CN110180610B

CN110180610B - Reagent sequential loading method and structure and microfluidic device

Info

Publication number: CN110180610B
Application number: CN201910530656.7A
Authority: CN
Inventors: 魏珊珊
Original assignee: Shenzhen Gangzhu Medical Technology Co ltd
Current assignee: Shenzhen Chenghui Medical Technology Co ltd
Priority date: 2019-06-19
Filing date: 2019-06-19
Publication date: 2021-09-03
Anticipated expiration: 2039-06-19
Also published as: CN110180610A

Abstract

The application relates to a reagent sequential loading method, a reagent sequential loading structure and a microfluidic device, wherein the reagent sequential loading structure comprises a loading communicating port, a target communicating port, at least one target chamber and at least two loading chambers which are sequentially arranged, the first loading chamber is communicated with the loading communicating port and is communicated with the corresponding target chamber through a first loading pipeline, the position, close to a target rotation center, of the latter loading chamber is communicated with the former loading pipeline through a communicating pipeline at the target position of the former loading pipeline, and the position, far away from the target rotation center, of the latter loading chamber is communicated with the corresponding target chamber through a loading pipeline. The sequential loading of the reagent can be realized by the sequential reagent loading structure at a constant centrifugal rotating speed, the realization is simple, and the related cost of the valve is saved; the time interval for loading different reagents is controlled by controlling the centrifugal rotating speed, so that the time is reserved for the reaction of the reagents, and the method is particularly suitable for the situation that a plurality of reagents are loaded respectively in sequence.

Description

Reagent sequential loading method and structure and microfluidic device

Technical Field

The application relates to the field of centrifugal microfluidics, in particular to a reagent sequential loading method, a reagent sequential loading structure and a microfluidic device.

Background

Microfluidics (Microfluidics) refers to the manipulation of liquids on a sub-millimeter scale. It integrates the basic operation units related to the biological and chemical fields, even the functions of the whole laboratory, including sampling, diluting, reacting, separating, detecting, etc. on a small Chip, so it is also called Lab-on-a-Chip. Centrifugal microfluidics is a branch of microfluidics, where centrifugal force is used to drive the flow of liquids, particularly devices that use centrifugal force to manipulate liquids on a sub-millimeter scale by rotating the centrifugal microfluidic device. It integrates the basic operation units involved in the fields of biology and chemistry on a small disc-shaped (disc-shaped) chip. In addition to the advantages specific to microfluidics, the overall device is more compact since only one motor is required for centrifugal microfluidics to provide the force required for liquid manipulation. Centrifugal microfluidics is increasingly being used for point-of-care diagnostics. When the microfluidic method is applied to the field of in vitro diagnosis, an important operation is to enable reagents to react according to a certain sequence, and finally obtain a diagnosis result. To realize the reaction of multiple reagents in a certain order, the multiple reagents are loaded into a designated reaction chamber in a certain order. In conventional centrifugal microfluidics, sequential loading of reagents is mainly achieved by means of valves, such as capillary valves, siphon valves, paraffin valves, etc. When the micro-fluidic chip rotates according to the set rotating speed time sequence, different reagents can break through different valves in sequence, and therefore sequential loading of the reagents is achieved. However, in centrifugal microfluidics, these valves are not easy to implement, the use of valves tends to increase the processing cost of the entire microfluidic chip, and the sequential loading achieved with valves tends to be difficult to guarantee with repeatability and reliability. The traditional capillary valve has high requirements on the processing precision of a pipeline, the capillary valve is related to the contact angle of a liquid reagent on the surface of a material, and different reagents need different pipeline sizes to realize the effect of the capillary valve; the traditional siphon valve needs hydrophilic treatment on a siphon pipeline, and the treatment process has high requirements and often greatly increases the processing cost of a chip; the melting of paraffin in the traditional paraffin valve usually needs the instrument to carry out corresponding temperature control, and the design difficulty and the cost of the instrument are increased.

Disclosure of Invention

In view of the above, there is a need for a sequential reagent loading method, structure and microfluidic device.

A method of sequential reagent loading comprising the steps of: in the microfluidic device, each loading chamber is communicated with a corresponding target chamber through a loading pipeline of the loading chamber, wherein the first loading chamber is communicated with the external environment; under the centrifugal state, loading the reagent in the first loading chamber into the first target chamber through the first loading pipeline; when the liquid level of the reagent in the first loading chamber is lower than the first target position in the first loading pipeline, the second loading chamber is communicated with the first loading chamber through the first target position to be communicated with the external environment, the reagent loaded in the second loading chamber enters the second target chamber through the second loading pipeline, and the minimum distance between the second loading chamber and the target rotation center is smaller than the distance between the first target position and the target rotation center; the sequence is executed until the reagent in the last loading chamber enters the last target chamber.

A reagent sequential loading structure comprises a loading communicating port, a target communicating port, at least one target chamber and at least two loading chambers which are sequentially arranged, wherein each loading chamber corresponds to one target chamber; the reagent sequential loading structure has a target center of rotation; in each loading chamber, a first loading chamber is communicated with the loading communicating port and is communicated with a corresponding target chamber through a first loading pipeline, a later loading chamber is communicated with a previous loading pipeline at a target position of the previous loading pipeline through a communicating pipeline at a position close to the target rotation center, and the later loading chamber is communicated with a corresponding target chamber through a loading pipeline at a position far away from the target rotation center; each target chamber is communicated with the target communication port.

A microfluidic device comprising a reagent sequential loading structure according to any one of the preceding claims.

The reagent sequential loading method and the structure are applied to the microfluidic device, on one hand, the sequential loading of the reagent can be realized only by a carrier of the reagent sequential loading structure, such as the microfluidic device, under a constant centrifugal rotating speed, no additional valve device is needed, the realization is simple, and the related cost of the valve is saved; on the other hand, the centrifugal rotating speed can be controlled, so that the time interval for loading different reagents is controlled, the time is reserved for the occurrence of reagent reaction, and the method is particularly suitable for the situation that a plurality of reagents are loaded respectively in sequence; on the other hand, the method is beneficial to simplifying the processing technology and the processing quality control cost of the microfluidic chip.

Drawings

FIG. 1 is a schematic structural diagram of an embodiment of a sequential reagent loading structure according to the present application. Fig. 2 is an enlarged schematic view of the embodiment shown in fig. 1 at a. FIG. 3 is a schematic structural diagram of an embodiment of a microfluidic device according to the present application employing the sequential reagent loading structure shown in FIG. 1. FIG. 4 is a schematic cross-sectional view along the direction B-B of the embodiment shown in FIG. 3. Fig. 5 is an enlarged schematic view of the embodiment of fig. 4 at C. Fig. 6 is another schematic view of the embodiment shown in fig. 3. Fig. 7 is another schematic view of the embodiment of fig. 3. Fig. 8 is an enlarged schematic view at D of the embodiment shown in fig. 7. Fig. 9 is another schematic view of the embodiment of fig. 7. FIG. 10 is a schematic structural diagram of another embodiment of the microfluidic device according to the present application employing the sequential reagent loading structure shown in FIG. 1. Fig. 11 is an enlarged schematic view at E of the embodiment shown in fig. 10. FIG. 12 is a schematic structural diagram of another embodiment of a sequential reagent loading structure according to the present application. Fig. 13 is an enlarged schematic view at F of the embodiment shown in fig. 12. Fig. 14 is an enlarged view at G of the embodiment shown in fig. 12. FIG. 15 is a schematic structural diagram of an embodiment of a microfluidic device according to the present application employing the sequential reagent loading structure shown in FIG. 12. Fig. 16 is an enlarged view at H of the embodiment shown in fig. 15.

Detailed Description

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. This application is capable of embodiments in many different forms than those described herein and that modifications may be made by one skilled in the art without departing from the spirit and scope of the application and it is therefore not intended to be limited to the specific embodiments disclosed below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

In one embodiment of the present application, a method for sequential loading of reagents comprises the steps of: in the microfluidic device, each loading chamber is communicated with a corresponding target chamber through a loading pipeline of the loading chamber, wherein the first loading chamber is communicated with the external environment; under the centrifugal state, loading the reagent in the first loading chamber into the first target chamber through the first loading pipeline; when the liquid level of the reagent in the first loading chamber is lower than the first target position in the first loading pipeline, the second loading chamber is communicated with the first loading chamber through the first target position to be communicated with the external environment, the reagent loaded in the second loading chamber enters the second target chamber through the second loading pipeline, and the minimum distance between the second loading chamber and the target rotation center is smaller than the distance between the first target position and the target rotation center; the sequence is executed until the reagent in the last loading chamber enters the last target chamber. The reagent sequential loading method is applied to the microfluidic device, on one hand, the sequential loading of the reagent can be realized only by the carrier of the reagent sequential loading structure, such as the microfluidic device, at a constant centrifugal rotating speed, no additional valve device is needed, the realization is simple, and the related cost of the valve is saved; on the other hand, the centrifugal rotating speed can be controlled, so that the time interval for loading different reagents is controlled, the time is reserved for the occurrence of reagent reaction, and the method is particularly suitable for the situation that a plurality of reagents are loaded respectively in sequence; on the other hand, the method is beneficial to simplifying the processing technology and the processing quality control cost of the microfluidic chip. In one embodiment, a reagent sequential loading method comprises some or all of the steps of the following embodiments; that is, the reagent sequential loading method includes some or all of the following technical features. In one embodiment, the reagent sequential loading method is implemented by using the reagent sequential loading structure of any one of the following embodiments.

In the microfluidic device, each loading chamber is communicated with a corresponding target chamber through a loading pipeline of the loading chamber, wherein the first loading chamber is communicated with the external environment; in one embodiment, in the microfluidic device, each loading chamber is communicated with a corresponding target chamber through a loading pipeline thereof, and the microfluidic device includes: in the microfluidic device with the reagent sequential loading structure of any embodiment, each loading chamber is communicated with the corresponding target chamber through the loading pipeline. Wherein, corresponding reagents are preset in each loading chamber. Further, in one embodiment, the position of the first loading chamber is set according to a target centrifugation direction to control the force of the centrifugal force on the reagent in the first loading chamber. Under the centrifugal state, loading the reagent in the first loading chamber into the first target chamber through the first loading pipeline; in one embodiment, a predetermined centrifugation speed is used to control the reagent loaded in the first loading chamber through the first loading conduit into the first target chamber.

When the liquid level of the reagent in the first loading chamber is lower than the first target position in the first loading pipeline, the second loading chamber is communicated with the first loading chamber through the first target position to be communicated with the external environment, the reagent loaded in the second loading chamber enters the second target chamber through the second loading pipeline, and the minimum distance between the second loading chamber and the target rotation center is smaller than the distance between the first target position and the target rotation center; that is, when the liquid level of the reagent in the previous loading chamber is lower than the target position in the loading pipeline, the reagent in the next loading chamber is communicated with the first loading chamber through the first target position to communicate with the external environment, so that the reagent can enter the target chamber corresponding to the next loading chamber through the loading pipeline of the next loading chamber. The sequence is executed until the reagent in the last loading chamber enters the last target chamber. Therefore, sequential loading of reagents can be realized, an additional valve device is not needed, the realization is simple, the related cost of the valve is saved, the centrifugal rotating speed can be controlled, the loading time interval of different reagents can be controlled, the time is reserved for the occurrence of reagent reaction, and the device is particularly suitable for the situation that a plurality of reagents are respectively loaded in sequence. In one embodiment, the target chambers are arranged in combination; alternatively, the rate of loading of the reagent in each loading chamber is controlled by controlling the rotational speed of the centrifuge, the length of each loading conduit and/or the area through which each loading conduit passes. In one embodiment, the target chambers are arranged in combination; the loading rate of the reagent in each loading chamber is controlled by controlling the centrifugal speed, the length of each loading conduit and/or the passage area of each loading conduit. The design is beneficial to controlling and adjusting the loading rate of the reagent in each loading chamber, so that the device is suitable for different reaction environments.

In one embodiment of the present application, a sequential reagent loading structure includes a loading communication port, a target communication port, at least one target chamber, and at least two loading chambers sequentially disposed, each loading chamber corresponding to a target chamber; the reagent sequential loading structure has a target center of rotation; in each loading chamber, the first loading chamber is communicated with the loading communicating port and is communicated with a corresponding target chamber through a first loading pipeline, the position, close to the target rotation center, of the latter loading chamber is communicated with the former loading pipeline at the target position of the former loading pipeline through a communicating pipeline of the latter loading chamber, and the position, far away from the target rotation center, of the latter loading chamber is communicated with the corresponding target chamber through a loading pipeline of the latter loading chamber; each target chamber is communicated with a target communicating port. The reagent sequential loading structure is applied to the microfluidic device, on one hand, the sequential loading of the reagent can be realized only by the carrier of the reagent sequential loading structure, such as the microfluidic device, at a constant centrifugal rotating speed, no additional valve device is needed, the realization is simple, and the related cost of the valve is saved; on the other hand, the centrifugal rotating speed can be controlled, so that the time interval for loading different reagents is controlled, the time is reserved for the occurrence of reagent reaction, and the method is particularly suitable for the situation that a plurality of reagents are loaded respectively in sequence; on the other hand, the method is beneficial to simplifying the processing technology and the processing quality control cost of the microfluidic chip. In one embodiment, a reagent sequential loading structure comprises a part of or the whole structure of the following embodiments; that is, the reagent sequential loading structure includes some or all of the following technical features. In one embodiment, a sequential reagent loading structure includes a loading communication port, a target communication port, at least one target chamber, and at least two loading chambers sequentially arranged, wherein each loading chamber corresponds to one target chamber. In one embodiment, a target chamber may correspond to multiple loading chambers, or each target chamber may be arranged in combination, i.e. only one target chamber is arranged; in one embodiment, the number of loading chambers and target chambers is the same, and each loading chamber is arranged in one-to-one correspondence with each target chamber. In one embodiment, the reagent sequential loading structure includes only one target chamber. With this design, all the reagents of the loading chamber are loaded into the target chamber sequentially.

The reagent sequential loading structure has a target center of rotation; the target rotation center can be a solid or an imaginary bit; the target rotation center may be inside or outside the reagent sequential loading structure, but is usually assumed to be outside the reagent sequential loading structure, i.e. the target rotation center is an external relative reference. In one embodiment, the reagent sequential loading structure is configured to be disposed in a microfluidic device, and the target rotation center is a centrifugal center of the microfluidic device. Further, in one embodiment, the reagent sequential loading structure is provided with loading pipelines at positions of each loading chamber far away from the target rotation center, and the loading chambers are communicated with a target chamber corresponding to the loading chambers through the loading pipelines; besides the first loading cavity, the reagent sequential loading structure is provided with communicating pipelines at the positions of the other loading cavities close to the target rotation center respectively, and the loading cavities are communicated with the loading pipeline of the previous loading cavity through the communicating pipelines.

In one embodiment, at least one loading pipe is arranged in a bent mode; in one embodiment, the length of each loading conduit and/or the passing area of each loading conduit is set according to the target loading rate of each loading chamber. In one embodiment, at least one loading pipe is arranged in a bent mode; the length of each loading duct and/or the passing area of each loading duct is set according to the target loading rate of each loading chamber. The design can conveniently control or adjust the time intervals of loading different reagents according to reaction requirements or application requirements, reserve good time for the occurrence of reagent reaction, and is particularly suitable for the situation that a plurality of reagents are loaded respectively in sequence.

In each loading chamber, the first loading chamber is communicated with the loading communicating port and is communicated with a corresponding target chamber through a first loading pipeline, the position, close to the target rotation center, of the latter loading chamber is communicated with the former loading pipeline at the target position of the former loading pipeline through a communicating pipeline of the latter loading chamber, and the position, far away from the target rotation center, of the latter loading chamber is communicated with the corresponding target chamber through a loading pipeline of the latter loading chamber; in one embodiment, the loading communication port may serve as a pouring port for pouring the reagent into the first loading chamber, and in one embodiment, each loading chamber is provided with a pouring port, and after the reagent is poured, the pouring ports other than the pouring port of the first loading chamber are sealed so that the outside environment can be communicated only through the loading communication port, i.e., the pouring port of the first loading chamber, except for the target communication port. Alternatively, in one embodiment, each loading chamber is provided with a loading opening and a loading cover thereof, and when reagent needs to be added, the loading cover is opened to expose the loading opening, the reagent is added into the loading chamber, and then the loading cover is covered to seal the loading opening. Further, in one embodiment, the second loading chamber is communicated with the first loading chamber at a first target position close to the target rotation center through a second communication pipeline, and the second loading chamber is communicated with a corresponding target chamber at a position far away from the target rotation center through a second loading pipeline, the first target position is located in the first loading pipeline, the minimum distance between the second loading chamber and the target rotation center is smaller than the distance between the first target position and the target rotation center, and the maximum distance between the first loading chamber and the target rotation center is smaller than the distance between the first target position and the target rotation center; the other loading chambers are communicated with the previous loading chamber and a corresponding target chamber in the sequence. The loading chambers arranged in sequence are matched to realize the effect of sequential loading control, so that the reagents in the other loading chambers except the first loading chamber enter the corresponding target chambers after the reagents in the previous loading chamber are loaded to a certain degree or even after the loading is finished.

Each target chamber is communicated with a target communicating port. That is, the other loading chambers than the first loading chamber communicate not only with the loading communicating port through the communicating channel thereof and the first loading chamber but also with the target communicating port through the loading channel and the corresponding target chamber. Further, in one embodiment, each target chamber is in communication with a respective target communication port. In one embodiment, each target chamber communicates with a target communication port through the same target communication channel. In one embodiment, the number of the target communication ports is multiple, and each target chamber is communicated with one target communication port. By adopting the design, a reagent sequential loading system from far to near relative to the target rotation center is formed, and the reagent sequentially enters the target chamber from each loading chamber under the action of centrifugal force.

In one embodiment, the loading communication port is arranged at a position of the first loading chamber closest to the target rotation center, or the first loading chamber is communicated with the loading communication port through the first vent pipeline, and the maximum distance between the loading communication port and the target rotation center is larger than or equal to the distance between the position of the first loading chamber communicated with the first vent pipeline and the target rotation center; that is, the loading communication port may be disposed in the first loading chamber, or may be disposed outside the first loading chamber. In one embodiment, the reagent sequential loading structure is provided with a target communicating port in each target chamber, or each target chamber is communicated with the target communicating port through a target communicating channel, and the maximum distance between the target communicating port and the target rotation center is greater than or equal to the distance between the position where each target chamber is communicated with the target communicating channel and the target rotation center. That is, the target communication port may be disposed in the target chamber or may be disposed outside the target chamber. In one embodiment, the loading communication port is arranged at a position of the first loading chamber closest to the target rotation center, or the first loading chamber is communicated with the loading communication port through the first vent pipeline, and the maximum distance between the loading communication port and the target rotation center is larger than or equal to the distance between the position of the first loading chamber communicated with the first vent pipeline and the target rotation center; the reagent sequential loading structure is characterized in that a target communicating port is respectively arranged in each target chamber, or each target chamber is respectively communicated with the target communicating ports through a target communicating pipeline, and the maximum distance between each target communicating port and the target rotation center is larger than or equal to the distance between the position where each target chamber is communicated with the target communicating pipeline and the target rotation center.

In one embodiment, each loading chamber is communicated with a corresponding target chamber at a position, close to the target rotation center, of the corresponding target chamber through a loading pipeline of the loading chamber; that is, the loading chamber is communicated with the target chamber at a position closer to the target rotation center, which is beneficial for outputting the reagent to the target chamber at a position with relatively small centrifugal force and collecting the reagent at a position away from the target rotation center of the target chamber under the action of the centrifugal force. In one embodiment, each loading chamber has a contracted shape at a position away from the target rotation center; in one embodiment, each loading chamber is in the shape of an inverted triangle, circle, shuttle, or oval, among others. Such a design is advantageous in that the reagent is transported out of the contracted shape to allow full loading. In one embodiment, each target chamber has a contracted shape at a location away from the target center of rotation; in one embodiment, each target chamber is in the shape of an inverted triangle, circle, shuttle, or oval, among others. Such a design is advantageous for cleaning or delivering the reagent in the target chamber on the one hand, and for achieving the reagent delivery function in cooperation with the embodiment having the collection chamber and the waste liquid chamber on the other hand. In one embodiment, the center of each loading chamber is located at the same or similar distance from the target rotation center, or the position of each loading chamber closest to the target rotation center is located at the same or similar distance from the target rotation center; wherein, the similarity is that the maximum value is not more than 111% of the average value and the minimum value is not less than 90% of the average value; in one embodiment, the closeness is such that the maximum is no greater than 108% of the average and the minimum is no less than 91% of the average. Such a design facilitates control of the centrifugal force conditions experienced by the reagent in the loading chamber, thereby assisting in controlling reagent loading in conjunction with centrifugal rate. In one embodiment, the communication pipeline comprises an ascending pipeline, a transition pipeline, a descending pipeline and a connecting pipeline which are sequentially arranged, and the connecting pipeline is communicated with the previous loading pipeline at the target position of the previous loading pipeline; the maximum distance between the transition pipeline and the target rotation center is smaller than the maximum distance between the ascending pipeline and the target rotation center, the maximum distance between the descending pipeline and the target rotation center and the minimum distance between the connecting pipeline and the target rotation center. The design is beneficial to forming the control effect of the air pressure valve by matching with the target position, so that the reagent in the loading chamber can enter the corresponding target chamber only after the reagent in the previous loading chamber enters the corresponding target chamber. Further, the target location is formed with a bending zone. The design is beneficial to increasing the length and resistance of the corresponding loading pipeline, and the effect of increasing the flow resistance of the liquid is achieved. In one embodiment, each loading chamber is communicated with a corresponding target chamber at a position, close to the target rotation center, of the corresponding target chamber through a loading pipeline of the loading chamber; and/or each loading chamber has a contracted shape at a position away from the target rotation center; and/or each target chamber has a contracted shape at a position away from the target rotation center; and/or the distance between the center position of each loading chamber and the target rotation center is the same or similar, or the distance between the position of each loading chamber closest to the target rotation center and the target rotation center is the same or similar; wherein, the similarity is that the maximum value is not more than 111% of the average value and the minimum value is not less than 90% of the average value; and/or the communication pipeline comprises an ascending pipeline, a transition pipeline, a descending pipeline and a connecting pipeline which are sequentially arranged, and the connecting pipeline is communicated with the previous loading pipeline at the target position of the previous loading pipeline; the maximum distance between the transition pipeline and the target rotation center is smaller than the maximum distance between the ascending pipeline and the target rotation center, the maximum distance between the descending pipeline and the target rotation center and the minimum distance between the connecting pipeline and the target rotation center.

In one embodiment, the reagent sequential loading structure further comprises a collection chamber and a waste liquid chamber, wherein the minimum distance between the collection chamber and the target rotation center is greater than the maximum distance between the target chamber and the target rotation center, and the minimum distance between the waste liquid chamber and the target rotation center is greater than the maximum distance between the target chamber and the target rotation center; the bottom of the target chamber is provided with a filtering area, namely the bottom is far away from the target rotation center; the collecting chamber is communicated with the filtering area through a collecting pipeline, and the waste liquid chamber is communicated with the filtering area through a waste liquid pipeline; the target communicating port comprises a collecting communicating port and a waste liquid communicating port; relative to a connecting line of the center of the filtering area and the target rotation center, the collecting chamber and the waste liquid chamber are respectively positioned at two sides of the connecting line, and the collecting communicating port and the waste liquid communicating port are also respectively positioned at two sides of the connecting line; the collecting communicating port is arranged at the position, closest to the target rotation center, of the collecting chamber, or the collecting communicating port is communicated with the collecting chamber through the collecting vent pipe, and the maximum distance between the collecting communicating port and the target rotation center is larger than or equal to the distance between the position, communicated with the collecting vent pipe, of the collecting chamber and the target rotation center; the waste liquid communicating port is arranged at the position, closest to the target rotation center, of the waste liquid chamber, or the waste liquid communicating port is communicated with the waste liquid chamber through a waste liquid ventilating pipeline, and the maximum distance between the waste liquid communicating port and the target rotation center is larger than or equal to the distance between the position, communicated with the waste liquid ventilating pipeline, of the waste liquid chamber and the target rotation center; the target chamber is communicated with the collecting communicating port through the collecting pipeline and the collecting chamber in sequence, and is communicated with the waste liquid communicating port through the waste liquid pipeline and the waste liquid chamber in sequence. Further, in one embodiment, a filter membrane such as a silica gel membrane is disposed inside the filtering region.

In one embodiment, the reagent sequential loading structure comprises only one target chamber, and the reagent sequential loading structure comprises four loading chambers; in each loading chamber, the first loading chamber is communicated with the loading communicating port and is communicated with the target chamber through a first loading pipeline; the second loading chamber is communicated with the first loading pipeline at a first target position close to the target rotation center through a second communication pipeline and is communicated with the target chamber at a position far away from the target rotation center through a second loading pipeline, wherein the first target position is positioned in the first loading pipeline, the minimum distance between the second loading chamber and the target rotation center is smaller than the distance between the first target position and the target rotation center, and the maximum distance between the first loading chamber and the target rotation center is smaller than the distance between the first target position and the target rotation center; the third loading chamber is communicated with a second loading pipeline at a second target position close to the target rotation center through a third communication pipeline, and the third loading chamber is communicated with the target chamber at a position far away from the target rotation center through a third loading pipeline, wherein the second target position is positioned in the second loading pipeline, the minimum distance between the third loading chamber and the target rotation center is smaller than the distance between the second target position and the target rotation center, and the maximum distance between the second loading chamber and the target rotation center is smaller than the distance between the second target position and the target rotation center; the fourth loading chamber is communicated with the third loading pipeline at a third target position close to the target rotation center through a fourth communication pipeline, and the fourth loading chamber is communicated with the target chamber at a position far away from the target rotation center through a fourth loading pipeline, wherein the third target position is located in the third loading pipeline, the minimum distance between the fourth loading chamber and the target rotation center is smaller than the distance between the third target position and the target rotation center, and the maximum distance between the third loading chamber and the target rotation center is smaller than the distance between the third target position and the target rotation center. This design is exemplified by four loading chambers and one target chamber, but is equally applicable to other numbers of loading chambers and/or reagent sequential loading configurations of multiple target chambers.

In one embodiment, a microfluidic device comprises a reagent sequential loading structure according to any of the embodiments. In one embodiment, the microfluidic device is a microfluidic chip, i.e. a centrifugal microfluidic chip. In one embodiment, the microfluidic device comprises at least two reagent sequential loading structures.

In one embodiment, a sequential reagent loading structure is shown in fig. 1, which includes a loading communicating port 219, a target communicating port 259, a target chamber 250, and four loading chambers sequentially arranged, where the four loading chambers include a first loading chamber 210, a second loading chamber 220, a third loading chamber 230, and a fourth loading chamber 240, each loading chamber corresponds to the same target chamber 250, please refer to fig. 2, 3, 4, and 5, the sequential reagent loading structure 200 is disposed in the microfluidic device 100 or the sequential reagent loading structure 200 is disposed on the microfluidic device 100, and the sequential reagent loading structure 200 has a target rotation center 300; the target center of rotation 300 is the center of centrifugation of the microfluidic device 100. The reagent sequential loading structure is provided with loading pipelines respectively and correspondingly at positions of each loading chamber far away from the target rotation center 300, wherein each loading pipeline comprises a first loading pipeline 212, a second loading pipeline 222, a third loading pipeline 232 and a fourth loading pipeline 242; of the four loading chambers, the first loading chamber 210 communicates with the loading communication port 219 through the first vent conduit 211 and communicates with the target chamber 250 through the first loading conduit 212; the second loading chamber 220 communicates with the first loading chamber 212 at a first target position 214 near the target rotation center 300 through a second communication channel 221 and the second loading chamber 220 communicates with the target chamber 250 at a second target position away from the target rotation center 300 through a second loading channel 222, wherein the first target position 214 is located in the first loading channel 212, the minimum distance between the second loading chamber 220 and the target rotation center 300 is smaller than the distance between the first target position 214 and the target rotation center 300, and the maximum distance between the first loading chamber 210 and the target rotation center 300 is smaller than the distance between the first target position 214 and the target rotation center 300; the third loading chamber 230 communicates with the second loading channel 222 at a second target position 224 through a third communication channel 231 near the target rotation center 300 and the third loading chamber 230 communicates with the target chamber 250 at a third target position 232 far from the target rotation center 300, wherein the second target position 224 is located in the second loading channel 222, the minimum distance between the third loading chamber 230 and the target rotation center 300 is smaller than the distance between the second target position 224 and the target rotation center 300, and the maximum distance between the second loading chamber 220 and the target rotation center 300 is smaller than the distance between the second target position 224 and the target rotation center 300; the fourth loading chamber 240 communicates with the third loading channel 232 at a third target position 234 near the target rotation center 300 through a fourth communication channel 241 and the fourth loading chamber 240 communicates with the target chamber 250 at a fourth target position away from the target rotation center 300 through a fourth loading channel 242, wherein the third target position 234 is located in the third loading channel 232, the minimum distance between the fourth loading chamber 240 and the target rotation center 300 is smaller than the distance between the third target position 234 and the target rotation center 300, and the maximum distance between the third loading chamber 230 and the target rotation center 300 is smaller than the distance between the third target position 234 and the target rotation center 300. The first loading pipe 212, the third loading pipe 232 and the fourth loading pipe 242 are bent. The target chamber 250 communicates with the target communication port 259 through a target communication conduit 251 at its target communication location 252 near the target rotation center 300. The connecting channels include an ascending channel, a transition channel, a descending channel and a connecting channel, for example, as shown in fig. 1 and 2, the fourth connecting channel 241 includes a fourth ascending channel 2411, a fourth transition channel 2412, a fourth descending channel 2413 and a fourth connecting channel 2414, the fourth connecting channel 2414 is communicated with the third loading channel 232 at the third target position 234, the fourth ascending channel 2411 is communicated with the fourth loading chamber 240, the maximum distance between the fourth transition channel 2412 and the target rotation center 300 is smaller than the maximum distance between the fourth ascending channel 2411 and the target rotation center 300, the maximum distance between the fourth descending channel 2413 and the target rotation center 300 and the minimum distance between the fourth connecting channel 2414 and the target rotation center 300. With further reference to fig. 6 and 7, the reagent sequence loading structure 200 may be located in the microfluidic device 100, and it is understood that one, two or more reagent sequence loading structures 200 may be provided in one microfluidic device 100. Referring to fig. 8, the first loading channel 212 communicates with the target chamber 250 at the first communication position 213, the second loading channel 222 communicates with the target chamber 250 at the second communication position 223, the third loading channel 232 communicates with the fourth loading channel 242 at the third communication position 233, and the fourth loading channel 242 communicates with the target chamber 250 at the fourth communication position 243. The positions of the loading communication port 219 and the target communication port 259 in the microfluidic device 100 are shown in fig. 9. In one embodiment, as shown in fig. 10, the sequential reagent loading structure 200 is located at a non-central position of the microfluidic device 100, and referring to fig. 11, the first loading chamber 210, the second loading chamber 220, the third loading chamber 230, the fourth loading chamber 240 and the target chamber 250 are all circular.

An example of a specific application is given below, in which a liquid reagent is injected into the first loading chamber 210 through the loading communication port 219 as an injection hole. Liquid reagents are pre-arranged in the second loading chamber 220, the third loading chamber 230 and the fourth loading chamber 240, or liquid injection holes are reserved in the second loading chamber 220, the third loading chamber 230 and the fourth loading chamber 240, the liquid injection holes are sealed after liquid injection is completed, and the hole sealing mode includes but is not limited to paraffin sealing, glue sealing, adhesive tape sealing and the like. In one embodiment, reservoirs are pre-disposed in the second loading chamber 220, the third loading chamber 230 and the fourth loading chamber 240, and reagents are pre-disposed in the reservoirs. In one embodiment, the reagent is disposed in a heat-stake wrap disposed in the loading chamber. In one embodiment, the reagent is disposed in a wrap disposed in the loading chamber and the wrap is provided with an opening that is closed with a hot melt layer. In one embodiment, the reservoir is affixed to the loading chamber. In one embodiment, the reservoir has an aluminum foil layer. In one embodiment, the liquid storage container is provided with an opening, a puncturing piece, an elastic piece and a sealing membrane, wherein the sealing membrane is used for sealing the opening, one end of the elastic piece is connected with the puncturing piece, the other end of the elastic piece is fixed in the loading chamber, and the puncturing piece is used for cooperating with the elastic piece to generate displacement to puncture the sealing membrane during centrifugation so as to release a reagent. First, in conjunction with the loading chamber reservoir, the liquid in the first loading chamber 210, i.e., the reagent, is released into the target chamber 250 using heat or higher speed centrifugation. Initially, since the connection between the second communication channel 221 and the first loading chamber 210, i.e., the first target position 214, is not directly communicated with the atmosphere, the stored liquid in the second loading chamber 220, the third loading chamber 230, and the fourth loading chamber 240 cannot be released into the target chamber 250. Subsequently, when the liquid in the first loading chamber 210 flows into the target chamber 250 through the first loading pipe 212 by medium-high speed centrifugation until the liquid level in the first loading pipe 212 is lower than the junction of the first loading pipe 212 and the second communication pipe 221, the second loading chamber 220 communicates with the outside atmosphere. During medium and high speed centrifugation, the liquid in the second loading chamber 220 flows into the target chamber 250 through the second loading pipe 222 until the third loading chamber 230 is communicated with the atmosphere when the liquid level in the second loading pipe 222 is lower than the second target position 224, which is the connection point of the second loading pipe 222 and the third communication pipe 231. During medium and high speed centrifugation, the liquid in the third loading chamber 230 flows into the target chamber 250 through the third loading pipe 232 until the fourth loading chamber 240 is communicated with the atmosphere when the liquid level in the third loading pipe 232 is lower than the third target position 234 which is the connection point of the third loading pipe 232 and the fourth communication pipe 241. During medium and high speed centrifugation, the liquid in the fourth loading chamber 240 flows into the target chamber 250 through the fourth loading conduit 242. Through the sequence of the first loading chamber 210, the second loading chamber 220, the third loading chamber 230 and the fourth loading chamber 240 communicating with the outside atmosphere, the liquid reagents in the loading chambers are sequentially released into the target chamber 250, and thus the sequential loading of the reagents is realized. In order to control the time interval of liquid release of each loading chamber, each loading pipeline can be designed into a continuously bent serpentine pipeline, and it needs to be noted that the design of the serpentine pipeline can play a role in increasing the liquid flow resistance, and the size of each loading pipeline can also be set to be small. Or in the centrifugal process, the centrifugal rotating speed can be controlled, so that the flowing speed of the liquid in each loading chamber is controlled, and the time interval of the release of the liquid in each loading chamber is further controlled. The specific centrifugal speed may be designed or adjusted according to actual conditions, and should not be considered as an additional limitation to the embodiments of the present application. It should be noted that the sequential reagent loading device can realize sequential loading of a plurality of reagents, including but not limited to four.

In one embodiment, a sequential reagent loading structure is shown in fig. 12, the sequential reagent loading structure further includes a collection chamber 260 and a waste chamber 270, a minimum distance between the collection chamber 260 and the target rotation center 300 is greater than a maximum distance between the target chamber 250 and the target rotation center 300, and a minimum distance between the waste chamber 270 and the target rotation center 300 is greater than a maximum distance between the target chamber 250 and the target rotation center 300; the first loading duct 212, the second loading duct 222, the third loading duct 232, and the fourth loading duct 242 are all disposed in a bent manner, taking the fourth loading duct 242 as an example, the fourth loading duct 242 includes a first bent duct 2421, a second bent duct 2422, a third bent duct 2423, a fourth bent duct 2424, and a fifth bent duct 2425 disposed in sequence, wherein the fifth bent duct 2425 is communicated with the target chamber 250; referring to fig. 13, the third target position 234 of the third loading pipe 232 is located between the sixth bending pipe 2321 and the seventh bending pipe 2322, which are communicated with the third loading pipe 232, the fourth connecting pipe 2414 is communicated with the third loading pipe 232 at the third target position 234, and a bending region is formed at the third target position 234. With continuing reference to fig. 12, the target chamber 250 is in the shape of an inverted triangle, and with further reference to fig. 14, the contracted shape of the target chamber 250, i.e., the bottom thereof, is provided with a filtering area 253, the collecting chamber 260 is communicated with the filtering area 253 through a collecting pipe 261, and the waste liquid chamber 270 is communicated with the filtering area 253 through a waste liquid pipe 271; the target communication port 259 includes a collection communication port 269 and a waste communication port 279; relative to the line connecting the center of the filtering zone 253 and the target rotation center 300, the collection chamber 260 and the waste chamber 270 are respectively located at both sides of the line, and the collection communication port 269 and the waste communication port 279 are also respectively located at both sides of the line; the collection communication port 269 is provided at a position where the collection chamber 260 is closest to the target rotation center 300, or the collection communication port 269 communicates with the collection chamber 260 through a collection ventilation pipe and the maximum distance of the collection communication port 269 from the target rotation center 300 is greater than or equal to the distance of the collection chamber 260 from the target rotation center 300; the waste communication port 279 is provided at a position where the waste chamber 270 is closest to the target rotation center 300, or the waste communication port 279 communicates with the waste chamber 270 through a waste vent pipe and the maximum distance of the waste communication port 279 from the target rotation center 300 is greater than or equal to the distance of the waste chamber 270 from the target rotation center 300; target chamber 250 communicates with collection communication port 269 sequentially through collection channel 261 and collection chamber 260, and with waste communication port 279 sequentially through waste channel 271 and waste chamber 270.

In one embodiment, as shown in fig. 15, referring to fig. 16, the first loading channel 212 communicates with the target chamber 250 at the first communication position 213, the second loading channel 222 communicates with the target chamber 250 at the second communication position 223, the third loading channel 232 communicates with the target chamber 250 at the third communication position 233, and the fourth loading channel 242 communicates with the target chamber 250 at the fourth communication position 243. The first, second, third, and

fourth communication sites

213, 223, 233, and 243 are located at positions of the target chamber 250 near the target rotation center 300. The first, second, third and

fourth loading chambers

210, 220, 230, 240 are all circular, and the target chamber 250 is an inverted triangle.

A specific example is given below by taking the procedure of nucleic acid purification as an example. In the molecular diagnosis process, the purification of nucleic acid in the nucleic acid extraction step is very critical, and the nucleic acid purification is difficult to integrate into the microfluidic chip because the nucleic acid purification involves the sequential loading of several liquid reagents. First, the lysed sample is injected into the first loading chamber 210 through the loading communication port 219, the wash solution 1, the wash solution 2, and the eluent are respectively added into the second loading chamber 220, the third loading chamber 230, and the fourth loading chamber 240 through the injection holes or the loading openings, and then the injection holes or the loading openings are sealed. The cleaning solution 1, the cleaning solution 2 and the eluent can be respectively preset in three liquid storage containers by adopting a reagent presetting method, and the three liquid storage containers are respectively preset in the second loading chamber 220, the third loading chamber 230 and the fourth loading chamber 240. Then, the circle center of the centrifugal microfluidic chip is fixed on the rotating shaft of the motor, the centrifugal microfluidic chip is rotated clockwise at 1500rpm, and the first loading chamber 210 is communicated with the atmosphere through a loading communicating port 219. When the cracked sample in the first loading chamber 210 flows out and starts to pass through the silica gel membrane in the filtering area 253 through the serpentine first loading pipeline 212, the nucleic acid such as DNA or RNA in the cracked sample is adsorbed on the silica gel membrane, and the waste liquid completely enters the waste liquid chamber 270 under the action of the coriolis. When the liquid level in the first loading pipe 212 is lower than the joint of the second communication pipe 221 and the first loading pipe 212, the second loading chamber 220 is communicated with the atmosphere through the first loading chamber 210, the cleaning solution 1 in the second loading chamber 220 flows out, and when passing through the silica gel membrane in the filtering area 253, the nucleic acid on the silica gel membrane is cleaned, and the waste liquid completely enters the waste liquid chamber 270 under the action of the coriolis force. When the liquid level in the second loading pipe 222 is lower than the connection point between the second loading pipe 222 and the third communication pipe 231, the third loading chamber 230 is communicated with the atmosphere through the first loading chamber 210 and the second loading chamber 220, the cleaning solution 2 in the third loading chamber 230 flows out, and when passing through the silica gel membrane in the filtering zone 253, the nucleic acid on the silica gel membrane is cleaned, and the waste liquid completely enters the waste liquid chamber 270 under the action of coriolis force. When the liquid level in the third loading pipeline 232 is lower than the connecting port between the third loading pipeline 232 and the fourth communicating pipeline 241 by rotating the centrifugal microfluidic chip counterclockwise at 1500rpm, the fourth loading chamber 240 is communicated with the atmosphere through the first loading chamber 210, the second loading chamber 220 and the third loading chamber 230, the eluent in the fourth loading chamber 240 flows out, when passing through the silica gel membrane in the filtering area 253, the nucleic acid on the silica gel membrane is eluted, and the eluted nucleic acid solution completely enters the nucleic acid collecting chamber 260 under the action of coriolis force. It should be noted that, each loading channel is designed as a serpentine channel with a continuous curve, which can increase the flow resistance of the liquid, so as to control the release of the reagents in each loading chamber to be slow, and further control the release time interval of the liquid in each first loading chamber, so as to achieve the purpose of fully reacting each reagent with the nucleic acid. By the design, the centrifugal rotating speed can be controlled, so that the time interval for loading different reagents can be controlled, and the time for the reaction of the reagents to occur is reserved. The reagent sequential loading device applied to the centrifugal microfluidics can realize any kind of sequential loading only by simple change.

Other embodiments of the present application include a reagent sequential loading method, a reagent sequential loading structure, and a microfluidic device, which are capable of being implemented by combining technical features of the above embodiments.

The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, but should be considered as the scope of the present specification as long as there is no contradiction between the combinations of the technical features. The above examples only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the claims. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present application shall be subject to the appended claims.

Claims

1. A method for sequential loading of reagents, comprising the steps of:

in the microfluidic device, each loading chamber is communicated with a corresponding target chamber through a loading pipeline of the loading chamber, wherein the first loading chamber is communicated with the external environment;

under the centrifugal state, loading the reagent in the first loading chamber into the first target chamber through the first loading pipeline;

when the liquid level of the reagent in the first loading chamber is lower than the first target position in the first loading pipeline, the second loading chamber is communicated with the first loading chamber through the first target position to be communicated with the external environment, the reagent loaded in the second loading chamber enters the second target chamber through the second loading pipeline, and the minimum distance between the second loading chamber and the target rotation center is smaller than the distance between the first target position and the target rotation center;

sequentially executing until the reagent in the last loading chamber enters the last target chamber;

wherein the microfluidic device achieves sequential loading of reagents at a constant centrifugal rotational speed.

2. The method of claim 1, wherein the target chambers are arranged in a combined manner; and/or the presence of a gas in the gas,

the loading rate of the reagent in each loading chamber is controlled by controlling the centrifugal speed, the length of each loading conduit and/or the passage area of each loading conduit.

3. A reagent sequential loading structure is characterized by comprising a loading communicating port, a target communicating port, at least one target chamber and at least two loading chambers which are sequentially arranged, wherein each loading chamber corresponds to one target chamber;

the reagent sequential loading structure has a target center of rotation;

in each loading chamber, a first loading chamber is communicated with the loading communicating port and is communicated with a corresponding target chamber through a first loading pipeline, a later loading chamber is communicated with a previous loading pipeline at a target position of the previous loading pipeline through a communicating pipeline at a position close to the target rotation center, and the later loading chamber is communicated with a corresponding target chamber through a loading pipeline at a position far away from the target rotation center; wherein the first loading chamber is in communication with the external environment;

each target chamber is communicated with the target communication port.

4. The reagent sequential loading structure according to claim 3, wherein the loading communication port is disposed at a position of the first loading chamber closest to the target rotation center, or the first loading chamber is communicated with the loading communication port through a first vent pipe, and the maximum distance between the loading communication port and the target rotation center is greater than or equal to the distance between the position of the first loading chamber communicated with the first vent pipe and the target rotation center; and/or the reagent sequential loading structure is provided with one target communicating port in each target chamber, or each target chamber is communicated with the target communicating port through a target communicating pipeline, and the maximum distance between the target communicating port and the target rotation center is greater than or equal to the distance between the position where each target chamber is communicated with the target communicating pipeline and the target rotation center.

5. The sequential reagent loading structure of claim 3, wherein at least one of the loading tubes is bent; and/or setting the length of each loading pipeline and/or the passing area of each loading pipeline according to the target loading rate of each loading chamber.

6. The sequential reagent loading structure of claim 3, wherein each loading chamber is communicated with a corresponding target chamber through a loading pipeline thereof at a position of the corresponding target chamber near the target rotation center;

and/or each loading chamber has a contracted shape at a position away from the target rotation center;

and/or each of the target chambers has a contracted shape at a position thereof away from the target rotation center;

and/or the distance between the central position of each loading chamber and the target rotation center is the same or similar, or the distance between the position of each loading chamber closest to the target rotation center and the target rotation center is the same or similar; wherein the closeness is a maximum of no more than 111% and a minimum of no less than 90% of the average;

and/or the communication pipeline comprises an ascending pipeline, a transition pipeline, a descending pipeline and a connecting pipeline which are sequentially arranged, and the connecting pipeline is communicated with the previous loading pipeline at the target position of the previous loading pipeline; the maximum distance between the transition pipeline and the target rotation center is smaller than the maximum distance between the ascending pipeline and the target rotation center, the maximum distance between the descending pipeline and the target rotation center and the minimum distance between the connecting pipeline and the target rotation center.

7. The sequential reagent loading structure according to any one of claims 3 to 6, comprising only one of the target chambers.

8. The sequential reagent loading structure of claim 7, further comprising a collection chamber and a waste chamber, wherein the minimum distance between the collection chamber and the target rotation center is greater than the maximum distance between the target chamber and the target rotation center, and the minimum distance between the waste chamber and the target rotation center is greater than the maximum distance between the target chamber and the target rotation center;

the bottom of the target chamber is provided with a filtering area, the collecting chamber is communicated with the filtering area through a collecting pipeline, and the waste liquid chamber is communicated with the filtering area through a waste liquid pipeline;

the target communicating port comprises a collecting communicating port and a waste liquid communicating port; relative to a connecting line of the center of the filtering area and the target rotation center, the collecting chamber and the waste liquid chamber are respectively positioned at two sides of the connecting line, and the collecting communication port and the waste liquid communication port are also respectively positioned at two sides of the connecting line;

the collection communication port is arranged at a position, closest to the target rotation center, of the collection chamber, or the collection communication port is communicated with the collection chamber through a collection ventilation pipeline, and the maximum distance between the collection communication port and the target rotation center is larger than or equal to the distance between a position, communicated with the collection ventilation pipeline, of the collection chamber and the target rotation center;

the waste liquid communicating port is arranged at a position, closest to the target rotation center, of the waste liquid chamber, or the waste liquid communicating port is communicated with the waste liquid chamber through a waste liquid ventilating pipeline, and the maximum distance between the waste liquid communicating port and the target rotation center is larger than or equal to the distance between the waste liquid chamber and the target rotation center, wherein the position is communicated with the waste liquid ventilating pipeline;

the target chamber is communicated with the collection communicating port through the collection pipeline and the collection chamber in sequence, and is communicated with the waste liquid communicating port through the waste liquid pipeline and the waste liquid chamber in sequence.

9. The sequential reagent loading structure of claim 7, comprising four of said loading chambers;

in each loading chamber, a first loading chamber is communicated with the loading communicating port and is communicated with the target chamber through a first loading pipeline;

a second loading chamber is communicated with the first loading pipeline at a first target position close to the target rotation center through a second communication pipeline and is communicated with the target chamber at a position far away from the target rotation center through a second loading pipeline, wherein the first target position is positioned in the first loading pipeline, the minimum distance between the second loading chamber and the target rotation center is smaller than the distance between the first target position and the target rotation center, and the maximum distance between the first loading chamber and the target rotation center is smaller than the distance between the first target position and the target rotation center;

a third loading chamber is communicated with the second loading pipeline at a second target position through a third communication pipeline at a position close to the target rotation center and is communicated with the target chamber at a position far away from the target rotation center through a third loading pipeline, wherein the second target position is positioned in the second loading pipeline, the minimum distance between the third loading chamber and the target rotation center is smaller than the distance between the second target position and the target rotation center, and the maximum distance between the second loading chamber and the target rotation center is smaller than the distance between the second target position and the target rotation center;

a fourth loading chamber is communicated with the third loading pipeline at a third target position through a fourth communication pipeline at a position close to the target rotation center and is communicated with the target chamber at a position far away from the target rotation center through a fourth loading pipeline, wherein the third target position is positioned in the third loading pipeline, the minimum distance between the fourth loading chamber and the target rotation center is smaller than the distance between the third target position and the target rotation center, and the maximum distance between the third loading chamber and the target rotation center is smaller than the distance between the third target position and the target rotation center.

10. A microfluidic device comprising a reagent sequential loading structure according to any one of claims 3 to 9.