Integrated micro-fluidic chip for detecting alpha-synuclein related to Parkinson's disease

Abstract

The invention relates to the technical field of biomedical detection, in particular to an integrated micro-fluidic chip for detecting alpha-synuclein related to Parkinson's disease. The chip comprises a sample introduction module, a cell filtration module, a magnetic bead enrichment module, a mark detection module, a signal reading module and a microfluid driving control module which are integrated in a chip body to form a continuous microfluid channel system. Plasma separation is achieved through a double-layer filter membrane structure, target protein enrichment is completed through functionalized magnetic beads and a staged variable magnetic field strategy, a fluorescence labeling module is combined with a flow limiting structure and a constant-temperature cavity to achieve efficient signal reaction, and liquid directional transfer and automatic process control are completed in cooperation with positive and negative pressure cooperative driving. The chip has the advantages of compact structure, high processing efficiency, high sensitivity and the like, and is suitable for early screening and quantitative detection of Parkinson's disease.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to an integrated microfluidic chip for detecting Parkinson's disease-related α-synuclein. Background Technology

[0002] Parkinson's disease (PD), a common neurodegenerative disease, is characterized by movement disorders, resting tremor, and muscle rigidity. Studies have shown that abnormal aggregation and deposition of α-synuclein is a key biomarker in the pathogenesis of PD. Therefore, highly sensitive, specific, and rapid detection of α-synuclein in body fluid samples is of great significance for early screening, progression monitoring, and treatment evaluation of PD.

[0003] Traditional methods for detecting α-synuclein mainly include enzyme-linked immunosorbent assay (ELISA), Western blot, and mass spectrometry. While these methods possess certain detection capabilities, they still have many limitations in terms of sample volume, reaction time, operational complexity, and ease of on-site application, making it difficult to meet the clinical testing needs for rapid, high-throughput, and micro-sample processing. Furthermore, the sample processing steps during the testing process are cumbersome, and human error is significant, severely restricting testing efficiency and result stability.

[0004] In recent years, microfluidic chip technology has attracted widespread attention in the field of disease biomarker detection due to its advantages such as low sample consumption, high integration, fast response speed, and strong automation potential. Therefore, developing a compact, easy-to-operate, multifunctional integrated microfluidic chip suitable for the early diagnosis of Parkinson's disease has become one of the key technical problems that urgently need to be solved in this field. Summary of the Invention

[0005] To address the above problems, the present invention aims to provide an integrated microfluidic chip for detecting Parkinson's disease-related α-synuclein, comprising: The sample introduction module, cell filtering module, magnetic bead enrichment module, label detection module, signal reading module, and microfluidic drive control module are integrated into the chip body and form a continuous microfluidic path in sequence. Wherein: the sample introduction module includes a sample inlet, which is connected to a buffer for receiving unprocessed whole blood samples; The cell filtration module includes a first filter membrane layer and a second filter membrane layer, which are separated by a micro-spacing support structure, and are used to remove cellular components from the blood step by step to obtain plasma. The magnetic bead enrichment module is located downstream of the filtration module and is provided with an enrichment chamber and an external magnetic control area. The enrichment chamber is pre-filled with superparamagnetic magnetic beads with surface covalently modified anti-α-synuclein antibodies, and the magnetic control area is used to spatially position the magnetic beads. The labeling detection module includes a fluorescent probe injection port, a flow-limiting microchannel, and a labeling reaction chamber. The fluorescent probe is a fluorescent label conjugated with a secondary antibody. The flow-limiting microchannel is used to adjust the probe injection rate. The reaction chamber is connected to the enrichment chamber to realize the binding reaction between the fluorescent probe and the target complex. The signal reading module is located above the marking reaction chamber and is provided with an optically transparent window, which is suitable for the fluorescent signal acquisition device to receive light signals. The microfluidic drive control module includes a microchannel network and a pressure control interface. The interface is connected to positive and negative pressure generating devices and is used to control the liquid transfer, residence and cleaning process between different areas according to a set time sequence.

[0006] As a preferred technical solution, the first filter membrane layer is made of polycarbonate material with a pore size of 8-15 μm and a thickness of 20-30 μm; the second filter membrane layer is made of polyethersulfone material with a pore size of 0.2-1.0 μm and a thickness of 100-200 μm. A polypropylene support sheet with a spacing of 2.5 mm is provided between the first filter membrane layer and the second filter membrane layer. The edge of the support sheet is fitted into the chip cavity. The support sheet has multiple through holes with a diameter of 1 mm and an arrangement spacing of 2 mm.

[0007] As a preferred technical solution, the first filter membrane layer has a pore size of 10 μm and a thickness of 25 μm; the second filter membrane layer is made of polyethersulfone material with a pore size of 0.45 μm and a thickness of 150 μm.

[0008] As a preferred technical solution, the magnetic beads are magnetic particles with a particle size of 200-500 nanometers. The particle surface is modified with carboxyl groups and covalently linked to an anti-α-synuclein monoclonal antibody via carbodiimide coupling. The magnetic beads are dispersed and then injected into the enrichment chamber.

[0009] As a preferred technical solution, a magnetic field control component is provided outside the enrichment chamber, the magnetic field control component including a magnetic field generating module, a magnetic core array and a control circuit; The control circuit is used to execute a staged magnetic control program during the magnetic bead enrichment stage, the program including: In the first stage, the control circuit drives the magnetic field generating module to apply an alternating magnetic field that alternates between left and right along the cross-section of the chamber. The magnetic flux intensity increases from an initial low value to a medium level in a linear manner. The frequency of magnetic field changes is matched with the liquid flow rate to drive the magnetic beads to migrate laterally and increase their collision opportunities in the liquid, thereby increasing the number of magnetic beads that bind to the target protein. In the second stage, the control circuit switches the magnetic field to a steady-state directional magnetic field, and the magnetic flux density forms a spatial gradient distribution at the bottom or center of the enrichment chamber. The magnetic lines of force are always directed toward the settling surface of the enrichment area, which is used to guide the magnetic beads that have been bound to the target to quickly gather to the detection area and be stably positioned. The switching of the two-stage magnetic field is triggered by the feedback signal from the flow velocity sensor. The magnetic field change process remains continuous to avoid reverse drift or repositioning of the magnetic beads.

[0010] As a preferred technical solution, the first stage involves alternating magnetic fields driving the lateral migration of magnetic beads. In the time interval Inside, the magnetic field is an alternating field, and its magnetic flux density is... Expressed as: ; in, The initial magnetic flux density is expressed in T (Tesla). The rate of increase of magnetic flux intensity is expressed in T / s; The frequency of the alternating magnetic field is expressed in Hz, and satisfies the following conditions: , For liquid flow rate, The wavelength of the magnetic field; The transverse spatial phase distribution function makes the magnetic field alternately distributed on the left and right sides of the cavity cross section; These are the coordinates of the cross-section position; The movement of the magnetic bead in this stage is driven by the magnetic field gradient force, and its force Satisfying the formula: ; in, is the magnetic moment of the magnetic bead; To obtain the gradient; Second stage: Steady-state magnetic field guides the directional settling of magnetic beads: In the time interval Inside, the magnetic field switches to a constant directional field, and the magnetic flux density in space is: ; in, This represents the maximum magnetic flux density of the magnetic field; Indicates the central location of the enriched area; This represents the spatial scale factor that controls the magnetic flux density gradient. Represents three-dimensional spatial coordinates.

[0011] The magnetic field creates a magnetic flux density gradient inside the enrichment chamber, guiding the magnetic beads to sink along the magnetic field lines to the bottom region. The magnetic force acting on the magnetic beads is: ; in, Indicates the volume of the magnetic bead; Indicates magnetic susceptibility; Indicates the permeability of free space; This represents the gradient of the square of the magnetic flux density.

[0012] As a preferred technical solution, the fluorescent probe of the labeling detection module is a goat anti-mouse secondary antibody labeled with Cy5 fluorescent dye at a concentration of 0.5 μg / mL. The injection path is equipped with a flow-limiting channel with a cross-section of 60 μm × 50 μm and a length of 5 mm. The fluorescent probe enters the reaction chamber through the flow-limiting channel and binds to enrichment magnetic beads. The labeling reaction chamber is equipped with a temperature-controlled electrothermal film, which is heated to 37°C for 12 minutes. The chamber volume is 20 μL.

[0013] As a preferred technical solution, the microfluidic drive control module includes at least three positive pressure interfaces and at least three negative pressure interfaces. The positive pressure interfaces and negative pressure interfaces are respectively located at the sample inlet, the inlet of the magnetic bead enrichment module, the fluorescent probe injection port and downstream of the cell filtration module, the bottom of the magnetic control area, and the tail end of the labeling reaction chamber. They are connected to external positive and negative pressure sources through gas channels and indirectly connected to each liquid module through isolation membranes. Positive or negative pressure is applied independently at each drive node to realize multi-stage coordinated flow control of fluid between modules within the chip. As a preferred technical solution, the microfluidic drive control module is based on alternating positive and negative pressure and symmetrical pressure application mode, and adopts the following control flow: P1. During the sample loading stage, a first positive pressure is applied at the sample inlet, and a first negative pressure is applied downstream of the cell filtration module to form a positive and negative pressure synergistic driving state, which is used to directionally push the whole blood sample to the cell filtration module and complete the extraction of plasma components. P2, Target Enrichment Stage: A second positive pressure is applied at the entrance of the magnetic bead enrichment module, while a second negative pressure is applied at the bottom of the magnetic control area. The second negative pressure is used to guide the target protein to settle into the magnetic bead aggregation area, and the second positive pressure is used to maintain a constant flow rate. P3, the reagent injection stage, applies a third positive pressure at the fluorescent probe injection port and simultaneously applies a third negative pressure at the tail end of the labeling reaction chamber to promote uniform injection of the fluorescent probe and flow into the target area through the flow-limiting microchannel; P4. During the cleaning and drainage stage, cleaning solution is injected into the sample inlet and a fourth positive pressure is applied. A fourth negative pressure is applied at the tail end of the labeling reaction chamber to form a directional drainage path for removing unbound impurities and free probes.

[0014] As a preferred technical solution, a pretreatment buffer is provided between the sample introduction module and the cell filtration module. The buffer has a Y-shaped channel structure, with the left branch connected to PBS buffer at pH 7.4 and the right branch connected to diluent containing 1% Tween-20. The end of the Y-shaped channel is connected to a mixing chamber, which contains a spiral microcolumn structure with a diameter of 50 μm and a spacing of 100 μm. The bottom of the buffer is equipped with a Peltier temperature control element, with the temperature set at 28°C, for completing blood sample dilution, pH adjustment, and temperature stabilization operations.

[0015] As a preferred technical solution, the chip body is composed of PDMS and a glass substrate. The PDMS layer is 3mm thick. The microchannel structure is prepared using soft photolithography. The microchannel width is 200μm and the depth is 60μm. The reaction chamber is a planar cylindrical structure with a diameter of 3mm and a height of 400μm. The optical window of the signal reading module adopts a quartz glass embedded structure with a side length of 5mm and a light transmittance of not less than 95%. The edges are encapsulated with UV glue.

[0016] The beneficial effects of this invention are as follows: This invention provides an integrated microfluidic chip for detecting α-synuclein associated with Parkinson's disease. It features highly integrated functions including sample processing, separation and enrichment, fluorescent labeling, and signal readout, enabling highly sensitive detection of α-synuclein in whole blood samples. A cell filtration module employing a double-layer filter membrane and micro-septum support structure effectively removes blood cell components and stably outputs plasma. A magnetic bead enrichment module, combined with a staged magnetic field control strategy, achieves efficient capture even at low target molecule concentrations. A labeling detection module controls the labeling reaction conditions through a flow-limiting channel and a constant-temperature reaction chamber, ensuring reaction uniformity and repeatability. A microfluidic drive control module, combined with a multi-path positive and negative pressure collaborative drive mechanism, enables precise control and automatic switching of multiple process nodes within the chip. The chip has a compact overall structure, a rational flow path design, and programmable operation steps, making it suitable for early screening, dynamic monitoring, and clinical auxiliary diagnosis of Parkinson's disease. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the structural topology of the present invention; Figure 2 This is a schematic diagram of the buffer topology of the present invention; Figure 3 This is a schematic diagram of the cell filtration module of the present invention. Detailed Implementation

[0018] To enhance understanding of the present invention, the present invention will be further described in detail below with reference to embodiments. These embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0019] Example 1 This embodiment provides an integrated microfluidic chip system specifically designed for the rapid detection of α-synuclein, a protein associated with Parkinson's disease. The chip integrates key functional modules such as sample introduction, cell filtering, target protein enrichment, fluorescent labeling, signal readout, and fluid actuation on a single platform, achieving an integrated detection process of "instantaneous sample introduction—automatic processing—quantitative output" for whole blood samples. This system is widely applicable to early diagnosis, disease monitoring, and research.

[0020] Chip structure and composition: The microfluidic chip employs a PDMS (polydimethylsiloxane) elastic polymer laminated with a glass substrate. The PDMS layer is 3 mm thick, and a microchannel network is constructed internally using soft photolithography. The microchannels are 200 μm wide and 60 μm deep. Flow direction control is achieved between the main channels via microvalve and cross-control structures. Each functional module sequentially forms a continuous, closed liquid flow path from upstream to downstream, such as... Figure 1 As shown, it specifically includes the following parts: 1. Sample introduction module: This module is located at the sample inlet of the chip and consists of a standard injection interface, a buffer mixing structure, and a primary processing chamber.

[0021] The injection port has a diameter of 1.5 mm and is equipped with a hydrophobic coating to prevent sample residue and cross-contamination; like Figure 2 As shown, the buffer has a Y-shaped channel structure, with pH 7.4 PBS buffer and sample diluent containing 1% Tween-20 connected to the left and right sides, respectively; A spiral microcolumn mixing chamber is set at the end of the Y-shaped channel. The microcolumns are 50 μm in diameter, 100 μm apart, and 2 mm in length, which is used to enhance the rapid and uniform mixing of the sample and the buffer solution. The bottom of the buffer zone integrates a Peltier temperature control unit to maintain a constant temperature of 28°C in the mixing chamber, ensuring that the blood sample is in a stable state before entering the subsequent modules, preventing cell rupture or protein denaturation.

[0022] The main function of this module is to achieve pH adjustment, viscosity control and temperature stability of whole blood samples, providing a good pretreatment foundation for subsequent separation and enrichment.

[0023] 2. Cell Filtration Module: This module efficiently filters cellular components (red blood cells, white blood cells, platelets, etc.) from whole blood to obtain a clean plasma sample, such as... Figure 3 As shown, the design employs a double-layer filter membrane structure: The first filter membrane layer is made of polycarbonate (PC), with a pore size of 10μm and a thickness of 25μm, and is used to retain larger diameter cell components; The second filter membrane layer is made of polyethersulfone (PES) material with a pore size of 0.45μm and a thickness of 150μm, which further removes cell debris and large particulate impurities. A 2.5mm thick polypropylene support sheet is placed between the two filter membranes. The support sheet has 1mm diameter through holes (spaced 2mm apart). The through holes are arranged perpendicular to the channels to ensure smooth liquid transfer between the filter layers. The edge of the support plate is precisely fitted into the chip body cavity to ensure sealing and structural stability.

[0024] Under normal operation, this module can complete the stratification of a 20μL whole blood sample within 20 seconds, with an average plasma output of about 10~12μL, a filtration efficiency of over 90%, and a cell residue rate of less than 0.1%.

[0025] 3. Magnetic bead enrichment module: This module is the core detection component of the chip, employing an immunomagnetic enrichment strategy to target and capture α-synuclein in plasma.

[0026] The module's internal design features a cylindrical enrichment chamber, 3 mm in diameter and 400 μm in height. The chamber is pre-loaded with superparamagnetic beads, covalently linked to α-syn antibodies. These beads, with a particle size of 200–500 nm, are carboxyl-modified and covalently linked to the monoclonal antibody via EDC / NHS chemical coupling. The pretreatment concentration of the magnetic beads is 1 × 10⁻⁶. 7 Particles / mL, dispersed in PBS solution, injected into the chamber and held stationary until detection begins; an external magnetic control device is installed on the chip, including a programmable electromagnet, magnetic core array, and circuit control system, with the magnetic field switchable in two stages: The first stage is a transverse alternating magnetic field (magnetic field frequency 3Hz, initial magnetic flux intensity 0.1T linearly increasing to 0.3T), used to increase the probability of the magnetic beads moving laterally in the chamber and improve the encounter efficiency with the target protein. The second stage: a steady-state directional magnetic field (with a constant magnetic flux intensity of 0.4T) forms a vertical magnetic flux distribution, guiding the protein-bound magnetic beads to the bottom of the chamber and allowing them to settle stably; The switching of the magnetic control stage is triggered by the microflow sensor signal, and the whole process is completed automatically and continuously without manual intervention.

[0027] The module is designed to ensure efficient capture in a short time. Experimental data shows that the magnetic bead binding efficiency is as high as 85% or more within 10 minutes, and the concentration of the enriched target protein is significantly higher than the background value of untreated plasma.

[0028] 4. Labeling and detection module: Used to perform fluorescent labeling of the captured protein complex.

[0029] Equipped with a Cy5 fluorescent probe injection port and an internal injection system, this module injects 0.5 μg / mL of Cy5-labeled goat anti-mouse IgG. The probe is slowly injected into the chamber through a 60 μm × 50 μm flow-limiting channel (5 mm in length) to reduce the injection rate and prevent magnetic bead aggregation. After contact with the magnetic bead complex, the labeling reaction is facilitated under isothermal conditions. The total volume of the reaction chamber is 20 μL. An electrothermal film is embedded in the chamber sidewall to maintain a stable temperature of 37°C for 12 minutes to ensure complete fluorescent labeling. The chamber structure is cylindrical, with a diameter of 3 mm and a height of 400 μm. This module offers high labeling efficiency; unbound fluorescent probes can be removed by a downstream cleaning module, ensuring the specificity and accuracy of subsequent signal readings.

[0030] 5. Signal Reading Module: Located above the marking chamber, it features an embedded high-transmittance quartz glass optical window with a side length of 5mm and a transmittance greater than 95%, used for external fluorescence excitation and signal acquisition. The optical window is sealed and fixed in the PDMS layer with UV adhesive, providing excellent solvent resistance and thermal aging resistance. It can be connected to a standard fluorescence light source and a CCD detector to automatically acquire and quantitatively analyze the signal intensity in the Cy5 band (λmax≈670nm). The acquisition area is precisely located with low background signal, and it supports repeated readings for averaging correction.

[0031] 6. Microfluidic drive control module: such as Figure 1 As shown by the dashed lines, three pressure ports and three negative pressure ports are configured to act on the sample loading area, the magnetic control cavity inlet, and the probe injection port, respectively. The ports are indirectly connected to the channels inside the chip through isolation membranes to ensure that the gas does not come into direct contact with the liquid. All ports are controlled by an external multi-channel gas pressure generator with a pressure range of ±80 kPa. The system supports the following four working stages: P1 Sample loading: +60kPa is applied to the sample inlet and -50kPa is applied to the tail end of the filtration zone to create a pressure difference that propels blood through the filtration structure; P2 Target Enrichment: Apply +50kPa at the magnetic bead inlet and -40kPa at the magnetron bottom to ensure thorough mixing and binding of the magnetic beads; P3 Fluorescent probe injection: +30kPa is applied at the injection port and -20kPa is applied at the tail end of the reaction chamber to ensure stable entry of the fluorescent probe; P4 Cleaning and Drainage: Apply +50 kPa to the cleaning fluid inlet and -60 kPa to the waste fluid outlet to quickly flush away unbound residues.

[0032] By precisely matching timing and pressure, the orderly flow and effective switching of fluid paths within the chip are achieved, ensuring the accurate completion of each testing process.

[0033] Instructions for use: This integrated chip is designed to minimize sample requirements and maximize detection efficiency, requiring no manual intervention from sample introduction to signal output. The operation process is as follows: Step 1: Sample Preparation and Loading Freshly collected whole blood sample (recommended volume 20 μL) is slowly injected into the chip's inlet using a syringe. Simultaneously, the P1 stage positive and negative pressure control program is activated, and the system automatically introduces the blood sample from the injection port, while PBS and Tween-20 buffer are introduced from both sides. The three fluids rapidly fuse in the mixing chamber, forming a highly efficient shear mixture via the spiral microcolumns. The adjusted blood sample exhibits good fluidity and stability.

[0034] The temperature control unit ensures that the entire buffer zone is maintained at 28°C. This step can effectively prevent problems such as protein precipitation and cell aggregation in the blood, creating ideal conditions for subsequent separation and detection.

[0035] Step 2: Cell separation and plasma acquisition: The mixed blood sample enters the double-layered filtration area. First, it passes through the polycarbonate layer to intercept large-diameter red blood cells and white blood cells. Then, in the polyethersulfone filtration layer, secondary filtration of subcellular debris and protein complexes is completed. Only plasma and small protein molecules can pass through this structure to enter the downstream channel.

[0036] Example 2 This embodiment provides an integrated microfluidic chip for detecting Parkinson's disease-related α-synuclein, which adopts a multi-module integrated architecture and particularly highlights the application of a staged magnetic enrichment mechanism in the capture of low-titer target proteins.

[0037] Chip structure and overall integration: The chip employs a PDMS / glass substrate composite structure, with overall dimensions of 76 mm × 26 mm × 4 mm. The functional modules are interconnected via a microchannel network, forming a continuous fluid path. The layout of each module is as follows: Sample introduction module: Located at one end of the chip, it has a Φ2 mm sample inlet and is connected to the preprocessing buffer; Cell filtration module: Downstream is a dual-layer filter membrane structure. The first filter membrane layer is made of polycarbonate with a pore size of 10 μm and a thickness of 25 μm; the second filter membrane layer is made of polyethersulfone with a pore size of 0.45 μm and a thickness of 150 μm. Magnetic bead enrichment module: includes an enrichment chamber (3 mm in diameter and 400 μm in height), with an external magnetic core array and magnetic field coil; The labeling and detection module is equipped with a flow-limiting channel (60 μm wide, 50 μm deep, and 5 mm long) and a cylindrical reaction chamber. Signal reading module: An optical window is provided, with quartz glass embedded (5 mm on each side). Microfluidic drive module: It is equipped with 4 sets of positive pressure interfaces and 4 sets of negative pressure interfaces, and uses an isolation membrane structure to drive the liquid.

[0038] Sample preparation and injection: Take 1 mL of fresh anticoagulated peripheral whole blood sample, dilute it 1:4, and mix it with PBS buffer (pH 7.4) and diluent containing 1% Tween-20 at a volume ratio of 1:1. Inject the mixture through the chip delivery module. The sample is then homogenized in the pretreatment mixing zone using a spiral micropillar structure (50 μm in diameter, 100 μm spacing) and the temperature is maintained at 28°C.

[0039] Cellular component filtration: The sample first enters the cell filtration module, passing through the first filter membrane layer, where red blood cells and large-diameter white blood cells are blocked; then it enters the second filter membrane layer, which effectively traps small-diameter cells and debris, allowing only plasma and its dissolved substances to pass through, while α-synuclein is retained in the filtrate. The support layer has uniformly arranged pores (1 mm in diameter, 2 mm apart) to maintain structural stability and uniform fluid distribution between the upper and lower filter membrane layers.

[0040] Magnetic bead configuration: The pre-loaded magnetic beads in the enrichment module are 300 nm Fe3O4 nanoparticles with carboxyl-modified surfaces. They are covalently coupled to anti-α-synuclein monoclonal antibodies via EDC / NHS chemical reaction, with a coupling density of approximately 200 μg / mg (antibody / magnetic bead mass ratio). The magnetic bead concentration is set at 1 mg / mL, dispersed in PBS buffer, and injected into the chamber.

[0041] Staged Magnetically Controlled Enrichment Mechanism: This embodiment focuses on using a "staged magnetically controlled" strategy to achieve efficient enrichment of low-titer α-synuclein. The specific control process is as follows: Phase 1: Migration induced by alternating magnetic field Magnetic field direction: alternating laterally (X-axis direction); The magnetic field strength changes in a non-linear manner, increasing from its initial value. To the set value ,satisfy: , ;in, Let be the magnetic flux density at time t. The rate of increase of magnetic flux density The duration of the first phase; Alternating cycle: based on liquid flow rate Matching alternating frequencies ,in To enrich the cavity width.

[0042] The magnetic field induces the magnetic beads to repeatedly shift along the X-axis, promoting their diffusion in the fluid and increasing contact opportunities with the target protein, thereby increasing the binding rate. The average relative distance between the magnetic beads... The calculation is as follows: ; in, This represents the average relative distance the magnetic bead travels per unit time. This indicates the magnetic susceptibility of the ferrite bead; Indicates the viscosity of a liquid medium; Indicates the radius of the magnetic bead; By setting goals value (The average offset distance of the magnetic beads is 20%~50% of the channel width), and the set value of the magnetic induction intensity growth rate is calculated in reverse. : ; This optimizes the design objectives of the first-stage alternating magnetic field: like If the size is too small, the magnetic beads cannot effectively aggregate or will shift; if If the size is too large, the magnetic bead may deviate from the target area. It should meet the following requirements: ; Second stage: Steady-state magnetic field-oriented sedimentation Magnetic field direction: perpendicularly downwards along the Z-axis; Magnetic field characteristics: The magnetic induction intensity remains constant. The direction is fixed, and the magnetic flux density is concentrated in the central region at the bottom of the chamber; Magnetic field distribution: The symmetrical arrangement of the magnetic core array forms local magnetic wells, guiding the magnetic beads to sink and focus; During this stage, the magnetic field induces the movement of the magnetic beads, which is influenced by magnetic force. With gravity Overlay control: ; in, Indicates magnetic permeability; Indicates the volume of the magnetic bead; This represents the magnetic field gradient.

[0043] By applying a stable magnetic field with a spatial gradient, high-density aggregation of enriched magnetic beads is achieved at the bottom of the detection area. This, combined with the fixed position, reduces the fluorescence diffusion range and improves the accuracy of subsequent signal acquisition.

[0044] Stage switching method: Staged magnetic control is automatically switched by the control circuit, using a flow rate sensor to detect the time it takes for the liquid to flow through the enrichment chamber and the trigger time. Upon arrival, the magnetic field is immediately switched to the second stage steady-state magnetic field. The field strength is continuous during the magnetic field switching process to prevent the magnetic bead from floating or bouncing back.

[0045] Fluorescent probe injection and labeling reaction: The fluorescent labeling used was Cy5-labeled goat anti-mouse secondary antibody at a concentration of 0.5 μg / mL. The probe was injected into the reaction chamber through a flow-limiting channel. The flow-limiting structure controlled the injection rate and prevented probe dilution. An electrothermal membrane was installed inside the reaction chamber, maintaining a constant temperature of 37℃. The reaction time was set to 12 minutes to ensure complete labeling.

[0046] Washing and signal acquisition: The washing solution was PBS buffer containing 0.05% Tween-20. A fourth positive pressure was applied through the washing solution inlet, and a fourth negative pressure was applied through the waste solution outlet to drive the unbound probe out. Signal reading was performed by acquiring fluorescence images at the Cy5 excitation wavelength using a fluorescence microscope. The signal intensity was proportional to the concentration of the bound protein.

[0047] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. An integrated microfluidic chip for detecting Parkinson's disease-related α-synuclein, characterized in that, include: The sample introduction module, cell filtering module, magnetic bead enrichment module, label detection module, signal reading module, and microfluidic drive control module are integrated into the chip body and form a continuous microfluidic path in sequence. Wherein: the sample introduction module includes a sample inlet, which is connected to a buffer for receiving unprocessed whole blood samples; The cell filtration module includes a first filter membrane layer and a second filter membrane layer, which are separated by a micro-spacing support structure, and are used to remove cellular components from the blood step by step to obtain plasma. The magnetic bead enrichment module is located downstream of the filtration module and is provided with an enrichment chamber and an external magnetic control area. The enrichment chamber is pre-filled with superparamagnetic magnetic beads with surface covalently modified anti-α-synuclein antibodies, and the magnetic control area is used to spatially position the magnetic beads. The labeling detection module includes a fluorescent probe injection port, a flow-limiting microchannel, and a labeling reaction chamber. The fluorescent probe is a fluorescent label conjugated with a secondary antibody. The flow-limiting microchannel is used to adjust the probe injection rate. The reaction chamber is connected to the enrichment chamber to realize the binding reaction between the fluorescent probe and the target complex. The signal reading module is located above the marking reaction chamber and is provided with an optically transparent window, which is suitable for the fluorescent signal acquisition device to receive light signals. The microfluidic drive control module includes a microchannel network and a pressure control interface. The interface is connected to positive and negative pressure generating devices and is used to control the liquid transfer, residence and cleaning process between different areas according to a set time sequence.

2. The integrated microfluidic chip for detecting Parkinson's disease-related α-synuclein according to claim 1, characterized in that, The first filter membrane layer is made of polycarbonate material with a pore size of 8-15 μm and a thickness of 20-30 μm; the second filter membrane layer is made of polyethersulfone material with a pore size of 0.2-1.0 μm and a thickness of 100-200 μm. A polypropylene support sheet with a spacing of 2.5 mm is provided between the first filter membrane layer and the second filter membrane layer. The edge of the support sheet is fitted into the chip cavity. The support sheet has multiple through holes with a diameter of 1 mm and an arrangement spacing of 2 mm.

3. The integrated microfluidic chip for detecting Parkinson's disease-related α-synuclein according to claim 1, characterized in that, The first filter membrane layer has a pore size of 10 μm and a thickness of 25 μm; the second filter membrane layer is made of polyethersulfone material with a pore size of 0.45 μm and a thickness of 150 μm.

4. The integrated microfluidic chip for detecting Parkinson's disease-related α-synuclein according to claim 1, characterized in that, The magnetic beads are magnetic particles with a particle size of 200-500 nanometers. The particle surface is modified with carboxyl groups and covalently linked to anti-α-synuclein monoclonal antibody via carbodiimide coupling. The magnetic beads are dispersed and then injected into the enrichment chamber. The enrichment chamber is provided with a magnetic field control component, which includes a magnetic field generating module, a magnetic core array, and a control circuit. The control circuit is used to execute a staged magnetic control program during the magnetic bead enrichment stage, the program including: In the first stage, the control circuit drives the magnetic field generating module to apply an alternating magnetic field that alternates between left and right along the cross-section of the chamber. The magnetic flux intensity increases from an initial low value to a medium level in a linear manner. The frequency of magnetic field changes is matched with the liquid flow rate to drive the magnetic beads to migrate laterally and increase their collision opportunities in the liquid, thereby increasing the number of magnetic beads that bind to the target protein. In the second stage, the control circuit switches the magnetic field to a steady-state directional magnetic field, and the magnetic flux density forms a spatial gradient distribution at the bottom or center of the enrichment chamber. The magnetic field lines are always directed towards the settling surface of the enrichment area, which is used to guide the magnetic beads that have been bound to the target to quickly gather to the detection area and be stably positioned.

5. An integrated microfluidic chip for detecting Parkinson's disease-related α-synuclein according to claim 1, characterized in that, The fluorescent probe of the labeling detection module is a goat anti-mouse secondary antibody labeled with Cy5 fluorescent dye at a concentration of 0.5 μg / mL. The injection path is equipped with a flow-limiting channel with a cross-section of 60 μm × 50 μm and a length of 5 mm. The fluorescent probe enters the reaction chamber through the flow-limiting channel and binds to enrichment magnetic beads. The labeling reaction chamber is equipped with a temperature-controlled electrothermal film and is heated to 37°C for 12 minutes. The chamber volume is 20 μL.

6. An integrated microfluidic chip for detecting Parkinson's disease-related α-synuclein according to claim 1, characterized in that, The microfluidic drive control module includes at least three positive pressure interfaces and at least three negative pressure interfaces. The positive pressure interfaces and negative pressure interfaces are respectively located at the sample inlet, the inlet of the magnetic bead enrichment module, the fluorescent probe injection port and downstream of the cell filtration module, the bottom of the magnetic control area, and the tail end of the labeling reaction chamber. They are connected to external positive and negative pressure sources through gas channels and indirectly connected to each liquid module through isolation membranes. Positive or negative pressure is applied independently at each drive node to achieve multi-stage coordinated flow control of fluid between modules within the chip. The microfluidic drive control module is based on alternating positive and negative pressure and symmetrical pressure application mode, and adopts the following control process: P1. During the sample loading stage, a first positive pressure is applied at the sample inlet, and a first negative pressure is applied downstream of the cell filtration module to form a positive and negative pressure synergistic driving state, which is used to directionally push the whole blood sample to the cell filtration module and complete the extraction of plasma components. P2, Target enrichment stage: A second positive pressure is applied at the entrance of the magnetic bead enrichment module, and a second negative pressure is applied at the bottom of the magnetic control area. The second negative pressure is used to guide the target protein to settle into the magnetic bead aggregation area, and the second positive pressure is used to maintain a constant flow rate. P3, the reagent injection stage, applies a third positive pressure at the fluorescent probe injection port and simultaneously applies a third negative pressure at the tail end of the labeling reaction chamber to promote uniform injection of the fluorescent probe and flow into the target area through the flow-limiting microchannel; P4. During the cleaning and drainage stage, cleaning solution is injected into the sample inlet and a fourth positive pressure is applied. A fourth negative pressure is applied at the tail end of the labeling reaction chamber to form a directional drainage path for removing unbound impurities and free probes.

7. An integrated microfluidic chip for detecting Parkinson's disease-related α-synuclein according to claim 1, characterized in that, A pretreatment buffer is provided between the sample introduction module and the cell filtration module. The buffer has a Y-shaped channel structure, with the left branch connected to PBS buffer at pH 7.4 and the right branch connected to diluent containing 1% Tween-20. The end of the Y-shaped channel is connected to a mixing chamber, which contains spiral microcolumns with a diameter of 50 μm and a spacing of 100 μm. The bottom of the buffer is equipped with a Peltier temperature control element, with the temperature set at 28°C, for blood sample dilution, pH adjustment, and temperature stabilization.

8. An integrated microfluidic chip for detecting Parkinson's disease-related α-synuclein according to claim 1, characterized in that, The chip body is composed of PDMS and a glass substrate. The PDMS layer is 3mm thick. The microchannel structure is prepared using soft photolithography. The microchannel width is 200μm and the depth is 60μm. The reaction chamber is a planar cylindrical structure with a diameter of 3mm and a height of 400μm. The optical window of the signal reading module adopts a quartz glass embedded structure with a side length of 5mm and a light transmittance of not less than 95%. The edges are encapsulated with UV glue.

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