A microfluidic disc-based amanita phalloides detection device

Abstract

The application discloses a kind of based on microfluidic disc toxic mushroom detection equipment, including centrifugal temperature control analyzer and detection disc, centrifugal temperature control analyzer inside is provided with placing groove, rotating mechanism is provided in placing groove, optical detection assembly is provided on centrifugal temperature control analyzer, detection disc center position has sample adding cavity, detection disc is provided with expansion cavity, there are a plurality of independent detection cavities in the circumferential distribution on detection disc, first channel corresponding with detection cavity is provided on detection disc, and expansion cavity is embedded with amplification agent;The based on microfluidic disc for toxic mushroom detection equipment, by adopting wax valve physical isolation and denoising agent degradation primer double design, completely solve the false positive caused by free primer, greatly improve the detection signal-to-noise ratio and accuracy, optimize flow channel and chamber layout, prevent liquid backflow and cross contamination, reaction system is more stable, multiple independent detection cavities can synchronously detect multiple toxic mushrooms, matched with control system, and detection is efficient and reliable.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a poisonous mushroom detection device based on a microfluidic disc. Background Technology

[0002] Poisoning incidents caused by accidental ingestion of poisonous mushrooms are frequent. The toxins contained in highly poisonous mushrooms such as those in the genera *Amanita* and *Galerina* are characterized by their potent toxicity, rapid onset of action, and lack of specific antidotes. Ingestion can cause damage to multiple organs, including the liver, kidneys, and nervous system, within a short period, and in severe cases, can lead to death, posing a significant threat to public health. In the wild, poisonous mushrooms are highly similar in appearance to edible mushrooms, making accurate identification difficult for non-professionals and a major cause of accidental poisoning. The widespread use of outdoor recreational activities such as camping, hiking, and mushroom picking further increases the risk of accidental ingestion of highly poisonous mushrooms. Traditional detection of poisonous mushrooms relies on morphological identification by professionals or laboratory nucleic acid and toxin testing. Morphological identification is prone to misjudgment due to experience limitations, while laboratory testing suffers from time-consuming sample delivery, complex procedures, and the need for specialized equipment and facilities, making rapid screening in the wild impossible and often missing the optimal time for treatment after poisoning.

[0003] Existing technologies include rapid detection devices that combine the high-efficiency amplification advantages of recombinant polymerase amplification with the high specificity of clustered, regularly spaced, short palindromic repeat sequences. These devices are compatible with centrifugal temperature-controlled analyzers, and their detection discs typically have an amplification chamber and a detection chamber connected by a connecting channel. Controllable liquid transport is achieved through centrifugal force, allowing for stepwise amplification and signal detection. However, this technology still has significant drawbacks: RPA amplification requires the addition of single-stranded primers, and after amplification, unused free single-stranded primers remain in the reaction system. The CRISPR system is highly sensitive to single-stranded primers; these residual free single-stranded primers can non-specifically activate the trans-cleavage activity of Cas12a, causing the fluorescent reporter probe to be incorrectly cleaved, resulting in non-specific fluorescent signals without a target. This problem significantly reduces the signal-to-noise ratio, easily leading to false positives and hindering rapid and accurate identification of highly poisonous mushrooms.

[0004] Therefore, the present invention provides a poisonous mushroom detection device based on a microfluidic disc. Summary of the Invention

[0005] The purpose of this invention is to provide a poisonous mushroom detection device based on a microfluidic disc, so as to solve the problems mentioned in the background art.

[0006] A microfluidic disc-based poisonous mushroom detection device includes a centrifugal temperature control analyzer and a detection disc. The centrifugal temperature control analyzer has a placement slot inside, and a rotating mechanism is installed inside the placement slot. The detection disc is located on the rotating mechanism, and an optical detection component is installed on the centrifugal temperature control analyzer. The detection disc has a sample application cavity at its center, and an amplification cavity is provided inside the detection disc. The sample application cavity is connected to the amplification cavity. Several independent detection cavities are distributed circumferentially on the detection disc. A first channel corresponding to the detection cavity is provided on the detection disc, and the detection cavity and the amplification cavity are connected through the first channel. An amplification agent is embedded in the amplification cavity. A detection agent and probe specific to different poisonous mushroom species are embedded in the detection cavity. A first wax valve is provided in the first channel, and the inner wall of the first channel downstream of the first wax valve is coated with a noise-reducing agent for degrading the amplification agent. The centrifugal temperature control analyzer is equipped with an extraction mechanism for extracting different samples.

[0007] Preferably, the end of the first channel connected to the amplification cavity is lower than the end connected to the detection cavity, the first channel is connected to the lower part of the amplification cavity, and the first channel is connected to the upper part of the detection cavity.

[0008] Preferably, the first channel includes a wax sealing section and a noise reduction section. The wax sealing section is provided with a first wax valve inside. The noise reduction section is selected from one of a zigzag shape and a spiral shape. The inner wall of the noise reduction section is coated with a noise reduction agent. The wax sealing section and the noise reduction section are connected. The wax sealing section is U-shaped.

[0009] Preferably, the detection disc is arrayed with a waste liquid chamber corresponding to the detection chamber, the detection disc is arrayed with a second channel, and the waste liquid chamber and the detection chamber are connected through the second channel. The second channel is provided with a second wax valve, the melting point of the second wax valve is higher than that of the first wax valve, the inner side of the waste liquid chamber is concave, the second channel is connected to the lower part of the detection chamber and the upper part of the waste liquid chamber.

[0010] Preferably, the centrifugal temperature control analyzer has a display component on its front side, and a cover plate is connected to the centrifugal temperature control analyzer via a hinge. The cover plate is provided with a sealing block corresponding to the placement slot. The optical detection assembly includes an excitation light source and a photoelectric sensor. The excitation light source is located inside the sealed block, and the photoelectric sensor is located inside the placement slot. The centrifugal temperature control analyzer has a power supply module inside, and the power supply module is located on one side of the placement slot.

[0011] Preferably, the rotating mechanism includes a support plate, a drive motor, a power shaft, and a drive wheel. The support plate is disposed inside the placement groove, the drive motor is fixed inside the placement groove, one end of the power shaft is fixedly connected to the center position of the support plate, and the other end of the power shaft is fixedly connected to the output end of the drive motor. The drive wheel is fixed on the power shaft. An electric heating block is disposed inside the support plate, and a temperature sensor is disposed on the support plate.

[0012] Preferably, the support plate has a circumferential array of negative pressure plates, each negative pressure plate has a connecting hole at its center, a negative pressure cylinder is fixedly installed at the bottom of the negative pressure plate, and the connecting hole communicates with the negative pressure cylinder, a piston block is installed inside the negative pressure cylinder, a threaded cylinder is fixedly connected at the center of the piston block, an adjusting screw is threadedly connected inside the threaded cylinder, a power motor is fixedly installed inside the negative pressure cylinder, and the output end of the power motor is fixedly connected to the end of the adjusting screw, a guide cylinder is fixedly installed at the bottom of the piston block, a limiting rod passes through the guide cylinder, and one end of the limiting rod is fixedly connected to the inner wall of the negative pressure cylinder.

[0013] Preferably, the extraction mechanism includes a square box, an adjustment component, a limiting ring, a support block, a crushing cylinder, a crushing disc, and a pumping component. The square box is fixed inside the centrifugal temperature control analyzer and is located on one side of the placement slot. The limiting ring is mounted on the adjustment component, which is used to adjust the position of the limiting ring. The support block is mounted on the limiting ring, and a crushing cylinder is fixedly mounted on the support block. The crushing disc is rotatably mounted inside the crushing cylinder, and the pumping component is mounted inside the square box. The square box has a sealed door connected to its side by hinges; The adjustment assembly includes a square frame, a slider, a threaded rod, and a guide rod. The square frame array is arranged inside a square box, the slider is arranged inside the square frame, the threaded rod is rotatably arranged inside the square frame, and the outer surface of the threaded rod is threadedly connected to the inner surface of the slider. The guide rod is fixed inside the square frame and passes through the slider. The bottom of the limiting ring is fixedly connected to the top of the slider.

[0014] Preferably, a drive gear is rotatably arranged inside the slider, and a transmission gear is rotatably arranged inside the slider, with the transmission gear meshing with the drive gear. A locking block is fixedly connected to the center position of the drive gear. A magnet is arranged inside the limiting ring, and a positioning plate is arranged on the limiting ring. A positioning groove corresponding to the positioning plate is arranged on the support block, and the positioning plate engages with the positioning groove. A locking groove is arranged at the bottom end of the crushing disc, which engages with the locking block. A closed cover is arranged at the top of the crushing cylinder, and a sampling head is arranged on the closed cover, with one end of the sampling head extending into the crushing cylinder. The square box contains a protective box, which is connected to the square frame. Inside the protective box, there are rotating drive wheels arranged in an array, and adjacent drive wheels are connected by belt drive. A drive rod is fixedly connected to the center of one of the drive wheels, and a pulley is fixedly installed on the drive rod. The pulley and the drive wheel are connected by belt drive. Inside the protective box, there is a rotating drive gear arranged on a shaft. A driven wheel is fixedly connected to the center of the drive gear, and the driven wheel is connected to the drive wheel by belt drive. One side of the drive gear is located inside the square frame.

[0015] Preferably, the pumping assembly includes a support frame, a pumping pump, a delivery pipe, a support plate, and a liquid supply head. The support frame is fixed inside a square box, the pumping pump array is fixed inside the support frame, and the input end of the pumping pump is connected to the corresponding sampling head through a pipe. One end of the delivery pipe is connected to the output end of the pumping pump. The support plate is disposed inside the support frame, and the liquid supply head array corresponding to the pumping pump is disposed on the support plate. The delivery pipe is connected to the corresponding liquid supply head. A sliding block is fixedly disposed at the bottom of the support plate, and a threaded screw is threadedly connected inside the sliding block. The threaded screw is rotatably connected to the inside of the support frame. A servo motor is fixedly disposed inside the support frame, and the output end of the servo motor is fixedly connected to one end of the threaded screw.

[0016] Compared with the prior art, the beneficial effects of the present invention are: 1. The microfluidic disc-based poisonous mushroom detection device of this invention significantly improves the detection signal-to-noise ratio by employing a dual design of physical isolation with wax valves and primer degradation with noise-reducing agents. This completely eliminates false positives caused by free primers, greatly enhancing the detection signal-to-noise ratio and accuracy. The optimized flow channel and chamber layout prevents liquid backflow and cross-contamination, resulting in a more stable reaction system. Multiple independent detection chambers can simultaneously detect various poisonous mushrooms. Combined with a control system, the detection is highly efficient and reliable. Furthermore, the dual wax valves provide automatic timing control. With portable equipment, detection can be quickly completed in the field. The operation is simple and requires no professional laboratory.

[0017] 2. The poisonous mushroom detection device based on microfluidic discs described in this invention, by setting up three independent crushing cylinders, can control the start and stop of the crushing discs in each cylinder to simultaneously crush different mushroom samples, quickly prepare multiple test samples, and only need to replace the test discs to complete the sample transport during testing. The operation is simple and effectively improves the testing efficiency.

[0018] 3. The microfluidic disc-based poisonous mushroom detection device of this invention can efficiently drive the sample preparation process through the transmission rod and driven wheel, improving work efficiency. The movement of the slider can synchronously drive the displacement of the drive gear and the transmission gear. The crushing cylinder adopts a detachable design for easy cleaning and maintenance. The crushing cylinder is stably supported by the support block embedded in the limiting ring, which at the same time ensures precise engagement between the crushing disc and the clamping block, and reliable meshing between the transmission gear and the power gear. When the drive wheel rotates, it sequentially drives the pulley, the transmission rod, and the transmission wheel to rotate. The power is transmitted to the power gear through the driven wheel, which in turn drives the transmission gear and the drive gear to rotate. Finally, it drives the clamping block and the crushing disc to rotate at high speed to complete the sample crushing process. This structure can be adjusted and the power can be recycled as needed, stably and efficiently completing the sample preparation for detection, and significantly improving the overall work efficiency. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the structure of the microfluidic disk-based poisonous mushroom detection device of the present invention; Figure 2 This is a schematic diagram of the centrifugal temperature control analyzer of the present invention; Figure 3 This is a schematic diagram of the three-dimensional structure of the support disk of the present invention; Figure 4 This is a three-dimensional structural diagram of the electric heating block and support plate of the present invention; Figure 5 This is a schematic diagram of the three-dimensional structure of the screw negative pressure cylinder of the present invention; Figure 6 This is a top view schematic diagram of the disc being detected in Embodiment 1 of the present invention; Figure 7 This is a partial cross-sectional view of the disc being tested in Embodiment 1 of the present invention; Figure 8 This is a top view schematic diagram of the disc being detected in Embodiment 2 of the present invention; Figure 9 This is a cross-sectional schematic diagram of a portion of the disc being tested in Embodiment 3 of the present invention; Figure 10 This is a schematic diagram of the three-dimensional structure of the placement slot of the present invention; Figure 11 This is a three-dimensional structural diagram of the drive motor and power shaft of the present invention; Figure 12 This is a schematic diagram of the three-dimensional structure of the square box of the present invention; Figure 13 This is a schematic diagram of the three-dimensional structure of the square frame and crushing cylinder of the present invention; Figure 14 This is a schematic diagram of the three-dimensional structure of the protective box of the present invention; Figure 15 This is a schematic diagram of the support frame structure of the present invention.

[0020] In the diagram: 1. Amplification chamber; 2. Detection chamber; 3. First channel; 4. First wax valve; 5. Waste liquid chamber; 6. Second channel; 7. Second wax valve; 8. Sample loading chamber; 9. Centrifugal temperature control analyzer; 10. Display assembly; 11. Placement slot; 12. Cover plate; 13. Sealing block; 14. Excitation light source; 15. Photoelectric sensor; 16. Detection disc; 17. Support plate; 18. Temperature sensor; 19. Drive motor; 20. Electric heating block; 21. Negative pressure plate; 22. Connecting hole; 23. Negative pressure cylinder; 24. Piston block; 25. Guide cylinder; 26. Limiting rod; 27. Threaded cylinder; 28. Adjusting screw; 29. ​​Power motor; 30. Power supply module; 31. Wax sealing section; 32. Noise reduction section; 33. 34. Square box; 35. Sealed door; 36. Square frame; 37. Slider; 38. Drive gear; 39. Transmission gear; 40. Threaded rod; 41. Guide rod; 42. Limiting ring; 43. Locking block; 44. Magnet; 45. Positioning plate; 46. Support block; 47. Positioning groove; 48. Crushing cylinder; 49. Crushing disc; 50. Closing cover; 51. Sampling head; 52. Protective box; 53. Transmission rod; 54. Pulley; 55. Transmission wheel; 56. Driven wheel; 57. Support frame; 58. Pump; 59. Delivery pipe; 60. Support plate; 61. Liquid supply head; 62. Sliding block; 63. Threaded screw; 64. Servo motor; 65. Power shaft; 66. Drive wheel. Detailed Implementation

[0021] 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.

[0022] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0023] Example 1 like Figure 6 and 7 As shown, the microfluidic disc-based poisonous mushroom detection device of this embodiment includes a centrifugal temperature control analyzer 9 and a disposable multiplex detection disc 16 adapted thereto.

[0024] The centrifugal temperature control analyzer 9 has an internal rotating mechanism that can achieve stepless speed regulation from 0 to 3000 rpm and supports alternating forward and reverse rotation. The heating temperature can be precisely controlled by the electric heating block 20, with a temperature control range of 25-50℃ and an accuracy of ±0.5℃. The excitation light source 14 and photoelectric sensor 15 can scan the detection chamber 2 and collect fluorescence signals. The power supply module 30 is a rechargeable lithium battery with a battery life of ≥4 hours, which meets the needs of field use.

[0025] The detection disc 16 is made of polydimethylsiloxane as the substrate and is integrally formed by soft photolithography. The disc is circular with a diameter of 8cm and a thickness of 2mm. A snap-fit ​​structure is reserved in the center to match the rotating shaft of the centrifugal temperature control analyzer 9 to ensure coaxiality during rotation.

[0026] The detection disc 16 has a circular sample loading chamber 8 at its center. The sample loading chamber 8 is connected to an annular amplification chamber 1. Twelve independent detection chambers 2 are evenly distributed radially outside the amplification chamber 1. Twelve independent waste liquid chambers 5 are distributed correspondingly outside the detection chambers 2. The amplification chamber 1 is connected to each detection chamber 2 through an independent first channel 3. Each detection chamber 2 is connected to the corresponding waste liquid chamber 5 through an independent second channel 6. The connection between each chamber and the channel is rounded to avoid liquid retention. The amplification chamber 1 is pre-embedded with an amplification agent, specifically RPA lyophilized powder reagent. This reagent contains universal fungal primers or multiple specific primers and is embedded by vacuum freeze-drying process to ensure stability during room temperature storage.

[0027] Twelve detection chambers 2 are arranged in a ring array outside amplification chamber 1, with the center-to-center angle between adjacent detection chambers 2 being 30°. Each detection chamber 2 is pre-embedded with a detection reagent specific to different poisonous mushroom species and an ssDNA fluorescent reporter probe. The embedding system of each chamber is designed specifically for each species, as follows: First and seventh detection chambers: embedding Cas12a-crRNA ribonucleoprotein complex and ssDNA fluorescent reporter probes targeting specific gene targets of the deadly Amanita phalloides; The second and eighth detection chambers contain Cas12a-crRNA ribonucleoprotein complexes and ssDNA fluorescent reporter probes targeting specific gene targets of Amanita fulva. The third and ninth detection chambers: embed Cas12a-crRNA ribonucleoprotein complex and ssDNA fluorescent reporter probe targeting specific gene targets of Amanita muscaria. The fourth and tenth detection chambers are used to embed Cas12a-crRNA ribonucleoprotein complexes and ssDNA fluorescent reporter probes targeting specific gene targets of Amanita muscaria, serving as negative controls / exclusion items. The fifth and eleventh detection chambers are used to embed a Cas12a-crRNA-ribonucleoprotein complex and an ssDNA fluorescent reporter probe targeting Amanita muscaria; these serve as positive internal controls to confirm successful DNA extraction. The sixth and twelfth detection chambers served as blank controls.

[0028] The linear first channel 3 is arranged radially, with one end connected to the lower part of the amplification chamber 1 and the other end connected to the upper part of the detection chamber 2. The end connected to the amplification chamber 1 is 0.5 mm lower than the end connected to the detection chamber 2, forming an upward slope from the amplification chamber to the detection chamber 2. The first channel 3 is filled with paraffin material to form the first wax valve 4. The paraffin material is hot-melted and poured in, then cooled and shaped, and fits tightly against the U-shaped cavity wall to form a physical seal. On the side of the first wax valve 4 near the detection chamber 2, the inner wall of the first channel 3 is coated with DNA exonuclease as a noise-reducing agent. After coating, it is fixed by vacuum drying to ensure enzyme activity stability.

[0029] The waste liquid chamber 5 is an inner recessed waist-shaped chamber. Its recessed structure faces the center of the detection disc 16 and is connected to the second channel 6. The straight second channel 6 is arranged radially. One end is connected to the lower part of the detection chamber 2 and the other end is connected to the upper part of the waste liquid chamber 5. The interior is filled with paraffin material to form a second wax valve 7. The melting point of the second wax valve 7 is higher than that of the first wax valve 4 to ensure the effectiveness of timing control.

[0030] Example 2 like Figure 8 As shown, the microfluidic disk-based poisonous mushroom detection device in this embodiment is basically the same as that in Embodiment 1, except that: The first channel 3 is not linear; it includes a U-shaped wax-sealing section 31 near the amplification chamber 1 and a spiral noise-reducing section 32 near the detection chamber 2. The U-shaped bending radius of the wax-sealing section 31 is 1 mm, and it is filled with paraffin material to form the first wax valve 4. The paraffin material is hot-melted and poured in, then cooled and shaped to fit tightly against the U-shaped cavity wall, forming a physical seal. The noise-reducing section 32 has a three-turn spiral structure with a pitch of 1 mm and an inner diameter of 2 mm. Its inner wall is coated with DNA exonuclease as a noise-reducing agent. After coating, it is fixed by vacuum drying to ensure enzyme activity stability.

[0031] Example 3 like Figure 9 As shown, the microfluidic disk-based poisonous mushroom detection device in this embodiment is basically the same as that in Embodiment 1, except that: The first channel 3 is not straight. It includes a U-shaped wax-sealing section 31 near the amplification chamber 1 and a zigzag noise-reducing section 32 near the detection chamber 2. The U-shaped bending radius of the wax-sealing section 31 is 1 mm. The interior is filled with paraffin material to form the first wax valve 4. The paraffin material is hot-melted and poured in, then cooled and shaped to fit tightly against the U-shaped cavity wall, forming a physical seal. The noise-reducing section 32 has a four-fold structure. Its inner wall is coated with DNA exonuclease as a noise-reducing agent. After coating, it is vacuum dried and fixed to ensure enzyme activity stability.

[0032] Example 4 like Figure 1-15As shown, the present invention provides a technical solution: a poisonous mushroom detection device based on a microfluidic disc, comprising a centrifugal temperature control analyzer 9 and a detection disc 16. The centrifugal temperature control analyzer 9 has a placement slot 11 inside, and a rotating mechanism is installed inside the placement slot 11. The detection disc 16 is located on the rotating mechanism. An optical detection component is installed on the centrifugal temperature control analyzer 9. A sample application chamber 8 is located at the center of the detection disc 16. An amplification chamber 1 is installed inside the detection disc 16, and the sample application chamber 8 is connected to the amplification chamber 1. The detection disc 16 is circumferentially divided... The instrument has several independent detection chambers 2. The detection disc 16 is provided with a first channel 3 corresponding to the detection chamber 2. The detection chamber 2 and the amplification chamber 1 are connected through the first channel 3. The amplification chamber 1 contains an amplifying agent. The detection chamber 2 contains a detection agent and probe specific to different poisonous mushroom species. The first channel 3 is provided with a first wax valve 4. The inner wall of the first channel 3 downstream of the first wax valve 4 is coated with a noise-reducing agent for degrading the amplifying agent. The centrifugal temperature control analyzer 9 is provided with an extraction mechanism for extracting different samples. The rotating mechanism supports the detection disc 16 and drives its rotation. The optical detection component allows for sample inspection, and the sample addition chamber 8 facilitates sample addition. Utilizing the phase-change characteristics of the first wax valve 4, the amplification chamber 1 and detection chamber 2 are physically separated during the amplification stage, completely isolating the amplification and detection reactions in space. This fundamentally prevents residual free single-stranded primers from entering the detection chamber 2 during the amplification stage and non-specifically activating the Cas12a trans-cleavage activity, effectively avoiding false positive results. Furthermore, after the amplification reaction is complete, by adjusting the centrifugation speed and heating temperature, the blockage of the first wax valve 4 can be precisely breached, allowing the amplification product to enter the detection chamber 2 through the first channel 3. As it flows through the channel, it comes into full contact with the noise-reducing agent coated on the inner wall. The noise-reducing agent specifically degrades residual free single-stranded primers, further reducing background noise and improving the signal-to-noise ratio. Thus, by achieving controllable amplification and detection timing, the accuracy and reliability of poisonous mushroom detection are significantly improved.

[0033] Furthermore, the end of the first channel 3 connected to the amplification cavity 1 is lower than the end connected to the detection cavity 2. The first channel 3 is connected to the lower part of the amplification cavity 1 and to the upper part of the detection cavity 2. The first channel 3 forms an upward slope from the amplification chamber 1 to the detection chamber 2. During the amplification stage, the drive detection disc 16 rotates at a low speed to generate a certain centrifugal force. Since the tilt direction of the channel is opposite to the direction of the centrifugal force, the first wax valve 4 in the molten state can be stably maintained in the preset position under the combined action of its own gravity and the friction of the channel wall. It will not be pushed away from the first channel 3 by the low-speed centrifugal force. This effectively avoids the problem of wax seal failure caused by wax valve displacement or detachment, ensures reliable isolation between the two chambers during the amplification stage, and prevents the amplification reagent from entering the detection chamber 2 in advance, thereby ensuring the integrity of the amplification reaction and the accuracy of subsequent detection. The inlet of the first channel 3 is located at the lower part of the amplification chamber 1, and the outlet is located at the upper part of the detection chamber 2. After the amplification product is distributed into the detection chamber 2 by centrifugal force, the liquid level in the detection chamber 2 gradually rises from bottom to top. The channel outlet is always located above the liquid level, which can effectively prevent the liquid in the detection chamber 2 from flowing back to the amplification chamber 1 along the first channel 3 during subsequent reactions or centrifugation, avoiding cross-contamination, ensuring that the reaction system in each detection chamber 2 is independent and stable, and further improving the accuracy of detection.

[0034] Furthermore, the first channel 3 includes a wax sealing section 31 and a noise reduction section 32. The wax sealing section 31 is equipped with a first wax valve 4. The noise reduction section 32 is selected from one of a zigzag shape and a spiral shape. The inner wall of the noise reduction section 32 is coated with a noise reduction agent. The wax sealing section 31 and the noise reduction section 32 are connected. The wax sealing section 31 is U-shaped. The U-shaped structure forms a physical limit on the first wax valve 4. The centrifugal force generated by low-speed centrifugation during the amplification stage cannot push the wax valve out of the U-shaped limit area, effectively preventing the centrifugal force during amplification from throwing the wax valve out of the first channel 3, thus avoiding wax seal failure. The stable wax seal further ensures the spatiotemporal separation of the amplification and detection stages. Compared to a straight channel, the zigzag or spiral denoising section 32 significantly increases the effective length and flow channel complexity of the first channel 3. When the amplification product flows through this section, the actual fluid movement distance is greatly extended, compared to the section coated with denoising agent. The increased contact time with the inner wall provides more time for the enzymatic reaction. At the same time, the zigzag or helical structure induces turbulence or secondary flow in the fluid, enhancing the shear mixing between the liquid and the tube wall. This allows the free single-stranded primers to approach the surface of the noise-reducing agent coating more frequently, improving degradation efficiency. This dual mechanism of extended residence time and enhanced mixing ensures that the single-stranded primers are fully degraded before entering detection chamber 2, minimizing background noise and significantly improving the signal-to-noise ratio and detection sensitivity of the CRISPR detection system, effectively reducing the probability of false positive results.

[0035] Furthermore, the detection disc 16 is arrayed with waste liquid chambers 5 corresponding to the detection chamber 2, and the detection disc 16 is arrayed with a second channel 6. The waste liquid chamber 5 and the detection chamber 2 are connected through the second channel 6. A second wax valve 7 is provided in the second channel 6. The melting point of the second wax valve 7 is higher than that of the first wax valve 4. The inner side of the waste liquid chamber 5 is concave. The second channel 6 is connected to the lower part of the detection chamber 2 and to the upper part of the waste liquid chamber 5. The waste liquid chamber 5 and the second wax valve 7 are designed so that after the detection reaction is completed, the temperature can be increased to above the melting point of the second wax valve 7 to melt it. Combined with centrifugal force, the waste liquid in the detection chamber 2 can be transferred to the waste liquid chamber 5, realizing the automatic collection of the liquid after the reaction. This avoids the waste liquid from being left in the detection chamber 2 for a long time, which could lead to leakage and pollution. The design of the second wax valve 7 having a higher melting point than the first wax valve 4 ensures that the second wax valve 7 remains closed during the amplification product transfer stage. The second wax valve 7 is only opened at a higher temperature after the first wax valve 4 is opened, the amplification product enters the detection chamber 2 and the detection reaction is completed, thus realizing the timing control of the multi-step reaction liquid flow. The waste liquid chamber 5 is designed with an inner concave structure. When the waste liquid enters the waste liquid chamber 5 under the action of centrifugal force, the concave shape can physically lock the liquid. Even if centrifugation stops, the waste liquid is difficult to flow back to the detection chamber 2 along the second channel 6, thereby effectively preventing the collected waste liquid from contaminating the detection chamber 2. The inlet of the second channel 6 is located at the lower part of the detection chamber 2, and the outlet is located at the upper part of the waste liquid chamber 5. After the waste liquid is distributed into the waste liquid chamber 5 by centrifugal force, the liquid level in the waste liquid chamber 5 gradually rises from bottom to top. The channel outlet is always located above the liquid level, which can effectively prevent the liquid in the waste liquid chamber 5 from flowing back to the detection chamber 2 along the second channel 6.

[0036] Furthermore, the centrifugal temperature control analyzer 9 has a display component 10 on its front side, and a cover plate 12 is connected to the centrifugal temperature control analyzer 9 by a hinge. The cover plate 12 has a sealing block 13 corresponding to the placement slot 11. The optical detection component includes an excitation light source 14 and a photoelectric sensor 15. The excitation light source 14 is located inside the sealing block 13, and the photoelectric sensor 15 is located inside the placement slot 11. The centrifugal temperature control analyzer 9 has a power supply module 30 inside, and the power supply module 30 is located on one side of the placement slot 11. The cover plate 12 can move in a circular motion to shield and protect the upper part of the placement groove 11 from the sealing block 13. The detection results can be displayed through the display component 10. The excitation light source 14 and photoelectric sensor 15 can scan the detection cavity 2 and collect fluorescence signals.

[0037] Furthermore, the rotating mechanism includes a support plate 17, a drive motor 19, a power shaft 65, and a drive wheel 66. The support plate 17 is disposed inside the placement groove 11, the drive motor 19 is fixed inside the placement groove 11, one end of the power shaft 65 is fixedly connected to the center position of the support plate 17, and the other end of the power shaft 65 is fixedly connected to the output end of the drive motor 19. The drive wheel 66 is fixed on the power shaft 65. An electric heating block 20 is disposed inside the support plate 17, and a temperature sensor 18 is disposed on the support plate 17. After the detection disc 16 is placed on the support plate 17, when the detection disc 16 is driven to rotate, the drive motor 19 will work and drive the power shaft 65 to rotate. When the power shaft 65 rotates, it will drive the drive wheel 66 and the support plate 17 to rotate, which in turn will drive the detection disc 16 to rotate. The electric heating block 20 can adjust the temperature of the detection disc 16, and the temperature can be detected by the temperature sensor 18.

[0038] Furthermore, the support plate 17 has a circumferential array of negative pressure plates 21. A connecting hole 22 is provided at the center of the negative pressure plate 21. A negative pressure cylinder 23 is fixedly provided at the bottom of the negative pressure plate 21, and the connecting hole 22 is connected to the negative pressure cylinder 23. A piston block 24 is provided inside the negative pressure cylinder 23. A threaded cylinder 27 is fixedly connected at the center of the piston block 24. An adjusting screw 28 is threadedly connected inside the threaded cylinder 27. A power motor 29 is fixedly provided inside the negative pressure cylinder 23, and the output end of the power motor 29 is fixedly connected to the end of the adjusting screw 28. A guide cylinder 25 is fixedly provided at the bottom of the piston block 24. A limiting rod 26 passes through the guide cylinder 25, and one end of the limiting rod 26 is fixedly connected to the inner wall of the negative pressure cylinder 23. After the detection disc 16 is placed on the support plate 17, the bottom of the detection disc 16 is in contact with the top of the negative pressure plate 21. The control motor 29 will drive the adjusting screw 28 to rotate, which will drive the piston block 24 to move synchronously through the threaded cylinder 27. The guide cylinder 25 and the limiting rod 26 will guide the piston block 24 to move smoothly. When the piston block 24 moves down, the air inside the negative pressure plate 21 will be extracted through the connecting hole 22, thereby generating negative pressure inside the negative pressure plate 21, which will effectively limit the position of the detection disc 16.

[0039] Furthermore, the extraction mechanism includes a square box 33, an adjustment component, a limiting ring 41, a support block 45, a crushing cylinder 47, a crushing disc 48, and a pumping component. The square box 33 is fixed inside the centrifugal temperature control analyzer 9 and is located on one side of the placement slot 11. The limiting ring 41 is mounted on the adjustment component, which is used to adjust the position of the limiting ring 41. The support block 45 is mounted on the limiting ring 41, and the crushing cylinder 47 is fixedly mounted on the support block 45. The crushing disc 48 is rotatably mounted inside the crushing cylinder 47. The pumping component... The adjustment assembly includes a square frame 35, a slider 36, a threaded rod 39, and a guide rod 40. The square frame 35 is arranged in an array inside the square box 33. The slider 36 is arranged inside the square frame 35. The threaded rod 39 is rotatably arranged inside the square frame 35, and the outer surface of the threaded rod 39 is threadedly connected to the inner surface of the slider 36. The guide rod 40 is fixed inside the square frame 35 and passes through the slider 36. The bottom of the limiting ring 41 is fixedly connected to the top of the slider 36. Rotating the threaded rod 39 adjusts the position of the slider 36. The guide rod 40 guides the slider 36, allowing it to move smoothly. When the slider 36 moves, the limiting ring 41 and the support block 45 work together to move the crushing cylinder 47, which can adjust the position of the crushing cylinder 47. At the same time, the position of the transmission gear 38 can be adjusted, and whether the transmission gear 38 meshes with the power gear 56 can be adjusted. This allows the crushing disc 48 inside the crushing cylinder 47 to move and crush the mushrooms. There are three sets of crushing cylinders 47, which can be used to place different samples for crushing and preparing test samples. For subsequent testing, only the test disc 16 needs to be replaced, which facilitates the rapid transport of the prepared samples and improves testing efficiency.

[0040] Furthermore, a drive gear 37 is rotatably mounted inside the slider 36, and a transmission gear 38 is rotatably mounted inside the slider 36, with the transmission gear 38 meshing with the drive gear 37. A locking block 42 is fixedly connected to the center of the drive gear 37. A magnet 43 is mounted inside the limiting ring 41, and a positioning plate 44 is mounted on the limiting ring 41. A positioning groove 46 corresponding to the positioning plate 44 is mounted on the support block 45, and the positioning plate 44 engages with the positioning groove 46. A locking groove is provided at the bottom of the crushing disc 48, which engages with the locking block 42. A closing cover 49 is provided at the top of the crushing cylinder 47, and a sampling head 50 is mounted on the closing cover 49, with one end of the sampling head 50 extending into the crushing cylinder. Inside the square box 33, a protective box 51 is installed, and the protective box 51 is connected to the square frame 35. Inside the protective box 51, a transmission wheel 54 is arranged in an array and rotates. Adjacent transmission wheels 54 are connected by belt drive. A transmission rod 52 is fixedly connected to the center of one of the transmission wheels 54. A pulley 53 is fixedly installed on the transmission rod 52. The pulley 53 and the drive wheel 66 are connected by belt drive. Inside the protective box 51, a power gear 56 is rotatably installed via a shaft. A driven wheel 55 is fixedly connected to the center of the power gear 56. The driven wheel 55 and the transmission wheel 54 are connected by belt drive. One side of the power gear 56 is located inside the square frame 35. When the slider 36 moves, it drives the drive gear 37 and transmission gear 38 to move. The crushing cylinder 47 is detachable for cleaning or maintenance. The support block 45 is placed inside the limiting ring 41 to support the crushing cylinder 47. At the same time, it will cause the crushing disc 48 to engage with the locking block 42. When the transmission gear 38 meshes with the power gear 56, the drive wheel 66 rotates, which drives the pulley 53 to rotate. The pulley 53 rotates, which drives the transmission rod 52 to rotate. The transmission rod 52 rotates, which drives the transmission wheel 54 to rotate. When the transmission wheel 54 rotates, it drives the power gear 56 to rotate through the driven wheel 55. The power gear 56 drives the transmission gear 38 to rotate, which in turn drives the drive gear 37 to rotate. When the drive gear 37 rotates, it drives the locking block 42 to rotate, which in turn drives the crushing disc 48 to rotate. The crushing disc 48 can crush the sample. Before crushing, a reaction liquid can be added to the crushing cylinder 47. After the sample reacts, different reaction samples can be prepared, which improves the work efficiency.

[0041] Furthermore, the pumping assembly includes a support frame 57, a pumping pump 58, a delivery pipe 59, a support plate 60, and a liquid supply head 61. The support frame 57 is fixed inside the square box 33. The array of pumping pumps 58 is fixed inside the support frame 57, and the input end of the pumping pump 58 is connected to the corresponding sampling head 50 through a pipe. One end of the delivery pipe 59 is connected to the output end of the pumping pump 58. The support plate 60 is set inside the support frame 57. The array of liquid supply heads 61 corresponding to the pumping pump 58 is set on the support plate 60, and the delivery pipe 59 is connected to the corresponding liquid supply head 61. A sliding block 62 is fixedly set at the bottom of the support plate 60. A threaded screw 63 is threadedly connected inside the sliding block 62, and the threaded screw 63 is rotatably connected to the inside of the support frame 57. A servo motor 64 is fixedly set inside the support frame 57, and the output end of the servo motor 64 is fixedly connected to one end of the threaded screw 63. When different samples need to be tested, after replacing the corresponding test disc 16, the servo motor 64 is driven to rotate, which causes the threaded screw 63 to rotate. When the threaded screw 63 rotates, the sliding block 62 moves, which adjusts the position of the support plate 60, and in turn adjusts the position of the liquid supply head 61. After the liquid supply head 61 moves above the sample addition chamber 8, the corresponding pump 58 operates to extract the sample from inside the pulverizing cylinder 47 through the sampling head 50. The collected liquid sample is added to the test disc 16 through the liquid supply head 61, achieving the purpose of quickly changing different samples for testing and improving testing efficiency.

[0042] While the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the scope of the present invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the scope of the present invention, should fall within the protection scope of the present invention.

Claims

1. A poisonous mushroom detection device based on a microfluidic disc, characterized in that: It includes a centrifugal temperature control analyzer (9) and a detection disc (16). The centrifugal temperature control analyzer (9) has a placement slot (11) inside, and a rotating mechanism is provided inside the placement slot (11). The detection disc (16) is located on the rotating mechanism. An optical detection component is provided on the centrifugal temperature control analyzer (9). The detection disc (16) has a sample loading cavity (8) at its center. The detection disc (16) is provided with an amplification cavity (1), and the sample loading cavity (8) is connected to the amplification cavity (1). Several independent detection cavities (2) are distributed around the circumference of the detection disc (16). The detection disc (16) is provided with a first channel (3) corresponding to the detection cavity (2), and the detection cavity (2) is connected to the amplification cavity (1) through the first channel (3). The amplification cavity (1) is embedded with an amplifying agent. The detection cavity (2) is embedded with a detection agent and probe specific to different poisonous mushroom species. The first channel (3) is provided with a first wax valve (4), and the inner wall of the first channel (3) downstream of the first wax valve (4) is coated with a noise-reducing agent for degrading the amplifying agent. The noise-reducing agent is a DNA exonuclease. The centrifugal temperature control analyzer (9) is equipped with an extraction mechanism for extracting different samples; The first channel (3) is connected to the amplification cavity (1) at one end, which is lower than the end connected to the detection cavity (2). The first channel (3) is connected to the lower part of the amplification cavity (1) and to the upper part of the detection cavity (2). The first channel (3) includes a wax sealing section (31) and a noise reduction section (32). The wax sealing section (31) is provided with a first wax valve (4). The noise reduction section (32) is selected from one of the zigzag shape and the spiral shape. The inner wall of the noise reduction section (32) is coated with a noise reduction agent. The wax sealing section (31) and the noise reduction section (32) are connected. The wax sealing section (31) is U-shaped. The detection disc (16) is arranged with a waste liquid chamber (5) corresponding to the detection chamber (2). The detection disc (16) is arranged with a second channel (6). The waste liquid chamber (5) and the detection chamber (2) are connected through the second channel (6). The second channel (6) is provided with a second wax valve (7). The melting point of the second wax valve (7) is higher than that of the first wax valve (4). The inner side of the waste liquid chamber (5) is concave. The second channel (6) is connected to the lower part of the detection chamber (2) and to the upper part of the waste liquid chamber (5).

2. The poisonous mushroom detection device based on a microfluidic disk according to claim 1, characterized in that: The centrifugal temperature control analyzer (9) has a display component (10) on its front side. The centrifugal temperature control analyzer (9) is connected to a cover plate (12) by a hinge. The cover plate (12) is provided with a sealing block (13) corresponding to the placement groove (11). The optical detection assembly includes an excitation light source (14) and a photoelectric sensor (15). The excitation light source (14) is located inside the sealing block (13), and the photoelectric sensor (15) is located inside the placement slot (11). The centrifugal temperature control analyzer (9) is equipped with a power supply module (30), and the power supply module (30) is located on one side of the placement slot (11).

3. The poisonous mushroom detection device based on a microfluidic disk according to claim 2, characterized in that: The rotating mechanism includes a support plate (17), a drive motor (19), a power shaft (65), and a drive wheel (66). The support plate (17) is disposed inside the placement slot (11). The drive motor (19) is fixed inside the placement slot (11). One end of the power shaft (65) is fixedly connected to the center position of the support plate (17), and the other end of the power shaft (65) is fixedly connected to the output end of the drive motor (19). The drive wheel (66) is fixed on the power shaft (65). An electric heating block (20) is disposed inside the support plate (17), and a temperature sensor (18) is disposed on the support plate (17).

4. The poisonous mushroom detection device based on a microfluidic disk according to claim 3, characterized in that: The support plate (17) has a circumferential array of negative pressure plates (21). A connecting hole (22) is provided at the center of the negative pressure plate (21). A negative pressure cylinder (23) is fixedly provided at the bottom of the negative pressure plate (21), and the connecting hole (22) is connected to the negative pressure cylinder (23). A piston block (24) is provided inside the negative pressure cylinder (23). A threaded cylinder (27) is fixedly connected at the center of the piston block (24). An adjusting screw (28) is threadedly connected inside the threaded cylinder (27). A power motor (29) is fixedly provided inside the negative pressure cylinder (23), and the output end of the power motor (29) is fixedly connected to the end of the adjusting screw (28). A guide cylinder (25) is fixedly provided at the bottom of the piston block (24). A limiting rod (26) is passed through the guide cylinder (25), and one end of the limiting rod (26) is fixedly connected to the inner wall of the negative pressure cylinder (23).

5. The poisonous mushroom detection device based on a microfluidic disk according to claim 4, characterized in that: The extraction mechanism includes a square box (33), an adjustment component, a limiting ring (41), a support block (45), a crushing cylinder (47), a crushing disc (48), and a pumping component. The square box (33) is fixed inside the centrifugal temperature control analyzer (9), and the square box (33) is located on one side of the placement slot (11). The limiting ring (41) is set on the adjustment component, and the position of the limiting ring (41) is adjusted by the adjustment component. The support block (45) is set on the limiting ring (41), and the crushing cylinder (47) is fixedly set on the support block (45). The crushing disc (48) is rotatably set inside the crushing cylinder (47), and the pumping component is set inside the square box (33). The square box (33) has a sealed door (34) connected to its side by a hinge; The adjustment assembly includes a square frame (35), a slider (36), a threaded rod (39), and a guide rod (40). The square frames (35) are arranged in an array inside a square box (33). The slider (36) is arranged inside the square frame (35). The threaded rod (39) is rotatably arranged inside the square frame (35), and the outer surface of the threaded rod (39) is threadedly connected to the inner surface of the slider (36). The guide rod (40) is fixed inside the square frame (35) and passes through the slider (36). The bottom of the limiting ring (41) is fixedly connected to the top of the slider (36).

6. The poisonous mushroom detection device based on a microfluidic disk according to claim 5, characterized in that: A drive gear (37) is rotatably arranged inside the slider (36), and a transmission gear (38) is rotatably arranged inside the slider (36), and the transmission gear (38) meshes with the drive gear (37). A locking block (42) is fixedly connected to the center position of the drive gear (37). A magnet (43) is arranged inside the limiting ring (41), and a positioning plate (44) is arranged on the limiting ring (41). A positioning groove (46) corresponding to the positioning plate (44) is arranged on the support block (45), and the positioning plate (44) and the positioning groove (46) are engaged. A slot is arranged at the bottom of the crushing disc (48) and engages with the locking block (42). A closed cover (49) is arranged at the top of the crushing cylinder (47), and a sampling head (50) is arranged on the closed cover (49), and one end of the sampling head (50) extends into the crushing cylinder (47). The square box (33) is equipped with a protective box (51), and the protective box (51) is connected to the square frame (35). The protective box (51) is equipped with a rotating array of transmission wheels (54), and adjacent transmission wheels (54) are connected by belt drive. One of the transmission wheels (54) is fixedly connected to a transmission rod (52) at its center. The transmission rod (52) is fixedly equipped with a pulley (53), and the pulley (53) and the drive wheel (66) are connected by belt drive. The protective box (51) is equipped with a rotating shaft of a power gear (56), and the power gear (56) is fixedly connected to a driven wheel (55) at its center. The driven wheel (55) and the transmission wheel (54) are connected by belt drive, and one side of the power gear (56) is located inside the square frame (35).

7. A poisonous mushroom detection device based on a microfluidic disk according to claim 6, characterized in that: The pumping assembly includes a support frame (57), a pumping pump (58), a delivery pipe (59), a support plate (60), and a liquid supply head (61). The support frame (57) is fixed inside a square box (33). The pumping pumps (58) are arrayed and fixed inside the support frame (57), and the input end of the pumping pumps (58) is connected to the corresponding sampling head (50) through a pipe. One end of the delivery pipe (59) is connected to the output end of the pumping pumps (58). The support plate (60) is set inside the support frame (57) and connected to the pumping pumps (58). The corresponding liquid supply head (61) array is set on the support plate (60), and the delivery pipe (59) is connected to the corresponding liquid supply head (61). A sliding block (62) is fixedly set at the bottom of the support plate (60). A threaded screw (63) is threadedly connected inside the sliding block (62), and the threaded screw (63) is rotatably connected to the support frame (57). A servo motor (64) is fixedly set inside the support frame (57), and the output end of the servo motor (64) is fixedly connected to one end of the threaded screw (63).

Images