阅读说明：本技术 微流控基板、微流控装置及流体驱动方法 (Microfluidic substrate, microfluidic device, and fluid driving method ) 是由 胡涛 崔皓辰 袁春根 张湛 李婧 甘伟琼 胡立教 申晓贺 于 2019-09-26 设计创作，主要内容包括：公开了微流控基板、微流控装置及流体驱动方法。微流控基板包括反应部,其中所述反应部包括设有一种或多种检测位点的载体,并且所述一种或多种检测位点中的每种检测位点配置成检测一种特定的待检测物质,其中所述载体包括用于承载一种或多种检测位点的两个或更多个分部,并且所述两个或更多个分部的布置方向与所述反应部沿流体流动方向的延伸方向平行。(Disclosed are a microfluidic substrate, a microfluidic device, and a fluid driving method. The microfluidic substrate comprises a reaction part, wherein the reaction part comprises a carrier provided with one or more detection sites, and each of the one or more detection sites is configured to detect a specific substance to be detected, wherein the carrier comprises two or more subsections for carrying the one or more detection sites, and the arrangement direction of the two or more subsections is parallel to the extension direction of the reaction part in the fluid flow direction.)

1. A microfluidic substrate comprises a reaction part,

wherein the reaction part comprises a carrier provided with one or more detection sites, and each of the one or more detection sites is configured to detect a specific substance to be detected,

wherein the carrier comprises two or more sections for carrying one or more detection sites, and the two or more sections are arranged in a direction parallel to the extension direction of the reaction section in the fluid flow direction.

2. The microfluidic substrate of claim 1, further comprising a mixing section and a first feed section, the mixing section comprising a first port and a second port, the reaction section comprising a third port and a fourth port, the first port in fluid communication with the first feed section, and the second port in fluid communication with the third port.

3. The microfluidic substrate according to claim 2, wherein the mixing part comprises one or more mixing chambers in series communication with each other.

4. The microfluidic substrate of claim 3, wherein each of the one or more mixing chambers is tapered at two ends in a plane parallel to a surface of the microfluidic substrate, and one of the two ends is configured to flow a fluid in and the other of the two ends is configured to flow a fluid out.

5. The microfluidic substrate according to claim 4, further comprising a second feed portion, and the second feed portion is in fluid communication with the second port.

6. The microfluidic substrate of claim 5, further comprising a third feed, a fourth feed, and a fifth feed in fluid communication with each other and each in fluid communication with the second port of the mixing section.

7. The microfluidic substrate according to claim 6, further comprising:

a first extension between the first feed portion and the first port of the mixing portion, an

A second extension between the second port of the mixing section and the third port of the reaction section.

8. The microfluidic substrate according to claim 7, further comprising a main body portion and a first cover layer, wherein the main body portion and the first cover layer are attached to form the reaction portion, the mixing portion, the first feeding portion, the second feeding portion, the third feeding portion, the fourth feeding portion, the fifth feeding portion, the first extension portion and the second extension portion.

9. The microfluidic substrate according to claim 1, wherein the reaction part has a shape tapered at both ends in a plane parallel to a surface of the microfluidic substrate, and one of the two ends is configured to flow a fluid in and the other of the two ends is configured to flow a fluid out.

10. A microfluidic device comprising the microfluidic substrate of any of the preceding claims 1-9.

11. A fluid driving method for a microfluidic substrate comprising a reaction portion including a carrier provided with one or more detection sites each configured to detect a specific substance to be detected, wherein the carrier includes two or more subsections for carrying the one or more detection sites, and the two or more subsections are arranged in a direction parallel to an extension direction of the reaction portion in a fluid flow direction, the fluid driving method comprising:

preparing a mixture of a first fluid and a second fluid; and

introducing a mixture of a first fluid and a second fluid into the reaction portion along the arrangement direction of the two or more sections, and allowing the mixture to combine with the one or more detection sites within the reaction portion to form one or more first composite structures.

12. The fluid driving method according to claim 11, wherein preparing a mixture of the first fluid and the second fluid includes:

sealing the detection site of the reaction part with a second fluid; and

the first fluid is mixed with the second fluid in the mixing section,

and the fluid driving method further comprises, after introducing the mixture of the first fluid and the second fluid into the reaction section:

washing and removing the mixture of the first fluid and the second fluid which are not combined and remain in the reaction part by using a fourth fluid;

further combining a fifth fluid with the one or more first composite structures within the reaction portion to form one or more second composite structures; and

and removing the fifth fluid by using the third fluid cleaning.

13. The fluid driving method as defined in claim 12, further comprising, after removing the fifth fluid by the third fluid purge:

optically inspecting the one or more second composite structures.

Technical Field

The present disclosure relates to the field of biological detection, and more particularly to microfluidic substrates, microfluidic devices, and fluid driving methods.

Background

Microfluidic technology is a technology for accurately controlling and controlling micro-scale fluid, can integrate basic operation units such as sample adding, reaction, separation, detection and the like in the detection and analysis process into a device with micro-nano scale, and automatically completes the whole analysis process. The microfluidic technology has the advantages of less sample consumption, high detection speed, simple and convenient operation, multifunctional integration, small volume, convenience in carrying and the like, and has great application potential in the fields of biology, chemistry, medicine and the like.

Disclosure of Invention

In one aspect, embodiments of the present disclosure provide a microfluidic substrate comprising a reaction portion, wherein the reaction portion comprises a carrier provided with one or more detection sites, and each of the one or more detection sites is configured to detect a specific substance to be detected, wherein the carrier comprises two or more sections for carrying the one or more detection sites, and the two or more sections are arranged in a direction parallel to an extension direction of the reaction portion in a fluid flow direction.

In some embodiments, the microfluidic substrate further comprises a mixing section and a first feed section, the mixing section comprising a first port and a second port, the reaction section comprising a third port and a fourth port, the first port in fluid communication with the first feed section, and the second port in fluid communication with the third port.

In some embodiments, the mixing section comprises one or more mixing chambers in series communication with each other.

In some embodiments, each of the one or more mixing chambers is tapered in shape with two ends in a plane parallel to the microfluidic substrate surface, and one of the two ends is configured to flow fluid in and the other of the two ends is configured to flow fluid out.

In some embodiments, the microfluidic substrate further comprises a second feed, and the second feed is in fluid communication with the second port.

In some embodiments, the microfluidic substrate further comprises a third feed, a fourth feed, and a fifth feed in fluid communication with each other and each in fluid communication with the second port of the mixing section.

In some embodiments, the microfluidic substrate further comprises a first extension between the first feeding portion and the first port of the mixing portion, and a second extension between the second port of the mixing portion and the third port of the reaction portion.

In some embodiments, the microfluidic substrate further comprises a main body portion and a first covering layer, wherein the main body portion and the first covering layer are attached to form the reaction portion, the mixing portion, the first feeding portion, the second feeding portion, the third feeding portion, the fourth feeding portion, the fifth feeding portion, the first extension portion and the second extension portion.

In some embodiments, the reaction portion has a shape tapered at both ends in a plane parallel to the microfluidic substrate surface, and one of the two ends is configured to flow a fluid in and the other of the two ends is configured to flow a fluid out.

In another aspect, embodiments of the present disclosure also provide a microfluidic device including the above microfluidic substrate.

In yet another aspect, embodiments of the present disclosure also provide a fluid driving method for a microfluidic substrate, the microfluidic substrate including a reaction portion, the reaction portion including a carrier provided with one or more detection sites, each of the one or more detection sites being configured to detect a specific substance to be detected, wherein the carrier includes two or more sections for carrying the one or more detection sites, and an arrangement direction of the two or more sections is parallel to an extension direction of the reaction portion in a fluid flow direction, the fluid driving method including: preparing a mixture of a first fluid and a second fluid; and introducing a mixture of a first fluid and a second fluid into the reaction portion along the arrangement direction of the two or more sections, and allowing the mixture to combine with the one or more detection sites within the reaction portion to form one or more first composite structures.

In some embodiments, preparing the mixture of the first fluid and the second fluid comprises: sealing the detection site of the reaction part with a second fluid; and mixing the first fluid with the second fluid in a mixing section, and the fluid driving method further comprises, after introducing the mixture of the first fluid and the second fluid into the reaction section: washing and removing the mixture of the first fluid and the second fluid which are not combined and remain in the reaction part by using a fourth fluid; further combining a fifth fluid with the one or more first composite structures within the reaction portion to form one or more second composite structures; and removing the fifth fluid by cleaning with the third fluid.

In some embodiments, the fluid driving method further comprises, after removing the fifth fluid with the third fluid purge: optically inspecting the one or more second composite structures.

According to the microfluidic substrate, the microfluidic device and the fluid driving method provided by the embodiment of the invention, the detection sites are provided on the carrier of the reaction part, and can react and combine with the substance to be detected in the sample, so that the detection sites can be used for further detection. Meanwhile, the reaction part can comprise different carriers, and each carrier is provided with different detection sites, so that a plurality of items to be detected can be marked simultaneously to generate different detection signals, the simultaneous detection of multiple indexes of a single sample is realized, the repeated operation is reduced, the efficiency is improved, and the sample cost and the time cost are saved. At the same time, sufficient mixing of the first fluid (e.g., sample) and the second fluid (e.g., diluent) may be achieved with the mixing section. One or more mixing chambers are tapered at both ends in a plane parallel to the surface of the microfluidic substrate, so that the fluid can be drawn back and forth, mixed and the like in the mixing chambers without generating bubbles. By arranging the mixing part in parallel with the first flow channel, the fluid in the reaction part and the fluid in the first flow channel can be fully mixed with the first fluid, and the controllability of fluid driving is increased. In addition, the first fluid (e.g., sample) is detected by the second fluid, the third fluid, the fourth fluid and the fifth fluid, and the residual liquid of the previous fluid at the joint of the common flow channel can be washed away by adding new fluid every time, and the residual fluid in the reaction part can be washed away. The micro-fluidic substrate is simple in processing and preparation method, high in process and operation stability and capable of reducing processing and production cost. By utilizing the high integration of the microfluidic substrate, the automatic inspection of samples (such as blood) can be realized, most of manual operation steps are omitted, the detection system is simplified, and the image processing difficulty is reduced.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is apparent that the drawings in the following description are only some embodiments of the present disclosure.

Fig. 1A and 1B show perspective views of a body portion of a microfluidic substrate at different viewing angles according to an embodiment of the present disclosure;

fig. 2A shows a schematic top view of a reaction section according to an embodiment of the present disclosure;

FIG. 2B is a schematic cross-sectional view of the reaction section taken along line A-B in FIG. 2A;

FIG. 3A shows a schematic top view of a reaction section according to another embodiment of the present disclosure;

FIG. 3B is a schematic cross-sectional view of the reaction section taken along line C-D in FIG. 3A;

FIG. 4 is a schematic structural diagram of a second composite structure for inspection according to an embodiment of the present disclosure;

FIG. 5 is a perspective view of a first cover layer, according to an embodiment of the present disclosure, an

Fig. 6 is a flow chart of a fluid driving method according to an embodiment of the present disclosure.

The figures are merely schematic and are not necessarily to scale. The same reference numbers will be used throughout the drawings to refer to the same or like parts.

Detailed Description

To make the objects, technical solutions and advantages of the embodiments of the present disclosure more clear, the technical solutions of the embodiments of the present disclosure will be described in further detail below with reference to the accompanying drawings.

The products which are sold in the market at present and used for combining the fluid drive with the immunoassay have the defects of complex equipment and instruments, high manufacturing cost and the like, and are not beneficial to popularization in basic medical treatment and family medical treatment.

Therefore, it is a technical problem to be solved to develop a microfluidic device with low cost, easy processing, easy operation and simple structure. An object of the present disclosure is to provide a miniaturized and integrated microfluidic device, which can be further used for detecting a substance to be detected in a sample by various detection means such as optical means, and at the same time, the complexity and cost of instrument and optical system are reduced. In addition, the application field of the method can be expanded to other clinical and scientific research fields such as cell sorting control, microorganism detection and the like, and has wide application value and commercial prospect.

Fig. 1A and 1B show perspective views of a body portion of a microfluidic substrate at different viewing angles according to an embodiment of the present disclosure. Specifically, fig. 1A and 1B show schematic structural views of both side surfaces of the main body portion 100 in the form of perspective views. Illustratively, the main body portion 100 is, for example, a rectangular parallelepiped. The rectangular parallelepiped has a length of 1 to 100mm, for example 55mm, a width of 1 to 100mm, for example 35mm, and a height of 1 to 100mm, for example 15 mm. The microfluidic substrate may include a fluid space including the reaction part 110. Fig. 2A-3B illustrate an exemplary structure of the reaction portion 110 in further detail. Referring to fig. 2A-3B, reaction portion 110 may include one or more carriers 116, 118, and the like. The carriers 116, 118 are provided with one or more detection sites, and each of the one or more detection sites is configured to detect a specific substance to be detected. The carrier may include two or more sections for carrying one or more detection sites, and the arrangement direction of the two or more sections may be parallel to the extension direction of the reaction portion in the fluid flow direction. In some embodiments, as shown in fig. 2A, carrier 116 includes two or more strip sections for carrying one or more detection sites. Optionally, the extension direction of each of the two or more strip sections is parallel to the extension direction of the reaction section 110 in the fluid flow direction. In some embodiments, as shown in fig. 3A, carrier 118 includes two or more dot-shaped portions for carrying one or more detection sites. Alternatively, the arrangement direction of two or more dot portion portions is parallel to the extending direction of the reaction portion in the fluid flow direction. As shown in fig. 2B, a cross-section of the carrier 116 in a direction perpendicular to the fluid flow may include a rectangular shape or the like. As shown in fig. 3B, the cross-section of the carrier 118 in a direction perpendicular to the fluid flow may include a circular, arcuate, or the like shape. The cross section of the reaction part 110 in the direction parallel to the surface of the microfluidic substrate may be a regular or irregular pattern such as a rectangle, a square, an ellipse, a spindle, a diamond, a circular hole array, a square array, an ellipse array, a spindle array, a diamond array, etc. For example, the cross-section is rectangular. The rectangle is, for example, 0.1 to 100mm long and 0.1 to 100mm wide. Specifically, the rectangle is, for example, 15mm long and 3mm wide. Illustratively, the support of the reaction part 110 may comprise a polystyrene support plate, which may have a length of 0.1-100mm, e.g. 15mm, and a width of 0.1-100mm, e.g. 3 mm. The detection site may be located on the polystyrene carrier plate.

The detection principle of the detection site may be, for example, an immune reaction, i.e., specific capture of the target antigen or antibody is achieved in vitro using specific binding of antigen-antibody. Specifically, the fluorescent microsphere is combined with an antigen or an antibody in a specific manner to form the antigen or the antibody marked by the fluorescent microsphere, the antigen or the antibody marked by the fluorescent microsphere is further combined with a substance to be detected, and quantitative detection can be realized by detecting the optical signal intensity of the fluorescent microsphere. For example, as shown in fig. 4, the detection site includes a specific first antibody or first antigen 210, and the substance to be detected (e.g., a second antibody or second antigen 220) in the sample can be specifically bound to the first antibody or first antigen 210 and trapped and anchored to form a first complex structure. After the fluorescent microsphere-labeled third antibody or third antigen 230 is added, the substance to be detected (e.g., antibody or antigen 220) can also specifically bind to the fluorescent microsphere-labeled third antibody or third antigen 230, so that the fluorescent microsphere-labeled third antibody or third antigen 230 is trapped and anchored, and is gathered at the detection site, thereby forming the second composite structure 200. The fluorescent microsphere-labeled third antibody or third antigen 230 may include a third antibody or third antigen 232 and a fluorescent microsphere 234. The fluorescent microspheres can be, for example, latex microspheres, colored microspheres, ordinary fluorescent microspheres, time-resolved fluorescent microspheres. The surface of the microsphere can be modified by different chemical groups and biological groups. Finally, the detection device may further detect, for example optically, the second composite structure 200, emit light of a specific wavelength, excite the second composite structure to emit excitation light (for example, fluorescence) of a specific wavelength, and receive the emitted excitation light to obtain a detection result. In some embodiments, the detection site and the substance to be detected, the fluorescent microsphere, form a sandwich structure of first antibody 210-second antigen 220-third antibody 232 (fluorescent microsphere 234 labeled) in the substance to be detected, or a sandwich structure of first antigen 210-second antibody 220-third antigen 232 (fluorescent microsphere 234 labeled) in the substance to be detected. Through the formation of sandwich structure, can carry out the anchoring combination with detection site, antigen, antibody and the fluorescence microballon three in the sample, wherein the fluorescence microballon can be optical nanoparticle, and light signal stability is strong, and sensitivity is high, thereby can effectively avoid sample self interference to carry out quantitative fluorescence detection, has improved the accuracy of result, simple process.

It should be understood that specific binding of an antibody antigen is only one example of a detection principle of the present disclosure. The present disclosure is not limited to the principle and type of binding and the principle and type of further detection, as long as it is capable of binding or reacting with the detection site and further detection can be performed.

In the embodiment of the present disclosure, by setting different carriers 116, 118, etc., the reaction portion 110 may include different carriers, each of which is provided with one or more detection sites, so that antigens/antibodies of a plurality of items to be detected may be labeled simultaneously to generate different fluorescent signals, thereby realizing simultaneous detection of multiple indexes of a single sample, reducing repetitive operations, improving efficiency, and saving sample cost and time cost.

Fig. 5 is a perspective view of a first cover layer according to an embodiment of the present disclosure. In some embodiments, the fluid space is formed by the main body 100 and the first cover layer 300. For example, as shown in fig. 1A to 1B, an open groove having a certain depth is formed on a surface of one side of the main body portion 100, and the open groove includes each functional portion, a flow channel, and the like. Illustratively, the grooves are 0.1 to 100mm wide, e.g., 0.3mm, and 0.1 to 100mm deep, e.g., 0.2 mm. Meanwhile, on the other side surface of the main body 100, there are formed respective feeding and discharging portions, etc. opened to the outside air and communicating with the grooves. In some embodiments, the first cover layer 300 has a flat surface, and encloses a fluid space with the main body 100 for performing multiple functions of loading, flowing, mixing, diluting, reacting, and the like. In some embodiments, the surface of the first cover layer 300 may also be grooved to form a fluid space with the body portion 100. In some embodiments, a bonding layer (not shown) may also be included between the first cover layer 300 and the body portion 100 for providing a tight bond of the first cover layer 300 and the body portion 100. In some embodiments, a relief space corresponding to the fluid space is disposed on the bonding layer to prevent interference with the fluid space. The body part 100 may be made of, for example, PS plastic or PMMA, and the body part 100 may be formed at one time through an injection molding process, and has a simple structure and is easily mass-produced.

The first cover layer 300 may include an elastic film. The elastic film may be a composite material comprising a hydrophilic layer and an elastic layer, for example, the hydrophilic layer may be made of PS and the elastic layer may be made of PET. In this case, the first cover layer 300 may be vibrated at a high frequency to perform a fluid mixing function in addition to forming a fluid space for the attachment.

In some embodiments, referring to fig. 2B and 3B, the fluid space of the reaction part 110 may be formed by the main body part 100 and the first cover 300 being attached.

Referring again to fig. 1A-1B, in some embodiments, the fluid space further includes a mixing section 120 and a first feed section 130 for adding a first fluid. Mixing section 120 includes a first port 126 and a second port 128, reaction section 110 includes a third port 112 and a fourth port 114, first port 126 is in fluid communication with a first feed section 130, and second port 128 is in fluid communication with third port 112. The first feed portion 130 may have a feed diameter of 0.1 to 100mm, for example 2mm, and a depth of 0.1 to 100mm, for example 2 mm.

In some embodiments, the fluid space further comprises a second feed 140, the second feed 140 being in fluid communication with the second port 128. In some embodiments, the fluid space further comprises a third feed 150, a fourth feed 160, and a fifth feed 170, wherein the second feed 140, the third feed 150, the fourth feed 160, and the fifth feed 170 are in fluid communication with each other and with the second port 128 of the mixing section. The second feed 140, third feed 150, fourth feed 160, and fifth feed 170 are used to add the second fluid, third fluid, fourth fluid, and fifth fluid, respectively. In some embodiments, the fluid space further includes a first flow channel 162. The second feed 140, the third feed 150, the fourth feed 160, and the fifth feed 170 are fluidly connected to the second port 128 of the mixing section 120 through the first flow passage 162, i.e., the mixing section 120 is arranged in parallel with the first flow passage 162. In addition, the communication ports of the second feeding portion 140, the third feeding portion 150, the fourth feeding portion 160 and the fifth feeding portion 170 with the first flow channel 162 are a first port 142, a second port 152, a third port 162 and a fourth port 172, respectively, and the first port 142, the second port 152, the third port 162 and the fourth port 172 are sequentially arranged in a direction away from the second port 128. With this arrangement, sufficient mixing of the first fluid (e.g., sample) and the second fluid (e.g., diluent) may be achieved with the mixing portion 120. By arranging the mixing part 120 in parallel with the first flow channel 162, it is possible to achieve sufficient mixing of the fluid in the reaction part 110 and the first fluid, and sufficient mixing of the fluid in the first flow channel 162 and the first fluid, respectively, and controllability of fluid driving is increased. In addition, the first fluid (e.g., sample) is detected by the second fluid, the third fluid, the fourth fluid and the fifth fluid, and the residual liquid of the previous fluid at the joint of the common flow channel can be washed away by adding new fluid every time, and the residual fluid in the reaction part can be washed away. The diameter and depth of the second, third, fourth and fifth feeds 140, 150, 160 and 170 may be the same as the first feed 130, for example.

Optionally, a filtering part (not shown) may be provided corresponding to one or more of the first, second, third, fourth and fifth feeding parts 130, 140, 150, 160 and 170, respectively, for filtering the first, second, third, fourth and fifth fluids, etc. For example, if the first fluid is blood, a blood filtering membrane for filtering cells and the like in the blood may be disposed between the first feeding part 130 and the mixing part 120.

In some embodiments, the fluid space further comprises: a first extension 190 located between the first feed section 130 and the first port 126 of the mixing section 120; and a second extension part 195 positioned between the second port 128 of the mixing part 120 and the third port 112 of the reaction part 110. The first and second extensions 190, 195 may be used to store a quantity of fluid and may be used to distinguish between different functional sections. Illustratively, the first and second extensions 190, 195 may be 1-100mm long, such as 30 mm.

In some embodiments, the main body 100 and the first cover layer 300 are attached to form the reaction part 110, the mixing part 120, the first feeding part 130, the second feeding part 140, the third feeding part 150, the fourth feeding part 160, the fifth feeding part 170, the first flow channel 162, the first extension 190, and the second extension 195.

In some embodiments, the mixing section 120 may include one or more mixing chambers in series communication with each other, two mixing chambers 122, 124 being exemplarily shown in fig. 1A-1B. The provision of one or more mixing chambers 122, 124 may allow for a higher degree of controllability of the mixing effect. The mixing chambers 122, 124 may be diamond shaped in cross-section in a direction parallel to the microfluidic substrate surface. Illustratively, the center diagonal of the diamond shape is 1-100mm long and 0.1-10mm wide. To facilitate rapid back and forth drawing of fluid without the generation of bubbles in the diamond-shaped mixing chambers 122, 124 during operation of the microfluidic substrate, the diagonals of the diamond shape are, for example, 16mm long and 1.5mm wide.

In some embodiments, the reaction part 110 has a tapered shape with two ends in a plane parallel to the microfluidic substrate surface, wherein one of the two ends is configured to let the fluid in and the other of the two ends is configured to let the fluid out. For example, the reaction part 110 may have a diamond-shaped cross section in a plane parallel to the microfluidic substrate surface. In some embodiments, one or more mixing chambers 122, 124 are tapered at two ends in a plane parallel to the microfluidic substrate surface, wherein one of the two ends is configured to flow fluid in and the other of the two ends is configured to flow fluid out. For example, the cross-section of one or more mixing chambers 122, 124 in a plane parallel to the microfluidic substrate surface may be diamond shaped. Illustratively, the angle formed by the front end of the reaction part 110 tapering is 1 to 30 degrees, for example 6.5 degrees; the back end of the reaction part 110 is tapered to form an angle of 1-30 degrees, for example, 14.5 degrees, so that the fluid can be drawn back and forth in the reaction part 110 without generating bubbles. Illustratively, the angle formed by the front and back end tapers of the mixing chambers 122, 124 may be the same as the reaction portion 110, i.e., the angle formed by the front end tapers is 1-30 degrees, such as 6.5 degrees, and the angle formed by the back end tapers is 1-30 degrees, such as 14.5 degrees, to allow the fluid to be drawn back and forth, mixed, etc., in the mixing chambers 122, 124 without generating bubbles.

In some embodiments, the fluid space further comprises a drain 180. The discharge part 180 may store a certain amount of liquid and has a backflow prevention function. In some embodiments, the exhaust part 180 penetrates one side surface of the main body part 100 and is in fluid communication with the reaction part 110.

Embodiments of the present disclosure also provide a microfluidic device including the aforementioned microfluidic substrate. The microfluidic device may further include a sealing member for sealing different functional portions of the microfluidic substrate, a driving device for driving the fluid to flow, and the like, which are known to those skilled in the art and will not be described herein.

Embodiments of the present disclosure also provide a fluid driving method for a microfluidic substrate including a reaction portion including a carrier provided with one or more detection sites each configured to detect a specific substance to be detected, wherein the carrier includes two or more sections for carrying the one or more detection sites, and an arrangement direction of the two or more sections is parallel to an extension direction of the reaction portion in a fluid flow direction, the fluid driving method including:

s410: preparing a mixture of a first fluid and a second fluid; and

s420: a mixture of a first fluid and a second fluid is introduced into the reaction portion along the arrangement direction of the two or more subsections, and the mixture is combined with one or more detection sites within the reaction portion to form one or more first composite structures.

Specifically, fig. 6 is a flow chart of a fluid driving method according to an embodiment of the present disclosure. As shown in fig. 6, wherein S410 includes:

s510: sealing the detection site of the reaction part with a second fluid;

s520: mixing the first fluid with the second fluid in a mixing section;

and the fluid driving method further includes, after S420:

s540: washing and removing the mixture of the first fluid and the second fluid which are not combined and remain in the reaction part by using a fourth fluid;

s550: further combining the fifth fluid with the one or more first composite structures within the reaction section to form one or more second composite structures; and

s560: and removing the fifth fluid by using the third fluid cleaning.

The fluid driving method further includes, after S560:

s570: the one or more second composite structures are optically inspected.

The method of fluid actuation and the process of detection will be described, for example, in connection with the structures shown in fig. 1A-5.

The second fluid may be a confining liquid. The blocking solution functions to block empty sites on the carrier (e.g., polystyrene plate) that are not occupied by the detected sites, to prevent the antigen or antibody from intercalating into these empty sites, resulting in false positive results. The confining liquid may for example be a mixture comprising one or more of the following fluids: deionized water, 0.1 × PBS (phosphate buffered saline), 0.01-20% Tween-20, 0.01-20% BSA, 0.01-20% casein, 0.01-20% PEG3000, 0.01-20% trehalose, 0.01-20% animal serum, etc. The sealing liquid is not limited in the disclosure, and all fluids capable of playing a sealing role can realize the method of the disclosure.

The third fluid may be a cleaning fluid for cleaning the fluid remaining in the flow channel. The third fluid may for example be a mixture comprising one or more of the following fluids: deionized water, 0.1 × PBS, 0.01-20% Tween-20, 0.01-20% BSA, 0.01-20% casein, 0.01-20% PEG3000, 0.01-20% trehalose, 0.01-20% animal serum, etc. The cleaning liquid is not limited in the disclosure, and all fluids capable of playing an effective cleaning role can realize the method of the disclosure.

The fourth fluid may be a cleaning liquid for cleaning the liquid remaining in the flow channel. The fourth fluid may for example be a mixture comprising one or more of the following fluids: deionized water, 0.1 × PBS, 0.01-20% Tween-20, 0.01-20% BSA, 0.01-20% casein, 0.01-20% PEG3000, 0.01-20% trehalose, 0.01-20% animal serum, etc. The cleaning liquid is not limited in the disclosure, and all fluids capable of playing an effective cleaning role can realize the method of the disclosure. In general, the composition ratio of the third fluid to the fourth fluid may be different. If the third fluid and the fourth fluid are identical, the third charging section and the fourth charging section can be combined into one charging section.

The fifth fluid may be fluorescent microspheres labeled with antibodies or antigens for binding to the antigens or antibodies in the sample. The fifth fluid may for example be a fluorescent microsphere labelled with cTnI antibody at a concentration of 0.001-100% and having a diameter of 1-100000 nm, for example 300 nm. The fifth fluid may also comprise a mixture of one or more of the following fluids: deionized water, 0.1 × PBS, 0.01-20% Tween-20, 0.01-20% BSA, 0.01-20% casein, 0.01-20% PEG3000, 0.01-20% trehalose, 0.01-20% animal serum, etc. The fluorescent microspheres are not limited by the present disclosure, and any fluorescent microspheres that can serve a labeling function should be protected.

First, the second fluid in the second feeding part 140 is injected into the reaction part at a certain injection rate (e.g., 8 μ l/s) using the microfluidic substrate, and is reciprocated in the reaction part, and incubated for 1-1300 seconds, thereby closing blank sites on the carrier (e.g., polystyrene plate). Then, a first fluid is injected into the fluid space at a certain injection speed (for example, 8 μ l/s) by using the microfluidic substrate, and the first fluid sequentially enters the first extension part 190 and the mixing part 120. With the microfluidic substrate, the first fluid is pushed and pulled back and forth in the mixing section 120 at a certain speed and progress (e.g., 15 μ l/s and 10mm progress) for 1-1300 seconds, thereby being sufficiently mixed with the second fluid. Subsequently, the mixture of the first fluid and the second fluid continues into the second extension 195 and the reaction part 110. The reaction part is pushed and pulled back and forth for 1-1300 seconds at a certain speed and process (for example, 2 mu l/s speed and 10mm process), so that the target protein (for example, the second antibody or the second antigen 220) in the first fluid is specifically combined with the detection site (for example, the first antibody or the first antigen 210) on the carrier to form a first composite structure. Thereafter, the fourth feeding part 150 is opened, and the fourth fluid is injected into the reaction part 110 at a certain injection rate (e.g., 8 μ l/s) for cleaning the first fluid and the second fluid remaining in the reaction part 110. Thereafter, the fifth feeding part 160 is opened, the fifth fluid is injected into the reaction part 110 at a certain injection rate (for example, 8. mu.l/s), and push-pull is performed at a certain speed and progress (for example, 2 mul/s speed and 10mm progress) for 1-1300 seconds, the first composite structure is further specifically bound with the fluorescent microsphere-labeled third antibody or third antigen 230, so that the fluorescent microsphere-labeled third antibody or third antigen 230 is trapped, anchored and aggregated at the detection site to form a second composite structure 200 (e.g., a first antibody 210-second antigen 220-third antibody 232 (fluorescent microsphere 234-labeled) sandwich structure in the substance to be detected, or a first antigen 210-second antibody 220-third antigen 232 (fluorescent microsphere 234-labeled) sandwich structure in the substance to be detected). Thereafter, the third feeding part 140 is opened, and a third fluid is injected into the reaction part 110 at a certain injection rate (e.g., 8 μ l/s) for washing the fluorescent microsphere-labeled third antibody or third antigen 230 remaining in the reaction part 110. Finally, the formed second composite structure 200 is further detected, for example, optically detected, and the second composite structure 200 is excited to emit fluorescence of a specific wavelength, and the detection result is obtained by receiving the fluorescence. In some embodiments, the injection of the first, second, third, fourth, and fifth fluids may be driven by various methods known in the art, for example, a syringe pump in the device may be utilized as a driver to inject the fluids into the fluid space; or by being compressively deformed under a sealed condition by a flexible sealing member for closing each feeding portion.

It should be noted that this embodiment is intended to illustrate one operation of the device, and for different detection types, it is fully possible to design other different operation modes based on the structure of the device.

According to the microfluidic substrate, the microfluidic device and the fluid driving method provided by the embodiment of the invention, the detection sites are provided on the carrier of the reaction part, and can react and combine with the substance to be detected in the sample, so that the detection sites can be used for further detection. Meanwhile, the reaction part can comprise different states, and each state is provided with different detection sites, so that a plurality of items to be detected can be marked simultaneously to generate different detection signals, the simultaneous detection of multiple indexes of a single sample is realized, the repeated operation is reduced, the efficiency is improved, and the sample cost and the time cost are saved. At the same time, sufficient mixing of the first fluid (e.g., sample) and the second fluid (e.g., diluent) may be achieved with the mixing section. One or more mixing chambers are tapered at both ends in a plane parallel to the surface of the microfluidic substrate, so that the fluid can be drawn back and forth, mixed and the like in the mixing chambers without generating bubbles. By arranging the mixing part in parallel with the first flow channel, the fluid in the reaction part and the fluid in the first flow channel can be fully mixed with the first fluid, and the controllability of fluid driving is increased. In addition, the first fluid (e.g., sample) is detected by the second fluid, the third fluid, the fourth fluid and the fifth fluid, and the residual liquid of the previous fluid at the joint of the common flow channel can be washed away by adding new fluid every time, and the residual fluid in the reaction part can be washed away. The micro-fluidic substrate is simple in processing and preparation method, high in process and operation stability and capable of reducing processing and production cost. By utilizing the high integration of the microfluidic substrate, the automatic inspection of samples (such as blood) can be realized, most of manual operation steps are omitted, the detection system is simplified, and the image processing difficulty is reduced.

It should be understood that in the description of the present disclosure, "fluid communication" refers to a flow channel connection or arrangement between functional portions that may be in communication by fluid. Unless expressly indicated to the contrary, there are no other functional moieties between two functional moieties that are "in fluid communication".

As will be apparent to those skilled in the art, many different ways of performing the methods of the embodiments of the present disclosure are possible. For example, the order of the steps may be changed, or some of the steps may be performed in parallel. In addition, other method steps may be inserted between the steps. The intervening steps may represent modifications to the methods, such as those described herein, or may be unrelated to the methods. Furthermore, a given step may not have been completely completed before the next step begins.

Various modifications and alterations of this disclosure may be made by those skilled in the art without departing from the spirit and scope of this disclosure. Thus, if such modifications and variations of the present disclosure fall within the scope of the claims of the present disclosure and their equivalents, the present disclosure is intended to include such modifications and variations as well.