ceramic 33 is electrically connected to the boost drive module via pluggable wires, facilitating device maintenance, replacement, and modular assembly.

[0100] The boost drive module provides the driving electrical signal for the ultrasonic piezoelectric ceramic 33. The boost drive module is powered by 220V AC, with a maximum output voltage of 400V and a rated power of 35W. It connects to an external power source via a standard 220V two-pin power plug 34 to ensure stable power supply to the device. The boost drive module outputs a high-frequency AC signal suitable for the operation of the ultrasonic piezoelectric ceramic 33, driving it to generate mechanical vibrations and produce ultrasonic waves.

[0101] The control module is used to control the boost drive module. The control module includes at least a switch control unit and a timing control unit, enabling the start-up, shutdown, and operation time setting of the ultrasonic device. The control module connects to the boost drive module via a pluggable signal adapter cable, thereby controlling the output status of the drive module.

[0102] The base 21 supports the various modules of the ultrasonic equipment and the microfluidic chip. The base 21 was designed in three dimensions using CAD software and manufactured using 3D printing technology. The main body of the base 21 is made of PC polymer material to achieve good mechanical strength and structural stability. A 50mm diameter piece is fixedly mounted on the upper layer of the base 21. 50 A 1mm thick aluminum alloy plate 23. An ultrasonic piezoelectric ceramic 33 is adhered to the lower surface of the aluminum alloy plate 23 with a special polymer adhesive to ensure that ultrasonic vibrations can be effectively transmitted to the aluminum alloy plate 23.

[0103] The upper shell of the base 21 is provided with multiple chip fixing holes 24 for fixing the microfluidic chip, so as to ensure that the microfluidic chip maintains stable contact with the aluminum alloy plate 23 during operation, thereby improving the transmission efficiency of ultrasonic vibration to the microchannel inside the chip; the ultrasonic vibration generated by the ultrasonic piezoelectric ceramic 33 is transmitted to the microfluidic chip through the aluminum alloy plate 23, causing microscale disturbance of the sample in the area of ​​the serpentine micro-serrated mixing channel 4 and the micro-pillar filter array 5, thereby realizing ultrasonic-assisted mixing of the sample and reducing the risk of channel blockage.

[0104] The present invention also provides a protein detection method for use with a microfluidic chip; including a rapid protein detection technology based on the principle of enzyme-linked immunosorbent assay (ELISA), and a real-time data reading method.

[0105] In rapid protein detection technologies based on the principle of enzyme-linked immunosorbent assay (ELISA), the specific binding reaction between antigen and antibody is used to identify and detect target proteins. This invention immobilizes the capture antibody 42 on a detachable solid carrier and integrates it into the immunoreaction chamber of a microfluidic chip, thereby enabling replaceable detection modules and multi-target detection capabilities.

[0106] Capture antibody 42 is immobilized on the surface of a plastic carrier sheet, preferably made of polycarbonate (PC). To improve the immobilization ability of protein molecules, the surface of the plastic carrier sheet is modified with polydopamine to form an adsorption layer with active functional groups on the material surface, thereby improving the adsorption efficiency of antibody molecules. Through this surface modification, capture antibody 42 can be stably immobilized on the carrier sheet surface and maintain good biological activity. The carrier sheet after immobilization of the capture antibody is then blocked to reduce non-specific adsorption.

[0107] Since the capture antibody 42 is immobilized on an independent plastic carrier sheet, different types of capture antibody 42 can be replaced according to different detection needs, thereby enabling parallel detection of different target proteins on the same microfluidic chip platform, improving the flexibility and customization of the detection system.

[0108] Regarding antibody detection, this invention employs a pre-stored, dried enzyme-labeled antibody method for reagent integration. The detection antibody is a horseradish peroxidase (HRP)-labeled antibody. To improve the stability of the enzyme-labeled antibody in the dried state, trehalose is added to the antibody solution as a protective agent, and the antibody solution is then dried and fixed onto the surface of a plastic carrier sheet. Trehalose can form a protective structure during the drying process, thereby maintaining the stability of the antibody protein and enzyme molecules.

[0109] The stability of enzyme-labeled antibodies during drying and preservation is affected by the concentration of the protective agent. Comparative experiments were conducted on antibody samples under five conditions with trehalose concentrations ranging from 1% to 5% to observe the retention of antibody activity and the reconstitution rate after drying and preservation. The results showed that adjusting the trehalose concentration can alter the protective effect of the antibody during drying and the solubility of the dried layer. When the trehalose concentration is low, the protective structure is insufficient, and the antibody is prone to structural changes during drying, leading to reduced enzyme activity. When the trehalose concentration is too high, the protective layer formed after drying is thicker, which may reduce the dissolution rate of the antibody after it enters the reaction system. Considering both antibody activity retention and reconstitution efficiency, a trehalose concentration of 3% was selected as the optimal protective condition for the drying and preservation of enzyme-labeled antibodies.

[0110] During the detection process, when the liquid sample enters the microfluidic chip and comes into contact with the carrier plate, the pre-dried and preserved enzyme-labeled antibody redissolves under the action of the liquid and is released into the reaction system, thereby participating in the subsequent immune reaction. The target protein in the sample first specifically binds to the capture antibody 42 immobilized on the carrier plate, and then forms a sandwich immune complex with the released enzyme-labeled antibody. Under the action of the chromogenic substrate, the enzyme-labeled antibody catalyzes the substrate to produce a color change signal, thereby realizing the detection of the target protein.

[0111] Real-time data acquisition methods are used to quantitatively analyze the colorimetric signals generated by immune reactions. The method employs a mobile terminal to capture colorimetric images, and then uses image processing algorithms to analyze the colorimetric intensity, thereby obtaining a pseudo-OD value corresponding to the optical density (OD value) of a traditional microplate reader, enabling quantitative analysis of the target protein concentration.

[0112] Specifically, after the immune reaction is completed and a colorimetric reaction occurs, an image data is acquired by photographing the immune reaction area of ​​the microfluidic chip using a mobile terminal device. The mobile terminal is preferably a smartphone with a camera. To improve the stability of the detection results, during the photographing process, a fixed shooting distance, stable and uniform lighting conditions, and a horizontal shooting angle are set, combined with a specific color (OD) solution in the sample calibration chamber, to reduce the impact of ambient light variations on the image acquisition results.

[0113] After acquiring image data, image recognition and color information extraction are performed on the immune reaction area and sample calibration chamber. RGB color channel data of the color development area are extracted, and the RGB data are processed to obtain grayscale or comprehensive color values ​​related to color intensity. By establishing the correspondence between the comprehensive color value and the standard OD value, the pseudo-OD value is calculated.

[0114] By conducting comparative experiments on samples with different color development intensities, a fitting relationship was established between the RGB values ​​of the color development region and the OD values ​​measured by the microplate reader, thereby constructing a standard curve model for calculating the pseudo-OD value. In actual detection, by substituting the color development image data acquired by photography into the model, the pseudo-OD value of the sample can be quickly calculated, and further quantitative analysis of the target protein concentration can be achieved based on the standard curve.

[0115] In the description of this invention, it should be understood that the terms "longitudinal", "lateral", "up", "down", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this invention, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this invention.

[0116] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An ultrasound-assisted integrated microfluidic ELISA rapid detection system, characterized in that: The device includes an ultrasonic device with a microfluidic chip, which is used to apply ultrasonic vibration to the microfluidic chip during the detection process to assist in sample mixing and filtration. The microfluidic chip includes a chip body, in which a buffer injection chamber (1) is formed. The buffer injection chamber (1) is connected to a sample chamber (3) through a first-stage fishbone burst valve (2). The sample chamber (3) is connected to a microcolumn filter array (5) through a serpentine micro-serrated mixing channel (4). The microcolumn filter array (5) is connected to a three-parallel immune reaction chamber (9) through a Tesla one-way valve (6). The three-parallel immune reaction chamber (9) is connected to a waste liquid chamber (11). The chip body also has multiple calibration chambers (12).

2. The ultrasound-assisted integrated microfluidic ELISA rapid detection system according to claim 1, characterized in that: The three parallel immune reaction chambers (9) include three immune reaction chambers, which are respectively connected to the waste liquid chamber (11) through a three-stage fishbone burst valve (10).

3. The ultrasound-assisted integrated microfluidic ELISA rapid detection system according to claim 1, characterized in that: The channel between the Tesla one-way valve (6) and the three parallel immune reaction chambers (9) is connected to a buffer chamber (7) through a secondary fishbone burst valve (8), and the secondary fishbone burst valve (8) is located close to the Tesla one-way valve (6).

4. The ultrasound-assisted integrated microfluidic ELISA rapid detection system according to claim 1, characterized in that: The microfluidic chip is prepared by replication molding of PDMS material. The PDMS prepolymer and curing agent are mixed at a mass ratio of 10:1 and fully degassed before being poured onto a microstructure mold. The mixture is then cured at 60-80℃ to form a PDMS layer with a microchannel structure.

5. The ultrasound-assisted integrated microfluidic ELISA rapid detection system according to claim 4, characterized in that: The microstructure mold is formed by spin-coating SU-8 photoresist on a 4-inch silicon wafer and then performing an ultraviolet exposure and development process.

6. The ultrasound-assisted integrated microfluidic ELISA rapid detection system according to claim 4, characterized in that: The PDMS layer is bonded to the glass substrate after being treated with oxygen plasma, thereby forming a closed microfluidic chip structure.

7. The ultrasound-assisted integrated microfluidic ELISA rapid detection system according to claim 1, characterized in that: The internal channels of the microfluidic chip are hydrophobic to form a hydrophobic surface.

8. The ultrasound-assisted integrated microfluidic ELISA rapid detection system according to claim 1, characterized in that: The ultrasonic device includes a base (21), a cover plate (22) is installed on the top surface of the base (21), an aluminum alloy plate (23) is installed on one end of the cover plate (22) away from the base (21), a plurality of chip fixing holes (24) are provided around the aluminum alloy plate (23), the plurality of chip fixing holes (24) are opened on the cover plate (22), and the microfluidic chip is installed on the cover plate (22) through the plurality of chip fixing holes (24).

9. The ultrasound-assisted integrated microfluidic ELISA rapid detection system according to claim 8, characterized in that: The base (21) is equipped with a piezoelectric ceramic drive board (31), which is electrically connected to a program control board (32), an ultrasonic piezoelectric ceramic (33), and a two-prong power plug (34).

10. The ultrasound-assisted integrated microfluidic ELISA rapid detection system according to claim 9, characterized in that: The ultrasonic piezoelectric ceramic (33) penetrates the cover plate (22) and is mounted on one end of the aluminum alloy plate (23) facing the base (21).

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