Air pressure driven non-wetting multi-step time sequence release centrifugal microfluidic chip and method

Abstract

The application discloses a kind of gas pressure drive wetting-free multi-step timing release centrifugal microfluidic chip and method, it is related to enzymology or microbiology device field, it solves the problem that siphon valve exists complex process and lower reliability in prior art, with the beneficial effect of reasonable structure setting, specific scheme is as follows: a kind of gas pressure drive wetting-free multi-step timing release centrifugal microfluidic chip, buffer main channel includes first ascending section and first descending section communicated in turn, first ascending section gradually approaches center hole, first descending section gradually away from center hole, first descending section is communicated with function chamber, the side of first descending section is communicated with at least one liquid storage cavity, first descending section is communicated with corresponding liquid storage cavity by U-shaped pipe, the side of U-shaped pipe close to first descending section is communicated with the middle part of the side of waste liquid cavity by air pressure balance passage, to make that liquid in liquid storage cavity can enter buffer main channel after waste liquid in waste liquid cavity closes air pressure balance passage.

Description

Technical Field

[0001] This invention relates to the field of microfluidic chip technology, and in particular to a pneumatically driven, time-release centrifugal microfluidic chip and method. Background Technology

[0002] The statements in this section are merely background information related to the present invention and do not necessarily constitute prior art.

[0003] Centrifugal microfluidic chips, with their advantages of requiring no external pump source and ease of integration, are widely used in biochemical analysis, point-of-care diagnostics, and other fields. Compared with other microfluidic technologies, the core advantage of centrifugal microfluidics lies in using centrifugal force as the main driving force for chip functionality, enabling extreme simplicity and high integration at the system level. Retaining this advantage requires precise valve design to ensure that samples from each chamber are released sequentially and transported in a directional manner as needed for the experiment. Currently, mainstream passive valves in centrifugal microfluidics include siphon valves and capillary valves, which utilize the balance between capillary and centrifugal forces to control liquid flow. However, these valves are highly dependent on the wettability of the chip surface and the stability of its wettability. In practical applications, the wettability of the chip material is easily affected by batch variations, environmental conditions, and usage history, often requiring frequent surface modifications to maintain valve performance. This not only increases process complexity but also easily leads to uncontrolled valve opening (opening too early or too late), reducing the reliability of detection results.

[0004] On the other hand, when the sample needs to be shaken (e.g., for mixing or incubation) within the chamber, traditional siphon valves are prone to automatically triggering siphoning due to weakened centrifugal force during the oscillation process at reduced speeds. This causes premature liquid release, preventing the sample from being temporarily stored in the original chamber and disrupting the time sequence of multi-step reactions. Furthermore, once the siphon valve opens, it empties the liquid from the upstream chamber, requiring separate chambers and valves for each cleaning step. This results in complex chip structures and increased manufacturing difficulty. Moreover, multi-valve, multi-chamber systems often rely on manual or robotic arms to open or close valves, reducing the level of automation and increasing the complexity of the chip's operating system.

[0005] More importantly, the design of siphon valves faces significant challenges when multiple chambers need to be opened sequentially. The opening conditions of a siphon valve are determined by a combination of parameters, including the cross-sectional dimensions of the channel, the wettability of the material surface, the radius of the valve from the center, and the rotational speed. If any of these factors fluctuates (e.g., dimensional deviations due to machining tolerances or changes in material surface energy), the actual opening time of the valve will deviate from the design sequence, resulting in premature opening or delayed failure, thus disrupting the entire multi-step reaction process. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide a pneumatically driven, non-wetting, multi-step, time-sequential release centrifugal microfluidic chip, which solves the problems of complex manufacturing processes and low reliability of existing siphon valves.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution: A pneumatically driven, wettability-free, multi-step, time-sequential release centrifugal microfluidic chip is disclosed. The opening and closing of the chip's valves are independent of the chip surface wettability, being pneumatically driven. The chip has one or more first liquid storage units around its central aperture. Each first liquid storage unit includes a buffer solution storage chamber. A release liquid temporary storage chamber is located on one side of the buffer solution storage chamber, communicating with the buffer solution storage chamber to allow buffer solution to gradually enter the release liquid temporary storage chamber during chip rotation. A buffer solution main channel is located on the side of the release liquid temporary storage chamber away from the central aperture. The buffer solution main channel includes a first rising section and a first falling section connected sequentially. The first rising section... As the first descending segment gradually moves closer to the central hole, it gradually moves away from the central hole. The end of the first descending segment away from the first ascending segment is connected to the functional chamber, which is connected to the waste liquid chamber. The side of the first descending segment is connected to at least one storage chamber. The first descending segment is connected to the corresponding storage chamber through a U-shaped pipe. The side of the U-shaped pipe near the first descending segment is connected to the middle of the side of the waste liquid chamber through a pressure balance passage. This allows the liquid in the storage chamber to enter the buffer solution main channel after the waste liquid seals the pressure balance passage in the waste liquid chamber. The chip surface has a first opening at the position of each corresponding chamber for liquid and / or air intake.

[0008] In the pneumatically driven, non-wetting, multi-step, time-release centrifugal microfluidic chip described above, the distance between the waste liquid chamber and the central hole is greater than the distance between the storage chamber and the central hole. The functional chamber is connected to the waste liquid chamber through a functional chamber drainage channel. The connection point between the functional chamber drainage channel and the waste liquid chamber is located at the distal end of the waste liquid chamber, and the connection point between the pneumatically balanced channel and the waste liquid chamber is located at the proximal end of the waste liquid chamber.

[0009] In the pneumatically driven, moisture-free, multi-step, time-release centrifugal microfluidic chip described above, when multiple liquid storage chambers are provided, the distance between each liquid storage chamber and the central hole gradually increases along the radial direction outward of the chip. The U-shaped pipe includes a second rising section and a second falling section, the second rising section is connected to the second falling section, and the second falling section is connected to the side of the liquid storage chamber away from the central hole.

[0010] As described above, a pneumatically driven, moisture-free, multi-step, sequentially released centrifugal microfluidic chip has multiple waste liquid chambers, with adjacent waste liquid chambers connected. The multiple waste liquid chambers are arranged in the same circumferential plane along the clockwise direction of the chip. The functional chamber drainage channel is connected to the first waste liquid chamber. Each waste liquid chamber has two of the first openings. Along the radial direction of the chip, the side of the i-th liquid storage chamber away from the waste liquid chamber is connected to the (i+1)-th waste liquid chamber in the clockwise direction, and the U-shaped pipe on the front side of the i-th liquid storage chamber is connected to the i-th waste liquid chamber in the clockwise direction.

[0011] As described above, in a pneumatically driven, moisture-free, multi-step sequential release centrifugal microfluidic chip, the buffer storage chamber, the release liquid temporary storage chamber, and the liquid storage chamber are arranged sequentially along the counterclockwise direction of the chip; Along the counterclockwise direction of the chip, the buffer storage cavity gets closer and closer to the central hole on the side near the central hole, and the buffer storage cavity is connected to the release liquid temporary storage cavity on the side near the central hole.

[0012] As described above, in a pneumatically driven, non-wetting, multi-step sequential release centrifugal microfluidic chip, an extension section is provided on the side of the release liquid temporary storage chamber away from the buffer storage chamber, and the extension section is connected to the side of the first descending section near the first ascending section. The distance between the connection point of the release fluid storage chamber and the buffer solution storage chamber and the central hole is less than the distance between the storage chamber and the central hole. The distance between the connection point of the release fluid storage chamber and the buffer solution storage chamber and the central hole is less than the distance between the connection point of the first rising section and the first falling section and the central hole.

[0013] The pneumatically driven, moisture-free, multi-step, time-release centrifugal microfluidic chip described above further includes a second liquid storage unit, which is disposed on one side of the first liquid storage unit. The second liquid storage unit includes an upstream liquid storage chamber and a pressurizing chamber. An anti-backflow channel is provided between the outlets of the upstream liquid storage chamber and the pressurizing chamber. The pressurizing chamber is connected to the functional chamber through a downstream liquid channel. The downstream liquid channel is curved toward the central hole. The distance between the pressurizing chamber and the central hole is greater than the distance between the upstream liquid storage chamber and the central hole. The upstream liquid storage chamber and the pressurizing chamber are each provided with a second opening.

[0014] In the pneumatically driven, non-wetting, multi-step sequential release centrifugal microfluidic chip described above, the distance between the bend apex of the downstream liquid channel and the central hole is less than the distance between the liquid surface in the upstream storage chamber and the central hole. Along the counterclockwise direction of the chip, the width of the pressurization chamber gradually decreases on the side away from the downstream liquid channel; The probability of liquid transfer in the downstream liquid channel is positively correlated with the chip rotation speed and negatively correlated with the deceleration ramp slope of the rotating equipment. The chip rotation speed threshold is positively correlated with the volume of the upstream liquid storage chamber.

[0015] As described above, in a pneumatically driven, non-wetting, multi-step sequential release centrifugal microfluidic chip, a flow guide is provided between the buffer storage chamber and the release liquid temporary storage chamber, and the side of the flow guide facing the buffer storage chamber is arc-shaped.

[0016] Secondly, the present invention also provides a method for operating a pneumatically driven, non-wetting, multi-step, time-sequential release centrifugal microfluidic chip, comprising the following: The chip is mounted on a rotating device, which drives the chip to rotate at a set speed, so that the buffer solution in the buffer storage chamber enters the release solution temporary storage chamber. Under the perfusion siphon effect, the liquid in the release fluid storage chamber enters the buffer main channel, and the liquid in the release fluid storage chamber gradually enters the waste liquid chamber. When the liquid in the waste liquid chamber is higher than the connection between the gas pressure balance passage and the waste liquid chamber, the gas pressure balance passage is sealed. As the liquid in the buffer main channel moves, the corresponding U-shaped pipe descending section of the storage chamber generates a pressure drop. The liquid in the storage chamber breaks through the top of the U-shaped pipe and enters the buffer main channel, and gradually flows to the functional chamber. Once the liquid in the release fluid storage chamber is emptied, the drainage process is automatically interrupted, enabling multiple step-by-step releases from a single chamber.

[0017] The beneficial effects of the present invention are as follows: The chip provided by this invention has a first liquid storage unit, which includes a buffer storage chamber and a release liquid temporary storage chamber on one side. During chip rotation, the buffer solution gradually enters the release liquid temporary storage chamber. The main channel of the buffer solution is reasonably set to realize the opening of the perfusion siphon valve, so as to realize the multiple step-by-step release of the buffer solution in the buffer storage chamber. This avoids the situation in the prior art where the capillary siphon valve is opened and all the liquid in the chamber is emptied. It also avoids the failure or premature release of the buffer solution due to changes in the wettability of the valve. As the chip operates, the liquid in each chamber is eventually discharged into the waste liquid chamber. When the liquid in the waste liquid chamber is higher than the connection between the gas pressure balance passage and the waste liquid chamber, the liquid seals the gas pressure balance passage. As the liquid in the buffer main channel moves, the corresponding U-shaped pipe descending section of the storage chamber generates a pressure drop. The liquid in the storage chamber breaks through the top of the U-shaped pipe and enters the buffer main channel, and gradually flows to the functional chamber. The structure of the buffer storage chamber and the release buffer chamber allows the buffer solution in the buffer storage chamber to be released in multiple steps through the release buffer chamber, unlike the traditional valve that is directly connected to the buffer storage chamber. Once the valve is opened, all the liquid in the chamber is emptied. This eliminates the need to configure separate chambers and valves for each cleaning step, thus avoiding an overly complex chip structure. When multiple liquid storage chambers are set up, the reasonable setting of the overall structure allows multiple liquid storage chambers to be opened sequentially, no longer affected by factors such as channel cross-sectional dimensions, material surface wettability, and overlapping start-up speeds of valves in different chambers, thus avoiding affecting the design sequence and ensuring the smooth progress of the multi-step reaction process. Attached Figure Description

[0018] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0019] Figure 1 This is a front view of a pneumatically driven, moisture-free, multi-step, time-release centrifugal microfluidic chip according to one or more embodiments of the present invention.

[0020] Figure 2 This is a graph showing the change of liquid transfer state with rotational speed when the solution volume in the upstream storage chamber of a pneumatically driven, non-wetting, multi-step, sequentially released centrifugal microfluidic chip is 100µL, according to one or more embodiments of the present invention.

[0021] Figure 3 This is a graph showing the change of liquid transfer state with rotational speed when the solution volume in the upstream storage chamber of a pneumatically driven, non-wetting, multi-step, sequentially released centrifugal microfluidic chip is 200µL, according to one or more embodiments of the present invention.

[0022] Figure 4 This is a graph showing the change in liquid transfer state with rotational speed when the solution volume in the upstream storage chamber of a pneumatically driven, non-wetting, multi-step, sequentially released centrifugal microfluidic chip is 300µL, according to one or more embodiments of the present invention.

[0023] Figure 5 This is a graph showing the change in liquid transfer state of a 100µL solution in the upstream reservoir of a pneumatically driven, non-wetting, multi-step, sequentially released centrifugal microfluidic chip according to one or more embodiments of the present invention, as a function of deceleration ramp time.

[0024] Figure 6 This is a graph showing the change in liquid transfer state with deceleration ramp time when the solution volume in the upstream reservoir of a pneumatically driven, non-wetting, multi-step, sequentially released centrifugal microfluidic chip, according to one or more embodiments of the present invention, is 200 µL.

[0025] Figure 7 This is a graph showing the change of liquid transfer state with deceleration ramp time when the solution volume in the upstream reservoir of a pneumatically driven, non-wetting, multi-step, sequentially released centrifugal microfluidic chip, according to one or more embodiments of the present invention, is 300µL.

[0026] Figure 8 This is a diagram showing the effect of deceleration ramp time on the start-up of a pulse infusion siphon valve in a pneumatically driven, non-wetting, multi-step sequential release centrifugal microfluidic chip according to one or more embodiments of the present invention.

[0027] Figure 9 This is a spatial diagram of the rotational speed-volume activation threshold parameter of a centrifugal pneumatic valve in a pneumatically driven, non-wetting, multi-step, time-sequentially released centrifugal microfluidic chip according to one or more embodiments of the present invention.

[0028] Figure 10 This is a Raman spectroscopy result of bacteria captured by a pneumatically driven, non-wetting, multi-step, time-sequential release centrifugal microfluidic chip according to one or more embodiments of the present invention.

[0029] The diagram exaggerates the spacing or dimensions between parts to show their positions; the diagram is for illustrative purposes only.

[0030] The components are: 1. Downstream liquid channel, 2. Functional chamber, 3. Anti-backflow channel, 4. Pressurization chamber, 5. Upstream liquid storage chamber, 6. First pressure balance path, 7. First liquid storage chamber, 8. Second pressure balance path, 9. Second liquid storage chamber, 10. Third pressure balance path, 11. Third liquid storage chamber, 12. First waste liquid chamber, 13. Second waste liquid chamber, 14. Buffer main channel, 15. Third waste liquid chamber, 16. Fourth waste liquid chamber, 17. Gas path, 18. Guide section, 19. Buffer storage chamber, 20. Release liquid temporary storage chamber, 21. Central hole, 22. Extension section, 23. First opening, 24. Second opening, 25. U-shaped pipe, 26. Drainage channel, 27. Third opening. Detailed Implementation

[0031] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0032] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, unless otherwise expressly indicated by the invention, the singular form is also intended to include the plural form. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. As introduced in the background section, existing technologies are prone to problems such as accidental triggering of the siphon valve leading to premature liquid release and the need to configure an independent chamber for each step, resulting in complex structural settings. In order to solve the above technical problems, this invention proposes a pneumatically driven, non-wetting, multi-step sequential release centrifugal microfluidic chip.

[0033] Example 1 In a typical embodiment of the present invention, reference is made to Figure 1 As shown, a pneumatically driven, wettable, multi-step, time-sequential release centrifugal microfluidic chip has a central hole 21 at its center. The central hole 21 is hexagonal or rectangular in shape and can be installed in rotating devices such as centrifuges. One or more first liquid storage units are located around the central hole 21. Each first liquid storage unit includes a buffer storage chamber 19. A release liquid temporary storage chamber 20 is located on one side of the buffer storage chamber 19 and is connected to the buffer storage chamber 19 to allow the buffer solution to gradually enter the release liquid temporary storage chamber 20 during chip rotation. A buffer main channel 14 is located on the side of the release liquid temporary storage chamber 20 away from the central hole 21. 4 includes a first rising section and a first falling section connected in sequence. The first rising section gradually approaches the central hole, and the first falling section gradually moves away from the central hole. The end of the first falling section away from the first rising section is connected to the functional chamber 2. The functional chamber 2 is connected to the waste liquid chamber. The side of the first falling section is connected to at least one liquid storage chamber. The first falling section is connected to the corresponding liquid storage chamber through a U-shaped pipe 25. The side of the U-shaped pipe 25 near the first falling section is connected to the middle of the side of the waste liquid chamber through a gas pressure balance passage, so that after the waste liquid closes the gas pressure balance passage in the waste liquid chamber, the liquid in the liquid storage chamber can enter the buffer solution main channel. The chip surface is provided with a first opening at the position of each corresponding cavity for liquid and / or gas intake. It should be noted that the buffer storage chamber 19, the release fluid temporary storage chamber 20, and the buffer main channel 14 form a pulse perfusion siphon valve. At high speed, the Euler force drives the buffer along the guide plate to the buffer temporary storage chamber. When the liquid in the buffer temporary storage chamber is emptied, the siphon is automatically interrupted, realizing multiple step-by-step releases in a single chamber. This solves the problem of emptying the traditional siphon chamber upon startup, eliminating the need to set up an independent chamber for each cleaning step. After the liquid enters the functional chamber 2, it can react. The U-shaped pipe 25 is inverted U-shaped. The functional chamber 2 is equipped with a gas path, and a third opening 27 is provided at the gas path.

[0034] In addition, the first descending section is connected to the corresponding liquid storage chamber through a U-shaped pipe 25. The side of the U-shaped pipe 25 near the first descending section is connected to the middle of the waste liquid chamber through a pressure balance passage. This allows the liquid in the liquid storage chamber to enter the main channel of the buffer solution after the waste liquid seals the pressure balance passage in the waste liquid chamber. The chip surface is provided with a first opening 23 at the position of each corresponding cavity for liquid and / or air intake. One or two first openings 23 can be provided at each cavity. Liquid can be injected into the corresponding cavity through the first opening 23. The first opening 23 can also ensure the communication between the cavity and the external air. For some first openings, the first openings can be sealed with tape after liquid injection, according to experimental requirements.

[0035] Specifically, the U-shaped pipe 25 is a wave crest pipe, which includes a second rising section and a second falling section. The second rising section is connected to the second falling section, and the second falling section is connected to the side of the storage chamber away from the central hole. The inner diameter of the U-shaped pipe is smaller than the inner diameter of the buffer main channel, so as to ensure that the liquid flow in the buffer main channel 14 can drive the liquid flow in the U-shaped pipe. Moreover, the second falling section in the wave crest pipe is connected to the corresponding waste liquid chamber through the air pressure balance passage, so as to realize the liquid seal air pressure balance passage during the gradual increase of waste liquid, thereby triggering the release of liquid in the storage chamber into the buffer main channel 14.

[0036] It should be noted that the second rising section and the second falling section have a smooth transition. In the U-shaped pipe closest to the central hole 21, the distance between the connection point of the second rising section and the second falling section and the central hole 21 is less than the distance between the connection point of the first rising section and the first falling section and the central hole 21. In other U-shaped pipes, the distance between the connection point of the second rising section and the second falling section and the central hole 21 is greater than the distance between the connection point of the first rising section and the first falling section and the central hole. To ensure the smooth operation of the liquid storage unit detection and to ensure a reasonable structural layout, the buffer storage chamber 19, the release liquid temporary storage chamber 20, and the liquid storage chamber are arranged in sequence along the counterclockwise direction of the chip. The buffer storage chamber 19 is connected to the side of the release liquid temporary storage chamber 20 near the central hole, which facilitates the entry of the buffer solution in the buffer storage chamber into the release liquid temporary storage chamber 20 during chip rotation.

[0037] In this embodiment, a flow guide 18 is provided between the buffer storage chamber 19 and the release liquid temporary storage chamber 20. The flow guide 18 is specifically a flow guide plate. The side of the flow guide 18 facing the buffer storage chamber 19 is arc-shaped, and the side of the buffer storage chamber 19 away from the central hole 21 is arc-shaped, which facilitates the transport of liquid in the buffer storage chamber 19 to the release liquid temporary storage chamber through the flow guide plate provided between the buffer storage chamber 19 and the release liquid temporary storage chamber 20.

[0038] In some examples, an extension section 22 is provided on the side of the release fluid storage chamber 20 away from the buffer storage chamber 19. The extension section 22 is connected to the side of the first descending section near the first ascending section. The extension section 22 is lower than the connection between the release fluid storage chamber 20 and the buffer storage chamber 19. The extension section 22 facilitates the flow of buffer solution to the main buffer channel 14 through the extension section 22, which helps to accelerate the release of buffer solution in the release fluid storage chamber 20. In addition, to ensure the smooth progress of the reaction, the distance between the connection between the release fluid storage chamber 20 and the buffer storage chamber 19 and the central hole 21 is less than the distance between the storage chamber and the central hole 21, and the distance between the connection between the release fluid storage chamber 20 and the buffer storage chamber 19 and the central hole 21 is less than the distance between the connection between the first rising section and the first falling section and the central hole, so as to ensure the smooth opening of the perfusion siphon effect.

[0039] Considering that the liquid in all chambers can eventually be discharged into the waste liquid chamber, the distance between the waste liquid chamber and the central hole 21 is greater than the distance between the storage chamber and the central hole. The buffer main channel 14 is connected to the functional chamber 2. The drainage channel of the functional chamber 2 is connected to the side of the waste liquid chamber away from the central hole to gradually fill the waste liquid chamber. The connection between the drainage channel 26 of the functional chamber 2 and the waste liquid chamber and the connection between the pressure balance channel and the waste liquid chamber are located on opposite sides of the waste liquid chamber, so that the liquid-sealed pressure balance channel can be gradually realized after the liquid enters the waste liquid chamber.

[0040] In this embodiment, there are two or three liquid storage cavities. Of course, four or other quantities can also be set according to the size of the chip. Along the radial direction of the chip, the distance between each liquid storage cavity and the central hole gradually increases. That is, along the radial direction of the chip, multiple liquid storage cavities are arranged sequentially from the inside to the outside, namely the first liquid storage cavity 7, the second liquid storage cavity 9 and the third liquid storage cavity 11. Correspondingly, there are multiple waste liquid chambers. Specifically, there are four waste liquid chambers arranged in the same circumferential plane along the clockwise direction of the chip. The multiple waste liquid chambers are the first waste liquid chamber 12, the second waste liquid chamber 13, the third waste liquid chamber 15, and the fourth waste liquid chamber 16. Adjacent waste liquid chambers are connected by a bend tube. For example, the side of the second waste liquid chamber 13 away from the central hole is connected to the side of the third waste liquid chamber 15 near the central hole. The functional chamber is connected to the first waste liquid chamber through the functional chamber drainage channel 26. Each waste liquid chamber is provided with two first openings 23 and each waste liquid chamber is provided with a gas passage 17. One of the first openings 23 is located at the gas passage 17. The first opening 23 can also be used for rinsing each chamber. Considering the release sequence of liquids in each storage chamber, the first waste liquid chamber 12 is connected to the drainage channel 26 of the functional chamber 2. Waste liquid sequentially fills the first waste liquid chamber 12, the second waste liquid chamber 13, the third waste liquid chamber 15 and the fourth waste liquid chamber 16, thereby sequentially liquid sealing the first pressure balance passage 6, the second pressure balance passage 8 and the third pressure balance passage 10, so that the liquids in each storage chamber are released into the functional chamber 2 in the order of reaction.

[0041] Specifically, along the radial direction of the chip, the side of the i-th (i ≥ 1, i is a natural number) liquid storage chamber away from the waste liquid chamber is connected to the (i+1)-th waste liquid chamber in a clockwise direction. The U-shaped pipe 25 on the front side of the i-th liquid storage chamber is connected to the i-th waste liquid chamber in a clockwise direction. For example, the first liquid storage chamber 7 is connected to the first waste liquid chamber 12 through the first air pressure balance passage 6. A first pipe is provided on the side of the first liquid storage chamber 7 near the central hole 21. The first pipe is connected to the side of the second waste liquid chamber 13 away from the first waste liquid chamber 12. The second rising section in the U-shaped pipe at the inlet side of cavity 9 is connected to the first pipe, so that the second rising section in the second liquid storage cavity 9 is connected to the second waste liquid cavity 13 through the second air pressure balance passage 8; a second pipe is provided on the side of the second liquid storage cavity 9 near the central hole, and the second pipe is connected to the side of the third waste liquid cavity away from the second waste liquid cavity 13; the second rising section in the U-shaped pipe 25 at the inlet side of the third liquid storage cavity 11 is connected to the second pipe, so that the second rising section in the third liquid storage cavity 11 is connected to the second waste liquid cavity through the third air pressure balance passage 10; In this embodiment, one or more sets of first liquid storage units are set in the chip. According to the test requirements, one set of first liquid storage units is used to carry out the test. The above-described method for operating a pneumatically driven, moisture-free, multi-step, time-sequentially released centrifugal microfluidic chip includes the following: The chip is mounted on a rotating device, which drives the chip to rotate at a set speed, so that the buffer solution in the buffer storage chamber enters the release solution temporary storage chamber. Under the perfusion siphon, the liquid in the release liquid storage chamber 20 enters the buffer main channel 14, and the liquid in the release liquid storage chamber 20 gradually enters the first waste liquid chamber 12. When the first waste liquid chamber 12 is not yet full, the first pressure balance passage 6 is in the open state (indirectly connected to the atmosphere through the first pressure balance passage). At this time, the liquid in the buffer main channel will not exert an attraction on the liquid in the U-shaped channel of the first liquid storage chamber, and the first liquid storage chamber remains closed. As the cleaning waste liquid continuously enters the first waste liquid chamber 12, the liquid level in the first waste liquid chamber 12 gradually rises, and the liquid seal closes the first pressure balance passage 6. With the first pressure balance passage 6 closed, the migrating liquid in the buffer main channel 14 thus exerts an attraction on the liquid in the U-shaped channel of the first storage chamber 7. The liquid climbs along the rising section of the U-shaped channel and breaks through the arch, and the reagent in the first storage chamber 7 is released into the buffer main channel 14 and enters the functional chamber 2 (i.e., the reaction chamber). During the emptying process of the first liquid storage chamber 7, since its vent is indirectly connected to the atmosphere through the second pressure balance passage 8 of the second liquid storage chamber 9 (at this time, the second waste liquid chamber is not yet full, and the passage is still open), the pressure inside and outside the chamber remains balanced, and no negative pressure is generated in the first liquid storage chamber to hinder the release of liquid in the chamber, so the liquid can be drained stably. After the first liquid storage chamber is emptied, no additional operation is required. At this time, the second waste liquid chamber 13 is not yet full, and the second pressure balance passage of the second liquid storage chamber 9 is still open. The second liquid storage chamber 9 will not be accidentally triggered. After the cleaning is completed, the waste liquid continues to enter the second waste liquid chamber 13 and finally the second pressure balance passage 8 connected to the second liquid storage chamber 9 is closed by liquid seal, triggering the release of the second liquid storage chamber 9, and so on.

[0042] In the above design, the vent of the previous liquid storage chamber is indirectly connected to the atmosphere by borrowing the pressure balance path of the next liquid storage chamber. This ensures pressure balance during the emptying phase and also enables the vent to be automatically "discarded" after emptying. When the next waste liquid chamber is filled and the pressure balance path is closed by a liquid seal, the vent of the previous emptied chamber is also indirectly closed, without manual intervention. This cascading structure naturally couples the start-up sequence of multiple liquid storage chambers with the waste liquid accumulation process, achieving fully automatic multi-step reaction timing control without the need for external valves or manual operation.

[0043] Depend on Figure 8 As can be seen, the buffer chamber valve did not activate when the deceleration ramp time was 2000, 1800, 1600, 1400, 1200, 1000, 800, and 600 ms; however, the valve successfully activated when the deceleration ramp time was shortened to 400, 200, and 100 ms. The threshold transition range was 400–600 ms, meaning the valve stably opened when the deceleration time was ≤400 ms and stably closed when it was ≥600 ms. This result indicates that, with fixed parameters such as chip rotation speed and sample volume, the activation of the U-valve can be precisely controlled by adjusting the deceleration ramp time, without relying on the multiple coupling parameters required by traditional siphon valves, such as chip surface wettability, channel size, or valve radial position.

[0044] The first liquid storage unit provided in this embodiment allows the buffer solution to gradually enter the release liquid storage chamber during chip rotation. The main channel of the buffer solution is reasonably set to enable the opening of the perfusion siphon valve, realizing the multiple step-by-step release of the buffer solution in the buffer storage chamber. This avoids the situation in the prior art where the opening of the capillary siphon valve empties all the liquid in the chamber, and also avoids buffer solution release failure or premature release due to changes in valve wettability. When the liquid in the waste liquid chamber is higher than the connection between the gas pressure balance passage and the waste liquid chamber, the gas pressure balance passage is sealed by liquid. Subsequently, the movement of liquid in the main channel of the buffer solution causes a pressure drop in the descending section of the U-shaped tube, driving the liquid in the storage chamber to break through the apex of the U-shaped tube and enter the main channel of the buffer solution, and gradually flow to the functional chamber. In this way, it is not necessary to configure an independent chamber and valve for each cleaning step.

[0045] Assuming the main buffer channel has a cross-sectional size of 800μm × 500μm, and the siphon valves in each reservoir have a cross-sectional size of 500μm × 500μm, and the chip material is untreated natural polycarbonate (PC), with a water contact angle of approximately 82°, the capillary pressure ΔPcap ≈ 800μm × 500μm for the main buffer channel. 65 Pa; for the siphon channel of the liquid storage chamber (500 μm × 500 μm), ΔPcap ≈ 80 Pa. The capillary pressure in both channels is less than 100 Pa and is negative, indicating that the capillary force exhibits a repulsive effect on the liquid; In this embodiment, the sequential triggering of the valves in each storage chamber of the multi-chamber timing start-up chip in the first storage unit is achieved by the sequential closure of the gas pressure balance path of each storage chamber by the rising liquid level in the waste liquid chamber. The entire timing control logic relies on the accumulation of waste liquid volume and the opening and closing of each gas pressure path, rather than the surface wettability differences of each siphon channel. In summary, the capillary pressure of the buffer main channel and the siphon channels of each storage chamber in the multi-chamber timing start-up chip are much lower than the centrifugal pressure at the actual operating speed. The opening sequence of each chamber valve is triggered by the step-by-step liquid seal of the gas pressure balance path, without relying on the capillary effect or wettability differences of the channel surface.

[0046] Example 2 The difference between this embodiment and Embodiment 1 is that: A second liquid storage unit is provided at the chip, and the second liquid storage unit is located on one side of the first liquid storage unit. The second liquid storage unit is connected to the first liquid storage unit or is set independently of each other. The second liquid storage unit includes an upstream liquid storage chamber 5 and a pressurizing chamber 4. An anti-backflow channel 3 is provided between the liquid outlets of the upstream liquid storage chamber 5 and the pressurizing chamber 4. The pressurizing chamber 4 is connected to the functional chamber 2 through a downstream liquid channel 1. The downstream liquid channel 1 is curved towards the center. The upstream liquid storage chamber 5 and the pressurizing chamber 4 are respectively provided with second openings 24. Air or liquid is injected into each chamber through the second openings. The second openings 24 at the upstream liquid storage chamber 5 and the pressurizing chamber 4 need to be closed after liquid injection. In this embodiment, the upstream liquid storage chamber 5, the pressurization chamber 4, and the anti-backflow channel 3 form a centrifugal pneumatic valve. The liquid transfer driving force of this valve comes from the synergistic effect of the compressed air expansion force and the centrifugal force, as well as the structural resistance difference between the upstream and downstream channels. It does not depend on the capillary force on the channel surface, and therefore is not sensitive to the wettability of the chip material surface. To rationally arrange the cavities, the upstream liquid storage cavity 5 and the pressurization cavity 4 are arranged outward in sequence along the radial direction of the chip. The distance between the bend of the downstream liquid channel 1 and the central hole is less than the distance between the liquid surface in the upstream liquid storage cavity 5 and the central hole. Along the counterclockwise direction of the chip, the width of the pressurization cavity 4 on the side away from the downstream liquid channel 1 gradually decreases, and the side of the pressurization cavity 4 away from the downstream liquid channel 1 is arc-shaped to facilitate the emptying of the liquid in the pressurization cavity during the liquid drainage stage.

[0047] It should be explained that the downstream liquid channel 1 includes a connecting pipe, which is connected to the pressurization chamber 4 and the anti-backflow channel 3 respectively. The connecting pipe is also connected to the fourth rising section, which is connected to the fourth falling section. The fourth rising section faces the direction of the central hole 21, and the fourth falling section is set away from the direction of the central hole, so that the downstream liquid channel 1 is in an inverted U-shape.

[0048] Specifically, the inner diameter of the anti-backflow channel 3 is smaller than that of the downstream liquid channel 1 to facilitate the flow of liquid to the downstream liquid channel. During the chip acceleration stage, because the top of the inverted U-shaped channel of the downstream liquid channel 1 is closer to the center than the liquid surface in the upstream storage chamber 5, the injection siphon is not activated, allowing liquid to enter the pressurization chamber. The liquid continuously enters the pressurization chamber and compresses the air inside. During the chip deceleration stage, the compressed air expands, pushing the liquid simultaneously in both directions of the anti-backflow channel and the downstream liquid channel. Because the anti-backflow channel is equipped with an anti-backflow structure (specifically a Tesla valve structure), and the channel cross-section (500μm×500μm) is smaller than the cross-section of the downstream liquid channel (800μm×500μm), its reverse flow resistance is much greater than that of the direct flow channel of the downstream liquid channel. Based on the resistance difference between the two channels, the liquid preferentially flows along the downstream liquid channel, breaking through the inverted U-shaped injection siphon dome and completing the directional transfer from upstream to the functional chamber. The valve's opening is controlled by a series of two parameters: rotational speed and deceleration ramp time. The valve will only open when the rotational speed exceeds a threshold for the corresponding volume and the deceleration ramp time is sufficiently short. The liquid transfer driving force of this valve originates from the combined effect of compressed air expansion pressure and centrifugal force, as well as the structural resistance difference between the upstream and downstream channels. It does not rely on capillary forces on the channel surface and is therefore insensitive to the wettability of the chip material surface. Furthermore, this functional structure enables liquid transfer from the distal to the proximal end, providing an option for highly integrated microfluidic chip designs.

[0049] Throughout the process, the driving force for liquid transfer in the second storage unit originates from the combined effect of compressed air pressure and centrifugal force, as well as the structural resistance difference between the upstream and downstream channels, rather than the capillary force relied upon by traditional siphon valves. The following quantitative calculations demonstrate that, given the materials and channel dimensions used in the chip, the contribution of capillary action to liquid transport is negligible; therefore, valve performance is unaffected by the wettability of the chip surface.

[0050] The chip material is polycarbonate (PC). The water contact angle of untreated PC in its natural state is about 82°, corresponding to cos82°≈0.14, which shows weak hydrophobicity. The capillary driving force of aqueous solution on its surface is extremely weak.

[0051] For a rectangular cross-section microchannel, the capillary pressure ΔPcap is given by the Young-Laplace equation: ΔPcap= 2γcosθ×(1 / w+1 / h) In the formula, γ is the surface tension of water (72 × 10⁻⁶). - ³N / m), θ is the contact angle, and w and h are the width and height of the channel cross-section, respectively.

[0052] Specifically, the cross-sectional dimensions of the anti-backflow channel 3 in the chip are 500μm × 500μm, and the cross-sectional dimensions of the downstream liquid channel are 800μm × 500μm. Substituting these parameters, the calculation yields: Capillary pressure of the anti-backflow channel (500μm×500μm): ΔPcap= 2×72×10 - ³×cos82°×(1 / (500×10 -6 )+1 / (500×10 -6 )) ≈ 80 Pa Capillary pressure in the downstream liquid channel (800μm × 500μm): ΔPcap= 2×72×10 - ³×cos82°×(1 / (800×10 -6 )+1 / (500×10 -6 )) ≈ 65 Pa It is evident that, in the natural state of PC material, the capillary pressure in both channels is less than 100 Pa and is negative, indicating that the capillary force exhibits a repulsive effect rather than driving liquid aspiration, and the aqueous solution does not spontaneously capillary wetting and climbing in the channels.

[0053] For comparison, based on the experimental results of Example 1, the minimum operating speed (i.e., the minimum speed required for successful liquid transfer) of the centrifugal pneumatic valve for each sample volume is as follows: 1500 rpm for 100 µL, 2300 rpm for 200 µL, and 4500 rpm for 300 µL. In this example, the radius of the valve inlet from the rotation center is 3.278 cm, the radius of the outlet from the rotation center is 4.689 cm, the radial span is 1.411 cm, and the average radius is 3.98 cm. Taking the minimum operating speed of 1500 rpm (corresponding to a 100 µL sample) for conservative estimation, the centrifugal pressure Pcent on the liquid column is: Pcent=ρ·ω²· ·Δr =1000×(1500×2π / 60)²×0.0398×0.01411 ≈ 1.39×10 4 Pa In the formula, ρ is the liquid density, and ω is the chip rotation speed. Let be the average radius, and Δr be the radius difference between the valve inlet and outlet.

[0054] At this minimum operating speed, the centrifugal pressure (approximately 1.39 × 10⁻⁶) is... 4The centrifugal pressure (Pa) exceeds the capillary pressure (approximately 80 Pa) by 173 times. In actual operation, the capillary effect is completely overwhelmed by the centrifugal force, and it has no observable impact on the liquid transport behavior. For higher operating speeds (2300 rpm and 4500 rpm) at 200 µL and 300 µL, the centrifugal pressure is approximately 3.26 × 10⁻⁶ Pa, respectively. 4 Pa and 1.25×10 5 The ratio of Pa to capillary pressure is 408 times and 1560 times, respectively, showing a more significant advantage.

[0055] To further evaluate the potential impact of changes in surface wettability on valve performance, an extreme degradation scenario is considered: It is assumed that under long-term use or environmental factors, the chip's surface contact angle drops significantly from 82° to 40° (cos40°≈0.77), and the capillary pressure in the anti-backflow channel increases to approximately [missing value]. 440 Pa). Even under the most unfavorable operating conditions—minimum operating speed of 1500 rpm and minimum centrifugal pressure of approximately 1.39 × 10⁻⁶ Pa. 4 Under the condition of Pa, the centrifugal pressure still exceeds the capillary pressure by about 31 times. Therefore, fluctuations in the wettability of the chip surface within a reasonable range will not affect the normal opening and closing function of the valve.

[0056] In summary, the directional transfer of liquid in the second reservoir unit does not rely on capillary action on the channel surface. Even under the lowest operating speed conditions, the centrifugal pressure still far exceeds the capillary pressure (173 times that of normal conditions and 31 times that of extreme degradation conditions), and the valve performance is insensitive to the wettability of the chip material surface. This characteristic eliminates the need for surface modification treatments (such as plasma treatment or hydrophilic coatings) during chip fabrication, significantly simplifying the manufacturing process; it also avoids the impact of changes in surface wettability caused by batch variations in materials, environmental aging, or repeated use on valve performance, ensuring the chip's process robustness and operational reliability.

[0057] It should be noted that whether liquid transfer can occur is related to the chip rotation speed, and the chip rotation speed is positively correlated with the volume of the buffer storage chamber. (See reference...) Figure 2 , Figure 3 and Figure 4 As shown, through experiments, the chip's rotational speed threshold is 1200~1500 rpm when the buffer storage chamber volume is 100µL; the chip's rotational speed threshold is 2100~2300 rpm when the buffer storage chamber volume is 200µL; and the chip's rotational speed threshold is 4000~4500 rpm when the buffer storage chamber volume is 300µL.

[0058] Additionally, whether liquid transfer can occur is related to the deceleration ramp time of the rotating equipment (referring to the acceleration of the rotating equipment, determined by settings of the rotating equipment), see reference. Figure 5 , Figure 6 and Figure 7 As shown, the shorter the deceleration ramp time (i.e., the larger the absolute value of the negative acceleration), the faster the centrifugal force decays. The compressed air in the pressurization chamber generates a large expansion pressure difference in a short time, pushing the liquid to break through the top of the U-shaped valve.

[0059] The above results indicate that the opening of the second liquid storage unit is controlled by two parameters in series: rotational speed and deceleration ramp time. The valve will only open when the rotational speed exceeds the threshold for the corresponding volume and the deceleration ramp time is sufficiently short. This dual-parameter control mechanism provides ample parameter modulation space for the independent timing control of different chambers in the multifunctional chip. This characteristic allows the chip to be unaffected by batch differences in materials and surface condition variations during mass production, exhibiting good process robustness and operational reliability. It provides crucial technical support for the automated timing control of complex multi-step biochemical reaction processes.

[0060] Figure 9 This is a speed-volume parameter space diagram for centrifugal pneumatic valves. The threshold speed increases non-linearly with sample volume: from 100 µL to 200 µL, the threshold increases by approximately 850 rpm; from 200 µL to 300 µL, the threshold increases by approximately 2050 rpm, showing a significant increase. This parameter space diagram provides chip designers with an intuitive operating window reference: for a given chamber volume, the required speed operating range can be directly read from the diagram, providing a design basis for differentiated speed programming of different chamber volumes in multi-chamber chips.

[0061] The pneumatically driven, moisture-free, multi-step sequential release centrifugal microfluidic chip can be used for bacterial capture and Raman detection applications. Specifically, the buffer storage chamber 19 is pre-filled with 800 μL of 0.1×PBS buffer (pH 7.2-7.4). During chip rotation, the buffer gradually enters the main buffer channel 14 through the release liquid temporary storage chamber 20, thereby sequentially releasing the liquid from each storage chamber.

[0062] Functional chamber 2: Pre-filled with 100 μg of LL-37 antimicrobial peptide-functionalized magnetic bead suspension (magnetic bead size approximately 200 nm, surface covalently coupled with maleimide-thiol group to LL-37 peptide, coupling density approximately 20 μg peptide / mg magnetic bead; suspension matrix is ​​0.1×PBS).

[0063] The N-terminus of the LL-37 polypeptide is introduced with a cysteine ​​residue (Cys-LL-37 sequence: Cys-LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES), which electrostatically binds to the negatively charged bacterial membrane surface (lipoteichoic acid / lipopolysaccharide) through the positively charged side chains of its lysine and arginine residues, thereby achieving broad-spectrum bacterial capture. First reservoir 7: Pre-contains 50 μL of sample suspension containing the bacteria to be tested (matrix is ​​0.1×PBS, pH 7.2). Second reservoir 9: Pre-contains 50 μL of washing buffer (0.02% Tween-20 polysorbate-20) to wash away non-specifically adsorbed impurities and unbound bacteria. Third reservoir 11: Pre-contains 50 μL of Raman signal enhancement solution. The Raman signal enhancement solution is an aqueous solution of silver nanoparticles with a particle size of 50-60 nm.

[0064] Upstream storage chamber 5: Pre-filled with 300uL of bacterial release solution with a concentration of 1×PBS + 150mM NaCl (sodium chloride).

[0065] Workflow: Install the chip on the rotating device and run the following procedure: Step 1 (Magnetic Bead Washing): The chip is rapidly started with an acceleration ramp time of 100 ms, and rotated clockwise to 2500 rpm for 15 seconds. The PBS (phosphate buffer solution) in the buffer storage chamber 19 is driven by centrifugal force and flows through the guide plate into the release liquid storage chamber 20. Under the action of the perfusion U-valve, it gradually flows to the functional chamber 2 along the rising and falling sections of the main buffer channel 14. After the magnetic bead washing is completed, the perfusion U-valve is opened, and the liquid enters the first waste liquid chamber 12. The magnetic beads are trapped in the functional chamber 2 under the action of centrifugal force, the liquid level in the waste liquid chamber rises, and the first gas pressure balance passage 6 is blocked by liquid seal. Step 2 (Release of the sample to be tested): Re-initiate the release of liquid in the buffer storage chamber. Under the negative pressure suction generated in the descending section of the first storage chamber, the bacterial sample suspension in the first storage chamber 7 is released into the functional chamber 2 through the U-shaped tube.

[0066] Step 3 (Magnetic Beads Capturing Bacteria): Activate the chip's clockwise-counterclockwise oscillation mode. At this time, the LL-37 functionalized magnetic beads are in full contact with the bacterial suspension in the functional chamber. The positively charged residues of the LL-37 peptides generate strong electrostatic binding with the negatively charged groups on the bacterial membrane surface, forming a magnetic bead-bacteria complex, thus achieving efficient bacterial capture.

[0067] Step 4 (Release of Functional Chamber Solution): The release of liquid from the buffer storage chamber is initiated again. The newly injected buffer raises the liquid level in functional chamber 2, thereby opening the perfusion U-valve. The liquid in the functional chamber is discharged into the waste liquid chamber. The magnetic bead-bacterial complex is trapped at the distal end of the functional chamber, i.e., the bottom of the chamber, under the action of centrifugal force. As waste liquid continues to accumulate, the second waste liquid chamber 13 is filled and the second pressure balance passage 8 is closed by liquid seal.

[0068] Step 5 (Releasing Raman Enhancement Buffer): Re-initiate the release of liquid from the buffer storage chamber, triggering the release of Raman enhancement buffer from the second storage chamber 9. The Raman enhancement buffer flows through the main buffer channel 14, mixes with the magnetic bead-bacteria complex, runs at 3000 rpm for 20 seconds, and then a drop of the magnetic bead-bacteria and Raman signal enhancement buffer complex from the distal end of the functional chamber is added to a glass slide for Raman detection.

[0069] Step 6 (Rinsing the functional chamber to release bacteria from the magnetic beads): Attach a magnetic patch to the drain port of functional chamber 2. Start the chip rotation clockwise to 5000 rpm with a 5000 ms ramp time, run for 20 seconds, then decelerate to 500 rpm with a 100 ms ramp time, run for 20 seconds, completing the transfer of the release liquid from the upstream outlet chamber 5 to functional chamber 2, and then discharge it into the waste liquid chamber through the drain channel 26. Under the action of the high-concentration salt solution, bacteria are released from the surface of the magnetic beads. The reduced centrifugal force at low speed makes it impossible to retain the bacteria and magnetic beads in functional chamber 2. As the liquid flows towards the waste liquid chamber, the magnetic beads are attracted by the magnetic iron sheet, and the bacteria enter the waste liquid chamber.

[0070] refer to Figure 10 As shown, taking *Pseudomonas aeruginosa* as an example, bacteria captured by this microfluidic chip can be detected with stable Raman signals. This approach avoids the 24-72 hour culture time required by traditional blood cultures, providing a culture-free technical route for rapid clinical pathogen identification.

[0071] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A pneumatically driven, moisture-free, multi-step, time-release centrifugal microfluidic chip, wherein the chip has a central hole for mounting in a rotating device, characterized in that, The chip has one or more first liquid storage units around the central hole. Each first liquid storage unit includes a buffer storage chamber. A release liquid temporary storage chamber is provided on one side of the buffer storage chamber. The release liquid temporary storage chamber is connected to the buffer storage chamber so that the buffer solution gradually enters the release liquid temporary storage chamber during chip rotation. A buffer main channel is provided on the side of the release liquid temporary storage chamber away from the central hole. The buffer main channel includes a first rising section and a first falling section connected in sequence. The first rising section gradually approaches the central hole, and the first falling section gradually moves away from the central hole. The end of the first falling section away from the first rising section is connected to a functional chamber. The functional chamber is connected to a waste liquid chamber. The side of the first falling section is connected to at least one liquid storage chamber. The first falling section is connected to the corresponding liquid storage chamber through a U-shaped pipe. The side of the U-shaped pipe near the first falling section is connected to the middle of the side of the waste liquid chamber through a pressure balance passage so that after the waste liquid in the waste liquid chamber closes the pressure balance passage, the liquid in the liquid storage chamber can enter the buffer main channel. The chip surface has a first opening at the position of each corresponding cavity for liquid and / or air intake.

2. The pneumatically driven, non-wetting, multi-step, time-sequential release centrifugal microfluidic chip according to claim 1, characterized in that, The distance between the waste liquid chamber and the central hole is greater than the distance between the storage chamber and the central hole. The functional chamber is connected to the waste liquid chamber through the functional chamber drainage channel. The connection point between the functional chamber drainage channel and the waste liquid chamber is located at the distal end of the waste liquid chamber. The connection point between the pressure balance channel and the waste liquid chamber is located at the proximal end of the waste liquid chamber.

3. The pneumatically driven, wettability-free, multi-step, time-sequential release centrifugal microfluidic chip according to claim 1, characterized in that, When multiple liquid storage cavities are provided, the distance between each liquid storage cavity and the central hole gradually increases along the radial direction outward of the chip; The U-shaped pipe includes a second rising section and a second falling section, the second rising section is connected to the second falling section, and the second falling section is connected to the side of the liquid storage chamber away from the central hole.

4. The pneumatically driven, non-wetting, multi-step, time-sequential release centrifugal microfluidic chip according to claim 3, characterized in that, The waste liquid chamber is provided in multiple ways, and two adjacent waste liquid chambers are connected. The multiple waste liquid chambers are arranged in the same circumferential plane along the clockwise direction of the chip. The functional chamber drainage channel is connected to the first waste liquid chamber. Each waste liquid chamber is provided with two of the first openings. Along the radial direction of the chip, the side of the i-th liquid storage chamber away from the waste liquid chamber is connected to the (i+1)-th waste liquid chamber in the clockwise direction, and the U-shaped pipe on the front side of the i-th liquid storage chamber is connected to the i-th waste liquid chamber in the clockwise direction.

5. The pneumatically driven, non-wetting, multi-step, time-sequential release centrifugal microfluidic chip according to claim 1, characterized in that, Along the counterclockwise direction of the chip, the buffer storage chamber, the release liquid temporary storage chamber, and the liquid storage chamber are arranged in sequence; Along the counterclockwise direction of the chip, the buffer storage cavity gets closer and closer to the central hole on the side near the central hole, and the buffer storage cavity is connected to the release liquid temporary storage cavity on the side near the central hole.

6. The pneumatically driven, wettability-free, multi-step, time-sequential release centrifugal microfluidic chip according to claim 1, characterized in that, An extension section is provided on the side of the release fluid temporary storage chamber away from the buffer storage chamber, and the extension section is connected to the side of the first descending section near the first ascending section. The distance between the connection point of the release fluid storage chamber and the buffer solution storage chamber and the central hole is less than the distance between the storage chamber and the central hole. The distance between the connection point of the release fluid storage chamber and the buffer solution storage chamber and the central hole is less than the distance between the connection point of the first rising section and the first falling section and the central hole.

7. The pneumatically driven, wettability-free, multi-step, time-sequential release centrifugal microfluidic chip according to claim 1, characterized in that, It also includes a second liquid storage unit, which is located on one side of the first liquid storage unit; The second liquid storage unit includes an upstream liquid storage chamber and a pressurizing chamber. An anti-backflow channel is provided between the outlets of the upstream liquid storage chamber and the pressurizing chamber. The pressurizing chamber is connected to the functional chamber through a downstream liquid channel. The downstream liquid channel is curved toward the central hole. The distance between the pressurizing chamber and the central hole is greater than the distance between the upstream liquid storage chamber and the central hole. The upstream liquid storage chamber and the pressurizing chamber are each provided with a second opening.

8. The pneumatically driven, wettability-free, multi-step, time-sequential release centrifugal microfluidic chip according to claim 7, characterized in that, The distance between the bend apex of the downstream liquid channel and the central hole is less than the distance between the liquid surface in the upstream storage cavity and the central hole; Along the counterclockwise direction of the chip, the width of the pressurization chamber gradually decreases on the side away from the downstream liquid channel; The probability of liquid transfer in the downstream liquid channel is positively correlated with the chip rotation speed and negatively correlated with the deceleration ramp slope of the rotating equipment. The chip rotation speed threshold is positively correlated with the volume of the upstream liquid storage chamber.

9. The pneumatically driven, wettability-free, multi-step, time-sequential release centrifugal microfluidic chip according to claim 1, characterized in that, A flow guide is provided between the buffer storage chamber and the release fluid temporary storage chamber, and the side of the flow guide facing the buffer storage chamber is arc-shaped.

10. The method for operating a pneumatically driven, non-wetting, multi-step, time-sequentially released centrifugal microfluidic chip according to any one of claims 1-9, characterized in that, Includes the following: The chip is mounted on a rotating device, which drives the chip to rotate at a set speed, so that the buffer solution in the buffer storage chamber enters the release solution temporary storage chamber. Under the perfusion siphon effect, the liquid in the release fluid storage chamber enters the buffer main channel, and the liquid in the release fluid storage chamber gradually enters the waste liquid chamber. When the liquid in the waste liquid chamber is higher than the connection between the gas pressure balance passage and the waste liquid chamber, the gas pressure balance passage is sealed. As the liquid in the buffer main channel moves, the corresponding U-shaped pipe descending section of the storage chamber generates a pressure drop. The liquid in the storage chamber breaks through the top of the U-shaped pipe and enters the buffer main channel, and gradually flows to the functional chamber. Once the liquid in the release fluid storage chamber is emptied, the drainage process is automatically interrupted, enabling multiple step-by-step releases from a single chamber.

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