Microfluidic substrate, microfluidic chip and its operation method

Abstract

A microfluidic substrate (10), a microfluidic chip, and a method for operating the same are disclosed. The microfluidic substrate (10) includes a flow channel structure comprising a delivery channel (100) and a plurality of detection groups (200). Each detection group (200) includes a first fluid groove (210), a first microchannel (240), a buffer groove (220), a second microchannel (250), and a second fluid groove (230) connected in sequence. The first fluid groove (210) is connected to the delivery channel (100), and a reagent is disposed in the second fluid groove (230) of at least one detection group (200). The microfluidic substrate (10) has a rotation axis (11), and the detection groups (200) are located on the side of the delivery channel (100) opposite to the rotation axis (11). The distances from the first fluid groove (210), the buffer groove (220), and the second fluid groove (230) in each detection group (200) to the rotation axis (11) increase sequentially.

Description

Technical Field

[0001] This disclosure relates to the field of analytical testing, and more specifically, to a microfluidic substrate, a microfluidic chip, and a method for operating the same. Background Technology

[0002] Microfluidics integrates the basic operational units of biological, chemical, and medical analysis processes, such as sample preparation, reaction, separation, and detection, onto a single chip, automating the entire analytical process. Due to its enormous potential in biology, chemistry, and medicine, it has developed into a new interdisciplinary research field encompassing biology, chemistry, medicine, fluid dynamics, electronics, materials science, and mechanics.

[0003] Centrifugal microfluidics, which drives and controls fluid flow in microchannels using centrifugal force, boasts advantages such as high integration, automation, miniaturization, and the ability to detect multiple samples or indicators in parallel, making it an important branch of microfluidic chip technology. However, current microfluidic chips, limited by their structural design, are prone to cross-contamination and difficulty in controlling reaction time during detection, leading to inaccurate results and failing to meet user needs. Summary of the Invention

[0004] In view of this, the present disclosure provides a microfluidic substrate, a microfluidic chip and its operation method. By setting a buffer tank before the reaction tank with pre-loaded reagents, the control of the fluid is enhanced, ensuring that the reagents pre-loaded in different reaction tanks react with the fluid at the same time, avoiding cross-contamination of pre-loaded reagents in different reaction tanks, thereby ensuring reliable detection results.

[0005] This disclosure provides a microfluidic substrate comprising a flow channel structure, including a delivery channel and multiple detection groups. Each detection group includes a first fluid tank, a first microchannel, a buffer tank, a second microchannel, and a second fluid tank connected in sequence. The first fluid tank is connected to the delivery channel, and a reagent is disposed in the second fluid tank of at least one detection group. The microfluidic substrate has a rotation axis, and the detection groups are located on the side of the delivery channel opposite to the rotation axis. The distances from the first fluid tank, buffer tank, and second fluid tank in each detection group to the rotation axis increase sequentially. This microfluidic substrate can prevent the liquid in the first fluid tank from prematurely contacting the pre-filled reagent in the second fluid tank, precisely control the reaction time of the reagent in the second fluid tank, and also avoid the risk of cross-contamination of reagents in different detection groups.

[0006] In a specific embodiment of the microfluidic substrate provided in the first aspect of this disclosure, when there is fluid in the first microchannel, the fluid in the first microchannel, the buffer groove, the second microchannel, and the second fluid groove are formed into a closed groove. Thus, after the microfluidic substrate is encapsulated (e.g., mated with a cover plate), the buffer groove, the second microchannel, and the second fluid groove communicate with the first fluid groove only through the first microchannel.

[0007] In the above scheme, when the fluid flows along the sidewall of the first fluid tank through the delivery channel to the bottom of the first fluid tank (the part of the first fluid tank away from the rotation axis), that is, during the stage where the fluid delivery channel enters the first fluid tank to achieve quantitative measurement (this stage requires preventing the fluid from entering the second fluid tank), the inlet of the first microchannel at the bottom of the first fluid tank (the opening of the first microchannel connected to the first fluid tank) will be sealed due to the interfacial tension of the fluid. The fluid continuing to flow into the first fluid tank is further driven by centrifugal force into the first microchannel, and the air sealed in the buffer tank and the second fluid tank will be compressed, generating reverse pressure. When the reverse pressure and the surface tension of the fluid reach equilibrium with the centrifugal force, the fluid will stop flowing, thereby forming a stable gas-liquid interface. This prevents the fluid from directly entering the second fluid tank during this stage and avoids premature reaction between the fluid and the pre-filled reagent in the second fluid tank.

[0008] In a specific embodiment of the first aspect of this disclosure, a microfluidic substrate is provided, wherein a first microchannel is configured to have a first length such that, at a first rotational frequency not greater than that of the microfluidic substrate, a fluid from a first fluid tank and a gas present in a buffer tank form a gas-liquid interface, and such a gas-liquid interface exists in the first microchannel or at the connection between the first microchannel and the buffer tank.

[0009] In a specific embodiment of the first aspect of this disclosure, the microfluidic substrate has a first length of 0.1 to 5 mm, a width of 0.1 to 1 mm, and a depth of 0.1 to 0.5 mm.

[0010] In a specific embodiment of the first aspect of this disclosure, the microfluidic substrate includes a buffer groove comprising a first sidewall and a second sidewall. The first sidewall includes a first inlet communicating with a first microchannel, and the second sidewall is opposite to the first sidewall and includes a first outlet communicating with a second microchannel.

[0011] In a specific embodiment of the first aspect of this disclosure, the microfluidic substrate has a second sidewall that is planar and perpendicular to the direction along the rotation axis to the first inlet.

[0012] In another specific embodiment of the first aspect of this disclosure, the microfluidic substrate provided has a second sidewall area without a first outlet, which includes at least one recessed portion. The distance from the bottom of the at least one recessed portion to the rotation axis is greater than the distance from the first outlet to the rotation axis.

[0013] In the above scheme, during the stage where the fluid in the delivery channel fills the first fluid tank, if some fluid flows into the buffer tank, because the microfluidic substrate is in a rotating state, this part of the fluid will accumulate in the recessed part, and the distance from the bottom of the recessed part to the rotation axis is greater than the distance from the first outlet to the rotation axis. The fluid accumulated in the recessed part will not enter the first outlet and thus will not enter the second fluid tank. That is, the risk of fluid entering the second fluid tank at this stage is further reduced.

[0014] In a specific embodiment of the first aspect of this disclosure, a microfluidic substrate is provided, in each buffer groove, a recessed portion is provided on both sides of the first outlet, and the first outlet is located in the middle of the second sidewall.

[0015] In the above scheme, regardless of whether the preset rotation direction of the microfluidic chip is clockwise or counterclockwise, during the stage when the fluid in the delivery channel fills the first fluid groove, the recessed part can be used to gather the fluid entering the buffer groove, thereby reducing the risk of the fluid entering the second fluid groove during this stage.

[0016] In another specific embodiment of the first aspect of this disclosure, a microfluidic substrate is provided, in each buffer groove, a recessed portion is provided on one side of the first outlet, the first outlet is located in the middle of the second sidewall, and the recessed portion and the first outlet are arranged sequentially along the preset rotation direction of the microfluidic substrate.

[0017] In another specific embodiment of the first aspect of this disclosure, a microfluidic substrate is provided in each buffer groove, a recessed portion is provided on one side of the first outlet, the first outlet is located at one end of the second sidewall and along the preset rotation direction of the microfluidic substrate, and the recessed portion and the first outlet are arranged sequentially.

[0018] In the above scheme, since the first outlet is located at one end of the second sidewall, it is equivalent to increasing the design area of ​​the recessed part. That is, compared with the first outlet being located in the middle of the second sidewall, the design area of ​​the recessed part in the second sidewall can be increased, and the design volume of the recessed part can be increased. In other words, the amount of fluid that the recessed part can store is increased. During the stage when the fluid in the conveying channel fills the first fluid tank, the risk of fluid flowing into the second fluid tank is further reduced.

[0019] In a specific embodiment of the first aspect of this disclosure, a microfluidic substrate is provided, in each buffer groove, a first inlet is located in the middle of a first sidewall.

[0020] In another specific embodiment of the first aspect of this disclosure, in the microfluidic substrate, in each buffer groove, the first inlet is located at one end of the first sidewall, and the first inlet and the first outlet are arranged sequentially along the preset rotation direction of the microfluidic substrate.

[0021] In the above scheme, compared with the first inlet being located in the middle of the first sidewall, the distance between the first inlet and the first outlet is increased along the preset rotation direction. During the stage when the fluid in the conveying channel fills the first fluid tank, the risk of the fluid falling directly onto the first outlet is reduced when the fluid flows into the buffer tank through the first inlet, thereby further reducing the risk of the fluid flowing into the second fluid tank during this stage.

[0022] In a specific embodiment of the microfluidic substrate provided in the first aspect of this disclosure, each detection group further includes a first siphon channel in each buffer groove. One end of the first siphon channel is connected to a recessed portion to communicate with the buffer groove, and the other end of the first siphon channel is connected to a second fluid groove. The inner diameter of the first siphon channel is smaller than the inner diameter of the second microchannel, and the distance from a portion of the first siphon channel to the rotation axis is smaller than the distance from the first outlet to the rotation axis.

[0023] After the fluid in the first fluid tank is introduced into the second fluid tank, some of the fluid in the buffer tank may be confined in the recessed part. In this case, the first siphon channel can introduce the fluid in the recessed part into the second fluid tank, thereby improving the utilization rate of the fluid.

[0024] In a specific embodiment of the first aspect of this disclosure, the volume of the first fluid channel is smaller than the volume of the second fluid channel.

[0025] In another specific embodiment of the microfluidic substrate provided in the first aspect of this disclosure, the volume of the first fluid channel is greater than the volume of the second fluid channel, and the volume of the first fluid channel is less than or equal to the sum of the volumes of the second fluid channel and the buffer channel.

[0026] In the above scheme, residual fluid in the first fluid tank can be avoided, thereby ensuring that the fluid metered by the first fluid tank can enter the buffer tank and the second fluid tank, and preventing fluid from accumulating in the first fluid tank. By making the volume of the first fluid tank larger than the volume of the second fluid tank, it can be ensured that the fluid metered by the first fluid tank can fill the second fluid tank.

[0027] In another specific embodiment of the first aspect of this disclosure, the volume of the first fluid channel is greater than the sum of the volumes of the second fluid channel and the buffer channel.

[0028] For example, in some embodiments of this disclosure, the volumes of the first fluid tanks may be the same or different, the volumes of the buffer tanks may be the same or different, the volumes of the second fluid tanks may be the same or different, and the volumes of the first fluid tank, the buffer tank, and the second fluid tank may range from 1 microliter to 50 microliters.

[0029] In a specific embodiment of the first aspect of this disclosure, the microfluidic substrate has a non-closed annular shape, with the center of the circle containing the annulus being the axis of rotation; or, the transport channel has a non-closed annular shape, with the distance from the first end of the transport channel to the axis of rotation being less than the distance from the second end of the transport channel to the axis of rotation, and the distance from the first end to the second end increasing sequentially.

[0030] In the above scheme, when the microfluidic substrate is rotated, it is beneficial for the fluid to be evenly distributed in the delivery channel, so that the fluid flows evenly into the first fluid tank in each detection group.

[0031] A specific embodiment of the microfluidic substrate provided in the first aspect of this disclosure may further include a first waste liquid tank. The first waste liquid tank is connected to one end of a delivery channel. The first waste liquid tank and the delivery channel are arranged sequentially along a preset rotation direction of the microfluidic substrate.

[0032] In the above scheme, after the fluid in the conveying channel fills the first fluid tank, it flows into the first waste liquid tank to avoid the fluid in the conveying channel being compressed and continuing to enter the first fluid tank in the detection group, which would cause the fluid pressure to be too high and damage the gas-liquid interface. That is, in the stage of making the fluid in the conveying channel fill the first fluid tank, the risk of the fluid breaking through the buffer tank and entering the second fluid tank is reduced.

[0033] In a specific embodiment of the microfluidic substrate provided in the first aspect of this disclosure, when the first waste liquid tank and the delivery channel are arranged sequentially along a preset rotation direction of the microfluidic substrate, the distance of the first waste liquid tank from the rotation axis is greater than the distance from any first fluid tank to the rotation axis. Furthermore, because the first fluid tank is located on the side of the delivery channel opposite to the rotation axis, the actual distance from the first waste liquid tank to the rotation axis is also greater than the distance from the delivery channel to the rotation axis.

[0034] In the above scheme, the fluid in the conveying channel, while rotating, will first pass through all the first fluid tanks and then through the first waste liquid tank. During this process, the first fluid tanks are filled with fluid first, thus ensuring the quantitative function of the first fluid tanks. Moreover, among the first waste liquid tank, the conveying channel, and the first fluid tanks, the first waste liquid tank is the one furthest from the axis of rotation. Under the action of centrifugal force, the fluid in the conveying channel that has not entered the first fluid tanks will all flow into the first waste liquid tank, thus ensuring that there is no fluid accumulation in the conveying channel, reducing the risk of fluid mixing in different first fluid tanks, and not affecting the quantitative function of the first fluid tanks. This helps to avoid cross-contamination and improve the accuracy of the first fluid tanks in quantitatively measuring the fluid.

[0035] A specific embodiment of the microfluidic substrate provided in the first aspect of this disclosure may further include a mixing tank and a second siphon channel. The mixing tank includes two inlets and one outlet, one end of the second siphon channel is connected to the outlet of the mixing tank, and the other end of the second siphon channel is connected to a delivery channel. The distance from a portion of the second siphon channel to the rotation axis is less than the distance from the mixing tank to the rotation axis.

[0036] A specific embodiment of the microfluidic substrate provided in the first aspect of this disclosure may further include a sample tank, a sample metering tank, a sample overflow tank, a third siphon channel, a diluent tank, a diluent metering tank, a diluent overflow tank, and a fourth siphon channel. The sample metering tank is connected to the sample tank, and the distance from the sample metering tank to the rotation axis is greater than the distance from the sample tank to the rotation axis. The sample overflow tank is connected to the sample tank, and the distance from the sample overflow tank to the rotation axis is greater than the distance from the sample metering tank to the rotation axis. One end of the third siphon channel is connected to the sample metering tank, and the other end of the third siphon channel is connected to one of the two inlets of the mixing tank, and the distance from a portion of the second siphon channel to the rotation axis is less than the distance from the sample metering tank to the rotation axis. The diluent metering tank is connected to the diluent tank, and the distance from the diluent metering tank to the rotation axis is greater than the distance from the diluent tank to the rotation axis. The diluent overflow tank is connected to the diluent tank, and the distance from the diluent overflow tank to the rotation axis is greater than the distance from the diluent metering tank to the rotation axis. One end of the fourth siphon channel is connected to the diluent metering tank, and the other end of the fourth siphon channel is connected to the other of the two inlets of the mixing tank. The distance from part of the fourth siphon channel to the rotation axis is less than the distance from the diluent metering tank to the rotation axis.

[0037] The microfluidic substrate provided in one specific embodiment of the first aspect of this disclosure may further include a channel layer and a substrate. The channel structure is formed in the channel layer. The substrate is located on the opposite side of the channel layer from the side where the first fluid channel, the first microchannel, the buffer channel, the second microchannel, and the second fluid channel are disposed. The substrate is bonded to the channel layer or the substrate and the channel layer are integrally formed.

[0038] A second aspect of this disclosure provides a microfluidic chip, which includes a cover plate and a microfluidic substrate as described in the first aspect. The cover plate and the microfluidic chip are coupled together on one side of the microfluidic substrate where a first fluid channel, a first microchannel, a buffer channel, a second microchannel, and a second fluid channel are provided.

[0039] This disclosure provides a method for operating a microfluidic chip, wherein the microfluidic chip includes a cover plate and a microfluidic substrate. The microfluidic substrate includes a flow channel structure, which includes a delivery channel and multiple detection groups. Each detection group includes a first fluid tank, a first microchannel, a buffer tank, a second microchannel, and a second fluid tank connected in sequence. The first fluid tank is connected to the delivery channel. A reagent is disposed in the second fluid tank of at least one detection group. The microfluidic substrate has a rotation axis. The detection groups are located on the side of the delivery channel opposite to the rotation axis. The distances from the first fluid tank, buffer tank, and second fluid tank in each detection group to the rotation axis increase sequentially. The operating method includes: driving the microfluidic chip to rotate at a second rotation frequency so that fluid from the delivery channel enters the first fluid tank and forms a gas-liquid interface in the first microchannel or at the connection between the first microchannel and the buffer tank; after the first fluid tank in each detection group is filled with liquid, driving the microfluidic chip to rotate at a third rotation frequency so that fluid enters the buffer tank and the second fluid tank; wherein the third rotation frequency is greater than the second rotation frequency.

[0040] In a specific embodiment of the microfluidic chip operation method provided in the third aspect of this disclosure, a first microchannel is configured to have a first length, such that at a first rotation frequency not greater than that of the microfluidic substrate, fluid from a first fluid tank and gas present in a buffer tank form a gas-liquid interface, and such a gas-liquid interface exists in the first microchannel or at the connection between the first microchannel and the buffer tank. A second rotation frequency is not greater than the first rotation frequency, and a third rotation frequency is greater than the first rotation frequency; or the second rotation frequency is not greater than the first rotation frequency, and when the microfluidic chip is driven to rotate at the third rotation frequency, the rotation mode is reciprocating motion. Attached Figure Description

[0041] Figure 1 This is a schematic diagram of a partial area of ​​a microfluidic substrate provided in an embodiment of the present disclosure;

[0042] Figure 2 For example Figure 1 A schematic diagram of a planar structure of a detection group in a microfluidic chip is shown.

[0043] Figure 3 A schematic diagram of a planar structure of another detection group in a microfluidic substrate provided in an embodiment of the present disclosure;

[0044] Figure 4A schematic diagram of a planar structure of another detection group in a microfluidic substrate provided in an embodiment of the present disclosure;

[0045] Figure 5 A schematic diagram of a planar structure of another detection group in a microfluidic substrate provided in an embodiment of the present disclosure;

[0046] Figure 6 A schematic diagram of a planar structure of another detection group in a microfluidic substrate provided in an embodiment of the present disclosure;

[0047] Figure 7 A schematic diagram of a planar structure of another detection group in a microfluidic substrate provided in an embodiment of the present disclosure;

[0048] Figure 8 This is a schematic diagram of a planar structure of a microfluidic chip provided in an embodiment of the present disclosure;

[0049] Figure 9 This is a cross-sectional schematic diagram of a portion of a microfluidic chip according to an embodiment of the present disclosure;

[0050] Figure 10 A cross-sectional schematic diagram of a portion of a microfluidic chip provided in an embodiment of this disclosure;

[0051] Figures 11-15 for Figure 8 The diagram shows the operation flow of the microfluidic chip. Detailed Implementation

[0052] 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.

[0053] Microfluidics refers to the science and technology involved in systems that use microchannels (tens to hundreds of micrometers in size) to process or manipulate tiny fluids (volumes ranging from nanoliters to microliters). It is an emerging interdisciplinary field involving chemistry, fluid physics, microelectronics, new materials, biology, and biomedical engineering. Due to their miniaturization and integration characteristics, microfluidic devices are often called microfluidic chips, and can also be referred to as lab-on-a-chip or micro-total analytical systems.

[0054] In a microfluidic chip, multiple detection slots (such as the second fluid slot in the embodiment below) are provided. These detection slots are pre-filled with reagents; for example, different detection slots may contain different reagents. This allows for multiple detections of a sample in a single detection process. Each detection slot is provided with a receiving slot (such as the first fluid slot in the embodiment below) to pre-store the fluid to be injected into each detection slot. In actual detection processes, before injecting the sample-containing fluid into the detection slots, the fluid needs to be injected into the receiving slots to pre-store the fluid for each detection slot. After fluid has been injected into each receiving slot, the fluid in the receiving slots can be injected into the detection slots by means such as increasing the rotation speed. However, in actual processes, during the stage of injecting fluid into the receiving slots to pre-store the fluid, the fluid in the receiving slots may flow into the detection slots and mix with the reagents in those slots, prematurely initiating the reaction, potentially leading to errors in the detection results.

[0055] The embodiments of this disclosure provide a microfluidic substrate, a microfluidic chip, and a method for operating the same, which can solve the aforementioned technical problems. The microfluidic substrate includes a flow channel structure, comprising a delivery channel and multiple detection groups. Each detection group includes a first fluid tank, a first microchannel, a buffer tank, a second microchannel, and a second fluid tank connected in sequence. The first fluid tank is connected to the delivery channel, and a reagent is disposed in the second fluid tank of at least one detection group. The microfluidic substrate has a rotation axis, and the detection groups are located on the side of the delivery channel opposite to the rotation axis. The distances from the first fluid tank, buffer tank, and second fluid tank in each detection group to the rotation axis increase sequentially. Thus, by setting a buffer tank to separate the first fluid tank and the second fluid tank, during the stage when the fluid in the delivery channel fills the first fluid tank, the fluid flowing out of the first fluid tank can be prevented from directly entering the second fluid tank. This reduces the risk of fluid entering the second fluid tank and re-entering the first fluid tank from the second fluid tank (at which point the fluid has already come into contact with the reagent), thereby reducing the risk of cross-contamination of reagents in each detection group. In addition, by setting a buffer tank, the fluid can be prevented from directly entering the second fluid tank during this stage to react with the reagent prematurely, thereby precisely controlling the reaction time of the reagent in the second fluid tank.

[0056] In actual processes, the flow of fluid from the delivery channel into the second fluid tank can be divided into two stages. In the first stage, at low speed, the fluid flows along the sidewall of the first fluid tank through the delivery channel towards the bottom of the first fluid tank (the part of the first fluid tank opposite to the axis of rotation). Due to interfacial tension, the inlet of the first microchannel at the bottom of the first fluid tank is sealed. The fluid continuing to flow into the first fluid tank is further driven by centrifugal force into the first microchannel, where the air sealed in the buffer tank and the second fluid tank is compressed, generating reverse pressure. When the reverse pressure and the surface tension of the fluid reach equilibrium with the centrifugal force, the fluid stops flowing, forming a stable gas-liquid interface in the first microchannel or at the junction of the first microchannel and the buffer tank. When all the first fluid tanks are full, excess fluid in the delivery channels is discharged (e.g., excess fluid flows into an overflow tank, which can be the first waste liquid tank in the following embodiment). A fixed amount of fluid is then independently pre-stored in each of the first fluid tanks. In the second stage, the rotation speed is increased, and the increased centrifugal force breaks the gas-liquid interface equilibrium, causing the fixed amount of fluid independently pre-stored in each of the first fluid tanks to simultaneously flow into the corresponding second fluid tank through the buffer tank connected to it. This allows the fluid to react with the reagent pre-loaded in the second fluid tank, and the trapped air is discharged through the first microchannel. This scheme ensures that the reagent pre-loaded in each of the second fluid tanks simultaneously comes into contact with the fluid and begins to react, while avoiding cross-contamination of the reagents in each of the second fluid tanks.

[0057] The buffer tank separating the first and second fluid tanks is designed to prevent fluid from flowing into the second fluid tank during the first stage and coming into contact with the reagents pre-loaded there, thus prematurely initiating the reaction and affecting the detection results. If the first and second fluid tanks are directly connected via microchannels, in practice, it was found that when the first fluid tank is filled in the first stage, before a stable gas-liquid interface forms, some fluid may flow into certain second fluid tanks and prematurely initiate the reaction by contacting the pre-loaded reagents. Furthermore, in the implementation where the first and second fluid tanks are directly connected via microchannels, it was also found that when the first fluid tank is filled in the first stage, the gas-liquid interface formed in some microchannels can extend into the second fluid tank under centrifugal force, meaning the fluid contacts the pre-loaded reagents in the second fluid tank prematurely, initiating the reaction. This is especially true for lyophilized microsphere reagents with porous structures pre-loaded in the second fluid tank, whose microsphere size is only slightly smaller than the second fluid tank size, making them prone to prematurely initiating the reaction by contacting the fluid extending into the second fluid tank. In actual processes, using high-precision processing techniques or strictly controlling the processing technology of the microfluidic substrate to make all the first fluid channels, microchannels and second fluid channels uniform may reduce the risk of the above problems, but may significantly increase the processing difficulty and cost of the microfluidic substrate.

[0058] A buffer tank is placed between the first and second fluid tanks. A small amount of fluid flowing out of the first fluid tank will flow into the buffer tank, reducing the risk of it flowing into the second fluid tank. In addition, even if the gas-liquid interface extending from the first microchannel enters the buffer tank (the connection between the buffer tank and the first microchannel), the fluid will not come into contact with the reagent pre-loaded in the second fluid tank. The buffer tank can effectively prevent the fluid from prematurely contacting the reagent pre-loaded in the second fluid tank to start the reaction, significantly reducing the processing difficulty and cost of the microfluidic substrate.

[0059] The microfluidic substrate, microfluidic chip, and their operation method according to at least one embodiment of the present disclosure will now be described in detail with reference to the accompanying drawings. Furthermore, in these drawings, a spatial rectangular coordinate system is established with the plane of the microfluidic substrate as a reference to provide a detailed description of the positions of various components (e.g., buffer grooves) in the microfluidic substrate and microfluidic chip. In this spatial rectangular coordinate system, the X-axis and Y-axis are parallel to the plane of the microfluidic substrate, and the Z-axis is perpendicular to the plane of the microfluidic substrate.

[0060] In at least one embodiment of this disclosure, such as Figure 1 and Figure 2 As shown, the microfluidic substrate 10 includes a flow channel structure, which includes a delivery channel 100 and multiple detection groups 200. Each detection group 200 includes a first fluid tank 210, a first microchannel 240, a buffer tank 220, a second microchannel 250, and a second fluid tank 230 connected in sequence. The first fluid tank 210 is connected to the delivery channel 100, and a reagent 12 is disposed in the second fluid tank 230. The microfluidic substrate 10 has a rotation axis 11, and the detection groups 200 are located on the side of the delivery channel 100 away from the rotation axis 11. The distances from the first fluid tank 210, the buffer tank 220, and the second fluid tank 230 in each detection group 200 to the rotation axis 11 increase sequentially. That is, in each detection group 100, the first fluid tank 210, the buffer tank 220, and the second fluid tank 230 are arranged to move away from the rotation axis 11 in sequence. Thus, when the microfluidic substrate 10 rotates around the rotation axis 11, under the action of centrifugal force (inertial force), the fluid will sequentially enter the first fluid tank 210, the buffer tank 220 and the second fluid tank 230 from the delivery channel 100.

[0061] In the embodiments of this disclosure, the type of reagent is not limited and can be selected as needed. For example, the reagent can be a liquid reagent, a dry reagent, a solid microsphere containing the reagent, a microarray containing the reagent, a test strip containing the reagent, etc. Depending on the type of reagent pre-loaded in the second fluid tank, the microfluidic substrate (or a microfluidic chip including the microfluidic substrate) can analyze and detect components in the blood, body fluids, or tissues of humans, animals, microorganisms, or plants through methods such as PCR molecular detection, immunoassay, or biochemical detection.

[0062] In at least one embodiment of the microfluidic substrate provided in this disclosure, when fluid is present in the first microchannel, the fluid in the first microchannel, the buffer groove, the second microchannel, and the second fluid groove form a closed groove. Thus, after the microfluidic substrate is encapsulated (e.g., mated with a cover plate), when fluid is present in the first microchannel, the spaces in the buffer groove, the second microchannel, and the second fluid groove effectively form a sealed chamber. When fluid flows along the first microchannel towards the buffer groove, the fluid compresses the air in the chamber, causing an increase in the chamber's pressure, i.e., a back pressure is formed. This back pressure hinders the fluid from flowing towards the buffer groove. When the back pressure and the centrifugal force (inertial force) generated by the fluid's rotation reach equilibrium, a gas-liquid interface is formed in the first microchannel. Thus, fluid from the first fluid groove... The fluid is difficult to enter the buffer tank, and even if a small amount of fluid does enter the buffer tank, it will accumulate in the buffer tank during rotation and will be difficult to enter the second microchannel or the second fluid tank through the second microchannel, thereby reducing the risk of fluid entering the second fluid tank in the current situation. In addition, even if some of the fluid that entered the buffer tank enters the second fluid tank, because a gas-liquid interface is formed in the first microchannel, the fluid that has entered the second fluid tank (and has come into contact with the reagent) will not return to the first fluid tank through the first microchannel, that is, the fluid will not enter other detection groups. Thus, when the microfluidic chip formed using the microfluidic substrate of this disclosure performs detection, the risk of fluid from the first fluid tank entering the second fluid tank is avoided during the stage when the fluid in the delivery channel fills the first fluid tank, preventing premature reaction between the fluid and the reagent, and preventing cross-contamination of reagents from different detection groups (e.g., adjacent detection groups), which helps to ensure the reliability of the detection results.

[0063] For example, such as Figure 2As shown, the chamber composed of the buffer tank 220, the second microchannel 250, and the second fluid tank 230 has only an opening that communicates with the first microchannel 240. Thus, as the fluid from the first fluid tank 210 enters the first microchannel 240, the fluid effectively blocks the gas in the chamber. As the fluid flows along the first microchannel 240 toward the buffer tank 220, the gas-liquid interface P formed between the fluid and the gas also advances toward the buffer tank 220, causing the gas pressure in the chamber to increase, thereby hindering the fluid from flowing into the buffer tank. If the length of the first microchannel 240 is long enough, the gas pressure in the chamber will counteract the centrifugal force (inertial force) that causes the fluid to flow toward the first microchannel. That is, the pressure formed by the gas pressure and the centrifugal force reach a state of equilibrium at the gas-liquid interface P. The gas-liquid interface P exists in the first microchannel 240 or at the connection between the first microchannel 240 and the buffer tank 220 (for example, the inlet of the buffer tank mentioned in the following embodiment). Thus, the fluid in the first fluid tank 210 is unlikely to flow into the buffer tank 220, and even less likely to flow into the second fluid tank 230.

[0064] For example, in actual processes, at lower rotational speeds, fluid, driven by centrifugal force, first enters and fills the first fluid tank through a conveying channel connected to it, sealing the inlet of the first microchannel (the opening of the first microchannel communicating with the first fluid tank) between the first fluid tank and the buffer tank. Driven by centrifugal force, the fluid further enters the microchannel between the first fluid tank and the buffer tank (the first microchannel in this embodiment), compressing the air in the buffer tank and the second fluid tank. When the reverse pressure generated by the compressed air, combined with the surface tension of the fluid and the centrifugal force, reaches equilibrium, the fluid stops flowing, thus forming a gas-liquid interface in the microchannel (first microchannel). After excess fluid in the conveying channel flows into the overflow tank (the first waste liquid tank in this embodiment), a fixed amount of fluid is confined by the gas-liquid interface within the first fluid tank and the first microchannel. When a quantitative amount of fluid from the first fluid tank needs to be transferred to the second fluid tank (used as a reaction tank, or detection tank) to react with pre-filled reagents, increasing the rotation speed and the resulting centrifugal force will cause a Rayleigh-Taylor instability-like phenomenon at the gas-liquid interface between the air in the buffer tank and the fluid in the microchannel. That is, when two media of different densities are placed in the same container, with the denser medium above the less dense medium, the gas-liquid interface will become unstable under the influence of gravity (centrifugal force in this disclosure). Thus, the fluid enters the buffer tank and the second fluid tank intermittently, while the air in the buffer tank and the second fluid tank is discharged as bubbles through the first microchannel (e.g., subsequently discharged through the first fluid tank and the delivery channel) until all the gas trapped by the fluid is discharged. During this process, the reagents in the second fluid tank will not be cross-contaminated.

[0065] For example, in some embodiments of this disclosure, such as Figure 2 As shown, in actual processes, when the length of the first microchannel is not long enough or the rotation frequency is high, the gas-liquid interface P may exist at the connection between the first microchannel 240 and the buffer tank 220 (for example, the first inlet of the buffer tank mentioned in the following embodiment). In this case, the fluid may protrude out of the first inlet along the direction from the first microchannel 240 to the buffer tank 220. In this case, because the surface tension of the fluid surface exists, and the pressure (back pressure) formed by air compression and the centrifugal force reach equilibrium, the fluid will still maintain the protruding shape at the connection between the first microchannel 240 and the buffer tank 220 and will not continue to flow into the buffer tank. That is, the fluid will still form a stable gas-liquid interface P at the connection. The formation of a stable gas-liquid interface P requires a certain process. Before a stable gas-liquid interface P is formed, some fluid may have already entered the buffer tank 220. Due to the design of the buffer tank 220, in the rotating state, the fluid dripping into the buffer tank 220 is unlikely to enter the connection between the buffer tank 220 and the second microchannel 250 (e.g., the first outlet of the buffer tank mentioned in the following embodiment), thereby reducing the risk of the fluid entering the second fluid tank 230. Furthermore, even if the fluid flowing into the buffer tank 220 enters the second microchannel 250, because this part of the fluid is disconnected from the fluid stored in the first microchannel 240, it can only be... The gas in the second microchannel 250 and the second fluid tank 230 is compressed by the pressure generated by its own inertial force (centrifugal force). Because the fluid in the first microchannel 240 is additionally pressured by the fluid stored in the first fluid tank 240, the pressure provided by the fluid entering the second microchannel 250 is less than the pressure provided by the fluid in the first microchannel 240. Therefore, the fluid entering the second microchannel 250 is difficult to further compress the gas in the second microchannel 250 and the second fluid tank 230, and thus will not break through the second microchannel 250 and enter the second fluid tank 230.

[0066] In at least one embodiment of the microfluidic substrate provided in this disclosure, a first microchannel is configured to have a first length such that, at a first rotational frequency not greater than that of the microfluidic substrate, fluid from a first fluid tank and gas present in a buffer tank form a gas-liquid interface, and such a gas-liquid interface exists in the first microchannel or at the connection between the first microchannel and the buffer tank. The first microchannel has a cross-section perpendicular to its extension direction. With a fixed dimension of the cross-section (e.g., inner diameter, or width and depth, etc.), the larger the first length, the larger the volume proportion of the first microchannel in the entire cavity formed by the first fluid tank, the first microchannel, the buffer tank, the second microchannel, and the second fluid tank. The greater the gas pressure in the cavity formed by the first microchannel, the buffer tank, the second microchannel, and the second fluid tank before the fluid passes through the first microchannel, that is, greater resistance can be generated when compressing gas, thereby allowing for the design of a larger first rotational frequency. In the embodiments of this disclosure, the first rotation frequency can be designed according to the actual process. For example, the first rotation frequency can be the rotation frequency at which the fluid in the conveying channel enters the first fluid tank. Thus, the range of the first length can be designed according to the first rotation frequency.

[0067] In the microfluidic substrate provided in at least one embodiment of this disclosure, the first length of the first microchannel can be 0.1–5 mm, for example, further 0.2 mm, 0.5 mm, 1 mm, 1.5 mm, 2 mm, 3 mm, 4 mm, etc.; in addition, the width of the first microchannel is 0.1–1 mm, for example, further 0.2 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.8 mm, etc.; furthermore, the depth of the first microchannel is 0.1–0.5 mm, for example, further 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, etc. It should be noted that, in the embodiments of this disclosure, the parameters such as the first length, width, and depth of the first microchannel can be designed according to actual processes and are not limited to the above-mentioned numerical ranges.

[0068] In the microfluidic substrate provided in at least one embodiment of this disclosure, multiple buffer grooves can be provided in a detection group. When multiple buffer grooves are provided, these buffer grooves can be connected in series between the first fluid groove and the second fluid groove. In this way, when the fluid in the delivery channel fills the first fluid groove, the probability of fluid entering the second fluid groove can be further reduced.

[0069] In at least one embodiment of the microfluidic substrate provided in this disclosure, the buffer groove includes a first sidewall and a second sidewall. The first sidewall includes a first inlet communicating with a first microchannel, and the second sidewall is opposite to the first sidewall and includes a first outlet communicating with a second microchannel. For example, as shown... Figure 3As shown, the buffer tank 220 includes a first sidewall 221 and a second sidewall 222 that are opposite to each other. The first sidewall 221 includes a first inlet 2211 so that the buffer tank 220 is connected to the first fluid tank 210 through the first microchannel 240. The second sidewall 222 includes a first outlet 2221 so that the buffer tank 220 is connected to the second fluid tank 230 through the second microchannel 250.

[0070] For example, in some embodiments of this disclosure, in each detection group, a first microchannel can be provided, so that a first inlet can be provided on the first sidewall; or, in other embodiments of this disclosure, a first microchannel can be provided, so that a multiple first inlets can be provided on the first sidewall.

[0071] For example, in some embodiments of this disclosure, in each detection group, a second microchannel can be provided, so that a first inlet / outlet can be provided on the first sidewall; or, in other embodiments of this disclosure, a second microchannel can be provided, so that a multiple first inlet / outlet can be provided on the first sidewall.

[0072] During the stage where the fluid in the delivery channel fills the first fluid tank, some fluid may still enter the buffer tank. Therefore, the shape of the buffer tank can be designed to reduce the probability of this portion of fluid further entering the second microchannel. For example, in the embodiments of this disclosure, the distance from the portion of the second sidewall without the first outlet to the rotation axis is greater than the distance from the first outlet to the rotation axis. Thus, during rotational motion, the fluid entering the buffer tank tends to move away from the rotation axis under the action of centrifugal force, making it difficult for it to flow into the first outlet. That is, it increases the difficulty for this portion of fluid to enter the second microchannel from the second outlet.

[0073] In the embodiments disclosed herein, under the condition that the distance from the second sidewall of the buffer groove without the first outlet portion to the rotation axis is greater than the distance from the first outlet to the rotation axis, the specific shape of the buffer groove is not limited and can be designed according to the actual process requirements. The following is an explanation through several specific embodiments.

[0074] In some embodiments of the microfluidic substrate provided in this disclosure, the second sidewall is a plane, and the plane is perpendicular to the direction along the rotation axis to the first inlet. For details, please refer to... Figure 2 The buffer groove shown.

[0075] It should be noted that, in a rotating state, the trace fluid entering the buffer tank is affected by the Coriolis force (a description of the deviation of an object moving in a straight line in a rotating system due to inertia relative to the rotating system), and is prone to falling into the edge area of ​​the buffer tank and staying in the edge area.

[0076] In other embodiments of the microfluidic substrate provided in this disclosure, the area of ​​the second sidewall without the first outlet includes at least one recessed portion, and the distance from the bottom of the at least one recessed portion to the rotation axis is greater than the distance from the first outlet to the rotation axis. Thus, during the stage where the fluid in the delivery channel fills the first fluid tank, if some fluid flows into the buffer tank, because the microfluidic substrate is rotating, this portion of fluid will accumulate at the recessed portion, and since the distance from the bottom of the recessed portion to the rotation axis is greater than the distance from the first outlet to the rotation axis, the fluid accumulated in the recessed portion will not enter the first outlet and therefore will not enter the second fluid tank; that is, the risk of fluid entering the second fluid tank at this stage is further reduced. For example, as... Figure 3 As shown, when the microfluidic substrate is rotating counterclockwise, under the action of the Coriolis force, the fluid Q entering the buffer tank 220 is concentrated in the recessed portion 2222 located at the edge of the second sidewall 222. The recessed portion 2222 can store the fluid Q, allowing more fluid Q to enter; in addition, there is a height difference (distance from the rotation axis) between the bottom of the recessed portion 2222 and the first outlet 2221. After the fluid Q is concentrated in the recessed portion 2222, it becomes more difficult for it to enter the first outlet 2221. Thus, during the stage when the fluid in the delivery channel fills the first fluid tank, the probability of fluid Q entering the second fluid tank 230 is further reduced.

[0077] In the embodiments of this disclosure, the arrangement of the first inlet, first outlet, and recessed portion of the buffer groove can be determined according to the rotation mode of the microfluidic substrate (e.g., only clockwise rotation, only counterclockwise rotation, or switching between clockwise and counterclockwise rotation), so as to further reduce the probability of fluid entering the second fluid groove during the stage where the fluid in the delivery channel fills the first fluid groove. Specific methods can be designed according to actual process requirements; here, several specific implementation methods are illustrated through a few specific embodiments.

[0078] In the microfluidic substrate provided in some embodiments of this disclosure, recessed portions are provided on both sides of the first outlet in each buffer groove, and the first outlet is located in the middle of the second sidewall. Thus, regardless of whether the preset rotation direction of the microfluidic chip is clockwise or counterclockwise, during the stage where the fluid in the delivery channel fills the first fluid groove, the recessed portions can be used to collect the fluid entering the buffer groove, thereby reducing the risk of fluid entering the second fluid groove during this stage. For example, as... Figure 3As shown, along the preset rotation direction, recessed portions 2222 are provided on both the front and rear sides of the first outlet 2221. This allows fluid Q to accumulate in the recessed portion 2222 located behind the first outlet 2221 during rotation. During rotation, fluid Q will remain at the edge of the second sidewall, and fluid Q will lag behind the rotation; that is, along the rotation direction, the fluid and the first outlet are arranged sequentially, with fluid Q located behind the first outlet. For example, as... Figure 3 As shown, if the microfluidic substrate rotates counterclockwise, the fluid Q in the buffer groove 220 gathers in the recessed portion 2222 on the left side of the second sidewall 222; or, if the microfluidic substrate rotates clockwise, the fluid Q in the buffer groove 220 gathers in the recessed portion 2222 on the right side of the second sidewall 222.

[0079] In other embodiments of the microfluidic substrate provided in this disclosure, a recessed portion is provided on one side of the first outlet in each buffer groove. The first outlet is located in the middle of the second sidewall, and the recessed portion and the first outlet are arranged sequentially along a preset rotation direction of the microfluidic substrate. For example, as shown... Figure 4 As shown, the recessed portion 2222a of the second sidewall 222a of the buffer tank 220a is located to the left of the first outlet 2221a. The microfluidic substrate rotates counterclockwise around the rotation axis 11a. The fluid entering the buffer tank 220a from the first fluid tank 210a through the first microchannel 240a and the first inlet 2221a of the first sidewall 221a will fall into the recessed portion 2222a and will not enter the second fluid tank 230a through the second microchannel 250a.

[0080] In other embodiments of the microfluidic substrate provided in this disclosure, a recessed portion is provided on one side of the first outlet in each buffer groove. The first outlet is located at one end of the second sidewall and is arranged along a predetermined rotation direction of the microfluidic substrate, with the recessed portion and the first outlet arranged sequentially. Thus, because the first outlet is located at one end of the second sidewall, the design area of ​​the recessed portion is effectively increased. That is, compared to the first outlet being located in the middle of the second sidewall, the design area of ​​the recessed portion in the second sidewall can be increased, and the design volume of the recessed portion can be increased. This means the amount of fluid that the recessed portion can store is increased, further reducing the risk of fluid flowing into the second fluid groove during the stage where the fluid in the delivery channel fills the first fluid groove. For example, as... Figure 5As shown, the microfluidic substrate rotates counterclockwise around the rotation axis 11b. Along the rotation direction, a recessed portion 2222b is provided on the rear side of the first outlet 2221b of the second sidewall 222b. Thus, during rotation, fluid can accumulate in the recessed portion 2222b located on the rear side of the first outlet 2221b along the rotation direction. Furthermore, when fluid from the first fluid tank 210b enters the buffer tank 220b from the first microchannel 240b and the first inlet 2211b, during rotation, the fluid is already located on the rear side of the first outlet 2221b when it passes through the first inlet 2211b of the first sidewall 221b. That is, the fluid is more likely to accumulate in the recessed portion 2222b and less likely to enter the first outlet 2221b. In other words, during the stage when the fluid in the delivery channel fills the first fluid tank 210b, the probability of fluid entering the second microchannel 250b and the second fluid tank 230b is further reduced.

[0081] In other embodiments of the microfluidic substrate provided in this disclosure, in each buffer groove, the first inlet is located in the middle of the first sidewall. For example, as shown... Figure 5 As shown, the first entrance 2211b is located in the middle of the first sidewall 221b.

[0082] In other embodiments of the microfluidic substrate provided in this disclosure, in each buffer tank, a first inlet is located at one end of a first sidewall, and the first inlet and first outlet are arranged sequentially along a preset rotation direction of the microfluidic substrate. Thus, compared to having the first inlet located in the middle of the first sidewall, the increased spacing between the first inlet and first outlet along the preset rotation direction reduces the risk of fluid directly falling into the first outlet when the fluid flows into the buffer tank through the first inlet during the stage where the fluid in the delivery channel fills the first fluid tank, thereby further reducing the risk of fluid flowing into the second fluid tank during this stage. For example, as... Figure 6 As shown, the first inlet 2211c is located at one end of the first sidewall 221c. The microfluidic substrate rotates counterclockwise around the rotation axis 11c. Along the rotation direction, the first inlet 2211c is located behind the first outlet 2221c. Thus, when fluid from the first fluid tank 210c enters the buffer tank 220c from the first microchannel 240c and the first inlet 2211c, during rotation, the fluid is already located behind the first outlet 2221c when it passes through the first inlet 2211c of the first sidewall 221c, making it more difficult for the fluid to enter the first outlet 2221c. For example, further, as... Figure 6As shown, a recessed portion 2222c is provided on the rear side of the first outlet 2221c of the second sidewall 222c. This allows fluid to accumulate in the recessed portion 2222c located behind the first outlet 2221c during rotation. When fluid from the first fluid tank 210c enters the buffer tank 220c from the first microchannel 240c and the first inlet 2211c, during rotation, the fluid is already located behind the first outlet 2221c when it passes through the first inlet 2211c of the first sidewall 221c. This makes it easier for the fluid to accumulate in the recessed portion 2222c and more difficult for it to enter the first outlet 2221c. In other words, during the stage where the fluid in the delivery channel fills the first fluid tank 210c, the probability of fluid entering the second microchannel 250c and the second fluid tank 230c is further reduced.

[0083] In the microfluidic substrate provided in at least one embodiment of this disclosure, each detection group in each buffer tank further includes a first siphon channel. One end of the first siphon channel is connected to a recessed portion to communicate with the buffer tank, and the other end of the first siphon channel is connected to a second fluid tank. The inner diameter of the first siphon channel is smaller than the inner diameter of the second microchannel, and the distance from a portion of the first siphon channel to the rotation axis is smaller than the distance from the first outlet to the rotation axis. After fluid from the first fluid tank is introduced into the second fluid tank, some fluid in the buffer tank may be confined in the recessed portion. In this case, the first siphon channel can introduce the fluid in the recessed portion into the second fluid tank, improving fluid utilization. For example, as shown... Figure 7 As shown, one end of the first siphon channel 260d is connected to the recessed portion 2222d, and the other end of the first siphon channel 260d is connected to the second fluid tank 230d. In practical applications, during the stage where the fluid in the conveying channel fills the first fluid tank 210d, some fluid in the first fluid tank 210d may flow through the first microchannel 240d to the recessed portion 2222d of the second sidewall 222d of the buffer tank 220d; after increasing the rotation frequency (speed), the fluid in the first fluid tank 210d and the first microchannel 240d enters the buffer tank 220d and then enters the second fluid tank 230d through the second microchannel 250d. At this time, some fluid will accumulate in the recessed portion 2222d. During this process, because part of the first siphon channel 260d (such as...) Figure 7 The distance from the top (the part closest to the rotation axis 11d) to the rotation axis 11d is less than the distance from the first outlet 2221d to the rotation axis 11d. During this process, the fluid will not enter the second fluid tank 230d through the first siphon channel 260d. That is, the fluid will close the first siphon channel 260d so as not to affect the fluid entering the second fluid tank 230d from the second microchannel 250d. Then, after reducing the rotation frequency or stopping the rotation, these fluids are sucked into the second fluid tank 230d under the capillary force of the first siphon channel 260d.

[0084] For example, in some embodiments of this disclosure, the first siphon channel may be further configured as a capillary structure, thereby allowing the fluid gathered at the recessed portion to be drawn into the second fluid channel by capillary force.

[0085] In some embodiments of the microfluidic substrate provided in this disclosure, the volume of the first fluid channel is smaller than the volume of the second fluid channel.

[0086] In some other embodiments of the microfluidic substrate provided in this disclosure, the volume of the first fluid tank is larger than the volume of the second fluid tank, and the volume of the first fluid tank is less than or equal to the sum of the volumes of the second fluid tank and the buffer tank. In actual processes, considering factors such as the viscosity of the fluid itself, fluid residues may exist in various channels such as the buffer tank, the first microchannel, and the second microchannel. By making the volume of the first fluid tank less than or equal to the sum of the volumes of the second fluid tank and the buffer tank, residual fluid in the first fluid tank can be avoided, thereby ensuring that the fluid metered by the first fluid tank can enter the buffer tank and the second fluid tank, preventing fluid accumulation in the first fluid tank. By making the volume of the first fluid tank larger than the volume of the second fluid tank, it can be ensured that the fluid metered by the first fluid tank can completely fill the second fluid tank.

[0087] It should be noted that in the embodiments of this disclosure, fluid, "residue," and "aggregation" are different concepts. Fluid residue is caused by some uncontrollable factors, while fluid aggregation is expected in the process or structural design. For example, under ideal conditions without considering factors such as fluid viscosity and wettability, fluid may not leave any residue after passing through various flow channels (e.g., the first microchannel, the second microchannel, etc.) and grooves (e.g., the first fluid groove, the buffer groove, etc.). However, fluid aggregation may occur in specific structures of the flow channels or grooves (e.g., the recessed portion in the above embodiments).

[0088] In other embodiments of the microfluidic substrate provided in this disclosure, the volume of the first fluid channel is greater than the sum of the volumes of the second fluid channel and the buffer channel.

[0089] In embodiments of this disclosure, the shapes of the first fluid channel and the second fluid channel can be triangular, circular, rectangular, or polygonal, etc. For example, without considering the accumulation position of fluid in the buffer channel (e.g., without considering the setting of a recessed portion), the shape of the buffer channel can also be triangular, circular, rectangular, or polygonal, etc.

[0090] In the microfluidic substrate provided in at least one embodiment of this disclosure, within the same detection group, at least a portion (i.e., part or all) of the second fluid channel is at a distance from the rotation axis greater than the distances of the first fluid channel and the buffer channel from the rotation axis. For example, the distance of any portion of the second fluid channel from the rotation axis is greater than the distance of any portion of the first fluid channel and the buffer channel from the rotation axis; or, the distance of a portion of the second fluid channel from the rotation axis is greater than the distance of any portion of the first fluid channel and the buffer channel from the rotation axis, and the distance of another portion of the second fluid channel from the rotation axis is less than or equal to the distance of a portion of the first fluid channel and the buffer channel from the rotation axis.

[0091] In at least one embodiment of the microfluidic substrate provided in this disclosure, the transport channel is a non-closed ring, with the center of the ring being the axis of rotation; or, the transport channel is a non-closed ring, where the distance from the first end of the transport channel to the axis of rotation is less than the distance from the second end of the transport channel to the axis of rotation, and the distance from the first end to the second end increases sequentially. See also... Figure 1 The microfluidic substrate 10 shown has an arc-shaped (non-closed ring) delivery channel 100. The distance from the first end of the arc to the rotation axis is less than the distance from the second end of the delivery channel to the rotation axis. From the first end to the second end, the distance from the delivery channel to the rotation axis increases sequentially. This facilitates uniform fluid distribution within the delivery channel when the microfluidic substrate rotates, allowing the fluid to flow evenly into the first fluid tank in each detection group. For example, an inlet (for communication with a second siphon channel) can be provided at the first end for fluid injection. For example, a vent hole can be provided on the delivery channel. For example, the vent hole can be provided at the second end of the delivery channel; furthermore, a vent hole can also be provided at the first end.

[0092] The microfluidic substrate provided in at least one embodiment of this disclosure may further include a first waste liquid tank. The first waste liquid tank is connected to one end of the delivery channel. The first waste liquid tank and the delivery channel are arranged sequentially along a preset rotation direction of the microfluidic substrate. In actual processes, after all the first fluid tanks are filled with fluid, there may still be residual fluid in the delivery channel. This residual fluid may connect the fluid in some of the first fluid tanks (e.g., adjacent first fluid tanks) together, and also prevent a fixed amount of liquid from forming in the first fluid tanks. For example, during the process of increasing the rotation speed to allow the fluid in the first fluid tanks to enter the buffer tank and the second fluid tank, the residual fluid will still enter the first fluid tanks, ultimately resulting in an excessive amount of fluid entering the detection group. In this embodiment, after the fluid in the delivery channel fills the first fluid tank, it flows into the first waste liquid tank. This prevents the fluid in the delivery channel from being compressed and continuing to enter the first fluid tank in the detection group, which could lead to excessive fluid pressure and damage the gas-liquid interface. In other words, during the stage of filling the first fluid tank with fluid in the delivery channel, the risk of fluid overflowing the buffer tank and entering the second fluid tank is reduced. Furthermore, there is no residual fluid in the delivery channel; that is, the fluids in adjacent first fluid tanks are not interconnected, avoiding cross-contamination and ensuring the quantitative action of the first fluid tank on the fluid, preventing excessive fluid from flowing into the detection group. For example, as... Figure 1 As shown, the first waste liquid tank 110 is connected to the delivery channel 100. When fluid is injected into the delivery channel 100, the microfluidic substrate is configured to rotate counterclockwise based on the rotation axis 11. Along the rotation direction, the first waste liquid tank 110 is located behind the delivery channel 100. Thus, the first waste liquid tank 110 is also located behind the detection group 200 (first fluid tank 210) connected to the delivery channel 100. That is, after the fluid entering the delivery channel 100 fills the first fluid tank 210 of each detection group 200 in sequence, the excess fluid will enter the first waste liquid tank 110.

[0093] For example, in an embodiment of this disclosure, when the conveying channel is non-closed annular and the distance from the first end of the conveying channel to the rotation axis is less than the distance from the second end of the conveying channel to the rotation axis, the first waste liquid tank can be connected to the second end of the conveying channel. This facilitates emptying the fluid in the conveying channel at low speeds, allowing the fluid to enter the first waste liquid tank.

[0094] The microfluidic substrate provided in at least one embodiment of this disclosure may further include a mixing tank and a second siphon channel. The mixing tank includes two inlets and one outlet, one end of the second siphon channel is connected to the outlet of the mixing tank, and the other end of the second siphon channel is connected to a delivery channel. The distance from a portion of the second siphon channel to the rotation axis is less than the distance from the mixing tank to the rotation axis. For example, as... Figure 8As shown, the mixing tank 400 is connected to the delivery channel 100 through the second siphon channel 500. The two inlets of the mixing tank 400 can be used to introduce two types of fluids (e.g., sample and diluent) respectively, and the two fluids can be mixed evenly in the mixing tank. The mixed fluid enters the delivery channel 100 through the second siphon channel 500. For example, after the sample and diluent enter the mixing tank 400 through the two inlets, the microfluidic substrate continues to rotate. Because the distance from a portion of the second siphon channel 500 to the rotation axis is less than the distance from the mixing tank 400 to the rotation axis, the fluid in the mixing tank 400 will not enter the delivery channel 100. After the sample and diluent are mixed evenly in the mixing tank 400, the rotation frequency (speed) is reduced or the rotation is stopped. The fluid in the mixing tank 400 fills the second siphon channel 500 under the capillary force. The microfluidic substrate is rotated again, and the fluid in the mixing tank 400 enters the delivery channel 100 through the second siphon channel 500.

[0095] The microfluidic substrate provided in at least one embodiment of this disclosure may further include a sample tank, a sample metering tank, a sample overflow tank, a third siphon channel, a diluent tank, a diluent metering tank, a diluent overflow tank, and a fourth siphon channel. The sample metering tank is connected to the sample tank, and the distance from the sample metering tank to the rotation axis is greater than the distance from the sample tank to the rotation axis. The sample overflow tank is connected to the sample tank, and the distance from the sample overflow tank to the rotation axis is greater than the distance from the sample metering tank to the rotation axis. One end of the third siphon channel is connected to the sample metering tank, and the other end of the third siphon channel is connected to one of the two inlets of the mixing tank, and the distance from a portion of the third siphon channel to the rotation axis is less than the distance from the sample metering tank to the rotation axis. The diluent metering tank is connected to the diluent tank, and the distance from the diluent metering tank to the rotation axis is greater than the distance from the diluent tank to the rotation axis. The diluent overflow tank is connected to the diluent tank, and the distance from the diluent overflow tank to the rotation axis is greater than the distance from the diluent metering tank to the rotation axis. One end of the fourth siphon channel is connected to the diluent metering tank, and the other end of the fourth siphon channel is connected to the other of the two inlets of the mixing tank. The distance from part of the fourth siphon channel to the rotation axis is less than the distance from the diluent metering tank to the rotation axis.

[0096] For example, such as Figure 8 as well as Figures 11-15As shown, the microfluidic substrate includes a sample reservoir 210, a sample quantitative reservoir 220, a sample overflow reservoir 230, a third siphon channel 240, a diluent reservoir 310, a diluent quantitative reservoir 320, a diluent overflow reservoir 330, and a fourth siphon channel 340. When performing blood (sample) testing using the microfluidic substrate (or a microfluidic chip including the microfluidic substrate), 100 μL of whole blood can be injected into the sample reservoir 210, and 450 μL of diluent can be injected into the diluent reservoir 310. Then, by rotating the microfluidic chip, the sample flows into the sample quantitative reservoir 320, excess sample flows into the sample overflow reservoir 230, the diluent flows into the diluent quantitative reservoir 320, and excess diluent flows into the diluent overflow reservoir 330. When the microfluidic chip is stopped rotating, the sample (plasma in the supernatant) in sample metering tank 220 fills the third siphon channel 240 under capillary force, and the diluent in diluent metering tank 340 fills the fourth siphon channel 340 under capillary force. Rotating the microfluidic chip, the metered plasma in sample metering tank 220 and the metered diluent in diluent metering tank 320 are injected into mixing tank 400 through the third and fourth siphon channels 240 and 340, respectively. By controlling the rotation speed and reversal (clockwise and counterclockwise rotation) of the microfluidic chip, the sample (plasma) and diluent injected into mixing tank 400 are mixed evenly. When the microfluidic chip is stopped rotating, the fluid (mixture) in mixing tank 400 fills the second siphon channel 500 under capillary force. Rotating the microfluidic chip, the fluid (mixture) in the mixing tank 400 enters the delivery channel 100 through the second siphon channel 500, and the fluid (mixture) sequentially fills the first fluid tank 210 of each detection group through the delivery channel 100, while the excess fluid (mixture) flows into the first waste liquid tank.

[0097] The microfluidic substrate provided in at least one embodiment of this disclosure may further include a channel layer and a substrate. The channel structure is formed in the channel layer. The substrate is located on the opposite side of the channel layer from the side where the first fluid channel, the first microchannel, the buffer channel, the second microchannel, and the second fluid channel are disposed. The substrate is bonded to the channel layer or the substrate and the channel layer are integrally formed. For example, in some embodiments, the channel layer includes opposing first and second main surfaces; when the channel layer is disposed in a microfluidic chip, the first main surface of the channel layer faces the cover plate, and the second main surface of the channel layer faces the substrate.

[0098] The microfluidic substrate disclosed herein can be fabricated using conventional methods such as injection molding, machining, etching, engraving, and imprinting. The materials used can be plastics, ceramics, glass, silicon wafers, and silicone, among others.

[0099] In one example of this disclosure, such as Figure 9As shown, the microfluidic substrate 10 has a one-piece structure, that is, the substrate and the flow channel layer are one-piece.

[0100] In another example of this disclosure, such as Figure 10 As shown, the microfluidic substrate 10 includes a channel layer 12 and a substrate 13. A delivery channel 100, a first fluid channel 210, a buffer channel 220, and a second fluid channel 230 are formed in the channel layer 12. When the delivery channel 100, the first fluid channel 210, the buffer channel 220, and the second fluid channel 230 are formed in the channel layer 12 but do not penetrate the channel layer 12, the delivery channel 100, the first fluid channel 210, the buffer channel 220, and the second fluid channel 230 are formed on the side of the channel layer 12 facing the cover plate 20, and the substrate 13 is located on the side of the channel layer 12 away from the cover plate 20.

[0101] For example, in some embodiments of this disclosure, the second fluid tank is configured as a through-flow channel layer, while the conveying channel, first fluid tank, buffer tank, first siphon channel, second siphon channel, etc., can be configured as non-through-flow channel layers. The substrate is configured as a transparent substrate, thereby allowing optical detection and comparison of the second fluid tank (detection tank, reaction tank) through the substrate. For example, the substrate can be a sheet material (generally with a thickness of 0.5 mm or more) or a film material (generally with a thickness of less than 0.5 mm), and the material is a thermoplastic polymer, including one or more of polymethyl methacrylate, polycarbonate, polystyrene, polyamide, and polyethylene terephthalate, to ensure good light transmittance.

[0102] At least one embodiment of this disclosure provides a microfluidic chip, which includes a cover plate and a microfluidic substrate as described in the first aspect above. The cover plate and the microfluidic chip are coupled together on one side of the microfluidic substrate where a first fluid groove, a first microchannel, a buffer groove, a second microchannel, and a second fluid groove are provided. For example, as shown... Figure 9 and Figure 10 As shown, the cover plate 20 is mated with the microfluidic substrate 10, thus forming a fluid-containing chamber with the first fluid tank, the first microchannel, the buffer tank, the second microchannel, and the second fluid tank. For example, the cover plate can be transparent, allowing optical detection and comparison of the second fluid tank (detection tank, reaction tank) through the cover plate. For example, the cover plate can be made of a sheet material (generally with a thickness of 0.5 mm or more) or a film material (generally with a thickness of less than 0.5 mm), and the material is a thermoplastic polymer, including one or more of polymethyl methacrylate, polycarbonate, polystyrene, polyamide, and polyethylene terephthalate, to ensure good light transmittance.

[0103] At least one embodiment of this disclosure provides a method for operating a microfluidic chip, wherein the microfluidic chip includes a cover plate and a microfluidic substrate. The microfluidic substrate includes a flow channel structure, which includes a delivery channel and multiple detection groups. Each detection group includes a first fluid tank, a first microchannel, a buffer tank, a second microchannel, and a second fluid tank connected in sequence. The first fluid tank is connected to the delivery channel. A reagent is disposed in the second fluid tank of at least one detection group. The microfluidic substrate has a rotation axis. The detection groups are located on the side of the delivery channel opposite to the rotation axis. The distances from the first fluid tank, buffer tank, and second fluid tank in each detection group to the rotation axis increase sequentially. The operation method includes: driving the microfluidic chip to rotate at a second rotation frequency so that fluid from the delivery channel enters the first fluid tank and forms a gas-liquid interface in the first microchannel; after the first fluid tank in each detection group is filled with liquid, driving the microfluidic chip to rotate at a third rotation frequency so that fluid enters the buffer tank and the second fluid tank; wherein the third rotation frequency is greater than the second rotation frequency. In this operating method, the microfluidic chip utilizes a microfluidic substrate with a buffer groove separating the first fluid groove and the second fluid groove. During the stage where the fluid in the delivery channel fills the first fluid groove, this prevents the fluid flowing out of the first fluid groove from directly entering the second fluid groove, reducing the risk of fluid entering the second fluid groove and re-entering the first fluid groove from the second fluid groove (at which point the fluid has already come into contact with the reagent). This reduces the risk of cross-contamination of reagents in different detection groups. Furthermore, the buffer groove prevents fluid from directly entering the second fluid groove during this stage and reacting prematurely with the reagent, thereby precisely controlling the reaction time (detection time) of the reagent in the second fluid groove. The specific structure of the microfluidic chip and its microfluidic substrate used in this operating method can be found in the aforementioned... Figures 1-10 The relevant descriptions in the illustrated embodiments will not be repeated here.

[0104] In the microfluidic chip operation method provided in at least one embodiment of this disclosure, a first microchannel is configured to have a first length, such that at a first rotation frequency not greater than that of the microfluidic substrate, fluid from a first fluid tank and gas present in a buffer tank form a gas-liquid interface, and the gas-liquid interface exists in the first microchannel or at the connection between the first microchannel and the buffer tank. A second rotation frequency is not greater than the first rotation frequency, and a third rotation frequency is greater than the first rotation frequency; or the second rotation frequency is not greater than the first rotation frequency, and when the microfluidic chip is driven to rotate at the third rotation frequency, the rotation mode is reciprocating motion. In this operation method, the gas-liquid interface in the first microchannel can be disrupted, allowing fluid to enter the buffer tank and then the second fluid tank to react with the reagent.

[0105] For example, in the microfluidic chip operation method provided in at least one embodiment of this disclosure, the microfluidic substrate of the microfluidic chip may further include structures such as a sample groove, a sample metering groove, a sample overflow groove, a third siphon channel, a diluent groove, a diluent metering groove, a diluent overflow groove, and a fourth siphon channel. Specific designs of these structures and operation methods of the microfluidic chip including these structures can be found in the foregoing embodiments (regarding...). Figure 8 , Figures 11-15 The specific descriptions in the illustrated embodiments are not repeated here.

[0106] In the embodiments of this disclosure, parameters such as rotation frequency in the operation method of the microfluidic chip need to be designed according to the specific structure of the microfluidic chip. Below, several specific examples will be used to illustrate several microfluidic chips and their operation methods as disclosed in this disclosure.

[0107] In one embodiment of this disclosure, the microfluidic chip has a two-layer structure (the microfluidic chip includes a microfluidic substrate and a cover plate; the microfluidic substrate is integrally formed, see [reference]). Figure 9The upper layer is a cover layer containing sample loading holes and diluent loading holes (the cover plate in the above embodiment), and the lower layer is a microfluidic layer (the microfluidic substrate in the above embodiment). The two layers are watertightly bonded together. The liquid delivery channel (the delivery channel in the above embodiment) is annular, gradually decreasing in size away from the rotation axis, and is connected to 23 buffer tanks (i.e., 23 detection groups). An overflow tank (the first waste liquid tank in the above embodiment) is connected to the end away from the rotation axis. The first fluid tank is rectangular, with a volume ranging from approximately 7 μL to 20 μL. The second fluid tank is circular, with its center approximately 40 mm from the rotation axis and a diameter of approximately 2 mm. By designing different depths, its volume is 2 μL smaller than that of the corresponding first fluid tank. The buffer tank is rectangular, with a volume of approximately 2.5 μL. The length of the first microchannel between the first fluid tank and the buffer tank is 1 mm, the width is 0.4 mm, and the depth is 0.2 mm. The length of the second microchannel between the buffer tank and the second fluid tank is 1.5 mm, the width is 0.4 mm, and the depth is 0.2 mm. Part of the second fluid tank is pre-loaded with various types of lyophilized biochemical reagents in spherical shapes with a diameter slightly less than 2 mm. These reagents can be used to detect biochemical items in samples such as plasma, serum, or other body fluids, including at least one or a combination of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (γ-GT), alkaline phosphatase (ALP), total bilirubin (TBIL), direct bilirubin (DBIt), total protein (TP), albumin (Alb), urea (Urea), inositol (Cr), uric acid (UA), glucose (Glu), total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (VLDL), very low-density lipoprotein (LDL), serum magnesium (Mg), serum potassium (K), serum sodium (Na), serum chloride (Cl), serum calcium (Ca), and serum phosphorus (P).

[0108] For example, 100 μL of whole blood (i.e., the sample is blood) is injected into the sample volume indicator structure in the sample well through the sample loading port, and 450 μL of diluent is injected into the diluent tank through the diluent loading port. Then, the microfluidic chip is fixed on the motor and rotated for 2 minutes at a rotation frequency of 5000 rpm. The sample flows into the sample quantitative tank, and excess sample flows into the sample overflow tank. The diluent flows into the diluent quantitative tank, and excess diluent flows into the diluent overflow tank. The motor stops, and the plasma in the supernatant of the sample quantitative tank fills the sample siphon microchannel (the third siphon channel in the above embodiment) under capillary action. The diluent in the diluent quantitative tank fills the diluent siphon microchannel (the fourth siphon channel in the above embodiment) under capillary action. The motor is then restarted and rotated for 30 seconds at a speed of 5000 rpm. The plasma quantified by the sample quantitative tank and the diluent quantified by the diluent quantitative tank are injected into the mixing tank through the siphon microchannels (the third and fourth siphon channels in the above embodiment). The motor is kept running, and its speed is adjusted 20 times between 4500 rpm and 1500 rpm to ensure uniform mixing of the plasma and diluent injected into the mixing tank. When the motor stops, the mixture in the mixing tank fills the siphon microchannel (the second siphon channel in the above embodiment) under capillary action. The motor is then restarted and rotated for 60 seconds, with the speed gradually increasing from 600 rpm to 1200 rpm (e.g., the second rotation frequency in the above embodiment). The fluid formed by the mixture sequentially fills the first fluid tank through the delivery channel, and excess fluid flows into the overflow tank (the first waste tank in the above embodiment). The motor is then stopped, and observation reveals that all the spherical freeze-dried biochemical reagents pre-loaded in the second fluid tank remain intact, with no fluid contacting the reagents. A few buffer tanks contain trace amounts of fluid, indicating that the presence of the buffer tanks prevents premature reaction initiation by contact between the fluid and the biochemical reagents. The motor is then restarted, and its speed is controlled to accelerate and decelerate 30 times between 4500 rpm and 1500 rpm (as in the third rotation frequency described in the above embodiment). The fluid in the first fluid tank enters the second fluid tank and mixes with the biochemical reagents to react. According to the above operating method, the applicant detected the reaction results in the second fluid tank by spectrophotometry and found that the corresponding biochemical reaction results were normal, and no reagent contamination was found, i.e., no cross-contamination occurred.

[0109] In another embodiment of this disclosure, the microfluidic chip has a three-layer structure (the microfluidic chip includes a microfluidic substrate and a cover plate; the microfluidic substrate includes a channel layer and a substrate, see [reference]). Figure 10The microfluidic chip consists of three layers: an upper cover layer containing sample loading holes and diluent loading holes (the cover plate in the above embodiment); a middle channel layer, in which the second fluid channel penetrates the channel layer; and other channels (such as the first fluid channel, buffer channel, etc.) and channels (such as the first microchannel, second microchannel, delivery channel, etc.) may or may not penetrate the channel layer. The lower cover layer is the substrate in the above embodiment. The three layers are watertight (i.e., fluids such as water will not enter the contact interface between the three layers). Three independent fluid modules are arranged on the microfluidic chip. The delivery channel in each fluid module is set as a ring that gradually moves away from the rotation axis, and 10 detection groups (i.e., 10 buffer channels) are connected to it. An overflow channel (the first waste liquid channel in the above embodiment) is connected to the end away from the rotation axis. The first fluid channel is rectangular and has a volume of 40 microliters. All the second fluid channels are circular, with the center about 60 mm away from the rotation axis, and have a volume of 50 microliters. The buffer tank is rectangular with a volume of 3 microliters. The first microchannel between the first fluid tank and the buffer tank is 3 mm long, 0.6 mm wide, and 0.1 mm deep. The second microchannel between the buffer tank and the second fluid tank is 2 mm long, 0.4 mm wide, and 0.1 mm deep. Part of the second fluid tank is pre-loaded with lyophilized antibody reagents, which can be used to detect immune parameters in samples such as plasma, serum, or other body fluids, including those related to infectious diseases, hormones, cardiac markers, tumor markers, and infection-related immune parameters.

[0110] For example, samples are injected into the sample well through the sample loading port into the sample well, and diluent is injected into the diluent well through the diluent loading port into the diluent well. The microfluidic chip is then fixed to a motor and rotated for 2 minutes at 5000 rpm. The sample flows into the sample quantification well, and excess sample flows into the sample overflow well. The diluent flows into the diluent quantification well, and excess diluent flows into the diluent overflow well. The motor stops, and the plasma in the supernatant of the sample quantification well fills the sample siphon microchannel (the third siphon channel in the above embodiment) under capillary action. The diluent in the diluent quantification well fills the diluent siphon microchannel (the fourth siphon channel in the above embodiment) under capillary action. The motor is then restarted and rotated for 30 seconds at 5000 rpm. The sample quantified by the sample quantification well and the diluent quantified by the diluent quantification well are injected into the mixing well through the siphon microchannels (the third and fourth siphon channels in the above embodiment). The motor continues to rotate, its speed adjusted 20 times between 4500 and 1500 rpm to ensure uniform mixing of the sample and diluent injected into the mixing tank. The motor then stops, and the mixture in the mixing tank fills the siphon microchannel (the second siphon channel in the above embodiment) under capillary action. The motor is then restarted and rotated for 60 seconds, gradually increasing the speed from 300 rpm to 800 rpm (e.g., the second rotation frequency in the above embodiment). The fluid formed by the above mixture sequentially fills the first fluid tank through the delivery channel, with excess fluid flowing into the overflow tank (the first waste tank in the above embodiment). The speed is then further increased to 3000 rpm (as in the third rotation frequency in the above embodiment), and the fluid in the first fluid tank enters the second fluid tank to mix and react with the pre-filled reagents. Based on the above operating method, the applicant, through testing the reaction results in the second fluid tank, found that the corresponding immunoreaction results were normal, and no reagent contamination was found, i.e., no cross-contamination occurred.

[0111] At least one embodiment of this disclosure provides a microfluidic chip holder, each holder capable of holding four independent microfluidic chips. Each microfluidic chip is equipped with a sample reservoir, a diluent reservoir, a sample quantification reservoir, a diluent quantification reservoir, an overflow reservoir (e.g., a first waste liquid reservoir), and a liquid delivery channel connecting 15 detection groups (including a buffer reservoir). The first fluid reservoir is rectangular with a volume of 25 μL. All second fluid reservoirs are circular, with their centers approximately 80 mm from the rotation axis, and also have a volume of 25 μL. The buffer reservoir is rectangular with a volume of 5 μL. The microchannel between the first fluid reservoir and the buffer reservoir (the first microchannel in the above embodiment) is 0.5 mm long, 0.3 mm wide, and 0.1 mm deep. The microchannel between the buffer reservoir and the second fluid reservoir (the second microchannel in the above embodiment) is 0.5 mm long, 0.3 mm wide, and 0.3 mm deep. Some of the second fluid reservoirs are pre-loaded with lyophilized primers and reagents required for nucleic acid amplification, which can be used for nucleic acid molecular detection.

[0112] For example, samples are injected into the sample tank through the sample loading port, and diluent is injected into the diluent tank through the diluent loading port. The microfluidic chip is then fixed to a motor and rotated for 2 minutes at 3000 rpm. The sample flows into the sample quantification tank, and excess sample flows into the sample overflow tank. The diluent flows into the diluent quantification tank, and excess diluent flows into the diluent overflow tank. The motor stops, and the plasma in the supernatant of the sample quantification tank fills the sample siphon microchannel (the third siphon channel in the above embodiment) under capillary action. The diluent in the diluent quantification tank fills the diluent siphon microchannel (the fourth siphon channel in the above embodiment) under capillary action. The motor is then restarted and rotated for 30 seconds at 3000 rpm. The sample quantified by the sample quantification tank and the diluent quantified by the diluent quantification tank are injected into the mixing tank through the siphon microchannels (the third and fourth siphon channels in the above embodiment). The motor continues to rotate, with its speed controlled between 4500 rpm and 1500 rpm, accelerating and decelerating 20 times per minute to ensure uniform mixing of the sample and diluent injected into the mixing tank. The motor then stops, and the mixture in the mixing tank fills the siphon microchannel (the second siphon channel in the above embodiment) under capillary action. The motor is then restarted and rotated for 60 seconds, with the speed gradually increasing from 300 rpm to 600 rpm (e.g., the second rotation frequency in the above embodiment). The fluid formed by the above mixture sequentially fills the first fluid tank through the delivery channel, with excess fluid flowing into the overflow tank (the first waste tank in the above embodiment). The motor is then restarted and rotated, with its speed controlled between 3000 rpm and 1500 rpm (e.g., the third rotation frequency in the above embodiment), accelerating and decelerating 20 times per minute. The fluid in the first fluid tank enters the second fluid tank and mixes with the pre-filled reagents. Based on the above operating method, the applicant, through testing the reaction results in the second fluid tank, found that the corresponding nucleic acid molecule detection results were normal, and no reagent contamination was found, i.e., no cross-contamination occurred.

[0113] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A microfluidic substrate, comprising a flow channel structure, wherein, The flow channel structure includes: Conveyor channel; Multiple detection groups, each of the multiple detection groups includes a first fluid tank, a first microchannel, a buffer tank, a second microchannel, and a second fluid tank connected in sequence, the first fluid tank being connected to the delivery channel, and at least one of the second fluid tanks of the detection group containing reagents; The microfluidic substrate has a rotation axis, and the detection group is located on the side of the delivery channel opposite to the rotation axis. The distances from the first fluid tank, buffer tank, and second fluid tank in each detection group to the rotation axis increase sequentially. When fluid is present in the first microchannel, the fluid in the first microchannel, the buffer tank, the second microchannel, and the second fluid tank form a closed channel to block fluid flow in the first microchannel by creating reverse pressure through air closure before all the first fluid tanks are filled with fluid. The buffer groove includes a first sidewall and a second sidewall. The first sidewall includes a first inlet communicating with the first microchannel. The second sidewall is opposite to the first sidewall and includes a first outlet communicating with the second microchannel. The area of ​​the second sidewall without the first outlet includes at least one recessed portion. The distance from the bottom of the at least one recessed portion to the rotation axis is greater than the distance from the first outlet to the rotation axis.

2. The microfluidic substrate according to claim 1, wherein, The first microchannel is configured to have a first length such that, at a first rotational frequency not greater than that of the microfluidic substrate, the fluid from the first fluid tank and the gas present in the buffer tank form a gas-liquid interface, and the gas-liquid interface exists in the first microchannel or at the connection between the first microchannel and the buffer tank.

3. The microfluidic substrate according to claim 2, wherein, The first length is 0.1 to 5 mm, the width of the first microchannel is 0.1 to 1 mm, and the depth of the first microchannel is 0.1 to 0.5 mm.

4. The microfluidic substrate according to any one of claims 1-3, wherein, In each of the aforementioned buffer slots, The first outlet has recessed portions on both sides, and the first outlet is located in the middle of the second sidewall; or The recessed portion is provided on one side of the first outlet, the first outlet is located in the middle of the second sidewall, and along the preset rotation direction of the microfluidic substrate, the recessed portion and the first outlet are arranged sequentially; or The recessed portion is provided on one side of the first outlet, the first outlet is located at one end of the second sidewall and along the preset rotation direction of the microfluidic substrate, and the recessed portion and the first outlet are arranged sequentially; and The first entrance is located in the middle of the first sidewall; or The first inlet is located at one end of the first sidewall and is arranged sequentially along the preset rotation direction of the microfluidic substrate.

5. The microfluidic substrate according to any one of claims 1-3, wherein, Each testing group also includes: The first siphon channel has one end connected to the recessed portion to communicate with the buffer groove, and the other end connected to the second fluid groove. Wherein, the inner diameter of the first siphon channel is smaller than the inner diameter of the second microchannel, and the distance from a portion of the first siphon channel to the rotation axis is smaller than the distance from the first outlet to the rotation axis.

6. The microfluidic substrate according to any one of claims 1-3, wherein, The volume of the first fluid tank is greater than the volume of the second fluid tank, and the volume of the first fluid tank is less than or equal to the sum of the volumes of the second fluid tank and the buffer tank.

7. The microfluidic substrate according to any one of claims 1-3, wherein, The conveying channel is non-closed annular in shape, and the center of the circle containing the annulus is the rotation axis; or The conveying channel is non-closed ring-shaped. The distance from the first end of the conveying channel to the rotation axis is less than the distance from the second end of the conveying channel to the rotation axis. From the first end to the second end, the distance from the conveying channel to the rotation axis increases sequentially.

8. The microfluidic substrate according to any one of claims 1-3, further comprising: The first waste liquid tank is connected to one end of the conveying channel, and the first waste liquid tank and the conveying channel are arranged in sequence along the preset rotation direction of the microfluidic substrate; The mixing tank includes two inlets and one outlet; The second siphon channel is connected at one end to the outlet of the mixing tank and at the other end to the conveying channel. The distance from a portion of the second siphon channel to the rotation axis is less than the distance from the mixing tank to the rotation axis. Sample slots; A sample quantitative cell is connected to the sample cell, and the distance from the sample quantitative cell to the rotation axis is greater than the distance from the sample cell to the rotation axis. A sample overflow trough is connected to the sample trough, and the distance from the sample overflow trough to the rotation axis is greater than the distance from the sample metering trough to the rotation axis. The third siphon channel has one end connected to the sample quantitative cell and the other end connected to one of the two inlets of the mixing cell, and the distance from a portion of the second siphon channel to the rotation axis is less than the distance from the sample quantitative cell to the rotation axis. Diluent tank; A diluent metering tank is connected to the diluent tank, and the distance from the diluent metering tank to the rotation axis is greater than the distance from the diluent tank to the rotation axis. A diluent overflow tank is connected to the diluent tank, and the distance from the diluent overflow tank to the rotation axis is greater than the distance from the diluent metering tank to the rotation axis. The fourth siphon channel is connected at one end to the diluent metering tank and at the other end to one of the two inlets of the mixing tank. The distance from a portion of the fourth siphon channel to the rotation axis is less than the distance from the diluent metering tank to the rotation axis. A flow channel layer, wherein the flow channel structure is formed in the flow channel layer; as well as The substrate is located on the opposite side of the flow channel layer from the side where the first fluid groove, the first microchannel, the buffer groove, the second microchannel and the second fluid groove are provided. The substrate is attached to the flow channel layer or the substrate and the flow channel layer are integrally formed.

9. A microfluidic chip, comprising a cover plate and a microfluidic substrate as described in any one of claims 1-8, wherein, The cover plate mates with the microfluidic chip and is located on one side of the microfluidic substrate where the first fluid groove, the first microchannel, the buffer groove, the second microchannel, and the second fluid groove are provided.

10. A method for operating a microfluidic chip, wherein, The microfluidic chip includes a cover plate and a microfluidic substrate. The microfluidic substrate includes a flow channel structure, which includes a delivery channel and multiple detection groups. Each detection group includes a first fluid tank, a first microchannel, a buffer tank, a second microchannel, and a second fluid tank connected in sequence. The first fluid tank is connected to the delivery channel. A reagent is disposed in the second fluid tank of at least one detection group. The microfluidic substrate has a rotation axis. The detection group is located on the side of the delivery channel opposite to the rotation axis. The distances from the first fluid tank, buffer tank, and second fluid tank in each detection group to the rotation axis increase sequentially. The operation method includes: The microfluidic chip is driven to rotate at a second rotation frequency so that the fluid in the delivery channel enters the first fluid tank and forms a gas-liquid interface in the first microchannel or at the connection between the first microchannel and the buffer tank. The fluid in the first microchannel, the buffer tank, the second microchannel and the second fluid tank form a closed tank so that the fluid flow in the first microchannel is blocked by air blocking to generate reverse pressure before all the first fluid tanks are filled with fluid. After the first fluid tank in each of the detection groups is filled with the liquid, the microfluidic chip is driven to rotate at a third rotation frequency so that the fluid enters the buffer tank and the second fluid tank. Wherein, the third rotation frequency is greater than the second rotation frequency, the buffer groove includes a first sidewall and a second sidewall, the first sidewall includes a first inlet communicating with the first microchannel, the second sidewall is opposite to the first sidewall and includes a first outlet communicating with the second microchannel, the area of ​​the second sidewall without the first outlet includes at least one recessed portion, and the distance from the bottom of the at least one recessed portion to the rotation axis is greater than the distance from the first outlet to the rotation axis.

11. The operating method according to claim 10, wherein, The first microchannel is configured to have a first length such that, at a first rotational frequency not greater than that of the microfluidic substrate, fluid from the first fluid tank and gas present in the buffer tank form a gas-liquid interface, and such gas-liquid interface exists in the first microchannel or at the connection between the first microchannel and the buffer tank. The second rotation frequency is not greater than the first rotation frequency, and the third rotation frequency is greater than the first rotation frequency; or The second rotation frequency is not greater than the first rotation frequency. When the microfluidic chip is driven to rotate at the third rotation frequency, the rotation mode is reciprocating motion.

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