Integrated alpha-synuclein hypersensitivity detector

Abstract

The invention discloses an integrated alpha-synuclein hypersensitive detector, which comprises a shell, and a cover plate is arranged at the top port of the shell; the processing part is arranged on the inner side of the cover plate in parallel; the micro-fluidic chip is positioned in the shell and is arranged below the processing part in parallel; a mounting through hole is formed in the center of the micro-fluidic chip, and a sample processing unit is formed in the micro-fluidic chip; the sample processing unit comprises a sample processing area and a transparent reaction detection area; the sample treatment area and the reaction detection area are arranged at an interval along the direction far away from the mounting through hole; the reaction detection area corresponds to the lower part; the detection part is arranged below the processing part. By integrating the functions of ultrasonic cracking, magnetic bead enrichment, fluorescence excitation, micro-fluidic partition detection and the like, high-sensitivity detection on the alpha-synuclein protein, especially the pathological aggregation state of the alpha-synuclein protein is realized.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to an integrated α-synuclein ultrasensitive detector. Background Technology

[0002] α-synuclein is a hydrophilic small protein widely distributed in the central nervous system, especially at the presynaptic terminals of neurons. Under normal physiological conditions, α-synuclein participates in regulating the vesicle storage and release of neurotransmitters, maintaining synaptic activity, and stabilizing cell structure, making it an important component of normal neuronal function. However, under pathological conditions, this protein is prone to abnormal aggregation, forming oligomers and fibrillary deposits, eventually developing into Lewy bodies. These aggregates are considered important pathogenic factors in neurodegenerative diseases such as Parkinson's disease and Lewy body dementia. Pathological α-synuclein not only damages cellular function but may also accelerate disease progression through intercellular transmission. Therefore, α-synuclein plays a crucial role in the development and progression of neurodegenerative diseases. Accurate detection of the content and aggregation status of α-synuclein in biological samples is of great value for early disease screening, progression monitoring, and evaluation of intervention effects. Especially in drug development and disease diagnosis and treatment research, α-synuclein, as a core biomarker, has become a key link in the research and translational application of neurological diseases due to the sensitivity and reliability of its detection methods.

[0003] Although various experimental methods for detecting α-synuclein have been developed, most techniques still have limitations in terms of sensitivity, specificity, ease of operation, and cost. Traditional Western blotting, while capable of detecting protein molecular weight and expression, is complex, cumbersome, and requires highly skilled personnel. It often requires large sample sizes to obtain reliable results, hindering high-throughput detection. Enzyme-linked immunosorbent assay (ELISA) is widely used due to its relative simplicity and suitability for routine sample screening; however, its detection efficiency for low-abundance, oligomeric, or conformationally specific α-synuclein is not ideal, posing a risk of false negatives. In recent years, RT-QuIC (Real-Time Quaking-Induced Conversion) technology has attracted attention due to its high sensitivity, capable of detecting trace amounts of pathological α-synuclein aggregates and amplifying them through induction. However, RT-QuIC detection still relies on long reaction times and large amounts of reagents, and has high requirements for instruments and operating environments, making it difficult to achieve rapid, high-throughput, and low-cost detection applications under routine laboratory conditions. Therefore, existing detection technologies still cannot fully meet the comprehensive needs for efficiency, sensitivity, and universality in early diagnosis, disease screening, and basic research, and there is an urgent need for more efficient and convenient alternatives.

[0004] In response to the aforementioned technical bottlenecks, it is particularly urgent to develop an α-synuclein detection and research system that combines high sensitivity, low cost, and rapid response capabilities. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an integrated α-synuclein ultrasensitive detector, the purpose of which is to solve the problems in the background technology.

[0006] To achieve the above objectives, the present invention provides the following technical solution: an integrated α-synuclein ultrasensitive detector, comprising: a housing, a processing unit, a microfluidic chip, and a detection unit; a cover plate is provided at the top port of the housing; The processing unit is arranged parallel to the inner side of the cover plate; the bottom of the processing unit is a light emitting component, an ultrasonic processing component, and a magnetic bead moving component; A microfluidic chip is located inside a housing and positioned parallel to the bottom of the processing unit. The microfluidic chip has a mounting through-hole at its center, and a sample processing unit is formed inside the microfluidic chip. The sample processing unit includes a sample processing area and a transparent reaction detection area. The sample processing area and the reaction detection area are spaced apart along a direction away from the mounting through-hole. The reaction detection area corresponds to the area below the light emitting component. The detection unit includes a servo motor and a photosensitive component. The servo motor is located below the microfluidic chip, and a connecting shaft is fixedly mounted on the output shaft of the servo motor. The connecting shaft is detachably fixed to the mounting through hole. The receiving end of the photosensitive component is located below the reaction detection area.

[0007] Furthermore, the sample processing area includes a sample loading chamber and a lysis buffer storage chamber; the sample loading chamber and the lysis buffer storage chamber are arranged at intervals along the surrounding mounting through holes; the microfluidic channel includes a first microfluidic channel, a second microfluidic channel, and a third microfluidic channel; The lysis chamber is located on the side of the sample loading chamber away from the mounting through hole, and the side of the lysis chamber near the mounting through hole is connected to the side of the sample loading chamber away from the mounting through hole through a first microfluidic channel; and the side of the lysis chamber near the mounting through hole is connected to the side of the lysis buffer storage chamber away from the mounting through hole through a second microfluidic channel. The reaction detection zone is located to the left of the pyrolysis chamber; and the right end of the reaction detection zone is connected to the left end of the pyrolysis chamber through a third microfluidic channel. The reaction detection area includes a manifold, a reaction chamber, and a waste liquid chamber; the third microfluidic channel is connected to the manifold at one end away from the lysis chamber, and six reaction chambers are provided on the manifold. A fourth microfluidic channel is provided between the manifold and the six reaction chambers, and a waste liquid chamber is provided at the other end of the manifold; the detection unit also includes a substrate; the substrate is arranged parallel to the bottom of the microfluidic chip; the substrate is fixed to the side plate of the housing with screws; a groove is provided at the top of the substrate; the photosensitive component is arranged on the bottom wall of the groove, located directly below the reaction chamber.

[0008] Furthermore, the middle part of the shell is surrounded and fixed by four side plates, the top of which is surrounded by the side plates has a top plate, and the bottom of the shell has a bottom plate. The periphery of the top plate is fixed to the top of the four side plates, and the periphery of the bottom plate is fixed to the bottom of the four side plates. One end of the cover plate is hinged to the top of a side plate, so that the cover plate can be rotated to one side of the shell to open.

[0009] Furthermore, the six reaction chambers are evenly distributed around the mounting holes; the light emitting component has six emitting ends, and the six emitting ends of the light emitting component are arranged in a one-to-one correspondence with the six reaction chambers; the photosensitive component is arranged in a one-to-one correspondence with the six reaction chambers.

[0010] Furthermore, the photosensitive component includes an optical detection plate and a printed circuit board. The bottom of the optical detection plate has a photosensitive element. The printed circuit board is parallel to and fixed to the corresponding position on the substrate with screws. The optical detection plate is parallel to and fixed to the bottom of the printed circuit board with screws. The photosensitive element is electrically connected to the printed circuit board.

[0011] Furthermore, a siphon valve one is installed on the second microfluidic channel; a siphon valve two is installed on the third microfluidic channel; Siphon valve three and siphon valve four are respectively installed on the first microfluidic flow channel and the fourth microfluidic flow channel.

[0012] Furthermore, the magnetic bead moving assembly contains an electromagnet, which is cylindrical and used to attract the magnetic beads in the lysis chamber to cut the cells in the sample solution and cause them to lyse.

[0013] Furthermore, the detection unit also includes heating ring one and heating ring two; heating ring one and heating ring two are arranged coaxially with the mounting through hole and are both fixed to the bottom of the groove by fixing column; heating ring one is located below the pyrolysis chamber; heating ring two is located below the reaction chamber.

[0014] Furthermore, the top of the microfluidic chip and the corresponding positions of multiple sample loading chambers and lysis buffer storage chambers are provided with corresponding sample loading holes; the top walls of the sample loading chamber, lysis buffer storage chamber, lysis chamber, manifold, and waste liquid chamber are all provided with exhaust holes.

[0015] Furthermore, the detection unit also includes a controller and a touch screen; the controller is located below the substrate and fixed to the bottom wall of the housing; the touch screen is located on the top of the cover plate; the processing unit, servo motor, light emission assembly, ultrasonic processing assembly, magnetic bead moving assembly, heating ring one, heating ring two and the touch screen are all electrically connected to the controller and are all controlled by the controller.

[0016] Compared with existing technologies, the present invention has the following advantages: (1) This invention integrates the functions of ultrasonic lysis, magnetic bead enrichment, fluorescence excitation and microfluidic partition detection into one, and realizes highly sensitive detection of α-synuclein protein, especially its pathological aggregation state. It effectively overcomes the shortcomings of traditional methods such as Western blot and ELISA in terms of sensitivity, operation complexity and sample requirements, and significantly improves detection efficiency and accuracy.

[0017] (2) This invention combines RT-QuIC technology to perform highly sensitive quantitative analysis of α-synuclein aggregates, which further enhances the specificity and sensitivity of the detection. It can realize real-time monitoring of pathological protein aggregation in trace samples, providing reliable technical support for early screening and intervention assessment of neurodegenerative diseases such as Parkinson's disease. Attached Figure Description

[0018] Figure 1 Exploded view of the overall structure of the integrated α-synuclein ultrasensitive detector of this invention.

[0019] Figure 2 This invention patent presents a schematic diagram of the internal structure of the microfluidic chip in the integrated α-synuclein ultrasensitive detector.

[0020] Figure 3 A top view schematic diagram of the microfluidic chip of the integrated α-synuclein ultrasensitive detector of this invention.

[0021] Figure 4 This invention patent shows a schematic diagram of the three layers of the integrated α-synuclein ultrasensitive detector microfluidic chip.

[0022] Reference numerals: 1. Shell; 11. Cover plate; 12. Top plate; 13. Side plate; 14. Bottom plate; 2. Processing unit; 21. Ultrasonic processing assembly; 22. Magnetic bead moving assembly; 23. Light emitting assembly; 3. Microfluidic chip; 31. Sample processing area; 311. Sample loading chamber; 312. Lysis buffer storage chamber; 313. Lysis chamber; 32. Mounting through hole; 33. Reaction detection area; 331. Manifold channel; 332. Reaction chamber 333. Waste liquid chamber; 34. Sample dispensing port; 35. Vent port; 36. Microfluidic channel; 361. First microfluidic channel; 362. Second microfluidic channel; 363. Third microfluidic channel; 364. Fourth microfluidic channel; 4. Detection unit; 41. Heating ring one; 42. Heating ring two; 43. Photosensitive component; 44. Substrate; 45. Servo motor; 451. Connecting shaft; 46. Controller; 47. Touch screen. Detailed Implementation

[0023] like Figures 1-4 As shown, the present invention provides a technical solution: an integrated α-synuclein ultrasensitive detector, comprising; The housing 1 is a cuboid structure. A cover plate 11 is provided at the top port of the housing 1. The middle part of the housing 1 is surrounded and fixed by four side plates 13. A top plate 12 is provided at the top of the area surrounded by the side plates 13. A bottom plate 14 is provided at the bottom of the housing 1. The top plate 12 is fixed to the top of the four side plates 13 around its periphery, and the bottom plate 14 is fixed to the bottom of the four side plates 13 around its periphery. One end of the cover plate 11 is hinged to the top of a side plate 13, so that the cover plate 11 can be rotated to one side of the housing 1 to open.

[0024] The processing unit 2 is arranged parallel to the inner side of the cover plate 11; the bottom of the processing unit 2 consists of a light emitting component 23, an ultrasonic processing component 21, and a magnetic bead moving component 22.

[0025] The microfluidic chip 3 is circular and located inside the housing 1, parallel to the lower part of the processing unit 2. The microfluidic chip 3 has a mounting through-hole 32 at its center, and a sample processing unit is formed within it. The sample processing unit includes a sample processing area 31 and a transparent reaction detection area 33. The sample processing area 31 and the reaction detection area 33 are spaced apart along a direction away from the mounting through-hole 32. The reaction detection area 33 corresponds to the area below the light emitting component 23. Light emitted by the light emitting component 23 can penetrate the reaction detection area 33 and be received by the photosensitive component 43 for photosensitive measurement. The reaction detection area 33 and the sample processing area 31 are connected through a microfluidic channel 36 inside the microfluidic chip 3. The detection unit 4 includes a servo motor 45 and a photosensitive component 43. The servo motor 45 is located below the microfluidic chip 3. A connecting shaft 451 is fixedly mounted on the output shaft of the servo motor 45. The connecting shaft 451 is detachably fixed to the mounting through hole 32 to facilitate the replacement of the microfluidic chip 3. The receiving end of the photosensitive component 43 is located below the reaction detection area 33. This invention patents an integrated α-synuclein ultrasensitive detector. A servo motor 45 drives a microfluidic chip 3 to rotate back and forth, oscillating and mixing the sample liquid in the sample processing area 31. By controlling the high-speed rotation of the microfluidic chip 3 driven by the servo motor 45 and controlling its rotation speed, the processed sample liquid in the sample processing area 31 can be transferred from the sample processing area 31 through the internal microfluidic channel 36 of the microfluidic chip 3 to the reaction detection area 33 under the action of centrifugal force. The light beam emitted by the light emitting component 23 interacts with the processed sample liquid as it passes through the reaction detection area 33, emitting fluorescence. The detection unit 4 receives the fluorescence signal fed back from the reaction detection area 33 through the photosensitive component 43, processes it to obtain fluorescence intensity information, and obtains the α-synuclein content by comparing it with a standard curve of fluorescence intensity information. This invention patents use fluorescence analysis to detect α-synuclein, offering convenient operation and achieving high detection sensitivity.

[0026] Specifically, the microfluidic chip 3 can be installed or removed by opening the cover plate 11; when installing the microfluidic chip 3, align the mounting through hole 32 with the connecting shaft 451 and fit the microfluidic chip 3 onto the connecting shaft 451.

[0027] In some specific embodiments, a reaction detection unit has six reaction chambers 332, which are evenly distributed around the mounting through-holes 32; a light emitting component 23 has six emitting ends, which are arranged one-to-one with the six reaction chambers 332; and a photosensitive component 43 is arranged one-to-one with the six reaction chambers 332. Thus, the six sample processing units can simultaneously complete the detection of samples, improving detection accuracy.

[0028] In some specific embodiments, the sample processing area 31 includes a sample loading chamber 311, a lysis buffer storage chamber 312, and a lysis chamber 313; the microfluidic channel 36 includes a first microfluidic channel 361, a second microfluidic channel 362, and a third microfluidic channel 363. Sample loading chamber 311 and lysis buffer storage chamber 312 are arranged at intervals along the surrounding mounting through holes 32; The lysis chamber 313 is located on the side of the sample loading chamber 311 away from the mounting through hole 32, and the side of the lysis chamber 313 near the mounting through hole 32 is connected to the side of the sample loading chamber 311 away from the mounting through hole 32 through a first microfluidic channel 362; and the side of the lysis chamber 313 near the mounting through hole 32 is connected to the side of the lysis buffer storage chamber 312 away from the mounting through hole 32 through a second microfluidic channel 361. The reaction detection zone 33 is located to the left of the pyrolysis chamber 313; and the right end of the reaction detection zone 33 is connected to the left end of the pyrolysis chamber 313 through the third microfluidic channel 363. The reaction detection area 33 includes a manifold channel 331, a reaction chamber 332, and a waste liquid chamber 333. A third microfluidic channel 363, located away from the pyrolysis chamber 313, is connected to the manifold channel 331. Six reaction chambers 332 are located on the manifold channel 331. A fourth microfluidic channel 364 is located between the manifold channel 331 and the six reaction chambers 332. The other end of the manifold channel 331 is equipped with a waste liquid chamber 333. The detection unit 4 also includes a substrate 44. The substrate 44 is parallel to the microfluidic chip 3 below it. The substrate 44 is fixed to the side plate 13 of the housing 1 with screws. A groove is formed at the top of the substrate 44. A photosensitive component 43 is located on the bottom wall of the groove, directly below the reaction chamber 332.

[0029] The photosensitive component 43 includes an optical detection plate and a printed circuit board. The bottom of the optical detection plate has a photosensitive element. The printed circuit board is parallel to and fixed to the corresponding position of the substrate 44 with screws. The optical detection plate is parallel to and fixed to the bottom of the printed circuit board with screws. The photosensitive element is electrically connected to the printed circuit board. The sample loading chamber 311 is used to add sample solution, and the lysis buffer storage chamber 312 is used to add lysis buffer. The sample solution can be ultrasonically treated by the ultrasonic treatment component 21 to disrupt the tissue structure within the sample solution. The sample solution and lysis buffer are mixed in the lysis chamber 313, which contains magnetic beads. The microfluidic chip 3 is controlled to rotate at a certain speed, and centrifugation causes the sample solution in the sample loading chamber 311 to flow into the lysis chamber 313 through the first microfluidic channel 361. The microfluidic chip 3 is also controlled to rotate at a certain speed, and centrifugation causes the lysis buffer in the lysis buffer storage chamber 312 to flow into the lysis chamber 313 through the second microfluidic channel 362 and mix with the sample solution. The magnetic bead moving component 22... The sample solution is attracted by magnetic beads to cut cells, causing the sample solution to mix and lyse. The ultrasonic treatment component 21 can ultrasonically treat the sample solution to destroy the lysed sample solution and mix with the remaining tissue structure. Then, the lysed sample solution in the lysis chamber 313 is centrifuged at a certain speed, so that the lysed sample solution flows into the confluence channel 331 through the third microfluidic channel 363, and the excess sample solution will flow into the waste liquid chamber 333. The microfluidic chip 3 is controlled to rotate at a certain speed, and the sample solution in the confluence channel 331 flows into the reaction detection area 33 through the fourth microfluidic channel 364 through centrifugation for reaction and fluorescence detection, and finally the α-synuclein content can be obtained.

[0030] In some specific embodiments, a siphon valve is provided on the second microfluidic channel 362; and a siphon valve is provided on the third microfluidic channel 363.

[0031] Both siphon valve one and siphon valve two have a certain centrifugal speed threshold; when the centrifugal speed threshold is exceeded, siphon valve one and siphon valve two can be centrifugally connected; the centrifugal speed threshold of siphon valve one can be set to be less than the centrifugal speed threshold of siphon valve two, so as to realize the control of the conduction state of sample liquid between sample loading chamber 311 and lysis chamber 313 and lysis solution between lysis solution storage chamber 312 and lysis chamber 313 by controlling the speed of microfluidic chip 3.

[0032] Among them, the first microfluidic flow channel 361 and the fourth microfluidic flow channel 364 are respectively equipped with siphon valve three and siphon valve four.

[0033] Specifically, the magnetic bead moving assembly 22 contains an electromagnet, which is cylindrical and is used to attract the magnetic beads in the lysis chamber 313 to cut the cells in the sample solution and cause them to lyse.

[0034] In some specific embodiments, the detection unit 4 further includes a heating ring 41 and a heating ring 42; the heating ring 41 and the heating ring 42 are coaxially arranged with the mounting through hole 32 and are both fixed to the bottom of the groove by a fixing post; the heating ring 41 is located below the pyrolysis chamber 313; the heating ring 42 is located below the reaction chamber 332.

[0035] After the sample solution flows into the lysis chamber 313, the corresponding lysis chamber 313 can be heated by turning on the heating ring 41; after the lysed sample solution enters the reaction chamber 332, the heating ring 42 can be turned on to control the temperature of the corresponding reaction chamber 332.

[0036] In some specific embodiments, the top of the microfluidic chip 3 and the corresponding positions of the multiple sample loading chambers 311 and the lysis buffer storage chamber 312 are all provided with corresponding sample loading holes 34; the top walls of the sample loading chamber 311, the lysis buffer storage chamber 312, the lysis chamber 313, the confluence channel 331, and the waste liquid chamber 333 are all provided with exhaust holes 35.

[0037] The sample loading chamber 311, lysis buffer storage chamber 312, lysis chamber 313, manifold 331, waste liquid chamber 333, and reaction chamber 332 are all through holes opened on the middle structural layer. The bottom encapsulation layer, top encapsulation layer, and middle structural layer encapsulate each chamber. The first microfluidic channel 361, the second microfluidic channel 362, the third microfluidic channel 363, and the fourth microfluidic channel 364 are all microfluidic channels opened on the top surface of the middle structural layer. The bottom encapsulation layer, top encapsulation layer, and middle structural layer encapsulate each microfluidic channel 36. The sample loading hole 34 and the vent hole 35 are through holes opened on the top encapsulation layer to connect the corresponding chambers.

[0038] Specifically, siphon valve one, siphon valve two, siphon valve three and siphon valve four are all microfluidic channels opened on the top surface of the middle structural layer. After the bottom encapsulation layer, top encapsulation layer and middle structural layer are encapsulated, they form the microfluidic channels of siphon valve one, siphon valve two, siphon valve three and siphon valve four.

[0039] In some specific embodiments, the detection unit 4 further includes a controller 46 and a touch screen 47; the controller 46 is disposed below the substrate 44 and fixed to the inner bottom wall of the housing 1; the touch screen 47 is disposed on the top of the cover plate 11; the processing unit 2, the servo motor 45, the light emitting component 23, the ultrasonic processing component 21, the magnetic bead moving component 22, the heating ring 1 41, the heating ring 2 42 and the touch screen 47 are all electrically connected to the controller 46 and are all controlled by the controller 46.

[0040] The working steps of the integrated α-synuclein ultrasensitive detector of this invention are as follows: Step 1: Inject sample solution and lysis buffer into the sample processing area 31 of the microfluidic chip 3 and the sample dispensing chamber 311 and the lysis buffer storage chamber 312 through the sample dispensing port 34, and then place the microfluidic chip 3 into the instrument. Step 2: Control the ultrasonic treatment component 21 to turn on and ultrasonically treat the sample solution in the sample loading chamber 311; Step 3: Control the motor to centrifuge the microfluidic chip 3 at the first centrifugation speed, so that the sample liquid in the sample loading chamber 311 enters the lysis chamber 313 through the first microfluidic channel 361; Step 4: Control the motor to centrifuge the microfluidic chip 3 at the second centrifugal speed, so that the lysis solution in the lysis solution storage chamber 312 enters the lysis chamber 313 through the second microfluidic channel 362; Step 5: Control the motor to rotate the microfluidic chip 3 back and forth at the first speed to mix the sample solution and lysis solution in the lysis chamber 313 and provide a shaking effect; Step 6: Control the start of the inner heating coil of the heater to heat the pyrolysis chamber 313; Step 7: Control the magnetic bead moving component 22 to open, attracting and cutting the cells in the lysis chamber 313 to lyse them; Step 8: Control the ultrasonic treatment component 21 to turn on and ultrasonically treat the sample solution in the lysis chamber 313. Step 9: Control the motor to centrifuge the microfluidic chip 3 at the third centrifugation speed, so that the sample liquid in the lysis chamber 313 enters the manifold channel 331 through the third microfluidic channel 363; Step 10: Control the motor to centrifuge the microfluidic chip 3 at the fourth centrifugation speed, so that the sample liquid in the manifold 331 enters the reaction chamber 332 through the fourth microfluidic channel 364; Step 11: Control the motor to rotate the microfluidic chip 3 back and forth at the first speed to mix the sample liquid and stored reactants in the reaction chamber 332 and provide a shaking effect; Step 12: Control the activation of the outer heating coil of the heater to heat the reaction chamber 332; Step 13: Use a fluorescence detection device (optical detection plate, printed circuit board, and microprogram controller 46) to detect the fluorescence intensity; the α-synuclein concentration can be obtained by comparing with the standard curve.

[0041] Specifically, the second centrifugal speed is greater than the centrifugal speed of siphon valve one, but less than the centrifugal speed of siphon valve two; the third centrifugal speed is greater than the centrifugal speed of siphon valve two; the third centrifugal speed is greater than the second centrifugal speed; the second centrifugal speed is greater than the first centrifugal speed; and the fourth centrifugal speed is greater than the third centrifugal speed. The specific values ​​need to be determined based on the chip structure.

[0042] This specification further describes the use of RT-QuIC (Real-Time Quaking-Induced Conversion) technology for highly sensitive quantitative analysis of α-synuclein aggregation seeding activity. This technology is based on the prion-like structure induction mechanism of pathological α-synuclein, utilizing its ability to induce conformational transitions in recombinant α-synuclein protein, forming fluorescently labeled aggregates, which are then monitored in real-time by changes in fluorescence intensity. Specifically, a trace amount of skin tissue lysate is added to the reaction system as a seed, and protein aggregation is induced under isothermal shaking conditions in a substrate system containing a fluorescent dye (such as ThT), with real-time reading of the fluorescence signal. A threshold is set to determine whether the reaction is positive, and quantitative analysis is performed in conjunction with known standard samples. RT-QuIC technology has high sensitivity and specificity in biomarker research for Parkinson's disease and other synuclein diseases, and its detection accuracy and stability have been fully validated through international peer-reviewed studies.

[0043] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An integrated α-synuclein ultrasensitive detector, characterized in that, include: The components include a housing, a processing unit, a microfluidic chip, and a detection unit; a cover plate is provided at the top port of the housing. The processing unit is arranged parallel to the inner side of the cover plate; the bottom of the processing unit is a light emitting component, an ultrasonic processing component, and a magnetic bead moving component; A microfluidic chip is located inside a housing and positioned parallel to the bottom of the processing unit. The microfluidic chip has a mounting through-hole at its center, and a sample processing unit is formed inside the microfluidic chip. The sample processing unit includes a sample processing area and a transparent reaction detection area. The sample processing area and the reaction detection area are spaced apart along a direction away from the mounting through-hole. The reaction detection area corresponds to the area below the light emitting component. The detection unit includes a servo motor and a photosensitive component. The servo motor is located below the microfluidic chip, and a connecting shaft is fixedly mounted on the output shaft of the servo motor. The connecting shaft is detachably fixed to the mounting through hole. The receiving end of the photosensitive component is located below the reaction detection area.

2. The integrated α-synuclein ultrasensitive detector according to claim 1, characterized in that: The sample processing area includes a sample loading chamber and a lysis buffer storage chamber; the sample loading chamber and the lysis buffer storage chamber are arranged at intervals along the surrounding mounting through-holes; the microfluidic channels include a first microfluidic channel, a second microfluidic channel, and a third microfluidic channel. The lysis chamber is located on the side of the sample loading chamber away from the mounting through hole, and the side of the lysis chamber near the mounting through hole is connected to the side of the sample loading chamber away from the mounting through hole through a first microfluidic channel; and the side of the lysis chamber near the mounting through hole is connected to the side of the lysis buffer storage chamber away from the mounting through hole through a second microfluidic channel. The reaction detection zone is located to the left of the pyrolysis chamber; and the right end of the reaction detection zone is connected to the left end of the pyrolysis chamber through a third microfluidic channel. The reaction detection area includes a manifold, a reaction chamber, and a waste liquid chamber; the third microfluidic channel is connected to the manifold at one end away from the lysis chamber, and six reaction chambers are provided on the manifold. A fourth microfluidic channel is provided between the manifold and the six reaction chambers, and a waste liquid chamber is provided at the other end of the manifold; the detection unit also includes a substrate; the substrate is arranged parallel to the bottom of the microfluidic chip; the substrate is fixed to the side plate of the housing with screws; a groove is provided at the top of the substrate; the photosensitive component is arranged on the bottom wall of the groove, located directly below the reaction chamber.

3. The integrated α-synuclein ultrasensitive detector according to claim 2, characterized in that: The middle part of the shell is surrounded and fixed by four side plates. The top of the side plates is a top plate, and the bottom of the shell is a bottom plate. The top plate is fixed to the top of the four side plates around its perimeter, and the bottom plate is fixed to the bottom of the four side plates around its perimeter. One end of the cover plate is hinged to the top of a side plate.

4. The integrated α-synuclein ultrasensitive detector according to claim 3, characterized in that: Six reaction chambers are evenly distributed with through holes around them; the light emitting component has six emitting ends, which are arranged one-to-one with the six reaction chambers; the photosensitive component is arranged one-to-one with the six reaction chambers.

5. The integrated α-synuclein ultrasensitive detector according to claim 4, characterized in that: The photosensitive assembly includes an optical detection plate and a printed circuit board. The bottom of the optical detection plate has a photosensitive element. The printed circuit board is parallel to and fixed to the corresponding position on the substrate with screws. The optical detection plate is parallel to and fixed to the bottom of the printed circuit board with screws. The photosensitive element is electrically connected to the printed circuit board.

6. The integrated α-synuclein ultrasensitive detector according to claim 5, characterized in that: A siphon valve is installed on the second microfluidic channel; a siphon valve is installed on the third microfluidic channel. Siphon valve three and siphon valve four are respectively installed on the first microfluidic flow channel and the fourth microfluidic flow channel.

7. The integrated α-synuclein ultrasensitive detector according to claim 6, characterized in that: The magnetic bead moving assembly contains an electromagnet, which is cylindrical and used to attract magnetic beads in the lysis chamber to cut cells in the sample solution and cause them to lyse.

8. The integrated α-synuclein ultrasensitive detector according to claim 7, characterized in that: The detection unit also includes heating ring one and heating ring two; heating ring one and heating ring two are arranged coaxially with the mounting through hole and are both fixed to the bottom of the groove by fixing column; heating ring one is located below the pyrolysis chamber; heating ring two is located below the reaction chamber.

9. The integrated α-synuclein ultrasensitive detector according to claim 8, characterized in that: The top of the microfluidic chip is provided with corresponding sample dispensing holes at the corresponding positions of multiple sample dispensing chambers and lysis buffer storage chambers; exhaust holes are provided on the top walls of the sample dispensing chamber, lysis buffer storage chamber, lysis chamber, manifold, and waste liquid chamber.

10. An integrated α-synuclein ultrasensitive detector according to claim 9, characterized in that: The detection unit also includes a controller and a touch screen; the controller is located below the substrate and fixed to the bottom wall of the housing; the touch screen is located on the top of the cover plate; the processing unit, servo motor, light emission assembly, ultrasonic processing assembly, magnetic bead moving assembly, heating ring one, heating ring two and the touch screen are all electrically connected to the controller and are all controlled by the controller.

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