CN117501126A

CN117501126A - Microfluidic substrate, microfluidic chip and operation method thereof

Info

Publication number: CN117501126A
Application number: CN202180003524.4A
Authority: CN
Inventors: 王战会; 王树相; 张天乐
Original assignee: Micro Nano Core Suzhou Technology Co ltd; TIANJIN MNCHIP TECHNOLOGIES CO LTD; Tianjin Nabaixin Technology Co ltd
Current assignee: Tianjin Nabaixin Technology Co ltd; Tianjin Weinanxin Technology Co ltd
Priority date: 2021-04-15
Filing date: 2021-04-15
Publication date: 2024-02-02
Also published as: WO2022217548A1

Abstract

A microfluidic substrate (10), a microfluidic chip and a method of operating the same, the microfluidic substrate (10) comprising a flow channel structure comprising a transport flow channel (100) and a plurality of detection groups (200). Each detection group (200) comprises a first fluid groove (210), a first micro-flow channel (240), a buffer groove (220), a second micro-flow channel (250) and a second fluid groove (230) which are communicated in sequence, wherein the first fluid groove (210) is communicated with the conveying flow channel (100), and reagents are arranged in the second fluid groove (230) of at least one detection group (200). The microfluidic substrate (10) is provided with a rotation axis (11), the detection groups (200) are positioned on one side of the conveying flow channel (100) which is away from the rotation axis (11), and 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) are sequentially increased.

Description

Microfluidic substrate, microfluidic chip and operation method thereof

Technical Field

The present disclosure relates to the field of analytical detection, and in particular, to a microfluidic substrate, a microfluidic chip, and methods of operating the same.

Background

The microfluidic chip technology (Microfluidics) integrates basic operation units of sample preparation, reaction, separation, detection and the like in biological, chemical and medical analysis processes on a chip, and automatically completes the whole analysis process. Because of its great potential in biological, chemical, medical and other fields, it has been developed into a new research field where the disciplines of biology, chemistry, medicine, fluids, electronics, materials, machinery and the like are crossed.

Centrifugal microfluidic, which drives fluid and controls the amount of fluid in a microfluidic channel by centrifugal force, has the advantages of high integration, automation, micromation and parallel detection of multiple samples or indexes, and has become an important branch in the technical field of microfluidic chips. However, the current microfluidic chip is limited to its own structural design, and when detection is performed, the problems of cross contamination, difficulty in controlling reaction time and the like are easy to exist, so that the detection result is inaccurate, and the requirement of a user is difficult to meet.

Disclosure of Invention

In view of the above, the disclosure provides a microfluidic substrate, a microfluidic chip and an operation method thereof, which enhance control of fluid by arranging a buffer tank before a reaction tank pre-filled with reagents, ensure that the reagents pre-filled in different reaction tanks simultaneously react with the fluid in a contact manner, and avoid cross contamination of the pre-filled reagents in different reaction tanks, thereby ensuring reliable detection results.

A first aspect of the present disclosure provides a microfluidic substrate comprising a flow channel structure comprising a transport flow channel and a plurality of detection groups. Each detection group comprises a first fluid groove, a first micro-channel, a buffer groove, a second micro-channel and a second fluid groove which are communicated in sequence, wherein the first fluid groove is communicated with the conveying channel, and a reagent is arranged in the second fluid groove of at least one detection group. The microfluidic substrate is provided with a rotation axis, the detection groups are positioned on one side of the conveying flow channel, which is away from the rotation axis, and the distances from the first fluid groove, the buffer groove and the second fluid groove in each detection group to the rotation axis are sequentially increased. The microfluidic substrate can avoid the liquid in the first fluid groove from contacting with the pre-loaded reagent in the second fluid groove in advance, accurately control the reaction time of the reagent in the second fluid groove, and avoid the risk of cross contamination of the reagents in each detection group.

In a particular embodiment of the first aspect of the present disclosure, in the case of having a fluid in the first fluidic channel, the buffer tank, the second fluidic channel and the second fluidic channel are formed into a closed tank. Thus, after the microfluidic substrate is packaged (e.g., aligned with the cover plate), the buffer channel, the second fluidic channel, and the second fluidic channel communicate with the first fluidic channel only through the first fluidic channel.

In the above-described arrangement, in the case where the fluid flows along the side wall of the first fluid tank through the delivery flow passage toward the bottom of the first fluid tank (the portion of the first fluid tank facing away from the rotation axis), that is, in the stage where the fluid delivery flow passage enters the first fluid tank to achieve the dosing in the first fluid tank (which stage requires avoiding the fluid entering the second fluid tank), the inlet of the first micro flow passage at the bottom of the first fluid tank (the opening of the first micro flow passage to which the first fluid tank is connected) will be blocked due to the interfacial tension of the fluid. The fluid which continuously flows into the first fluid groove is driven by centrifugal force to further enter the first micro-flow channel, and the air which is sealed in the buffer groove and the second fluid groove is compressed to generate reverse pressure. When the reverse pressure and the surface tension of the fluid reach equilibrium with the centrifugal force, the fluid stops flowing, so that a stable gas-liquid interface is formed, the fluid is prevented from directly entering the second fluid tank at the stage, and the fluid is prevented from reacting with the pre-loaded reagent in the second fluid tank in advance.

In a particular embodiment of the first aspect of the present disclosure, the first fluidic channel is configured to have a first length such that, at a first rotational frequency of the microfluidic substrate that is not greater than, the fluid from the first fluidic channel and the gas present in the buffer channel form a gas-liquid interface, and such that the gas-liquid interface is present in the first fluidic channel or at a communication of the first fluidic channel and the buffer channel.

In a microfluidic substrate provided in one specific embodiment of the first aspect of the present disclosure, the first length is 0.1 to 5mm, the width of the first micro flow channel is 0.1 to 1mm, and the depth of the first micro flow channel is 0.1 to 0.5mm.

In a particular embodiment of the first aspect of the present disclosure, the microfluidic substrate provided in the present disclosure, the buffer groove includes a first sidewall and a second sidewall. The first side wall comprises a first inlet communicated with the first micro-channel, and the second side wall is opposite to the first side wall and comprises a first outlet communicated with the second micro-channel.

In a specific embodiment of the first aspect of the present disclosure, the second side wall is a plane, and the plane is perpendicular to a direction along the rotation axis to the first inlet.

In another embodiment of the first aspect of the present disclosure, the area of the second sidewall not provided with the first outlet includes at least one concave portion, and a distance from a bottom of the at least one concave portion to the rotation axis is greater than a distance from the first outlet to the rotation axis.

In the above-described aspect, if a part of the fluid flows to the buffer tank in a stage in which the fluid of the transfer flow path is caused to fill the first fluid tank, the part of the fluid is collected at the concave portion because the microfluidic substrate is in a rotated state, and the distance from the bottom of the concave portion to the rotation axis is greater than the distance from the first outlet to the rotation axis, the fluid collected at the concave portion does not enter the first outlet and thus does not enter the second fluid tank, i.e., the risk that the fluid enters the second fluid tank in this stage is further reduced.

In a microfluidic substrate provided in one embodiment of the first aspect of the present disclosure, in each buffer tank, both sides of the first outlet are provided with concave portions, and the first outlet is located in the middle of the second sidewall.

In the above-mentioned scheme, no matter the preset rotation direction of the microfluidic chip is clockwise or anticlockwise, in the stage of enabling the fluid of the conveying flow channel to fill the first fluid groove, the concave part can be used for gathering the fluid entering the buffer groove, so that the risk of the fluid entering the second fluid groove in the stage is reduced.

In another embodiment of the first aspect of the present disclosure, in each buffer slot, a concave portion is disposed at one side of the first outlet, the first outlet is located in the middle of the second sidewall, and along a preset rotation direction of the microfluidic substrate, the concave portion and the first outlet are sequentially disposed.

In another embodiment of the first aspect of the present disclosure, in each buffer slot, a concave portion is disposed at one side of the first outlet, the first outlet is located at one end of the second sidewall, and along a preset rotation direction of the microfluidic substrate, and the concave portion and the first outlet are sequentially disposed.

In the above-described aspect, since the first outlet is located at one end of the second side wall, the design area of the concave portion is increased, that is, the design area of the concave portion in the second side wall can be increased as compared with the case where the first outlet is located in the middle of the second side wall, the design volume of the concave portion can be increased, that is, the amount of fluid that the concave portion can store is increased, and the risk of fluid flowing into the second fluid tank is further reduced at the stage of filling the first fluid tank with fluid that is to be delivered to the flow path.

In a particular embodiment of the first aspect of the present disclosure, in each buffer tank, the first inlet is located in the middle of the first sidewall.

In another embodiment of the first aspect of the present disclosure, in each buffer slot, the first inlet is located at one end of the first sidewall, and the first inlet and the first outlet are sequentially arranged along a preset rotation direction of the microfluidic substrate.

In the above-described aspect, the distance between the first inlet and the first outlet is increased in the preset rotational direction as compared with the first inlet being located in the middle of the first side wall, and the risk of the fluid falling directly at the first outlet is reduced at a stage where the fluid of the delivery flow path fills the first fluid tank 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 at this stage.

In a particular embodiment of the first aspect of the present disclosure, in each buffer tank, each detection group further includes a first siphon flow channel. One end of the first siphon runner is connected to the concave part to be communicated with the buffer groove, and the other end of the first siphon runner is communicated with the second fluid groove. The inner diameter of the first siphon runner is smaller than that of the second micro runner, and the distance from the part of the first siphon runner to the rotation axis is smaller than that from the first outlet to the rotation axis.

After the fluid of the first fluid tank is introduced into the second fluid tank, a portion of the fluid of the buffer tank may be restricted to the concave portion, in which case the first siphon flow passage may introduce the fluid of the concave portion into the second fluid tank, improving the utilization of the fluid.

In a particular embodiment of the first aspect of the present disclosure, there is provided a microfluidic substrate wherein the volume of the first fluid channel is smaller than the volume of the second fluid channel.

In another embodiment of the first aspect of the present disclosure, the microfluidic substrate is provided wherein 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.

In the above scheme, the residual fluid in the first fluid tank can be avoided, so that the fluid quantified by the first fluid tank can enter the buffer tank and the second fluid tank, the aggregation of the fluid in the first fluid tank is avoided, and the first fluid tank can be ensured to be filled with the fluid quantified by the first fluid tank by making the volume of the first fluid tank larger than that of the second fluid tank.

In another embodiment of the first aspect of the present disclosure, there is provided a microfluidic substrate wherein the volume of the first fluid channel is greater than the sum of the volumes of the second fluid channel and the buffer channel.

For example, in some embodiments of the present disclosure, the volumes of the first fluid tank may all be the same or different, the volumes of the buffer tank may all be the same or different, the volumes of the second fluid tank may all 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.

In the microfluidic substrate provided in one specific embodiment of the first aspect of the present disclosure, the conveying flow channel is in a non-closed ring shape, and the center of a circle where the ring is located is a rotation axis; or the conveying flow channel is in a non-closed ring shape, the distance from the first end of the conveying flow channel to the rotation axis is smaller than the distance from the second end of the conveying flow channel to the rotation axis, and the distance from the first end to the second end of the conveying flow channel to the rotation axis is sequentially increased.

In the above scheme, when the microfluidic substrate rotates, the fluid is uniformly distributed in the conveying flow channel, so that the fluid uniformly flows into the first fluid groove in each detection group.

The microfluidic substrate provided by one embodiment of the first aspect of the present disclosure may further include a first waste liquid tank. The first waste liquid tank is communicated to one end of the conveying flow channel. Along the preset rotation direction of the microfluidic substrate, the first waste liquid tank and the conveying flow channel are sequentially arranged.

In the above scheme, the fluid in the conveying flow channel flows into the first waste liquid tank after filling the first fluid tank, so that the risk that the fluid breaks through the buffer tank and enters the second fluid tank is reduced, wherein the risk that the fluid in the conveying flow channel continuously enters the first fluid tank in the detection group to cause the fluid pressure to be too high to damage the gas-liquid interface is avoided.

In the microfluidic substrate provided in one specific embodiment of the first aspect of the present disclosure, in a case that the first waste liquid tank and the conveying flow channel are sequentially arranged along a preset rotation direction of the microfluidic substrate, a distance from the rotation axis of the first waste liquid tank is greater than a distance from any one of the first fluid tanks to the rotation axis. In addition, because the first fluid tank is located at the side of the conveying flow channel, which is away from the rotation axis, the distance from the first waste liquid tank to the rotation axis is actually larger than the distance from the conveying flow channel to the rotation axis.

In the above scheme, the fluid in the conveying flow channel passes through all the first fluid grooves at first and then passes through the first waste liquid groove under the rotating state, in the process, the first fluid groove is filled with the fluid first so as to ensure the quantitative effect of the first fluid groove, and in the first waste liquid groove, the conveying flow channel and the first fluid groove, the first waste liquid groove is the side with the largest distance from the rotating axle center, and the fluid which does not enter the first fluid groove in the conveying flow channel can flow into the first waste liquid groove under the action of centrifugal force, so that the conveying flow channel is ensured not to have fluid aggregation, the risk of mixing the fluids in different first fluid grooves is reduced, the quantitative effect of the first fluid groove is not influenced, the cross contamination is avoided, and the quantitative accuracy of the fluid in the first fluid groove is improved.

The microfluidic substrate provided by one embodiment of the first aspect of the present disclosure may further include a mixing tank and a second siphon flow channel. The mixing tank comprises two inlets and an outlet, one end of the second siphon runner is communicated with the outlet of the mixing tank, and the other end of the second siphon runner is connected to the conveying runner. The distance from the part of the second siphon flow passage to the rotation axis is smaller than the distance from the mixing groove to the rotation axis.

A microfluidic substrate provided by one embodiment of the first aspect of the present disclosure may further include a sample well, a sample quantification well, a sample overflow well, a third siphon flow channel, a dilution liquid well, a dilution liquid quantification well, a dilution liquid overflow well, and a fourth siphon flow channel. The sample quantifying groove is communicated with the sample groove, and the distance from the sample quantifying groove to the rotation axis is greater than the distance from the sample groove to the rotation axis. The sample overflow groove is communicated with the sample groove, and the distance from the sample overflow groove to the rotation axis is larger than the distance from the sample quantifying groove to the rotation axis. One end of the third siphon flow passage is communicated with the sample quantifying groove, the other end of the third siphon flow passage is communicated with one of two inlets of the mixing groove, and the distance from the part of the second siphon flow passage to the rotation axis is smaller than the distance from the sample quantifying groove to the rotation axis. The diluent quantifying groove is communicated with the diluent groove, and the distance from the diluent quantifying groove to the rotation axis is greater than the distance from the diluent groove to the rotation axis. The diluent overflow groove is communicated with the diluent groove, and the distance from the diluent overflow groove to the rotation axis is greater than the distance from the diluent quantitative groove to the rotation axis. One end of the fourth siphon flow passage is communicated with the diluent quantifying groove, the other end of the fourth siphon flow passage is communicated with the other of the two inlets of the mixing groove, and the distance from the part of the fourth siphon flow passage to the rotation axis is smaller than the distance from the diluent quantifying groove to the rotation axis.

A microfluidic substrate provided by a specific embodiment of the first aspect of the present disclosure may further include a flow channel layer and a substrate. The runner structure is formed in the runner layer. The substrate is positioned on the other side of the runner layer opposite to the side provided with the first fluid groove, the first micro runner, the buffer groove, the second micro runner and the second fluid groove. The substrate and the runner layer are arranged in a laminating way or are integrally formed.

A second aspect of the present disclosure provides a microfluidic chip comprising a cover plate and a microfluidic substrate as in the first aspect described above. The cover plate and the microfluidic chip are combined and positioned on one side of the microfluidic substrate, on which the first fluid groove, the first micro-channel, the buffer groove, the second micro-channel and the second fluid groove are arranged.

The third aspect of the present disclosure provides an operation method of a microfluidic chip, where the microfluidic chip includes a cover plate and a microfluidic substrate, the microfluidic substrate includes a flow channel structure, the flow channel structure includes a conveying flow channel and a plurality of detection groups, each detection group of the plurality of detection groups includes a first fluid channel, a first micro flow channel, a buffer channel, a second micro flow channel and a second fluid channel that are sequentially communicated, the first fluid channel is communicated with the conveying flow channel, a reagent is disposed in the second fluid channel of at least one detection group, the microfluidic substrate has a rotation axis, the detection groups are located at a side of the conveying flow channel away from the rotation axis, and distances from the first fluid channel, the buffer channel and the second fluid channel in each detection group to the rotation axis are sequentially increased, and the operation method includes: driving the microfluidic chip to rotate at a second rotation frequency, so that fluid of the conveying flow channel enters the first fluid groove and a gas-liquid interface is formed in the first microfluidic channel or at the communication part of the first microfluidic channel and the buffer groove; after the first fluid groove in each detection group is filled with liquid, driving the micro-fluidic chip to rotate at a third rotation frequency so that fluid enters the buffer groove and the second fluid groove; wherein the third rotational frequency is greater than the first rotational frequency.

In a method of operating a microfluidic chip provided in one embodiment of the third aspect of the present disclosure, the first fluidic channel is configured to have a first length such that, at a first rotational frequency not greater than the microfluidic substrate, a fluid from the first fluidic channel and a gas present in the buffer channel form a gas-liquid interface, and such that the gas-liquid interface is present in the first fluidic channel or at a communication of the first fluidic channel and the buffer channel. 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, and the rotation mode is reciprocating motion when the micro-fluidic chip is driven to rotate at the third rotation frequency.

Drawings

Fig. 1 is a schematic plan view of a partial region of a microfluidic substrate according to an embodiment of the disclosure;

fig. 2 is a schematic plan view of a detection group in the microfluidic chip shown in fig. 1;

fig. 3 is a schematic plan view of another detection group in a microfluidic substrate according to an embodiment of the disclosure;

fig. 4 is a schematic plan view of another detection group in a microfluidic substrate according to an embodiment of the disclosure;

fig. 5 is a schematic plan view of another detection group in a microfluidic substrate according to an embodiment of the disclosure;

Fig. 6 is a schematic plan view of another detection group in a microfluidic substrate according to an embodiment of the disclosure;

fig. 7 is a schematic plan view of another detection group in a microfluidic substrate according to an embodiment of the disclosure;

fig. 8 is a schematic plan view of a microfluidic chip according to an embodiment of the disclosure;

fig. 9 is a schematic cross-sectional view of a partial region in a microfluidic chip according to an embodiment of the disclosure;

fig. 10 is a schematic cross-sectional view of a partial region in another microfluidic chip according to an embodiment of the disclosure;

fig. 11 to 15 are schematic operation flow diagrams of the microfluidic chip shown in fig. 8.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Microfluidic (Microfluidics) refers to the science and technology involved in systems that use microchannels (tens to hundreds of microns in size) to process or manipulate minute fluids (nanoliters to microliters in volume), and is an emerging intersection discipline involving chemical, fluid physics, microelectronics, new materials, biology, and biomedical engineering. Because of its features of miniaturization, integration, etc., microfluidic devices are commonly referred to as microfluidic chips, and may also be referred to as labs on a Chip (Lab a Chip) or micro-total analysis systems (micro-Total Analytical System).

In the microfluidic chip, a plurality of detection grooves (for example, a second fluid groove in the embodiment described below) are provided, and reagents are preloaded in the detection grooves, for example, different reagents are preloaded in different detection grooves, so that a plurality of kinds of detection on samples can be realized in one detection flow. Each of the detecting tanks is provided with a receiving tank (for example, a first fluid tank in the embodiment described below) to pre-store the fluid injected into each detecting tank, and in the actual detecting process, it is necessary to inject the fluid into the receiving tank to pre-store the fluid injected into each detecting tank before injecting the fluid containing the sample into the detecting tank, and after each receiving tank is injected with the fluid, the fluid of the receiving tank may be injected into the detecting tank by means such as increasing the rotation speed. However, in the actual process, in the stage of injecting the fluid into the accommodating tank to pre-store the fluid, the fluid in the accommodating tank may flow into the detecting tank to be mixed with the reagent in the detecting tank to start the reaction in advance, and errors may occur in the detection result.

The embodiments of the present disclosure provide a microfluidic substrate, a microfluidic chip, and an operation method thereof, which can solve the above technical problems. The microfluidic substrate comprises a flow channel structure, wherein the flow channel structure comprises a conveying flow channel and a plurality of detection groups. Each detection group comprises a first fluid groove, a first micro-channel, a buffer groove, a second micro-channel and a second fluid groove which are communicated in sequence, wherein the first fluid groove is communicated with the conveying channel, and a reagent is arranged in the second fluid groove of at least one detection group. The microfluidic substrate is provided with a rotation axis, the detection groups are positioned on one side of the conveying flow channel, which is away from the rotation axis, and the distances from the first fluid groove, the buffer groove and the second fluid groove in each detection group to the rotation axis are sequentially increased. Thus, by providing the buffer tank to space the first fluid tank and the second fluid tank, at a stage where the fluid of the transport flow channel fills the first fluid tank, it is possible to avoid that the fluid flowing out of the first fluid tank directly enters the second fluid tank, reducing the risk of fluid entering the second fluid tank and re-entering the first fluid tank from the second fluid tank (at this time the fluid has been in contact with the reagent) at this stage, i.e. reducing the risk of cross-contamination of the reagents in the respective detection groups; furthermore, by providing a buffer tank, it is possible to prevent the fluid from directly entering the second fluid tank in this stage to react with the reagent in advance, thereby precisely controlling the reaction time of the reagent in the second fluid tank.

In a practical process, the fluid passing from the delivery channel into the second fluid tank may comprise two stages. In the first stage, at a low rotation speed, fluid flows along the side wall of the first fluid groove to the bottom of the first fluid groove (the part of the first fluid groove facing away from the rotation axis) through the conveying flow channel, and the inlet of the first micro flow channel at the bottom of the first fluid groove is sealed due to interfacial tension. The fluid which continuously flows into the first fluid groove is driven by centrifugal force to further enter the first micro-flow channel, and the air which is sealed in the buffer groove and the second fluid groove is compressed to generate reverse pressure. When the back pressure and the surface tension of the fluid reach balance with the centrifugal force, the fluid stops flowing, and a stable gas-liquid interface is formed in the first micro-flow channel or at the communication part of the first micro-flow channel and the buffer tank. When all the first fluid tanks are filled with fluid, the excess fluid in the transfer flow path is discharged (e.g., the excess fluid flows into an overflow tank, which may be a first waste tank in the embodiments described below), and a fixed amount of fluid is independently pre-stored in each of the first fluid tanks; in the second stage, the rotating speed is increased, the increased centrifugal force breaks the gas-liquid interface balance, so that the quantitative fluid which is independently pre-stored in each first fluid groove flows into the corresponding second fluid groove through the buffer groove connected with the quantitative fluid, thereby reacting with the reagent pre-stored in the second fluid groove, and the sealed air is discharged through the first micro-flow channel. The scheme ensures that the reagents preloaded into the second fluid tanks start to react in contact with the fluid at the same time, and avoids the cross contamination of the reagents in the second fluid tanks.

The buffer tank is provided to separate the first fluid tank from the second fluid tank in order to prevent the fluid from flowing into the second fluid tank to contact the reagent preloaded in the second fluid tank in the first stage, and to initiate a reaction in advance to affect the detection result. If the first fluid tank and the second fluid tank are directly connected through the micro flow channel, it is found in practical implementation that when the first fluid tank is full of the first-stage fluid, part of the fluid may flow into the respective second fluid tank to contact with the preloaded reagent to start the reaction in advance before the stable gas-liquid interface is formed. In addition, in the practical implementation of the connection of the first fluid tank and the second fluid tank directly through the micro flow channels, it has also been found that, when the first fluid tank is filled with the first-stage fluid, the gas-liquid interface formed in the respective micro flow channel is driven by centrifugal force to extend into the second fluid tank, i.e. the fluid is brought into contact with the pre-filled reagent in the second fluid tank to initiate the reaction in advance. In particular, the freeze-dried microsphere reagent containing the porous structure, which is pre-loaded in the second fluid tank, has the microsphere size only slightly smaller than the second fluid tank, and is easy to contact with the fluid extending into the second fluid tank to start the reaction in advance. In practical processes, a high-precision processing technology or a strictly controlled processing technology of the microfluidic substrate may be used to make all the first fluid grooves, the micro-channels and the second fluid grooves uniform, which may reduce the risk of the occurrence of the above problems, but may significantly increase the processing difficulty and the processing cost of the microfluidic substrate.

A buffer groove is arranged between the first fluid groove and the second fluid groove, a small amount of fluid flowing out of the first fluid groove flows into the buffer groove, the risk of flowing into the second fluid groove is reduced, and in addition, even if a gas-liquid interface extending out of the first micro-flow channel extends into the buffer groove (the communicating position of the buffer groove and the first micro-flow channel), the fluid is not contacted with the reagent preloaded in the second fluid groove. The buffer groove can effectively prevent the fluid from being contacted with the reagent preloaded in the second fluid groove in advance to start the reaction, and the processing difficulty and the processing cost of the microfluidic substrate are obviously reduced.

Hereinafter, a microfluidic substrate, a microfluidic chip, and an operation method thereof according to at least one embodiment of the present disclosure will be described in detail with reference to the accompanying drawings. In these drawings, a space rectangular coordinate system is established with the surface of the microfluidic substrate as a reference, so that the positions of the respective components (e.g., buffer grooves, etc.) in the microfluidic substrate and the microfluidic chip are described in detail. In the space rectangular coordinate system, the X axis and the Y axis are parallel to the surface of the microfluidic substrate, and the Z axis is perpendicular to the surface of the microfluidic substrate.

In at least one embodiment of the present disclosure, as shown in fig. 1 and 2, a microfluidic substrate 10 includes a flow channel structure including a transport flow channel 100 and a plurality of detection groups 200. Each detection group 200 includes a first fluid channel 210, a first micro-channel 240, a buffer channel 220, a second micro-channel 250, and a second fluid channel 230 that are sequentially connected, where the first fluid channel 210 is in communication with the delivery channel 100, and the second fluid channel 230 is provided with the reagent 12. The microfluidic substrate 10 has a rotation axis 11, the detection groups 200 are located on a side of the conveying flow channel 100 facing away from the rotation axis 11, and 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 sequentially increase, that is, in each detection group 100, the first fluid groove 210, the buffer groove 220, and the second fluid groove 230 are arranged sequentially away from the rotation axis 11. In this way, when the microfluidic substrate 10 is rotated about the rotation axis 11, fluid enters the first fluid groove 210, the buffer groove 220, and the second fluid groove 230 in this order from the transfer flow path 100 by centrifugal force (inertial force).

In the embodiments of the present disclosure, the type of the reagent is not limited, and may be selected as needed. For example, the reagent may be a liquid reagent, a dry reagent, a solid microsphere containing a reagent, a microarray containing a reagent, a test strip containing a reagent, or the like. According to the types of the reagents preloaded in the second fluid tank, the microfluidic substrate (or the microfluidic chip comprising the microfluidic substrate) can analyze and detect the components in blood, body fluid or tissues of human, animals, microorganisms or plants through means such as PCR molecular detection, immunodetection or biochemical detection.

In the microfluidic substrate provided in at least one embodiment of the present disclosure, in the case where the first fluidic channel has a fluid therein, the fluid in the first fluidic channel, the buffer groove, the second fluidic channel, and the second fluidic channel are formed into a closed groove. Thus, when the microfluidic substrate is packaged (e.g. is matched with the cover plate), in the case that the first micro-flow channel is provided with fluid, a closed chamber is formed in the buffer tank, the second micro-flow channel and the space in the second fluid tank, so that when the fluid flows along the first micro-flow channel to the buffer tank, the fluid compresses the air of the chamber to cause the pressure of the chamber to increase, namely, back pressure is formed, the back pressure prevents the fluid from flowing to the buffer tank, so that when the back pressure and the centrifugal force (inertia force) of the fluid generated by rotation reach an equilibrium state, a gas-liquid interface is formed in the first micro-flow channel, so that the fluid from the first fluid tank is difficult to enter the buffer tank, and even if a small amount of fluid enters the buffer tank, the small amount of fluid is converged in the buffer tank to be difficult to enter the second micro-flow channel or enter the second fluid tank through the second micro-flow channel in the rotation state, so that the risk that the fluid enters the second fluid tank in the current state is reduced; in addition, even if a part of the fluid that has entered the buffer tank enters the second fluid tank again, because a gas-liquid interface is formed in the first microchannel, the fluid that entered the second fluid tank (which has been in contact with the reagent) does not return to the first fluid tank through the first microchannel, i.e., the fluid does not enter other detection groups. In this way, when the microfluidic chip formed by using the microfluidic substrate disclosed by the disclosure is used for detection, the risk that the fluid in the first fluid groove enters the second fluid groove is avoided in the stage of filling the fluid in the conveying flow channel, the fluid and the reagent are prevented from reacting in advance, and the occurrence of cross contamination of the reagents in different detection groups (such as adjacent detection groups) is avoided, so that the reliable detection result is guaranteed.

As illustrated in fig. 2, the chamber formed by the buffer tank 220, the second micro flow channel 250, and the second fluid tank 230 has only an opening communicating with the first micro flow channel 240, so that as the fluid of the first fluid tank 210 enters the first micro flow channel 240, the fluid has actually blocked the gas in the chamber, and as the fluid flows along the first micro flow channel 240 toward the buffer tank 220, that is, a gas-liquid interface P formed between the fluid and the gas also advances toward the buffer tank 220, resulting in an increase in the pressure of the gas in the chamber, thereby blocking the flow of the fluid into the buffer tank, and in the case that the length of the first micro flow channel 240 is long enough, the pressure of the gas in the chamber counteracts the centrifugal force (inertial force) that causes the fluid to flow toward the first micro flow channel, that is, the pressure of the gas pressure and the centrifugal force reach an equilibrium state at the gas-liquid interface P, which exists in the first micro flow channel 240 or at the communication of the first micro flow channel 240 with the buffer tank 220 (for example, the inlet of the buffer tank mentioned in the following embodiment). As such, it is difficult for the fluid in the first fluid tank 210 to flow into the buffer tank 220, and it is more difficult for the fluid to flow into the second fluid tank 230.

For example, in the actual process, under the lower rotation speed, the fluid enters and fills the first fluid groove through the conveying flow channel connected with the first fluid groove under the driving of the centrifugal force, and seals the inlet of the first micro flow channel (the opening of the first micro flow channel communicated with the first fluid groove) between the first fluid groove and the buffer groove, and the fluid further enters the micro flow channel (the first micro flow channel in the embodiment of the disclosure) between the first fluid groove and the buffer groove under the driving of the centrifugal force, so that the air in the buffer groove and the second fluid groove is compressed. When the reverse pressure of the compressed air plus the surface tension of the fluid and the centrifugal force reach equilibrium, the fluid stops flowing, so that a gas-liquid interface is formed in the micro flow channel (first micro flow channel), and after the redundant fluid in the conveying flow channel flows into the overflow groove (first waste liquid groove in the embodiment of the disclosure), the quantitative fluid is limited in the first fluid groove and the first micro flow channel by the gas-liquid interface. When it is desired to transfer the metered amounts of fluid from the first fluid cell to the second fluid cell (which is used as a reaction cell or referred to as a detection cell) to react with the preloaded reagent, the increased centrifugal force will cause a phenomenon similar to Rayleigh-taylor instability (Rayleigh-Taylor instability) in the air in the buffer cell and the gas-liquid interface of the fluid in the microchannel, i.e., when two media of different densities are placed in the same container and a medium of higher density is placed above a medium of lower density, the two-phase interface (gas-liquid interface) will be unstable due to gravity (centrifugal force in this disclosure). In this way, the fluid intermittently enters the buffer tank and the second fluid tank, and the air in the buffer tank and the second fluid tank is discharged through the first micro flow channel in the form of bubbles (for example, is further discharged through the first fluid tank and the conveying flow channel subsequently) until the gas sealed by the fluid is completely discharged. In the above process, the reagent in the second fluid tank does not cross-contaminate.

For example, in some embodiments of the present disclosure, as shown in fig. 2, in the actual process, in the case where the length of the first micro flow channel is not long enough, the rotation frequency is large, and the like, there may be a communication point of the first micro flow channel 240 with the buffer tank 220 (a first inlet of the buffer tank as mentioned in the embodiment described below), in which case the fluid may protrude from the first micro flow channel 240 to the buffer tank 220 in a direction from the first micro flow channel 240 to the buffer tank 220, in which case, because of the surface tension of the fluid, the pressure (back pressure) due to the compression of air and the centrifugal force are balanced, the fluid may still maintain a convex shape without continuing to flow into the buffer tank at the communication point of the first micro flow channel 240, that is, the fluid may still form a stable gas-liquid interface P at the communication point. The formation of the stable gas-liquid interface P requires a certain process, and before the stable gas-liquid interface P is not formed, part of the fluid may have entered the buffer tank 220, because of the provision of the buffer tank 220, in a rotated state, the fluid dropped into the buffer tank 220 is difficult to enter the communication between the buffer tank 220 and the second micro flow channel 250 (the first outlet of the buffer tank as mentioned in the following embodiments), thereby reducing the risk of the fluid entering the second fluid tank 230; in addition, even if the fluid flowing into the buffer tank 220 enters the second micro flow channel 250, since the portion of the fluid is disconnected from the fluid stored in the first micro flow channel 240, the gas in the second micro flow channel 250 and the second fluid tank 230 can be compressed only by the pressure generated by the own inertial force (centrifugal force), since the fluid in the first micro flow channel 240 is additionally supplied with the pressure supplied by the fluid additionally stored in the first fluid tank 240, the pressure supplied by the portion of the fluid entering the second micro flow channel 250 itself is smaller than the pressure supplied by the fluid in the first micro flow channel 240, and thus it is difficult for the portion of the fluid entering the second micro flow channel 250 to further compress the gas in the second micro flow channel 250 and the second fluid tank 230, so that the second micro flow channel 250 does not break through into the second fluid tank 230.

In a microfluidic substrate provided by at least one embodiment of the present disclosure, a first micro flow channel is configured to have a first length such that a fluid from a first fluid channel and a gas present in a buffer channel form a gas-liquid interface at a first rotational frequency of the microfluidic substrate, and such that the gas-liquid interface is present in the first micro flow channel or at a communication of the first micro flow channel and the buffer channel. The first micro flow path has a cross section perpendicular to the extending direction thereof, and in the case where the size (e.g., inner diameter, or width and depth, etc.) of the cross section is fixed, the larger the first length, the larger the volume ratio of the first micro flow path in the whole chamber constituted by the first fluid tank, the first micro flow path, the buffer tank, the second micro flow path and the second fluid tank, the larger the air pressure of the chamber constituted by the first micro flow path, the buffer tank, the second micro flow path and the second fluid tank before the fluid passes through the first micro flow path, that is, the larger resistance can be generated when compressing the gas, thereby allowing the design of a larger first rotation frequency. In embodiments of the present disclosure, the first rotational frequency may be designed according to an actual process, for example, the first rotational frequency may be a rotational frequency at which fluid in the delivery flow channel enters the first fluid tank, and thus, a range of the first length may be designed according to the first rotational frequency.

In the microfluidic substrate provided by at least one embodiment of the present disclosure, the first length of the first micro flow channel may be 0.1 to 5mm, for example, further may be 0.2mm, 0.5mm, 1mm, 1.5mm, 2mm, 3mm, 4mm, etc.; the width of the first micro flow channel may be 0.1 to 1mm, for example, 0.2mm, 0.4mm, 0.5mm, 0.6mm, 0.8mm, or the like; the depth of the first micro flow channel may be 0.1 to 0.5mm, for example, 0.2mm, 0.3mm, 0.4mm, 0.5mm, or the like. It should be noted that, in the embodiment of the present disclosure, parameters such as the first length, the width, the depth, etc. of the first micro flow channel may be designed according to an actual process, and may not be limited to the above numerical range.

In the microfluidic substrate provided in at least one embodiment of the present disclosure, a plurality of buffer grooves may be provided in one detection group, and in the case where a plurality of buffer grooves are provided, the buffer grooves may be connected in series between the first fluid groove and the second fluid groove, so that the probability of fluid entering the second fluid groove may be further reduced in a stage of filling the first fluid groove with fluid of the transport flow channel.

In a microfluidic substrate provided by at least one embodiment of the present disclosure, a buffer tank includes a first sidewall and a second sidewall. The first side wall comprises a first inlet communicated with the first micro-channel, and the second side wall is opposite to the first side wall and comprises a first outlet communicated with the second micro-channel. Illustratively, as shown in fig. 3, the buffer tank 220 includes a first side wall 221 and a second side wall 222 opposite to each other, the first side wall 221 including a first inlet 2211 to allow the buffer tank 220 to communicate with the first fluid tank 210 through the first micro flow channel 240, and the second side wall 222 including a first outlet 2221 to allow the buffer tank 220 to communicate with the second fluid tank 230 through the second micro flow channel 250.

For example, in some embodiments of the present disclosure, the first fluidic channel may be provided as one in each detection group, and thus, one first inlet may be provided on the first sidewall; alternatively, in other embodiments of the present disclosure, the first micro flow channel may be provided in plurality, and thus, a plurality of first inlets may be provided on the first sidewall.

For example, in some embodiments of the present disclosure, the second fluidic channel may be provided as one in each detection group, and thus, one first inlet and outlet may be provided on the first sidewall; alternatively, in other embodiments of the present disclosure, the second micro flow channels may be provided in plurality, and thus, a plurality of first inlets and outlets may be provided on the first sidewall.

At the stage of filling the first fluid groove with the fluid of the conveying flow channel, part of the fluid still possibly enters the buffer groove, so that the shape of the buffer groove can be designed to reduce the probability of the part of the fluid further entering the second micro flow channel. For example, in the embodiment of the present disclosure, the distance from the portion of the second side wall, which is not provided with the first outlet, to the rotation axis is greater than the distance from the first outlet to the rotation axis, so that, during the rotational movement, the fluid entering the buffer tank tends to move away from the position of the rotation axis by the centrifugal force, thereby making it difficult to flow into the first outlet, i.e., increasing the difficulty of the portion of the fluid entering the second micro flow channel from the second outlet.

In the embodiment of the present disclosure, the specific shape of the buffer tank is not limited under the condition that the distance from the second sidewall of the buffer tank, which is not provided with the first outlet portion, to the rotation axis is greater than the distance from the first outlet portion to the rotation axis, and may be designed according to the actual process requirements, and the following description will be made by means of several specific embodiments.

In the microfluidic substrate provided in some embodiments of the present disclosure, the second side wall is a plane, and the plane is perpendicular to a direction along the rotation axis to the first inlet, and may specifically refer to a buffer groove shown in fig. 2.

In the rotating state, a trace amount of fluid entering the buffer tank is affected by Coriolis force (a description of the displacement of an object moving linearly in the rotating system with respect to the linear motion generated by the rotating system due to inertia) and easily falls into and stays in the edge region of the buffer tank.

In other embodiments of the present disclosure, the area of the second sidewall where the first outlet is not disposed includes at least one concave portion, and a distance from a bottom of the at least one concave portion to the rotation axis is greater than a distance from the first outlet to the rotation axis. As such, if a portion of the fluid flows to the buffer tank in a stage in which the fluid of the transfer flow channel fills the first fluid tank, the portion of the fluid is collected at the recess portion because the microfluidic substrate is in a rotated state, and the distance from the bottom of the recess portion to the rotation axis is greater than the distance from the first outlet to the rotation axis, the fluid collected at the recess portion does not enter the first outlet and thus does not enter the second fluid tank, i.e., the risk of the fluid entering the second fluid tank in this stage is further reduced. Illustratively, as shown in fig. 3, when the microfluidic substrate is in a counterclockwise rotation state, the fluid Q entering the buffer tank 220 is concentrated in the recess 2222 located at the edge of the second sidewall 222 by the coriolis force. Recess 2222 may store fluid Q, may allow more fluid Q to enter; in addition, there is a difference in height (distance from the rotation axis) between the bottom of the concave portion 2222 and the first outlet 2221, and after the fluid Q gathers in the concave portion 2222, it becomes more difficult to enter the first outlet 2221, so that the probability of the fluid Q entering the second fluid tank 230 is further reduced at the stage of filling the first fluid tank with the fluid of the delivery flow path.

In embodiments of the present disclosure, the arrangement of the first inlet, the first outlet and the recessed portion of the buffer channel may be determined according to the rotational manner of the microfluidic substrate (e.g., only clockwise rotation, only counterclockwise rotation or need to be switched between clockwise rotation and counterclockwise rotation) to further reduce the probability of fluid entering the second fluid channel at a stage where the fluid of the transport channel fills the first fluid channel. The specific embodiments may be designed according to the needs of an actual process, and several specific embodiments are listed here by way of several specific examples.

In the microfluidic substrate provided in some embodiments of the present disclosure, in each buffer groove, both sides of the first outlet are provided with concave portions, and the first outlet is located in the middle of the second sidewall. In this way, regardless of whether the preset rotational direction of the microfluidic chip is clockwise or counterclockwise, the recessed portion can be used to collect fluid entering the buffer tank at a stage where the fluid of the transport flow channel fills the first fluid tank, thereby reducing the risk of fluid entering the second fluid tank at this stage. Illustratively, as shown in fig. 3, the first outlet 2221 is provided with recess portions 2222 on both front and rear sides in a preset rotational direction, so that the fluid Q can be collected in the recess portions 2222 located on the rear side of the first outlet 2221 in the rotational direction during rotation. During rotation, the fluid Q may remain at the edge of the second sidewall, and the fluid Q may lag the rotation, i.e., the fluid and the first outlet are sequentially arranged in front of and behind each other along the rotation direction, and the fluid Q is located at the rear side of the first outlet. Exemplarily, as shown in fig. 3, if the microfluidic substrate is rotated counterclockwise, the fluid Q in the buffer groove 220 is collected in the recess 2222 at the left side of the second sidewall 222; alternatively, if the microfluidic substrate is rotated clockwise, the fluid Q in the buffer tank 220 is collected in the recess 2222 on the right side of the second sidewall 222.

In the microfluidic substrate provided in other embodiments of the present disclosure, in each buffer slot, a concave portion is disposed at one side of the first outlet, the first outlet is located in the middle of the second sidewall, and the concave portion and the first outlet are sequentially disposed along a preset rotation direction of the microfluidic substrate. Illustratively, as shown in fig. 4, the concave portion 2222a of the second sidewall 222a of the buffer tank 220a is located at the left side of the first outlet 2221a, and the microfluidic substrate rotates counterclockwise about the rotation axis 11a, so that the fluid entering the buffer tank 220a from the first fluid tank 210a through the first micro flow channel 240a and the first inlet 2221a of the first sidewall 221a may fall into the concave portion 2222a and may not enter the second fluid tank 230a through the second micro flow channel 250 a.

In the microfluidic substrate provided in other embodiments of the present disclosure, in each buffer slot, a concave portion is disposed at one side of the first outlet, the first outlet is located at one end of the second sidewall, and along a preset rotation direction of the microfluidic substrate, and the concave portion and the first outlet are sequentially disposed. In this way, since the first outlet is located at one end of the second side wall, which corresponds to an increase in the design area of the recess portion, i.e. the design area of the recess portion in the second side wall may be increased compared to the first outlet being located in the middle of the second side wall, the design volume of the recess portion may be increased, i.e. the amount of fluid that the recess portion may store is increased, and the risk of fluid flowing into the second fluid tank is further reduced at a stage where the fluid of the delivery flow channel fills the first fluid tank. Illustratively, as shown in fig. 5, the microfluidic substrate rotates counterclockwise about the rotation axis 11b, and the recess 2222b is provided at the rear side of the first outlet 2221b of the second sidewall 222b along the rotation direction, so that fluid may be collected in the recess 2222b at the rear side of the first outlet 2221b along the rotation direction during rotation. Further, when the fluid from the first fluid groove 210b enters the buffer groove 220b from the first micro flow channel 240b and the first inlet 2211b, during rotation, the fluid is already located at the rear side of the first outlet 2221b when passing through the first inlet 2211b of the first side wall 221b, i.e., the fluid is more easily collected in the concave portion 2222b and more difficult to enter the first outlet 2221b, i.e., at the stage where the fluid of the transfer flow channel fills the first fluid groove 210b, the probability of the fluid entering the second micro flow channel 250b and the second fluid groove 230b is further reduced.

In other embodiments of the present disclosure, the first inlet is located in the middle of the first sidewall in each buffer tank. Illustratively, as shown in FIG. 5, the first inlet 2211b is located in the middle of the first sidewall 221 b.

In the microfluidic substrate provided in other embodiments of the present disclosure, in each buffer slot, the first inlet is located at one end of the first sidewall, and the first inlet and the first outlet are sequentially arranged along a preset rotation direction of the microfluidic substrate. In this way, the distance between the first inlet and the first outlet increases along the preset rotation direction compared to the first inlet being located in the middle of the first side wall, and the risk of fluid falling directly at the first outlet is reduced at a stage in which the fluid of the transport flow path fills the first fluid tank when the fluid flows into the buffer tank through the first inlet, thereby further reducing the risk of fluid flowing into the second fluid tank at this stage. Illustratively, as shown in fig. 6, the first inlet 2211c is positioned at one end of the first sidewall 221c, the microfluidic substrate rotates counterclockwise about the rotation axis 11c, and the first inlet 2211c is positioned at the rear side of the first outlet 2221c along the rotation direction. As such, when fluid from the first fluid groove 210c enters the buffer groove 220c from the first micro flow channel 240c and the first inlet 2211c, the fluid is more difficult to enter the first outlet 2221c during rotation while passing through the first inlet 2211c of the first side wall 221c, i.e., already located at the rear side of the first outlet 2221c. For example, further, as shown in fig. 6, the rear side of the first outlet 2221c of the second side wall 222c is provided with the concave portion 2222c, so that the fluid can be collected in the concave portion 2222c located at the rear side of the first outlet 2221c in the rotation direction during rotation. When the fluid from the first fluid groove 210c enters the buffer groove 220c from the first micro flow channel 240c and the first inlet 2211c, the fluid is easily collected in the concave portion 2222c and more difficult to enter the first outlet 2221c when passing through the first inlet 2211c of the first side wall 221c, i.e., already located at the rear side of the first outlet 2221c during rotation, i.e., at the stage where the fluid of the transfer flow channel fills the first fluid groove 210c, the probability of the fluid entering the second micro flow channel 250c and the second fluid groove 230c is further reduced.

In the microfluidic substrate provided in at least one embodiment of the present disclosure, in each buffer tank, each detection group further includes a first siphon flow channel. One end of the first siphon runner is connected to the concave part to be communicated with the buffer groove, and the other end of the first siphon runner is communicated with the second fluid groove. The inner diameter of the first siphon runner is smaller than that of the second micro runner, and the distance from the part of the first siphon runner to the rotation axis is smaller than that from the first outlet to the rotation axis. After the fluid of the first fluid tank is introduced into the second fluid tank, a portion of the fluid of the buffer tank may be restricted to the concave portion, in which case the first siphon flow passage may introduce the fluid of the concave portion into the second fluid tank, improving the utilization of the fluid. Illustratively, as shown in FIG. 7, one end of the first siphon flow passage 260d communicates with the recessed portion 2222d, and the other end of the first siphon flow passage 260d communicates with the second fluid groove 230d. In practical applications, at the stage of making the fluid of the transfer flow channel fill the first fluid groove 210d, part of the fluid of the first fluid groove 210d may flow to the concave portion 2222d of the second side wall 222d of the buffer groove 220d through the first micro flow channel 240 d; after increasing the rotational frequency (rotational speed), the fluid in the first fluid groove 210d and the first micro flow channel 240d enters the buffer groove 220d and enters the second fluid groove 230d from the second micro flow channel 250d, and some fluid is collected in the concave portion 2222d, in the process, because the distance from the portion of the first siphon flow channel 260d (the top portion as shown in fig. 7, i.e., the portion closest to the rotational axis 11 d) to the rotational axis 11d is smaller than the distance from the first outlet 2221d to the rotational axis 11d, in the process, the fluid does not enter the second fluid groove 230d through the first siphon flow channel 260d, i.e., the fluid closes the first siphon flow channel 260d so as not to affect the fluid entering the second fluid groove 230d from the second micro flow channel 250 d; after the rotation frequency is then reduced or rotation is stopped, the fluid is sucked into the second fluid groove 230d by the capillary force of the first siphon flow passage 260 d.

For example, in some embodiments of the present disclosure, the first siphon flow passage may be further provided in a capillary structure so that the fluid collected at the concave portion may be sucked into the second fluid groove by capillary force.

In some embodiments of the present disclosure, the first fluid channel has a smaller volume than the second fluid channel.

In other embodiments of the present disclosure, the microfluidic substrate is provided wherein 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. In a practical process, considering the viscosity of the fluid itself and other factors, there may be residues in the respective channels such as buffer tanks, first micro flow channels, second micro flow channels, and the like. Residual fluid in the first fluid tank can be avoided by making the volume of the first fluid tank smaller than or equal to the sum of the volumes of the second fluid tank and the buffer tank, so that the fluid quantified by the first fluid tank can enter the buffer tank and the second fluid tank, aggregation of the fluid in the first fluid tank is avoided, and the first fluid tank can be ensured to be filled with the fluid quantified by the first fluid tank by making the volume of the first fluid tank larger than the volume of the second fluid tank.

It should be noted that in embodiments of the present disclosure, fluid and "residue" and "build-up" are different concepts, with fluid residue being a result of some uncontrollable factors, and fluid build-up being contemplated in the process or structural design. For example, in an ideal state in which the viscosity, wettability, and the like of the fluid are not taken into consideration, there may be no residue after the fluid passes through the respective flow channels (e.g., first micro flow channel, second micro flow channel, and the like) and the grooves (e.g., first fluid grooves, buffer grooves, and the like), but there may be fluid accumulation in the specific structures of the flow channels or grooves (e.g., concave portions in the above-described embodiments).

In other embodiments of the present disclosure, a microfluidic substrate is provided in which the volume of the first fluid channel is greater than the sum of the volumes of the second fluid channel and the buffer channel.

In embodiments of the present disclosure, the first fluid groove and the second fluid groove may be triangular, circular, rectangular, polygonal, or the like in shape. For example, the shape of the buffer tank may be triangular, circular, rectangular, polygonal, or the like, regardless of the position of accumulation of the fluid in the buffer tank (e.g., regardless of the provision of the concave portion).

In the microfluidic substrate provided in at least one embodiment of the present disclosure, in the same detection group, at least part (i.e., part or all) of the second fluid groove is farther from the rotation axis than the first fluid groove and the buffer groove. For example, the distance from any part of the second fluid groove to the rotation axis is greater than the distance from any part of the first fluid groove and the buffer groove to the rotation axis; or, the distance from one part of the second fluid groove to the rotation axis is larger than the distance from any part of the first fluid groove and the buffer groove to the rotation axis, and the distance from the other part of the second fluid groove to the rotation axis is smaller than or equal to the distance from one part of the first fluid groove and the buffer groove to the rotation axis.

In the microfluidic substrate provided by at least one embodiment of the present disclosure, the conveying flow channel is in a non-closed ring shape, and the center of a circle where the ring is located is a rotation axis; or the conveying flow channel is in a non-closed ring shape, the distance from the first end of the conveying flow channel to the rotation axis is smaller than the distance from the second end of the conveying flow channel to the rotation axis, and the distance from the first end to the second end of the conveying flow channel to the rotation axis is sequentially increased. As shown in fig. 1, a circular arc-shaped (belonging to a non-closed ring shape) conveying flow channel 100 in the microfluidic substrate 10 can be seen, wherein the distance from the first end of the circular arc to the rotation axis is smaller than the distance from the second end of the conveying flow channel to the rotation axis, and the distance from the conveying flow channel to the rotation axis sequentially increases from the first end to the second end. . In this way, when the microfluidic substrate rotates, a uniform distribution of fluid in the transport channels is facilitated, so that fluid flows uniformly into the first fluid grooves in each detection group. For example, the first end may be provided with an inlet (for communication with the second siphon flow passage) for injecting fluid. For example, the delivery flow channel is provided with a vent. For example, the vent hole may be provided at the second end of the transport flow channel, for example, further, a vent hole may also be provided at the first end.

The microfluidic substrate provided by at least one embodiment of the present disclosure may further include a first waste liquid tank. The first waste liquid tank is communicated to one end of the conveying flow channel. Along the preset rotation direction of the microfluidic substrate, the first waste liquid tank and the conveying flow channel are sequentially arranged. In actual processes, after the fluid fills all of the first fluid cells, there may still be remaining fluid in the delivery flow channel, which may connect the fluids in some of the first fluid cells (e.g., adjacent first fluid cells) together and also prevent the formation of a certain amount of liquid in the first fluid cells, e.g., during an increase in rotational speed such that the fluid in the first fluid cell enters the buffer cell and the second fluid cell, the remaining fluid still enters the first fluid cell, eventually resulting in an excessive amount of fluid entering the test set. In this embodiment, the fluid in the conveying flow channel flows into the first waste liquid tank after filling the first fluid tank, so that the risk that the fluid breaks through the buffer tank and enters the second fluid tank is reduced; in addition, the residual fluid cannot exist in the conveying flow channel, namely, the fluid in the adjacent first fluid grooves cannot be communicated, so that cross contamination is avoided, the quantitative effect of the first fluid grooves on the fluid is ensured, and excessive flow of the fluid into the detection group is avoided. Illustratively, as shown in fig. 1, the first waste liquid tank 110 communicates with the transport flow channel 100, and when fluid is injected into the transport flow channel 100, the microfluidic substrate is configured to rotate counterclockwise based on the rotation axis 11, and the first waste liquid tank 110 is located at the rear side of the transport flow channel 100 in the rotation direction, so that the first waste liquid tank 110 is also located at the rear side of the detection group 200 (first fluid tank 210) communicating with the transport flow channel 100, that is, after the fluid entering the transport flow channel 100 fills the first fluid tank 210 of each detection group 200 in turn, the surplus fluid enters the first waste liquid tank 110.

For example, in an embodiment of the present disclosure, in a case where the conveying flow path is in a non-closed ring shape and a distance from a first end of the conveying flow path to the rotation axis is smaller than a distance from a second end of the conveying flow path to the rotation axis, the first waste liquid tank may communicate with the second end of the conveying flow path. In this way, it is advantageous to empty the fluid in the transfer channel at a low rotational speed so that the fluid enters the first waste liquid tank.

The microfluidic substrate provided by at least one embodiment of the present disclosure may further include a mixing tank and a second siphon flow channel. The mixing tank comprises two inlets and an outlet, one end of the second siphon runner is communicated with the outlet of the mixing tank, and the other end of the second siphon runner is connected to the conveying runner. The distance from the part of the second siphon flow passage to the rotation axis is smaller than the distance from the mixing groove to the rotation axis. Illustratively, as shown in fig. 8, the mixing tank 400 communicates with the delivery flow channel 100 through a second siphon flow channel 500. The two inlets of the mixing tank 400 may be used to introduce two types of fluids (e.g., sample and diluent) respectively, and the two fluids may be uniformly mixed in the mixing tank, and the mixed fluids enter the transfer flow channel 100 through the second siphon flow channel 500. For example, after the sample and the diluent enter the mixing channel 400 through the two inlets of the mixing channel 400, respectively, the microfluidic substrate keeps rotating because the distance from the portion of the second siphon flow channel 500 to the rotation axis is smaller than the distance from the mixing channel 400 to the rotation axis, and thus, the fluid in the mixing channel 400 does not enter the transport flow channel 100; after the sample and the diluent are uniformly mixed in the mixing tank 400, the rotation frequency (rotation speed) is reduced or the rotation is stopped, the fluid in the mixing tank 400 fills the second siphon flow channel 500 under the capillary force of the second siphon flow channel 500, the microfluidic substrate is rotated again, and the fluid in the mixing tank 400 enters the conveying flow channel 100 through the second siphon flow channel 500.

The microfluidic substrate provided by at least one embodiment of the present disclosure may further include a sample well, a sample quantification well, a sample overflow well, a third siphon flow channel, a dilution liquid well, a dilution liquid quantification well, a dilution liquid overflow well, and a fourth siphon flow channel. The sample quantifying groove is communicated with the sample groove, and the distance from the sample quantifying groove to the rotation axis is greater than the distance from the sample groove to the rotation axis. The sample overflow groove is communicated with the sample groove, and the distance from the sample overflow groove to the rotation axis is larger than the distance from the sample quantifying groove to the rotation axis. One end of the third siphon flow passage is communicated with the sample quantifying groove, the other end of the third siphon flow passage is communicated with one of two inlets of the mixing groove, and the distance from the part of the third siphon flow passage to the rotation axis is smaller than the distance from the sample quantifying groove to the rotation axis. The diluent quantifying groove is communicated with the diluent groove, and the distance from the diluent quantifying groove to the rotation axis is greater than the distance from the diluent groove to the rotation axis. The diluent overflow groove is communicated with the diluent groove, and the distance from the diluent overflow groove to the rotation axis is greater than the distance from the diluent quantitative groove to the rotation axis. One end of the fourth siphon flow passage is communicated with the diluent quantifying groove, the other end of the fourth siphon flow passage is communicated with the other of the two inlets of the mixing groove, and the distance from the part of the fourth siphon flow passage to the rotation axis is smaller than the distance from the diluent quantifying groove to the rotation axis.

As illustrated in fig. 8 and 11 to 15, the microfluidic substrate includes a sample well 210, a sample quantification well 220, a sample overflow well 230, a third siphon flow channel 240, a dilution liquid well 310, a dilution liquid quantification well 320, a dilution liquid overflow well 330, and a fourth siphon flow channel 340. In the case of performing blood (sample) detection using a microfluidic substrate (or a microfluidic chip including the microfluidic substrate), 100 microliters of whole blood may be injected into the sample tank 210, 450 microliters of diluent may be injected into the diluent tank 310, the microfluidic chip may then be rotated, the sample may flow into the sample quantifying tank 320, the excess sample may flow into the sample overflow tank 230, the diluent may flow into the diluent quantifying tank 320, and the excess diluent may flow into the diluent overflow tank 330. The microfluidic chip stops rotating, and the sample (in this case, the plasma in the supernatant fluid) in the sample quantifying well 220 fills the third siphon flow channel 240 by capillary force, and the diluent in the diluent quantifying well 340 fills the fourth siphon flow channel 340 by capillary force. The microfluidic chip is rotated, and the quantitative plasma in the sample quantitative tank 220 and the quantitative diluent in the diluent quantitative tank 320 are injected into the mixing tank 400 through the third siphon flow channel 240 and the fourth siphon flow channel 340, respectively. The sample (plasma) injected into the mixing tank 400 and the diluent are uniformly mixed by controlling the rotation speed change, forward and reverse conversion (clockwise rotation and counterclockwise rotation switching) and the like of the microfluidic chip. The rotation of the microfluidic chip is stopped, and the fluid (mixed liquid) in the mixing tank 400 fills the second siphon flow channel 500 under the capillary force of the second siphon flow channel 500. The microfluidic chip is rotated, the fluid (mixed solution) in the mixing tank 400 enters the transfer flow channel 100 via the second siphon flow channel 500, and the fluid (mixed solution) sequentially fills the first fluid tanks 210 of the respective detection groups through the transfer flow channel 100, and the surplus fluid (mixed solution) flows into the first waste liquid tanks.

The microfluidic substrate provided by at least one embodiment of the present disclosure may further include a flow channel layer and a substrate. The runner structure is formed in the runner layer. The substrate is positioned on the other side of the runner layer opposite to the side provided with the first fluid groove, the first micro runner, the buffer groove, the second micro runner and the second fluid groove. The substrate and the runner layer are arranged in a laminating way or are integrally formed. For example, in some embodiments, the flow channel layer includes opposing first and second major surfaces, the first major surface of the flow channel layer may face the cover plate and the second major surface of the flow channel layer may face the substrate when the flow channel layer is disposed in a microfluidic chip.

The microfluidic substrate of the present disclosure may be fabricated by conventional fabrication methods such as injection molding, machining, etching, knife etching, embossing, and the like. The materials used may be plastics, ceramics, glass, silicon wafers, silica gel, etc.

In one example of the present disclosure, as shown in fig. 9, the microfluidic substrate 10 is an integrally formed structure, i.e., the base and the flow channel layer are integrally formed.

In another example of the present disclosure, as shown in fig. 10, the microfluidic substrate 10 includes a flow channel layer 12 and a base 13, and a transport flow channel 100, a first fluid groove 210, a buffer groove 220, and a second fluid groove 230 are formed in the flow channel layer 12. In the case where the transfer flow path 100, the first fluid groove 210, the buffer groove 220, and the second fluid groove 230 are formed in the flow path layer 12 without penetrating the flow path layer 12, the transfer flow path 100, the first fluid groove 210, the buffer groove 220, and the second fluid groove 230 are formed on the side of the flow path layer 12 facing the cap plate 20, and the substrate 13 is positioned on the side of the flow path layer 12 facing away from the cap plate 20.

For example, in some embodiments of the present disclosure, the second fluid slot is disposed through the flow channel layer, and the delivery flow channel, the first fluid slot, the buffer slot, the first siphon flow channel, the second siphon flow channel, etc. may be disposed without passing through the flow channel layer. The substrate is provided as a transparent substrate so that the second fluid cell (detection cell, reaction cell) can be optically detected and compared to the like through the substrate. For example, the substrate may be a sheet (typically having a thickness of 0.5mm or more) or a film (typically having a thickness of 0.5mm or less) of a thermoplastic polymer including one or more of polymethyl methacrylate, polycarbonate, polystyrene, polyamide, and polyethylene terephthalate to ensure good light transmittance.

At least one embodiment of the present disclosure provides a microfluidic chip including a cover plate and the microfluidic substrate in the first aspect. The cover plate and the microfluidic chip are combined and positioned on one side of the microfluidic substrate, on which the first fluid groove, the first micro-channel, the buffer groove, the second micro-channel and the second fluid groove are arranged. As shown in fig. 9 and 10, the cover 20 is aligned with the microfluidic substrate 10, and thus, the cover 20 and the grooves such as the first fluid groove, the first micro flow channel, the buffer groove, the second micro flow channel, and the second fluid groove form a chamber capable of containing fluid. For example, the cover plate may be provided as a transparent cover plate, so that the second fluid cell (detection cell, reaction cell) can be optically detected and compared to the like through the cover plate. For example, the cover plate may be a plate (typically having a thickness of 0.5mm or more) or a film (typically having a thickness of 0.5mm or less) made of a thermoplastic polymer including one or more of polymethyl methacrylate, polycarbonate, polystyrene, polyamide, and polyethylene terephthalate to ensure good light transmittance.

The present disclosure provides an operation method of a microfluidic chip, where the microfluidic chip includes a cover plate and a microfluidic substrate, the microfluidic substrate includes a flow channel structure, the flow channel structure includes a conveying flow channel and a plurality of detection groups, each detection group of the plurality of detection groups includes a first fluid groove, a first micro flow channel, a buffer groove, a second micro flow channel and a second fluid groove that are sequentially communicated, the first fluid groove is communicated with the conveying flow channel, a reagent is disposed in the second fluid groove of at least one detection group, the microfluidic substrate has a rotation axis, the detection groups are located at a side of the conveying flow channel away from the rotation axis, and distances from the first fluid groove, the buffer groove and the second fluid groove in each detection group to the rotation axis are sequentially increased, and the operation method includes: driving the micro-fluidic chip to rotate at a second rotation frequency so that fluid of the conveying flow channel enters the first fluid groove and a gas-liquid interface is formed in the first micro-flow channel; after the first fluid groove in each detection group is filled with liquid, driving the micro-fluidic chip to rotate at a third rotation frequency so that fluid enters the buffer groove and the second fluid groove; wherein the third rotational frequency is greater than the first rotational frequency. In the operation method, a buffer groove is arranged in a microfluidic substrate in the utilized microfluidic chip to separate the first fluid groove and the second fluid groove, so that in the stage of filling the first fluid groove with fluid of a conveying flow channel, the fluid flowing out of the first fluid groove can be prevented from directly entering the second fluid groove, and the risk that the fluid enters the second fluid groove and re-enters the first fluid groove from the second fluid groove (at the moment, the fluid is contacted with the reagent) in the stage is reduced, namely, the risk of cross contamination of the reagents in each detection group is reduced; further, by providing the buffer tank, it is possible to prevent the fluid from directly entering the second fluid tank in this stage to react with the reagent in advance, thereby precisely controlling the reaction time (detection time) of the reagent in the second fluid tank. The specific structure of the microfluidic chip and the microfluidic substrate used in the operation method can be referred to the related description in the embodiments shown in fig. 1 to 10, and will not be described herein.

In the operation method of the microfluidic chip provided in at least one embodiment of the present disclosure, the first micro flow channel is configured to have a first length such that the fluid from the first fluid channel and the gas existing in the buffer channel form a gas-liquid interface at a first rotation frequency not greater than the microfluidic substrate, and such that the gas-liquid interface exists in the first micro flow channel or at a communication place of the first micro flow channel and the buffer channel. 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, and the rotation mode is reciprocating motion when the micro-fluidic chip is driven to rotate at the third rotation frequency. In this method of operation, the gas-liquid interface in the first microchannel may be disrupted so that fluid enters the buffer tank and, in turn, the second fluid tank to react with the reagent.

For example, in the operation method of the microfluidic chip provided in at least one embodiment of the present disclosure, the microfluidic substrate of the microfluidic chip may further include structures such as a sample well, a sample quantification well, a sample overflow well, a third siphon flow channel, a dilution liquid well, a dilution liquid quantification well, a dilution liquid overflow well, and a fourth siphon flow channel. The specific design of these structures and the operation method of the microfluidic chip including these structures can be referred to in the foregoing embodiments (the embodiments shown in fig. 8 and fig. 11 to fig. 15), and will not be described herein.

In the embodiments of the present disclosure, parameters such as rotational frequency in the operation method of the microfluidic chip need to be designed according to the specific structure of the microfluidic chip. In the following, several microfluidic chips of the present disclosure and methods of operating the same are described in connection with several specific examples.

In one embodiment of the disclosure, the microfluidic chip is of a double-layer structure (the microfluidic chip includes a microfluidic substrate and a cover plate, the microfluidic substrate is integrally formed, see fig. 9), the upper layer is a cover layer (the cover plate in the above embodiment) containing a sample loading hole and a diluent loading hole, the lower layer is a micro flow channel layer (the microfluidic substrate in the above embodiment), and the upper layer and the lower layer are bonded together in a watertight manner. The liquid transporting flow path (transporting flow path in the above embodiment) is in a ring shape gradually moving away from the rotation axis, to which 23 buffer tanks (i.e., 23 detection groups are provided) are connected, and to one end away from the rotation axis, an overflow tank (first waste liquid tank in the above embodiment) is connected. The first fluid cell is rectangular and varies in volume from about 7 microliters to about 20 microliters. The second fluid groove is circular, the center of the circle is about 40 mm away from the rotation axis, the diameter is about 2 mm, and the volume ratio and the volume of the corresponding first fluid groove are 2 microliters smaller by designing different depths. The buffer reservoir is rectangular and has a volume of about 2.5 microliters. The length of the first micro-channel between the first fluid groove and the buffer groove is 1 mm, the width is 0.4 mm, the depth is 0.2 mm, the length of the second micro-channel between the buffer groove and the second fluid groove is 1.5 mm, the width is 0.4 mm, and the depth is 0.2 mm. Some of the second fluid reservoirs are preloaded with different types of lyophilized biochemical reagents having a diameter of slightly less than 2 mm and may be used to detect biochemical events in a sample, such as plasma, serum or other body fluids, such as at least one or a combination of alanine Aminotransferase (ALT), aspartic acid Aminotransferase (AST), gamma-Gu Antai aminotransferase (gamma-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 Lipoproteins (HDL), low density low proteins (VLDL), very Low Density Lipoproteins (LDL), magnesium serum (Mg), potassium serum (K), sodium serum (Na), serum chloride (Cl), calcium serum (Ca), and phosphorus serum (P).

For example, 100 microliters of whole blood (i.e., the sample is blood) is injected from the sample injection well into the sample well and 450 microliters of diluent is injected from the diluent injection well into the diluent well, then the microfluidic chip is fixed to the motor and rotated for 2 minutes at a rotation frequency of 5000 revolutions per minute, the sample flows into the sample quantification well, the excess sample flows into the sample overflow well, the diluent flows into the diluent quantification well, and the excess diluent flows into the diluent overflow well. The motor is stopped, and the plasma in the supernatant liquid of the sample quantifying groove fills the sample siphon micro flow channel (the third siphon flow channel of the above embodiment) under the capillary force, and the diluent in the diluent quantifying groove fills the diluent siphon micro flow channel (the fourth siphon flow channel of the above embodiment) under the capillary force. The motor was then started to rotate for 30 seconds at 5000 rpm, and the plasma and the diluent were injected into the mixing tank through the siphon micro flow channels (third and fourth siphon flow channels in the above-described embodiment). The motor is not stopped, and the rotating speed of the motor is controlled to be accelerated and decelerated for 20 times between 4500 revolutions and 1500 revolutions per minute, so that the plasma and the diluent injected into the mixing tank are uniformly mixed. The motor is stopped, and the mixed liquid in the mixing tank fills the mixed liquid siphon micro flow passage (the second siphon flow passage of the above embodiment) under the capillary force. The motor is started again for 60 seconds, the rotation speed is gradually increased from 600 revolutions per minute to 1200 revolutions per minute (for example, the second rotation frequency in the above embodiment), the fluid formed by the mixed solution is sequentially filled in the first fluid tank through the conveying flow channel, and the surplus fluid flows into the overflow tank (the first waste liquid tank in the above embodiment). Stopping the motor, observing, and finding that all spherical freeze-dried biochemical reagents pre-loaded in the second fluid tank keep original appearance, the fluid is not contacted with the reagents, and the individual buffer tanks contain trace fluid, so that the existence of the buffer tanks prevents the fluid from contacting with the biochemical reagents to start reaction in advance. The motor is restarted to rotate, the motor speed is controlled to be accelerated and decelerated 30 times between 4500 revolutions and 1500 revolutions per minute (the third rotational frequency in the embodiment described above), and the fluid in the first fluid tank enters the second fluid tank to be mixed and reacted with the biochemical reagent. According to the above operation method, the applicant finds that the corresponding biochemical reaction results are normal by detecting the reaction results in the second fluid tank by spectrocolorimetry, and no inter-reagent pollution problem is found, i.e., no cross-contamination exists.

In another embodiment of the present disclosure, the microfluidic chip is of a three-layer structure (the microfluidic chip includes a microfluidic substrate including a flow channel layer and a base, and may be referred to as fig. 10), the upper layer is a cover layer (the cover plate in the above embodiment) including a sample loading hole and a diluent loading hole, the middle layer is a flow channel layer, wherein the second fluid groove penetrates the flow channel layer, other grooves (e.g., the first fluid groove, the buffer groove, etc.) and the flow channels (e.g., the first micro flow channel, the second micro flow channel, the transport flow channel, etc.) may penetrate the flow channel layer or not penetrate the flow channel layer, the lower layer is a cover layer (the base in the above embodiment), and the upper, middle and lower layers may be bonded together with water tightness (e.g., a fluid such as water may not enter a contact interface between the three layers). The microfluidic chip is provided with 3 independent fluid modules, the conveying flow channel in each fluid module is in a ring shape gradually far away from the rotation axis, 10 detection groups (namely, 10 buffer tanks) are connected to the conveying flow channel, and an overflow tank (the first waste liquid tank in the embodiment) is connected to one end far away from the rotation axis. The first fluid cell is rectangular and has a volume of 40 microliters. All the second fluid grooves are circular, the circle center is about 60 mm away from the rotation axis, and the volume is 50 microlitres. The buffer tank is rectangular and has a volume of 3 microlitres. The length of the first micro flow channel between the first fluid groove and the buffer groove is 3 mm, the width is 0.6 mm, the depth is 0.1 mm, the length of the second micro flow channel between the buffer groove and the second fluid groove is 2 mm, the width is 0.4 mm, and the depth is 0.1 mm. Part of the second fluid tank is preloaded with a lyophilized antibody reagent, which can be used to detect immune items in a sample such as plasma, serum, or other body fluids, such as infectious diseases, hormones, cardiac markers, tumor markers, and infection-related immune items.

For example, a sample is injected into the sample tank from the sample loading hole, a diluent is injected into the diluent tank from the diluent loading hole, and then the microfluidic chip is fixed on the motor and rotated for 2 minutes at a rotation speed of 5000 revolutions per minute, the sample flows into the sample quantifying tank, the excess sample flows into the sample overflow tank, the diluent flows into the diluent quantifying tank, and the excess diluent flows into the diluent overflow tank. The motor is stopped, and the plasma in the supernatant liquid of the sample quantifying groove fills the sample siphon micro flow channel (the third siphon flow channel of the above embodiment) under the capillary force, and the diluent in the diluent quantifying groove fills the diluent siphon micro flow channel (the fourth siphon flow channel of the above embodiment) under the capillary force. The motor was then started to rotate for 30 seconds at 5000 rpm, and the sample and the diluent were injected into the mixing tank through the siphon micro flow channels (third and fourth siphon flow channels in the above-described embodiment). The motor is not stopped, and the rotating speed of the motor is controlled to be accelerated and decelerated for 20 times between 4500 revolutions and 1500 revolutions per minute, so that the sample and the diluent injected into the mixing tank are uniformly mixed. The motor is stopped, and the mixed liquid in the mixing tank fills the mixed liquid siphon micro flow passage (the second siphon flow passage of the above embodiment) under the capillary force. The motor is started again for 60 seconds, the rotation speed is gradually increased from 300 revolutions per minute to 800 revolutions per minute (for example, the second rotation frequency in the above embodiment), the fluid formed by the above mixed solution sequentially fills the first fluid tank through the delivery flow channel, and the surplus fluid flows into the overflow tank (the first waste liquid tank in the above embodiment). The rotational speed is again increased to 3000 rpm (the third rotational frequency as in the previous embodiment) and the fluid in the first fluid tank is introduced into the second fluid tank and mixed with the pre-loaded reagent. According to the above-described operation method, the applicant found that the corresponding immunoreaction results were normal by detecting the reaction results in the second fluid tank, and did not find the problem of contamination between reagents, i.e., did not have cross contamination.

At least one embodiment of the present disclosure provides a microfluidic chip cartridge, on each of which 4 independent microfluidic chips can be mounted, and on each of which a sample tank, a dilution tank, a sample quantification tank, a dilution quantitative tank, an overflow tank (e.g., a first waste tank), and a liquid delivery flow channel to which 15 detection groups (including buffer tanks) are connected are arranged. The first fluid cell is rectangular and has a volume of 25 microliters. All the second fluid grooves are circular, the center of the circle is about 80 mm away from the rotation axis, and the volume is 25 microlitres. The buffer tank is rectangular and has a volume of 5 microlitres. The length of the micro flow channel between the first fluid tank and the buffer tank (the first micro flow channel in the above embodiment) is 0.5 mm, the width is 0.3 mm, the depth is 0.1 mm, and the length of the micro flow channel between the buffer tank and the second fluid tank (the second micro flow channel in the above embodiment) is 0.5 mm, the width is 0.3 mm, and the depth is 0.3 mm. Part of the second fluid tank is preloaded with the lyophilized primer and the reagents required for nucleic acid amplification, and can be used for nucleic acid molecule detection.

For example, a sample is injected into the sample tank from the sample loading hole, a diluent is injected into the diluent tank from the diluent loading hole, and then the microfluidic chip is fixed on the motor and rotated for 2 minutes at a rotation speed of 3000 revolutions per minute, the sample flows into the sample quantifying tank, the excess sample flows into the sample overflow tank, the diluent flows into the diluent quantifying tank, and the excess diluent flows into the diluent overflow tank. The motor is stopped, and the plasma in the supernatant liquid of the sample quantifying groove fills the sample siphon micro flow channel (the third siphon flow channel of the above embodiment) under the capillary force, and the diluent in the diluent quantifying groove fills the diluent siphon micro flow channel (the fourth siphon flow channel of the above embodiment) under the capillary force. The motor was then started to rotate for 30 seconds at a rotational speed of 3000 rpm, and the sample and the diluent, which were quantified by the sample and diluent quantifying tanks, were injected into the mixing tank through the siphon micro flow channels (the third and fourth siphon flow channels of the above-described embodiment). The motor is not stopped, and the rotating speed of the motor is controlled to be accelerated and decelerated for 20 times between 4500 revolutions and 1500 revolutions per minute, so that the sample and the diluent injected into the mixing tank are uniformly mixed. The motor is stopped, and the mixed liquid in the mixing tank fills the mixed liquid siphon micro flow passage (the second siphon flow passage of the above embodiment) under the capillary force. The motor is started again for 60 seconds, the rotation speed is gradually increased from 300 revolutions per minute to 600 revolutions per minute (for example, the second rotation frequency in the above embodiment), the fluid formed by the above mixed solution sequentially fills the first fluid tank through the delivery flow channel, and the surplus fluid flows into the overflow tank (the first waste liquid tank in the above embodiment). And then starting the motor to rotate, controlling the motor to rotate at a speed of between 3000 and 1500 revolutions per minute (such as the third rotation frequency in the embodiment) and accelerating for 20 times, wherein the fluid in the first fluid tank enters the second fluid tank to be mixed and reacted with the pre-loaded reagent. According to the above-described operation method, the applicant found that the detection result of the corresponding nucleic acid molecule was normal by detecting the reaction result in the second fluid tank, and did not find the problem of contamination between reagents, that is, did not have cross contamination.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is to be construed as including any modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims

A microfluidic substrate comprising a flow channel structure, wherein the flow channel structure comprises:

a delivery flow path;

each detection group comprises a first fluid groove, a first micro-channel, a buffer groove, a second micro-channel and a second fluid groove which are communicated in sequence, wherein the first fluid groove is communicated with the conveying flow channel, and a reagent is arranged in the second fluid groove of at least one detection group;

the microfluidic substrate is provided with a rotation axis, the detection groups are positioned on one side of the conveying flow channel, which is away from the rotation axis, and the distances from the first fluid groove, the buffer groove and the second fluid groove in each detection group to the rotation axis are sequentially increased.
The microfluidic substrate according to claim 1, wherein,

in the case where the first fluidic channel has a fluid therein, the fluid in the first fluidic channel, the buffer tank, the second fluidic channel, and the second fluid channel are formed into a closed tank.
The microfluidic substrate according to claim 1 or 2, wherein,

the first fluidic channel is configured to have a first length such that, at a first rotational frequency that is no greater than the microfluidic substrate, fluid from the first fluidic channel and gas present in the buffer channel form a gas-liquid interface, and such that the gas-liquid interface is present in the first fluidic channel or at a communication of the first fluidic channel and the buffer channel.
A microfluidic substrate according to any one of claims 1-3, wherein,

the first length is 0.1-5 mm, the width of the first micro flow channel is 0.1-1 mm, and the depth of the first micro flow channel is 0.1-0.5 mm.
The microfluidic substrate according to any one of claims 1-4, wherein the buffer tank comprises:

a first sidewall including a first inlet in communication with the first microchannel; and

and a second side wall opposite to the first side wall and comprising a first outlet communicated with the second micro flow channel.
The microfluidic substrate according to claim 5, wherein,

the second side wall is a plane which is perpendicular to the direction from the rotation axis to the first inlet.
The microfluidic substrate according to claim 5, wherein,

The region of the second side wall not provided with the first outlet comprises at least one concave portion, and the distance from the bottom of the at least one concave portion to the rotation axis is larger than the distance from the first outlet to the rotation axis.
The microfluidic substrate according to claim 7, wherein, in each of the buffer grooves,

the concave parts are arranged on two sides of the first outlet, and the first outlet is positioned in the middle of the second side wall; or alternatively

The first outlet is positioned in the middle of the second side wall, and the concave part and the first outlet are sequentially arranged along the preset rotation direction of the microfluidic substrate; or alternatively

One side of the first outlet is provided with the concave part, the first outlet is positioned at one end of the second side wall and along the preset rotation direction of the microfluidic substrate, and the concave part and the first outlet are sequentially arranged.
The microfluidic substrate according to claim 8, wherein, in each of the buffer grooves,

the first inlet is positioned in the middle of the first side wall; or alternatively

The first inlet is positioned at one end of the first side wall, and the first inlet and the first outlet are sequentially arranged along the preset rotation direction of the microfluidic substrate.
The microfluidic substrate according to any one of claims 7-9, wherein each detection group further comprises:

a first siphon runner having one end connected to the concave portion to communicate with the buffer tank and the other end communicating with the second fluid tank;

the inner diameter of the first siphon runner is smaller than that of the second micro runner, and the distance from the part of the first siphon runner to the rotation axis is smaller than that from the first outlet to the rotation axis.
The microfluidic substrate according to any one of claims 1-10, 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.
The microfluidic substrate according to any one of claims 1-11, wherein,

the conveying runner is in a non-closed ring shape, and the center of a circle where the ring is positioned is the rotation axis; or alternatively

The conveying runner is in a non-closed ring shape, the distance from the first end of the conveying runner to the rotation axis is smaller than the distance from the second end of the conveying runner to the rotation axis, and the distance from the first end to the second end of the conveying runner to the rotation axis is sequentially increased.
The microfluidic substrate according to any one of claims 1-12, further comprising:

the first waste liquid tank is communicated with one end of the conveying runner;

the first waste liquid tank and the conveying flow channel are sequentially arranged along the preset rotation direction of the microfluidic substrate.
The microfluidic substrate according to any one of claims 1-13, further comprising:

a mixing tank comprising two inlets and an outlet;

one end of the second siphon runner is communicated with the outlet of the mixing tank, and the other end of the second siphon runner is connected to the conveying runner;

the distance from the part of the second siphon flow passage to the rotation axis is smaller than the distance from the mixing groove to the rotation axis.
The microfluidic substrate according to any one of claims 1-14, further comprising:

a sample tank;

the sample quantifying groove is communicated with the sample groove, and the distance from the sample quantifying groove to the rotating shaft center is larger than the distance from the sample groove to the rotating shaft center;

the sample overflow groove is communicated with the sample groove, and the distance from the sample overflow groove to the rotation axis is larger than the distance from the sample quantifying groove to the rotation axis;

one end of the third siphon flow passage is communicated with the sample quantifying groove, the other end of the third siphon flow passage is communicated with one of two inlets of the mixing groove, and the distance from the part of the second siphon flow passage to the rotation axis is smaller than the distance from the sample quantifying groove to the rotation axis;

A dilution liquid tank;

the diluent quantifying groove is communicated with the diluent groove, and the distance from the diluent quantifying groove to the rotating shaft center is larger than the distance from the diluent groove to the rotating shaft center;

the diluent overflow groove is communicated with the diluent groove, and the distance from the diluent overflow groove to the rotating shaft center is larger than the distance from the diluent quantitative groove to the rotating shaft center;

and one end of the fourth siphon flow passage is communicated with the diluent quantifying groove, the other end of the fourth siphon flow passage is communicated with the other of the two inlets of the mixing groove, and the distance from the part of the fourth siphon flow passage to the rotation axis is smaller than the distance from the diluent quantifying groove to the rotation axis.
The microfluidic substrate according to any one of claims 1-15, comprising:

a runner layer in which the runner structure is formed;

the substrate is positioned on the other side of the runner layer opposite to the side provided with the first fluid groove, the first micro runner, the buffer groove, the second micro runner and the second fluid groove;

the substrate is attached to the runner layer or integrally formed with the runner layer.
A microfluidic chip comprising a cover plate and a microfluidic substrate according to any one of claims 1 to 16, wherein the cover plate is aligned with the microfluidic chip and is located on a side of the microfluidic substrate where the first fluidic channel, the buffer channel, the second fluidic channel and the second fluidic channel are located.
An operation method of a microfluidic chip, wherein the microfluidic chip includes a cover plate and a microfluidic substrate, the microfluidic substrate includes a flow channel structure, the flow channel structure includes a conveying flow channel and a plurality of detection groups, each detection group of the plurality of detection groups includes a first fluid groove, a first micro flow channel, a buffer groove, a second micro flow channel and a second fluid groove which are sequentially communicated, the first fluid groove is communicated with the conveying flow channel, a reagent is disposed in at least one second fluid groove of the detection group, the microfluidic substrate has a rotation axis, the detection group is located on one side of the conveying flow channel away from the rotation axis, and distances from the first fluid groove, the buffer groove and the second fluid groove in each detection group to the rotation axis are sequentially increased, the operation method includes:

Driving the micro-fluidic chip to rotate at a second rotation frequency, so that fluid of the conveying flow channel enters a first fluid groove and a gas-liquid interface is formed in the first micro flow channel or at the communication position of the first micro flow channel and the buffer groove;

after the first fluid groove in each detection group is filled with the liquid, driving the micro-fluidic chip to rotate at a third rotation frequency so that the fluid enters the buffer groove and the second fluid groove;

wherein the third rotational frequency is greater than the first rotational frequency.
The method of operation of claim 18, wherein the first fluidic channel is configured to have a first length such that the fluid from the first fluidic channel and the gas present in the buffer channel form a gas-liquid interface at a first rotational frequency that is no greater than the microfluidic substrate, and such that the gas-liquid interface is present in the first fluidic channel or at a communication of the first fluidic channel and the buffer channel, and

the second rotational frequency is not greater than the first rotational frequency, and the third rotational frequency is greater than the first rotational frequency; or alternatively

The second rotation frequency is not greater than the first rotation frequency, and the rotation mode is reciprocating motion when the micro-fluidic chip is driven to rotate at the third rotation frequency.