CN110452802B

CN110452802B - Full-extraction molecular diagnosis microfluidic chip and microfluidic system

Info

Publication number: CN110452802B
Application number: CN201910726228.1A
Authority: CN
Inventors: 汤明辉
Original assignee: Shenzhen Chenghui Medical Technology Co ltd
Current assignee: Shenzhen Chenghui Medical Technology Co ltd
Priority date: 2019-08-07
Filing date: 2019-08-07
Publication date: 2023-09-01
Anticipated expiration: 2039-08-07
Also published as: CN110452802A

Abstract

The application relates to a full-extraction molecular diagnosis microfluidic chip and a microfluidic system, wherein the full-extraction molecular diagnosis microfluidic chip comprises a storage cavity, a sample cavity, a first liquid adding cavity, a first buffer cavity, at least one controlled liquid adding cavity, a second liquid adding cavity, a distribution cavity, a first waste liquid cavity, a buffer cavity, a second waste liquid cavity, at least one measuring cavity and at least one PCR amplification cavity; the controlled liquid adding cavity is communicated with the second waste liquid cavity through a gas pipeline near the target rotation center, and a phase change valve is arranged in the gas pipeline, and the controlled liquid adding cavity is communicated with the distribution cavity through a liquid adding pipeline. The method integrates all processes of sample splitting, nucleic acid purification, equivalent distribution of reagents, amplification of multi-chamber nucleic acid under specific primer restraint and the like into one centrifugal microfluidic chip, and can realize subsequent analysis work such as fluorescent signal acquisition and analysis, realize a single-sample multi-index diagnosis mode and also provide possibility for molecular diagnosis and multi-pathogen screening facing diseases.

Description

Full-extraction molecular diagnosis microfluidic chip and microfluidic system

Technical Field

The application relates to the field of centrifugal microfluidics, in particular to a full-extraction molecular diagnosis microfluidic chip and a microfluidic system.

Background

Microfluidic (Microfluidics) refers to manipulation of liquids on a sub-millimeter scale. It integrates the basic operating units involved in the biological and chemical fields, even the functions of the whole laboratory, including sampling, dilution, reaction, separation, detection, etc., on a small Chip, so called Lab-on-a-Chip. The chip is generally composed of various liquid reservoirs and a micro-channel network which are connected with each other, so that the sample processing time can be shortened to a great extent, and the maximum utilization efficiency of reagent consumable materials can be realized by precisely controlling the liquid flow. Microfluidic provides a very broad prospect for application in a plurality of fields such as biomedical research, drug synthesis screening, environmental monitoring and protection, health quarantine, judicial identification, detection of biological reagents and the like. In particular, microfluidic is well suited to the demands of Point-of-care testing (POCT) miniaturized instruments, and thus is widely used in POCTs. In industrialization, microfluidics is generally divided into the following large categories: pressure (pneumatic or hydraulic) driven microfluidic, centrifugal microfluidic, droplet microfluidic, digital microfluidic, paper microfluidic, etc.

Microfluidic systems refer to devices that manipulate liquids on a sub-millimeter scale (typically a few micrometers to hundreds of micrometers). Centrifugal microfluidics belongs to one branch of microfluidics, and particularly relates to a centrifugal microfluidic chip which is rotated to control the flow of liquid on a submillimeter scale by using centrifugal force. It integrates the basic operating units involved in the biological and chemical fields on a small disc-shaped chip. Besides the advantages peculiar to the microfluidic, the whole device is more compact and compact since the centrifugal microfluidic requires only one motor to provide the force required for liquid manipulation. The ubiquitous centrifugal field on the disc chip can not only enable liquid to be driven more effectively and ensure that no residual liquid exists in the pipeline, but also effectively realize sample separation based on density difference and enable parallel processing to be simpler. Therefore, centrifugal microfluidics is also increasingly used in instant diagnostics.

Molecular diagnosis based on PCR amplification is to detect the existence of endogenous (genetic or variant) or exogenous (pathogen) target genes by primer-mediated specific amplification of target genes, thereby providing information and decision basis for diagnosis and treatment of diseases. The main application scenes of the kit include infectious disease diagnosis, blood screening, tumor mutation site detection, genetic disease diagnosis, prenatal diagnosis, tissue typing and the like. Molecular diagnostics based on PCR amplification generally comprise the steps of: sample lysis, nucleic acid purification, nucleic acid amplification under specific primer constraints, collection and analysis of fluorescent signals.

However, in molecular diagnostic systems based on PCR amplification, a partitioned laboratory is typically constructed to avoid cross-contamination between samples due to aerosol contamination during PCR amplification. The laboratory is required to realize the partitioning operation of sample processing, nucleic acid extraction and PCR amplification, and has a good ventilation system, so that the laboratory is high in construction cost, and only large medical institutions have the construction financial resources. On the other hand, laboratory operators need to support the guard, and the labor cost is greatly increased. Meanwhile, too much manual intervention can bring about human misoperation. This greatly improves the technological use threshold for PCR-based molecular diagnostics. In addition, in the current molecular diagnosis laboratory mode, the operation of multiple samples and multiple detection projects is finished in a centralized experimental field, and the process quality control requirement is high. Although molecular diagnosis has obvious technical advantages, the molecular diagnosis is also expensive because the steps are complicated, the process is time-consuming and requires professional operation, and the construction cost of a clinical molecular diagnosis laboratory is generally high. In addition, the current molecular diagnosis laboratory mode is generally a multi-sample single-index detection mode, the detection index is limited, and screening of single-sample multi-index infectious pathogens cannot be realized.

The applicant has proposed related microfluidic chip technology for nucleic acid purification-free, but there are many detection items for which nucleic acid purification is inevitably performed.

Disclosure of Invention

Based on this, it is necessary to provide a micro-fluidic chip and a micro-fluidic system for full-extraction molecular diagnosis, which mainly solve the problem of how to integrate full-extraction molecular diagnosis into one centrifugal micro-fluidic chip, integrate all the processes of sample splitting, nucleic acid purification, nucleic acid amplification under specific primer constraint, etc. into one centrifugal micro-fluidic chip, and thereby realize the collection and analysis of fluorescent signals, etc.

A full-extraction molecular diagnosis microfluidic chip, which has a target rotation center, and comprises a storage cavity, a sample cavity, a first liquid adding cavity, a first buffer cavity, at least one controlled liquid adding cavity, a second liquid adding cavity, a distribution cavity, a first waste liquid cavity, a buffer cavity, a second waste liquid cavity, at least one measuring cavity and at least one PCR amplification cavity;

the arrangement in the order of the distance from the target rotation center from small to large is as follows: the device comprises a storage cavity, a sample cavity, a first buffer cavity, a controlled liquid adding cavity, a distribution cavity, a first waste liquid cavity, a measurement cavity and a PCR amplification cavity;

The distance between the first liquid adding cavity and the target rotation center is greater than the distance between the storage cavity and the target rotation center and is smaller than the distance between the first buffer cavity and the target rotation center;

the distance between the second liquid adding cavity and the target rotation center is greater than the distance between the first buffer cavity and the target rotation center and less than the distance between the distribution cavity and the target rotation center;

the distance between the buffer cavity and the target rotation center is greater than or equal to the distance between the first waste liquid cavity and the target rotation center and is smaller than the distance between the measuring cavity and the target rotation center;

the distance between the second waste liquid cavity and the target rotation center is greater than that between the buffer cavity and the target rotation center;

a first sample adding hole is formed in the storage cavity near the target rotation center, and the storage cavity is communicated with the sample cavity through a first capillary valve;

a second sample adding hole is formed in the position, close to the target rotation center, of the sample cavity, and the sample cavity is communicated with the first buffer cavity through a first siphon pipeline;

the first liquid adding cavity is provided with a first liquid adding hole close to the target rotation center and is communicated with the first buffer cavity through a second siphon pipeline;

a first buffer hole is formed in the first buffer cavity, close to the target rotation center, and the first buffer cavity is communicated with the distribution cavity through a third siphon pipeline;

The second liquid adding cavity is provided with a second liquid adding hole near the target rotation center and is communicated with the distribution cavity through an elution pipeline;

the controlled liquid adding cavity is communicated with the second waste liquid cavity through a gas pipeline near the target rotation center, a phase change valve is arranged in the gas pipeline, and the controlled liquid adding cavity is communicated with the distribution cavity through a liquid adding pipeline;

the full-extraction molecular diagnosis microfluidic chip is provided with a filtering structure in or in front of the inlet of the distribution chamber;

one end of the distribution chamber, which is far away from the target rotation center, is communicated with the first waste liquid cavity through a waste liquid pipeline, the other end of the distribution chamber is communicated with the second buffer cavity through a collection pipeline, and the waste liquid pipeline and the collection pipeline are respectively positioned at two sides of the distribution chamber;

a waste liquid vent hole is arranged at the position, close to the target rotation center, of the first waste liquid cavity;

the second buffer cavity is communicated with the sample distribution pipeline through a fourth siphon pipeline;

the sample distribution pipeline is sequentially communicated with each measuring cavity, and the tail end of the sample distribution pipeline is communicated with the second waste liquid cavity; the number of the measuring cavities is the same as that of the PCR amplification cavities and the measuring cavities are correspondingly arranged, and each measuring cavity is communicated with one PCR amplification cavity;

and an air outlet hole is arranged at the position, close to the target rotation center, of the second waste liquid cavity.

The full-extraction molecular diagnosis microfluidic chip integrates all processes of sample splitting, nucleic acid purification, equivalent distribution of reagents, multi-chamber nucleic acid amplification under specific primer restraint and the like into one centrifugal microfluidic chip, and can realize subsequent analysis work such as fluorescent signal acquisition and analysis, realize a single-sample multi-index diagnosis mode and also provide possibility for molecular diagnosis to realize multi-pathogen screening facing diseases; in addition, the whole reaction process is in a sealed micro-fluidic chip, so that the burden of operators and the possibility of pollution are reduced, the whole molecular diagnosis process is not dependent on a molecular diagnosis laboratory any more, and is not dependent on professional operators any more, the requirement of rapid detection at any time and any place is met, and great help is brought to medical examination and disease prevention and control.

In one embodiment, the all-extraction molecular diagnosis microfluidic chip comprises two controlled liquid adding cavities, namely a first controlled liquid adding cavity and a second controlled liquid adding cavity;

the arrangement in the order of the distance from the target rotation center from small to large is as follows: the device comprises a storage cavity, a sample cavity, a first buffer cavity, a first controlled liquid adding cavity, a second controlled liquid adding cavity, a distribution cavity, a first waste liquid cavity, a measurement cavity and a PCR amplification cavity;

the first controlled liquid adding cavity is communicated with the second waste liquid cavity through a first gas pipeline near the target rotation center, a first paraffin valve is arranged in the first gas pipeline, and the first controlled liquid adding cavity is communicated with the distribution cavity through the first liquid adding pipeline;

the second controlled liquid adding cavity is communicated with the second waste liquid cavity through a second gas pipeline near the target rotation center, a second paraffin valve is arranged in the second gas pipeline, and the second controlled liquid adding cavity is communicated with the distribution cavity through the second liquid adding pipeline.

In one embodiment, the total-extraction molecular diagnosis microfluidic chip further includes a converging cavity arranged in order of a distance from the target rotation center from small to large as follows: a controlled filling chamber, a converging chamber, and a dispensing chamber;

the distance between the second liquid adding cavity and the target rotation center is greater than the distance between the first buffer cavity and the target rotation center and is smaller than the distance between the converging cavity and the target rotation center;

The first buffer cavity is communicated with the converging cavity through a third siphon pipeline;

the second liquid adding cavity is communicated with the converging cavity through an elution pipeline;

the controlled liquid adding cavity is communicated with the converging cavity through a liquid adding pipeline;

the converging cavity is communicated with the distributing cavity through a second capillary valve.

In one embodiment, the first buffer chamber is connected to the collecting channel via a third siphon channel, the second liquid feeding chamber is connected to the collecting channel via an elution channel, and the controlled liquid feeding chamber is connected to the collecting channel via a liquid feeding channel, the collecting channel being connected to the collecting chamber.

In one embodiment, a filter membrane is accommodated in the converging cavity as the filter structure; further, the filter membrane is a plurality of layers of silica gel membranes which are arranged in a laminated mode.

the arrangement in the order of the distance from the target rotation center from small to large is as follows: the device comprises a storage cavity, a sample cavity, a first buffer cavity, a first controlled liquid adding cavity, a second controlled liquid adding cavity, a converging cavity, a distributing cavity, a first waste liquid cavity, a measuring cavity and a PCR amplification cavity;

the first controlled liquid adding cavity is communicated with the second waste liquid cavity through a first gas pipeline near the target rotation center, a first paraffin valve is arranged in the first gas pipeline, and the first controlled liquid adding cavity is communicated with the converging cavity through the first liquid adding pipeline;

The second controlled liquid adding cavity is communicated with the second waste liquid cavity through a second gas pipeline near the target rotation center, a second paraffin valve is arranged in the second gas pipeline, and the second controlled liquid adding cavity is communicated with the converging cavity through a second liquid adding pipeline;

in one embodiment, the first buffer chamber is communicated with the collecting pipe through a third siphon pipe, the second liquid adding chamber is communicated with the collecting pipe through an elution pipe, the first controlled liquid adding chamber is communicated with the collecting pipe through the first liquid adding pipe, and the second controlled liquid adding chamber is communicated with the collecting pipe through the second liquid adding pipe; the converging pipeline is communicated with the converging cavity.

In one embodiment, the full-extraction molecular diagnosis microfluidic chip further comprises reagent conveying pipelines, the number of the measuring cavities is the same as that of the reagent conveying pipelines and the measuring cavities are correspondingly arranged, and each measuring cavity is communicated with a corresponding PCR amplification cavity through a corresponding reagent conveying pipeline; alternatively, the end of the sample distribution conduit communicates with the second waste chamber through a drain conduit.

In one embodiment, the all-extraction molecular diagnosis microfluidic chip has a body, where the body is provided with a part or all of a storage cavity, a first capillary valve, a sample cavity, a liquid discharge pipeline, a first siphon pipeline, a second siphon pipeline, a first liquid adding cavity, a second liquid adding cavity, a first buffer cavity, a third siphon pipeline, a collecting pipeline, a first controlled liquid adding cavity, a second controlled liquid adding cavity, a first gas pipeline, a second gas pipeline, a first liquid adding pipeline, a second liquid adding pipeline, an eluting pipeline, a collecting cavity, a second capillary valve, a distributing cavity, a waste liquid pipeline, a collecting pipeline, a second buffer cavity, a fourth siphon pipeline, a first waste liquid cavity, a sample distributing pipeline, each measuring cavity, each PCR amplification cavity, a second waste liquid cavity and a reagent conveying pipeline; the first sample adding hole, the second sample adding hole, the first liquid adding hole, the first buffer hole, the second liquid adding hole, the waste liquid vent hole and the air outlet hole respectively penetrate through the body and are communicated with the external environment; and/or a magnetic piece and/or a grinding piece are arranged in the sample cavity; and/or paraffin is preset in the PCR amplification chamber 135.

In one embodiment, the first sample adding hole, the second sample adding hole, the first liquid adding hole, the first buffer hole, the second liquid adding hole, the waste liquid vent hole and the air outlet hole respectively penetrate through the same side face of the body and are communicated with the external environment.

A microfluidic system comprising the whole extraction molecular diagnostic microfluidic chip of any one of the above.

The microfluidic system integrates all processes of sample splitting, nucleic acid purification, equivalent distribution of reagents, amplification of multi-chamber nucleic acid under the restraint of specific primers and the like into one centrifugal microfluidic chip, and can realize subsequent analysis work such as fluorescent signal acquisition and analysis, so that a single-sample multi-index diagnosis mode is realized, and possibility is provided for molecular diagnosis to realize multi-pathogen screening facing diseases; in addition, the whole reaction process is in a sealed micro-fluidic chip, so that the burden of operators and the possibility of pollution are reduced, the whole molecular diagnosis process is not dependent on a molecular diagnosis laboratory any more, and is not dependent on professional operators any more, the requirement of rapid detection at any time and any place is met, and great help is brought to medical examination and disease prevention and control.

Drawings

Fig. 1 is a schematic view illustrating an internal structure of an embodiment of the present application.

FIG. 2 is another schematic view of the embodiment of FIG. 1.

FIG. 3 is another schematic view of the embodiment of FIG. 1.

FIG. 4 is another schematic view of the embodiment of FIG. 1.

Fig. 5 is another schematic view of the embodiment shown in fig. 1.

Detailed Description

In order that the above objects, features and advantages of the application will be readily understood, a more particular description of the application will be rendered by reference to the appended drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. The present application may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit of the application, whereby the application is not limited to the specific embodiments disclosed below.

It will be understood that when an element is referred to as being "fixed" or "disposed" on another element, it can be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may also be present. The terms "vertical," "horizontal," "left," "right," and the like are used herein for illustrative purposes only and are not meant to be the only embodiment.

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 herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

In one embodiment of the application, a full extraction molecular diagnosis microfluidic chip has a target rotation center, and comprises a storage cavity, a sample cavity, a first liquid adding cavity, a first buffer cavity, at least one controlled liquid adding cavity, a second liquid adding cavity, a distribution cavity, a first waste liquid cavity, a buffer cavity, a second waste liquid cavity, at least one measuring cavity and at least one PCR amplification cavity; the arrangement in the order of the distance from the target rotation center from small to large is as follows: the device comprises a storage cavity, a sample cavity, a first buffer cavity, a controlled liquid adding cavity, a distribution cavity, a first waste liquid cavity, a measurement cavity and a PCR amplification cavity; the distance between the first liquid adding cavity and the target rotation center is greater than the distance between the storage cavity and the target rotation center and is smaller than the distance between the first buffer cavity and the target rotation center; the distance between the second liquid adding cavity and the target rotation center is greater than the distance between the first buffer cavity and the target rotation center and less than the distance between the distribution cavity and the target rotation center; the distance between the buffer cavity and the target rotation center is greater than or equal to the distance between the first waste liquid cavity and the target rotation center and is smaller than the distance between the measuring cavity and the target rotation center; the distance between the second waste liquid cavity and the target rotation center is greater than that between the buffer cavity and the target rotation center; a first sample adding hole is formed in the storage cavity near the target rotation center, and the storage cavity is communicated with the sample cavity through a first capillary valve; a second sample adding hole is formed in the position, close to the target rotation center, of the sample cavity, and the sample cavity is communicated with the first buffer cavity through a first siphon pipeline; the first liquid adding cavity is provided with a first liquid adding hole close to the target rotation center and is communicated with the first buffer cavity through a second siphon pipeline; a first buffer hole is formed in the first buffer cavity, close to the target rotation center, and the first buffer cavity is communicated with the distribution cavity through a third siphon pipeline; the second liquid adding cavity is provided with a second liquid adding hole near the target rotation center and is communicated with the distribution cavity through an elution pipeline; the controlled liquid adding cavity is communicated with the second waste liquid cavity through a gas pipeline near the target rotation center, a phase change valve is arranged in the gas pipeline, and the controlled liquid adding cavity is communicated with the distribution cavity through a liquid adding pipeline; the full-extraction molecular diagnosis microfluidic chip is provided with a filtering structure in or in front of the inlet of the distribution chamber; one end of the distribution chamber, which is far away from the target rotation center, is communicated with the first waste liquid cavity through a waste liquid pipeline, the other end of the distribution chamber is communicated with the second buffer cavity through a collection pipeline, and the waste liquid pipeline and the collection pipeline are respectively positioned at two sides of the distribution chamber; a waste liquid vent hole is arranged at the position, close to the target rotation center, of the first waste liquid cavity; the second buffer cavity is communicated with the sample distribution pipeline through a fourth siphon pipeline; the sample distribution pipeline is sequentially communicated with each measuring cavity, and the tail end of the sample distribution pipeline is communicated with the second waste liquid cavity; the number of the measuring cavities is the same as that of the PCR amplification cavities and the measuring cavities are correspondingly arranged, and each measuring cavity is communicated with one PCR amplification cavity; and an air outlet hole is arranged at the position, close to the target rotation center, of the second waste liquid cavity. The full-extraction molecular diagnosis microfluidic chip integrates all processes of sample splitting, nucleic acid purification, equivalent distribution of reagents, multi-chamber nucleic acid amplification under specific primer restraint and the like into one centrifugal microfluidic chip, and can realize subsequent analysis work such as fluorescent signal acquisition and analysis, realize a single-sample multi-index diagnosis mode and also provide possibility for molecular diagnosis to realize multi-pathogen screening facing diseases; in addition, the whole reaction process is in a sealed micro-fluidic chip, so that the burden of operators and the possibility of pollution are reduced, the whole molecular diagnosis process is not dependent on a molecular diagnosis laboratory any more, and is not dependent on professional operators any more, the requirement of rapid detection at any time and any place is met, and great help is brought to medical examination and disease prevention and control.

In one embodiment, a full extraction molecular diagnostic microfluidic chip includes part or all of the structures of the following embodiments; namely, the full extraction molecular diagnosis microfluidic chip includes some or all of the following technical features. In one embodiment, the full extraction molecular diagnosis microfluidic chip comprises a storage cavity, a sample cavity, a first liquid adding cavity, a first buffer cavity, at least one controlled liquid adding cavity, a second liquid adding cavity, a distribution cavity, a first waste liquid cavity, a buffer cavity, a second waste liquid cavity, at least one measuring cavity and at least one PCR amplification cavity. In one embodiment, the target rotation center is used as a rotation center when centrifuging. The target rotation center can be an entity or a virtual position; the target rotation center can be inside the full-extraction molecular diagnosis microfluidic chip or outside the full-extraction molecular diagnosis microfluidic chip, but is usually virtually arranged outside the full-extraction molecular diagnosis microfluidic chip, that is, the target rotation center is an external relative reference object. In one embodiment, the full-extraction molecular diagnosis microfluidic chip is configured to be disposed in a microfluidic system, and the target rotation center is a centrifugal center of the microfluidic system. Further, in one of the embodiments, the target rotation center is located inside the total extracted molecule diagnostic microfluidic chip or the target rotation center is located outside the total extracted molecule diagnostic microfluidic chip. Thus, the sequential loading of reagents can be achieved only by the microfluidic system or its body at a constant centrifugation speed.

In various embodiments, the distance from the target rotation center includes a minimum distance from the target rotation center or a distance from the target rotation center from a center position including a geometric center or a centroid, and the like. That is, the distance between the storage chamber and the target rotation center includes the minimum distance between the storage chamber and the target rotation center or the distance between the center position of the storage chamber and the target rotation center; in one embodiment, the distance to the target center of rotation is a minimum distance to the target center of rotation. In one embodiment, the distance from the target rotation center is a distance from a center position to the target rotation center. The embodiments of the application do not limit this in particular, but only can realize centrifugal micro-flow control; the distance from the target rotation center can be selected by a person skilled in the art according to actual requirements so as to realize the full-extraction molecular diagnosis micro-fluidic chip. It will be appreciated that in addition to being communicated to the external environment through the respective apertures, which are substantially closed to the external environment, the respective apertures include a first loading aperture, a second loading aperture, a first buffer aperture, a second loading aperture, a waste vent aperture, and a vent aperture, etc., and in view of centrifugal forces, the respective apertures are typically positioned near the target center of rotation and avoid the communication ports of the respective conduits; each pipeline comprises a first capillary valve, a liquid discharge pipeline, a first siphon pipeline, a second siphon pipeline, a third siphon pipeline, a collecting pipeline, a first gas pipeline, a second gas pipeline, a first liquid adding pipeline, a second liquid adding pipeline, an eluting pipeline, a second capillary valve, a waste liquid pipeline, a collecting pipeline, a fourth siphon pipeline, a sample distributing pipeline, a reagent conveying pipeline and the like; each chamber comprises a storage chamber, a sample chamber, a first liquid adding chamber, a second liquid adding chamber, a first buffer chamber, a first controlled liquid adding chamber, a second controlled liquid adding chamber, a converging chamber, a distributing chamber, a second buffer chamber, a first waste liquid chamber, each measuring chamber, each PCR amplifying chamber and part or all of the second waste liquid chamber. Alternatively, each chamber includes a portion or all of a storage chamber, a first capillary valve, a sample chamber, a drain chamber, a first siphon conduit, a second siphon conduit, a first liquid adding chamber, a second liquid adding chamber, a first buffer chamber, a third siphon conduit, a pooling conduit, a first controlled liquid adding chamber, a second controlled liquid adding chamber, a first gas conduit, a second gas conduit, a first liquid adding conduit, a second liquid adding conduit, an elution conduit, a pooling chamber, a second capillary valve, a dispensing chamber, a waste conduit, a collection conduit, a second buffer chamber, a fourth siphon conduit, a first waste chamber, a sample dispensing conduit, each measurement chamber, each PCR amplification chamber, a second waste chamber, and a reagent delivery conduit. The conduit is essentially an elongated small chamber. Further, in one embodiment, the dispensing chamber is rectangular, oval or elongated, in one embodiment, the length of the dispensing chamber in the centrifugal direction is greater than the length of the dispensing chamber in the rotational direction, and in one embodiment, the dispensing chamber is longer than the converging chamber design, and the dispensing chamber is present to increase the time that coriolis forces are applied to the liquid, thereby making the liquid more reliable in channel switching.

Further, in one of the embodiments, the storage chamber, the sample chamber, the buffer chambers, the filling chambers, the controlled filling chambers, and/or the measurement chambers each have a contracted shape at a position thereof remote from the target rotation center; in one embodiment, the shape of an inverted triangle, circle, fusiform, ellipse, or the like. Such a design is advantageous in that the liquid is output from the contracted shape to achieve a loading or output full effect. Further, in one embodiment, each chamber communicates with a corresponding chamber through a corresponding conduit at a location remote from the target center of rotation, and the corresponding chamber communicates with a corresponding conduit at a location near the target center of rotation, thereby facilitating the output and input of liquid.

In one embodiment, the full extraction molecular diagnosis microfluidic chip is provided with a filtering structure in the distribution chamber; or the whole extraction molecular diagnosis microfluidic chip is provided with a filtering structure at the inlet of the distribution chamber; or the whole extraction molecular diagnosis micro-fluidic chip is provided with a filtering structure in front of the inlet of the distribution chamber; further, in one embodiment, the filter structure comprises a filter membrane, cartridge, filter or the like. The filter structure is disposed in the dispensing chamber or the filter structure is disposed in front of the dispensing chamber so that the dispensing chamber can function as a post-filter dispenser.

Further, in one of the embodiments, the phase change valve is used for phase change through temperature control to conduct the gas pipe. In one embodiment, the full extraction molecular diagnosis microfluidic chip includes at least two controlled liquid adding cavities, and the phase change temperatures of the phase change valves of the different gas pipelines are set differently, that is, the phase change valves of the different gas pipelines are prepared by adopting materials with different phase change temperatures, so that the liquid of each controlled liquid adding cavity is orderly added into the distribution cavity or the convergence cavity. In one embodiment, the all-extraction molecular diagnosis microfluidic chip comprises two controlled liquid adding cavities, namely a first controlled liquid adding cavity and a second controlled liquid adding cavity; the arrangement in the order of the distance from the target rotation center from small to large is as follows: the device comprises a storage cavity, a sample cavity, a first buffer cavity, a first controlled liquid adding cavity, a second controlled liquid adding cavity, a distribution cavity, a first waste liquid cavity, a measurement cavity and a PCR amplification cavity; the first controlled liquid adding cavity is communicated with the second waste liquid cavity through a first gas pipeline near the target rotation center, a first paraffin valve is arranged in the first gas pipeline, and the first controlled liquid adding cavity is communicated with the distribution cavity through the first liquid adding pipeline; the second controlled liquid adding cavity is communicated with the second waste liquid cavity through a second gas pipeline near the target rotation center, a second paraffin valve is arranged in the second gas pipeline, and the second controlled liquid adding cavity is communicated with the distribution cavity through the second liquid adding pipeline. That is, in one embodiment, a full extraction molecular diagnostic microfluidic chip having a target center of rotation includes a storage chamber, a sample chamber, a first feeding chamber, a first buffer chamber, a first controlled feeding chamber, a second feeding chamber, a dispensing chamber, a first waste chamber, a buffer chamber, a second waste chamber, at least one measurement chamber, and at least one PCR amplification chamber; the arrangement in the order of the distance from the target rotation center from small to large is as follows: the device comprises a storage cavity, a sample cavity, a first buffer cavity, a first controlled liquid adding cavity, a second controlled liquid adding cavity, a distribution cavity, a first waste liquid cavity, a measurement cavity and a PCR amplification cavity; the distance between the first liquid adding cavity and the target rotation center is greater than the distance between the storage cavity and the target rotation center and is smaller than the distance between the first buffer cavity and the target rotation center; the distance between the second liquid adding cavity and the target rotation center is greater than the distance between the first buffer cavity and the target rotation center and less than the distance between the distribution cavity and the target rotation center; the distance between the buffer cavity and the target rotation center is greater than or equal to the distance between the first waste liquid cavity and the target rotation center and is smaller than the distance between the measuring cavity and the target rotation center; the distance between the second waste liquid cavity and the target rotation center is greater than that between the buffer cavity and the target rotation center; a first sample adding hole is formed in the storage cavity near the target rotation center, and the storage cavity is communicated with the sample cavity through a first capillary valve; a second sample adding hole is formed in the position, close to the target rotation center, of the sample cavity, and the sample cavity is communicated with the first buffer cavity through a first siphon pipeline; the first liquid adding cavity is provided with a first liquid adding hole close to the target rotation center and is communicated with the first buffer cavity through a second siphon pipeline; a first buffer hole is formed in the first buffer cavity, close to the target rotation center, and the first buffer cavity is communicated with the distribution cavity through a third siphon pipeline; the second liquid adding cavity is provided with a second liquid adding hole near the target rotation center and is communicated with the distribution cavity through an elution pipeline; the first controlled liquid adding cavity is communicated with the second waste liquid cavity through a first gas pipeline near the target rotation center, a first paraffin valve is arranged in the first gas pipeline, and the first controlled liquid adding cavity is communicated with the distribution cavity through the first liquid adding pipeline; the second controlled liquid adding cavity is communicated with the second waste liquid cavity through a second gas pipeline near the target rotation center, a second paraffin valve is arranged in the second gas pipeline, and the second controlled liquid adding cavity is communicated with the distribution cavity through the second liquid adding pipeline; the full-extraction molecular diagnosis microfluidic chip is provided with a filtering structure in or in front of the inlet of the distribution chamber; one end of the distribution chamber, which is far away from the target rotation center, is communicated with the first waste liquid cavity through a waste liquid pipeline, the other end of the distribution chamber is communicated with the second buffer cavity through a collection pipeline, and the waste liquid pipeline and the collection pipeline are respectively positioned at two sides of the distribution chamber; a waste liquid vent hole is arranged at the position, close to the target rotation center, of the first waste liquid cavity; the second buffer cavity is communicated with the sample distribution pipeline through a fourth siphon pipeline; the sample distribution pipeline is sequentially communicated with each measuring cavity, and the tail end of the sample distribution pipeline is communicated with the second waste liquid cavity; the number of the measuring cavities is the same as that of the PCR amplification cavities and the measuring cavities are correspondingly arranged, and each measuring cavity is communicated with one PCR amplification cavity; and an air outlet hole is arranged at the position, close to the target rotation center, of the second waste liquid cavity. The remaining embodiments and so on. Further, the total extraction molecular diagnosis microfluidic chip comprises a larger number of controlled liquid adding cavities, so that orderly and controlled liquid adding for a plurality of times is facilitated. The phase change valve is designed, and compared with a common passive valve, the phase change valve does not depend on the hydrophilicity and the hydrophobicity and the surface tension of a reagent, has universality, and can ensure repeatability and reliability; in this way, the phase change materials with different melting points are sequentially melted through temperature control, so that liquid reagents of the controlled liquid adding cavity are distributed into the cavity or the converging cavity according to a certain sequence, and the sequential loading of the reagents can be realized only under a constant centrifugal rotating speed; and the sequential loading of the reagents in any one of the controlled filling chambers can be realized by simple modification. Compared with common passive valves such as capillary valves, siphon valves, drain valves and the like, the phase-change valve is independent of the hydrophilicity and the hydrophobicity and the surface tension of reagents, has universality, can ensure repeatability and reliability, is realized by adopting phase-change materials in one embodiment, and is realized by adopting the phase-change materials to seal a loading pipeline or an air passage or an air outlet pipeline in one embodiment. Phase change materials include, but are not limited to, paraffin waxes, synthetic waxes, crystalline waxes, natural waxes, and the like, as well as various thermoplastic materials such as Polycarbonate (PC), PMMA, COC, and the like, as well as various materials that are solid at ordinary temperatures, melt upon appropriate temperature elevation, and the like. The phase change material is in a solid state at normal temperature, and is melted into a liquid state after being heated, so that the phase change material plays a role of a valve. It should be noted that the phase change valve is implemented in various ways, and the gas pipeline is blocked directly by the phase change material, and the controlled liquid adding cavity is conducted with the atmospheric pressure of the external environment through the air outlet hole of the second waste liquid cavity after the phase change material is melted by heating the phase change valve, so that the liquid in the controlled liquid adding cavity can be released into the distributing cavity or the converging cavity. By changing the temperature, the design melts the phase change materials with different melting points, so that the liquid breaks through different phase change valves at different times, and the sequential loading of liquid reagents is realized. Therefore, as long as the application mode of melting the phase change materials with different melting points through temperature control so as to realize the sequential loading of the liquid reagents of the controlled liquid adding cavity is adopted, the method is understood to belong to the protection scope of the embodiments of the application, and is not limited to the specific implementation mode of the phase change valve.

In one embodiment, the total-extraction molecular diagnosis microfluidic chip further includes a converging cavity arranged in order of a distance from the target rotation center from small to large as follows: a controlled filling chamber, a converging chamber, and a dispensing chamber; the distance between the second liquid adding cavity and the target rotation center is greater than the distance between the first buffer cavity and the target rotation center and is smaller than the distance between the converging cavity and the target rotation center; the first buffer cavity is communicated with the converging cavity through a third siphon pipeline; the second liquid adding cavity is communicated with the converging cavity through an elution pipeline; the controlled liquid adding cavity is communicated with the converging cavity through a liquid adding pipeline; the converging cavity is communicated with the distributing cavity through a second capillary valve. That is, the liquid in each chamber passes through the converging chamber into the dispensing chamber. In one embodiment, a filter membrane is accommodated in the converging cavity as the filter structure; further, in one embodiment, the filter membrane is a laminated multilayer silica gel membrane.

In one embodiment, the first buffer chamber is connected to the collecting channel via a third siphon channel, the second liquid feeding chamber is connected to the collecting channel via an elution channel, and the controlled liquid feeding chamber is connected to the collecting channel via a liquid feeding channel, the collecting channel being connected to the collecting chamber. The pipelines of the chambers are communicated with the converging pipelines and then are communicated with the converging cavity. In one embodiment, the all-extraction molecular diagnosis microfluidic chip comprises two controlled liquid adding cavities, namely a first controlled liquid adding cavity and a second controlled liquid adding cavity; the arrangement in the order of the distance from the target rotation center from small to large is as follows: the device comprises a storage cavity, a sample cavity, a first buffer cavity, a first controlled liquid adding cavity, a second controlled liquid adding cavity, a converging cavity, a distributing cavity, a first waste liquid cavity, a measuring cavity and a PCR amplification cavity; the first controlled liquid adding cavity is communicated with the second waste liquid cavity through a first gas pipeline near the target rotation center, a first paraffin valve is arranged in the first gas pipeline, and the first controlled liquid adding cavity is communicated with the converging cavity through the first liquid adding pipeline; the second controlled liquid adding cavity is communicated with the second waste liquid cavity through a second gas pipeline near the target rotation center, a second paraffin valve is arranged in the second gas pipeline, and the second controlled liquid adding cavity is communicated with the converging cavity through a second liquid adding pipeline; in one embodiment, the first buffer chamber is communicated with the collecting pipe through a third siphon pipe, the second liquid adding chamber is communicated with the collecting pipe through an elution pipe, the first controlled liquid adding chamber is communicated with the collecting pipe through the first liquid adding pipe, and the second controlled liquid adding chamber is communicated with the collecting pipe through the second liquid adding pipe; the converging pipeline is communicated with the converging cavity.

In one embodiment, the full-extraction molecular diagnosis microfluidic chip further comprises reagent conveying pipelines, the number of the measuring cavities is the same as that of the reagent conveying pipelines and the measuring cavities are correspondingly arranged, and each measuring cavity is communicated with a corresponding PCR amplification cavity through a corresponding reagent conveying pipeline; in one embodiment, the end of the sample distribution conduit communicates with the second waste chamber through a drain conduit. Further, in one of the embodiments, the minimum distance of the drain conduit from the center of rotation of the target is greater than or equal to the maximum distance of the sample distribution conduit from the center of rotation of the target. Further, the number of the measuring chambers, the reagent conveying pipelines and the PCR amplification chambers is 8. The application provides a full-extraction molecular diagnosis microfluidic chip, belongs to a centrifugal microfluidic chip, and is matched with a full-automatic nucleic acid analysis instrument to realize full automation of a full-extraction molecular diagnosis project. In this microfluidic chip, sample lysis, nucleic acid purification, and reagent equivalent distribution, multi-chamber PCR amplification are all performed sequentially. The whole reaction process is in a sealed micro-fluidic chip, so that the burden of operators and the possibility of pollution are reduced, the whole molecular diagnosis process is not dependent on a molecular diagnosis laboratory any more, and is not dependent on professional operators any more, the requirement of rapid detection at any time and any place is met, and great help is brought to medical examination and disease prevention and control. In the embodiment, the full-extraction molecular diagnosis microfluidic chip is provided with 8 PCR amplification cavities, and a nucleic acid analysis instrument corresponding to each amplification cavity is provided with 5 fluorescent channels, so that detection of 40 indexes at the same time can be realized at most. This single sample, multi-index approach also provides the potential for molecular diagnostics to achieve multi-pathogen screening for conditions.

In one embodiment, the all-extraction molecular diagnosis microfluidic chip has a body, where the body is provided with a part or all of a storage cavity, a first capillary valve, a sample cavity, a liquid discharge pipeline, a first siphon pipeline, a second siphon pipeline, a first liquid adding cavity, a second liquid adding cavity, a first buffer cavity, a third siphon pipeline, a collecting pipeline, a first controlled liquid adding cavity, a second controlled liquid adding cavity, a first gas pipeline, a second gas pipeline, a first liquid adding pipeline, a second liquid adding pipeline, an eluting pipeline, a collecting cavity, a second capillary valve, a distributing cavity, a waste liquid pipeline, a collecting pipeline, a second buffer cavity, a fourth siphon pipeline, a first waste liquid cavity, a sample distributing pipeline, each measuring cavity, each PCR amplification cavity, a second waste liquid cavity and a reagent conveying pipeline; the first sample adding hole, the second sample adding hole, the first liquid adding hole, the first buffer hole, the second liquid adding hole, the waste liquid vent hole and the air outlet hole respectively penetrate through the body and are communicated with the external environment. In one embodiment, a magnetic element is arranged in the sample cavity and is used for matching with an external magnetic environment to realize a cracking effect; or the sample cavity is provided with a magnetic part and an abrasive part. In one embodiment, the magnetic member includes a magnet or magnets, the shape of which is not limited; in one embodiment, the abrasive article comprises a glass body or stainless steel; in one embodiment, the abrasive member is a sphere, such as a glass bead or the like. Further, in one of the embodiments, paraffin is preset in the PCR amplification chamber for melting and sealing the PCR amplification chamber during PCR amplification.

Further, in one of the embodiments, the body has an integral cover portion for integrally covering the chambers and their associated conduits. In one embodiment, the integral cover portion is configured to entirely cover all of the storage chamber, the first capillary valve, the sample chamber, the drain chamber, the first siphon conduit, the second siphon conduit, the first addition chamber, the second addition chamber, the first buffer chamber, the third siphon conduit, the collection chamber, the first controlled addition chamber, the second controlled addition chamber, the first gas conduit, the second gas conduit, the first addition chamber, the second addition chamber, the elution chamber, the collection chamber, the second capillary valve, the distribution chamber, the waste liquid conduit, the collection conduit, the second buffer chamber, the fourth siphon conduit, the first waste liquid chamber, the sample distribution conduit, the measurement chambers, the PCR amplification chambers, the second waste liquid chamber, and the reagent delivery conduit. In one embodiment, the first sample adding hole, the second sample adding hole, the first liquid adding hole, the first buffer hole, the second liquid adding hole, the waste liquid vent hole and the air outlet hole respectively penetrate through the same side face of the body and are communicated with the external environment.

In one embodiment, the storage chamber is a proteinase K storage chamber for containing proteinase K; the first sample adding hole is used for adding proteinase K into the storage cavity; in one embodiment, the sample cavity is used for containing a sample to be detected, and the second sample adding hole is used for adding the sample into the sample cavity; in one embodiment, the first liquid adding cavity is a pyrolysis neutralization liquid storage cavity and is used for containing pyrolysis neutralization liquid, and the first liquid adding hole is used for adding the pyrolysis neutralization liquid into the pyrolysis neutralization liquid storage cavity; in one embodiment, the first buffer chamber is configured to receive a buffer solution, and the first buffer hole is configured to add the buffer solution to the first buffer chamber; in one embodiment, the first controlled addition chamber serves as a first cleaning liquid storage chamber for containing a first cleaning liquid and the second controlled addition chamber serves as a second cleaning liquid storage chamber for containing a second cleaning liquid; in one embodiment, the second liquid adding cavity is an eluent storage cavity and is used for containing eluent, and the second liquid adding hole is used for adding the eluent into the second buffer cavity; in one embodiment, the converging cavity is a silica gel membrane cavity for accommodating the laminated silica gel membranes; in one embodiment, the dispensing chamber is configured to dispense the pooled sample into the second buffer chamber or the waste into the first waste chamber depending on the direction of rotation; in one embodiment, the sample distribution pipeline is used for sequentially distributing the collected samples to each measuring cavity, and the redundant samples are output to the second waste liquid cavity, so that the samples in the measuring cavities enter the PCR amplification cavity through the reagent conveying pipeline under the action of centrifugal force, and the PCR amplification is completed. It will be appreciated that the passage area of each conduit may be set as required, for example, by centrifugal speed or the like. In one embodiment, proteinase K, the lysis neutralization solution, the first cleaning solution and the second cleaning solution are all preset in an aluminum foil container with one end encapsulated by low-melting paraffin and the other end glued by glue before use. The method comprises the steps of presetting proteinase K, cracking neutralization liquid, first cleaning liquid and adhering ends of an aluminum foil container of second cleaning liquid, and fixing the adhesive ends on the upper areas of a proteinase K storage cavity, a cracking neutralization liquid storage cavity, a first cleaning liquid storage cavity and a second cleaning liquid storage cavity by glue respectively, wherein the adhesive ends are close to a circle center, and paraffin sealing ends are far away from the circle center. On the other hand, the eluent is preset in an aluminum foil container with one end sealed by high-melting-point paraffin and the other end glued, the glued end of the aluminum foil container is fixed on the upper area of the eluent storage cavity by glue, wherein the glued end is close to the circle center, and the paraffin sealing end is far away from the circle center. When the temperature of the area where the aluminum foil container is positioned rises above the melting point of paraffin wax, the paraffin wax is melted, and the liquid stored in the aluminum foil container is released. The first gas pipeline is blocked by high-melting-point paraffin at the first paraffin valve; the second gas pipeline is blocked by low-melting-point paraffin at the second paraffin valve.

The following is a whole detection flow of the full-extraction molecular diagnosis micro-fluidic chip, which is described in detail below.

Proteinase k is added into the storage cavity through the first sample adding hole, and a sample is added into the sample cavity through the second sample adding hole;

centrifuging at 2000rpm anticlockwise, heating the inner temperature control area to 60 ℃, melting paraffin ends of aluminum foil containers in a proteinase k storage cavity (namely a storage cavity), a cracking neutralization liquid storage cavity, a first cleaning liquid storage cavity and a second cleaning liquid storage cavity, and releasing liquid reagents; meanwhile, the proteinase k solution in the proteinase k storage cavity breaks through the first capillary valve and enters the cracking cavity, namely the sample cavity;

the centrifugal speed is reduced to 600rpm anticlockwise, the rotating speed is kept for 10min, and meanwhile, the inner circular temperature zone is kept at 60 ℃ for 10min, and the magnetic stirring, grinding and cracking are carried out; when the micro-fluidic chip for full-extraction molecular diagnosis rotates to a position above a large magnet fixed on the instrument below the micro-fluidic chip, the small magnet is subjected to magnetic field force which points to the center of a circle and is larger than centrifugal force which is far away from the center of the circle because the position of the large magnet is closer to the center of the circle, and the small magnet moves to a position which is close to the center of the circle in the splitting cavity under the action of the magnetic field force in sequence; when the chip rotates to the point that the small magnet in the cracking cavity is far away from the large magnet, the magnetic field force is weaker and even disappears, and the small magnet returns to the bottom of the cracking cavity again under the action of centrifugal force; when the microfluidic chip rotates, the magnet continuously moves up and down in the splitting cavity, and the magnet is matched with glass beads preset in the splitting cavity to grind and split a sample in the splitting cavity;

The centrifugal speed is reduced to 200rpm anticlockwise and kept at the rotating speed for 30s, and the mixed liquid in the cracking cavity and the cracking neutralization liquid in the 11 cracking neutralization liquid storage cavity are subjected to hydrophilic treatment in advance under the action of capillary force respectively, so that larger capillary force exists to fill the first siphon pipeline and the second siphon pipeline;

the centrifugal speed is increased to 2000rpm anticlockwise and kept at the rotating speed for 1min, liquid in the first siphon pipeline and the second siphon pipeline breaks through capillary action and enters the cracking neutralization cavity, and then under the help of centrifugal force, the mixed liquid in the cracking cavity and the cracking neutralization liquid in the cracking neutralization liquid storage cavity completely flow into the first buffer cavity through the first siphon pipeline and the second siphon pipeline respectively;

maintaining at 2000rpm anticlockwise for 3min, and fully cracking and neutralizing the liquid in the first buffer cavity;

the centrifugal speed is reduced to 200rpm anticlockwise, the centrifugal speed is maintained for 30 seconds and then is increased to 2000rpm anticlockwise, the liquid in the first buffer cavity breaks through the third siphon pipeline and flows through the silica gel membrane of the converging cavity, and then enters the first waste liquid cavity under the action of the Coriolis force, and DNA in the liquid is adsorbed on the silica gel membrane;

And (3) maintaining 2000rpm anticlockwise, heating the external temperature to 60 ℃, melting the No. 54 paraffin at the first paraffin valve, opening the paraffin valve, and conducting the first cleaning liquid storage cavity with the air outlet hole of the second waste liquid cavity through the first gas pipeline. The first cleaning liquid is released, and after the DNA on the silica gel film is cleaned, the DNA enters the first waste liquid cavity under the action of the Coriolis force;

maintaining the counter-clockwise 2000rpm, heating the temperature of the outer temperature control area to 80 ℃, opening the second paraffin valve, releasing the second cleaning liquid, cleaning the DNA on the silica gel film, and then entering the first waste liquid cavity under the action of the Coriolis force;

the centrifugal speed is changed to clockwise rotation, and the speed is 500rpm clockwise;

the temperature of the inner circular temperature zone is increased to 70 ℃, paraffin of an aluminum foil container in an eluent preset cavity is melted, the eluent is released and flows into a converging cavity, a plurality of layers of silica gel films are preset on the eluent, and the eluent can not continue to flow downwards due to the existence of a second capillary valve at 500rpm, so that the eluent can soak the silica gel films, and the eluent is maintained at 500rpm clockwise for 5min;

the centrifugal speed is increased to 2000rpm clockwise, and the eluent after the silica gel film is eluted flows into the second buffer cavity under the action of the Coriolis force; it is noted that the dispensing chamber is present to increase the time that coriolis forces are applied to the liquid, thereby making the liquid more reliable in making channel changes, including left or right switching;

The centrifugal speed is reduced to 200rpm clockwise, the centrifugal speed is kept for 30 seconds, the centrifugal speed is accelerated to 1000rpm again, the eluent after the silica gel film is eluted breaks through a fourth siphon pipeline, enters a sample distribution pipeline and fills each measuring cavity one by one, and the excessive eluent enters a second waste liquid cavity;

the centrifugal speed is increased to 3000rpm clockwise, the liquid in each measuring cavity flows into each PCR amplification cavity respectively, and the reagent dry powder preset in the PCR amplification cavity is dissolved, and the special points are that paraffin is preset in the PCR amplification cavity, and the paraffin is melted and seals the PCR amplification cavity in the PCR amplification, so that the cross contamination caused by the escape of aerosol in the amplification of 8 amplification cavities is prevented;

the centrifugation speed is reduced to 500rpm, and PCR temperature cycle starts to amplify;

the PCR reaction sequence is started at 95 ℃ for 3min, denatured at 95 ℃ for 15s, annealed and extended at 60 ℃ for 40s, 40 temperature cycles are performed, and fluorescent signals are collected one by one in the 30 th s at 60 ℃.

In various embodiments, microfluidic chip materials include, but are not limited to PMMA, PDMS, PC, ABS, COC, COP, and the like. The packaging mode of the microfluidic chip includes, but is not limited to, hot pressing, ultrasonic welding, laser welding, gluing and the like.

In one embodiment, a full-extraction molecular diagnostic microfluidic chip having a target center of rotation and located outside the top of the full-extraction molecular diagnostic microfluidic chip, the full-extraction molecular diagnostic microfluidic chip comprising a storage chamber 102, a sample chamber 105, a first addition chamber 111, a first buffer chamber 113, a first controlled addition chamber 116, a second controlled addition chamber 117, a pooling chamber 124, a second addition chamber 110, a distribution chamber 126, a first waste chamber 132, a buffer chamber 129, a second waste chamber 139, at least one measurement chamber 134, and at least one PCR amplification chamber 135 is shown in fig. 1; referring to fig. 3, 4 and 5, the arrangement of the target rotation centers in order from small to large is as follows: a storage chamber 102, a sample chamber 105, a first buffer chamber 113, a first controlled addition chamber 116, a second controlled addition chamber 117, a pooling chamber 124, a dispensing chamber 126, a first waste chamber 132, a measurement chamber 134, a PCR amplification chamber 135; the distance between the first liquid adding cavity 111 and the target rotation center is greater than the distance between the storage cavity 102 and the target rotation center and less than the distance between the first buffer cavity 113 and the target rotation center; the distance between the second liquid adding cavity 110 and the target rotation center is greater than the distance between the first buffer cavity 113 and the target rotation center and less than the distance between the converging cavity 124 and the target rotation center; the distance between the buffer chamber 129 and the target rotation center is greater than or equal to the distance between the first waste liquid chamber 132 and the target rotation center and less than the distance between the measuring chamber 134 and the target rotation center; the distance between the second waste liquid chamber 139 and the target rotation center is greater than the distance between the buffer chamber 129 and the target rotation center; a first sample adding hole 101 is formed in the storage cavity 102 near the target rotation center, and the storage cavity 102 is communicated with a sample cavity 105 through a first capillary valve 103; a second sample adding hole 104 is formed in the position, close to the target rotation center, of the sample cavity 105, and the sample cavity 105 is communicated with a first buffer cavity 113 through a first siphon pipeline 107; a first liquid adding hole 109 is formed in the first liquid adding cavity 111 near the target rotation center, and the first liquid adding cavity 111 is communicated with a first buffer cavity 113 through a second siphon pipeline 108; a first buffer hole 112 is arranged in the first buffer cavity 113 near the target rotation center, and the first buffer cavity 113 is communicated with the converging cavity 124 through a third siphon pipeline 114; a second liquid adding hole 122 is formed in the second liquid adding cavity 110 near the target rotation center, and the second liquid adding cavity 110 is communicated with a converging cavity 124 through an elution pipeline 123; the first controlled filling cavity 116 is communicated with the second waste liquid cavity 139 through the first gas pipeline 118 near the target rotation center, a first paraffin valve 137 is arranged in the first gas pipeline 118, and the first controlled filling cavity 116 is communicated with the converging cavity 124 through the first filling pipeline 120; the second controlled filling cavity 117 is communicated with the second waste liquid cavity 139 through a second gas pipeline 119 near the target rotation center, a second paraffin valve 136 is arranged in the second gas pipeline 119, and the second controlled filling cavity 117 is communicated with the converging cavity 124 through a second filling pipeline 121; the converging cavity 124 is communicated with the distributing cavity 126 through the second capillary valve 125, and the total extraction molecular diagnosis microfluidic chip is provided with a filtering structure in the converging cavity 124; wherein the first buffer cavity 113 is communicated with the collecting pipeline 115 through a third siphon pipeline 114, the second liquid adding cavity 110 is communicated with the collecting pipeline 115 through an elution pipeline 123, the first controlled liquid adding cavity 116 is communicated with the collecting pipeline 115 through a first liquid adding pipeline 120, and the second controlled liquid adding cavity 117 is communicated with the collecting pipeline 115 through a second liquid adding pipeline 121; the converging duct 115 communicates with the converging chamber 124. One end of the distribution chamber 126, which is far away from the target rotation center, is communicated with a first waste liquid chamber 132 through a waste liquid pipeline 127, the other end of the distribution chamber is communicated with a second buffer chamber 129 through a collection pipeline 128, and the waste liquid pipeline 127 and the collection pipeline 128 are respectively positioned at two sides of the distribution chamber 126; the first waste liquid cavity 132 is provided with a waste liquid vent hole 131 near the target rotation center; the second buffer chamber 129 communicates with the sample dispensing conduit 133 through the fourth siphon conduit 130; the sample distribution pipe 133 is sequentially communicated with each measuring cavity 134, and the tail end of the sample distribution pipe 133 is communicated with the second waste liquid cavity 139 through the liquid discharge pipe 106; the number of the measuring cavities 134 is the same as that of the PCR amplification cavities 135 and the measuring cavities are correspondingly arranged, and each measuring cavity 134 is communicated with one PCR amplification cavity 135; in addition, the fully extracted molecular diagnosis microfluidic chip further comprises reagent conveying pipelines 140, the number of the measuring cavities 134 is the same as that of the reagent conveying pipelines 140 and the measuring cavities are correspondingly arranged, and each measuring cavity 134 is communicated with a corresponding PCR amplification cavity 135 through a corresponding reagent conveying pipeline 140. The second waste liquid chamber 139 is provided with an air outlet 138 near the target rotation center. It will be appreciated that in the embodiment shown in fig. 1, each chamber is closed, and has a structure similar to a lid to close the chambers and the pipes thereof, and only some holes are left, as shown in fig. 2, the first loading hole 101, the second loading hole 104, the first filling hole 109, the first buffer hole 112, the second filling hole 122, the waste air vent 131 and the air outlet 138 respectively pass through the same side of the body and are connected to the external environment.

As shown in fig. 1, the body is provided with a part of fan-shaped structure, and the target rotation center is positioned at the corresponding circle center of the part of fan-shaped structure; or the target rotation center is positioned between the circle center corresponding to the sector structure and the top of the body.

To enhance the siphoning effect, in one embodiment, each siphon line, including the first siphon line, the second siphon line, the third siphon line, and the fourth siphon line, has a hydrophilic layer or is hydrophilized on an inner surface thereof. As shown in fig. 1, the fourth siphon pipe 130 has a liquid inlet section 231, an ascending section 232, a top transition section 233 and a descending section 234, wherein the liquid inlet section 231 is used for inputting liquid, and the ascending section 232, the top transition section 233 and the descending section 234 cooperate to form a structure similar to a n shape or a n shape so as to form a siphon pipe and function as a siphon valve; in the design of the siphon structure, when the centrifugal force is small (low-speed centrifugal force) or no centrifugal force (rotation stop), the liquid in the cavity close to the target rotation center is pulled by capillary force to be in the position closest to the target rotation center, which is not close to the siphon pipeline with the siphon effect, until the siphon pipeline is full of the liquid; the centrifugal speed is then increased, and under the action of centrifugal force, a siphon flow occurs inside the siphon pipe, and the liquid entirely flows into the chamber located later away from the target rotation center.

In one embodiment of the application, a microfluidic system comprises the whole extraction molecule diagnostic microfluidic chip of any of the embodiments. The microfluidic system integrates all processes of sample splitting, nucleic acid purification, equivalent distribution of reagents, amplification of multi-chamber nucleic acid under the restraint of specific primers and the like into one centrifugal microfluidic chip, and can realize subsequent analysis work such as fluorescent signal acquisition and analysis, so that a single-sample multi-index diagnosis mode is realized, and possibility is provided for molecular diagnosis to realize multi-pathogen screening facing diseases; in addition, the whole reaction process is in a sealed micro-fluidic chip, so that the burden of operators and the possibility of pollution are reduced, the whole molecular diagnosis process is not dependent on a molecular diagnosis laboratory any more, and is not dependent on professional operators any more, the requirement of rapid detection at any time and any place is met, and great help is brought to medical examination and disease prevention and control.

The specific application of the microfluidic system or the full-extraction molecular diagnosis microfluidic chip is continuously provided below, and the mastitis is one of the most common diseases of the dairy cows and is also one of the diseases with the greatest harm to the dairy cow breeding industry. Mastitis refers to an inflammatory pathological change of dairy cows caused by stimulation of physical, chemical, microorganism and the like, and is characterized in that the physical and chemical properties of the milk change, the number of white blood cells increases, and the pathology of mammary gland tissues changes. Mastitis is classified into clinical type mastitis and recessive type mastitis. The clinical incidence rate of the mastitis of the Chinese cows is 33.41 percent (9.7 to 55.6 percent), the average positive rate of the recessive mastitis is 73.91 percent, and the incidence rate of the breast is 44.74 percent. Not only does the cow mastitis cause the milk yield of the cow to be reduced and the milk quality to be changed, but also the cow mastitis can cause suppuration and gangrene in a milk area and lose the lactation capacity, thereby causing huge economic loss. Meanwhile, as a large amount of antibiotics medicines are clinically applied, the risk of medicine residues exists, and the quality of cow milk is affected. Therefore, the prevention and treatment of cow mastitis is increasingly paid attention to by livestock workers. The bovine mastitis detection microfluidic consumable is matched with a full-automatic nucleic acid analysis system, and can provide detection results of up to 16 indexes for a single milk sample in about 70 minutes (because 8 PCR amplification cavities exist on a microfluidic chip, only two fluorescent channels are needed for 16 indexes).

The treatment procedure for the reagent for bovine mastitis detection is described below:

20. Mu.L of proteinase K (20 mg/ml) was added to the reservoir;

200. Mu.L of milk sample was added to the sample chamber;

200. Mu.L of Lysis Buffer was added as a lysate to the sample chamber;

200 mu L of absolute ethyl alcohol is added into the first liquid adding cavity as a neutralizing liquid;

500 mu L of Wash Buffer is added into the first controlled liquid adding cavity as a first cleaning liquid;

500 mu L of Wash Buffer is added into the second controlled liquid adding cavity as a second cleaning liquid;

100 mu L of the solution Buffer is added into the second liquid adding cavity as eluent;

each group of PCR amplification materials are respectively added into each PCR amplification cavity;

the time of each step is determined according to the traditional bovine mastitis detection mode, the above related embodiments are adopted and centrifugal control and temperature control are carried out, the application is simple and convenient, the requirement of rapid detection at any time and any place is met, the whole reaction process is in a closed microfluidic chip, and the burden of operators and the possibility of pollution are reduced.

It should be noted that other embodiments of the present application further include a micro-fluidic chip and a micro-fluidic system for full-extraction molecular diagnosis, which are formed by combining the technical features of the above embodiments.

The technical features of the above embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The foregoing examples illustrate only a few embodiments of the application, which are described in detail and are not to be construed as limiting the scope of the claims. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of the application should be determined from the following claims.

Claims

1. The full-extraction molecular diagnosis microfluidic chip is provided with a target rotation center and is characterized by comprising a storage cavity, a sample cavity, a first liquid adding cavity, a first buffer cavity, at least one controlled liquid adding cavity, a second liquid adding cavity, a distribution cavity, a first waste liquid cavity, a buffer cavity, a second waste liquid cavity, at least one measuring cavity and at least one PCR amplification cavity;

the controlled liquid adding cavity is communicated with the second waste liquid cavity through a gas pipeline near the target rotation center, a phase change valve is arranged in the gas pipeline, the controlled liquid adding cavity is communicated with the distribution cavity through the liquid adding pipeline, and the phase change valve is used for generating phase change through temperature control so as to conduct the gas pipeline;

an air outlet hole is arranged at the position, close to the target rotation center, of the second waste liquid cavity;

the full extraction molecular diagnosis microfluidic chip comprises two controlled liquid adding cavities, namely a first controlled liquid adding cavity and a second controlled liquid adding cavity;

The second controlled liquid adding cavity is communicated with the second waste liquid cavity through a second gas pipeline near the target rotation center, a second paraffin valve is arranged in the second gas pipeline, and the second controlled liquid adding cavity is communicated with the distribution cavity through the second liquid adding pipeline;

the full-extraction molecular diagnosis microfluidic chip further comprises a converging cavity;

the converging cavity is communicated with the distributing cavity through a second capillary valve;

a filter membrane is accommodated in the converging cavity as the filter structure;

the second controlled liquid adding cavity is communicated with the second waste liquid cavity through a second gas pipeline near the target rotation center, a second paraffin valve is arranged in the second gas pipeline, and the second controlled liquid adding cavity is communicated with the converging cavity through the second liquid adding pipeline.

2. The fully extracted molecular diagnostic microfluidic chip of claim 1, wherein the first buffer chamber is connected to the collection conduit via a third siphon conduit, the second liquid adding chamber is connected to the collection conduit via an elution conduit, the controlled liquid adding chamber is connected to the collection conduit via a liquid adding conduit, and the collection conduit is connected to the collection chamber.

3. The total extracted molecule diagnostic microfluidic chip of claim 2, wherein a filter membrane is accommodated in the converging chamber as the filter structure.

4. The total extracted molecular diagnostic microfluidic chip of claim 3, wherein said filter membrane is a laminated multilayer silica gel membrane.

5. The fully extracted molecular diagnostic microfluidic chip of claim 1, wherein the first buffer chamber is connected to the collection conduit via a third siphon conduit, the second liquid-feeding chamber is connected to the collection conduit via an elution conduit, the first controlled liquid-feeding chamber is connected to the collection conduit via a first liquid-feeding conduit, and the second controlled liquid-feeding chamber is connected to the collection conduit via a second liquid-feeding conduit; the converging pipeline is communicated with the converging cavity.

6. The fully extracted molecular diagnostic microfluidic chip of claim 1, further comprising reagent delivery conduits, the number of measurement chambers being the same as and corresponding to the number of reagent delivery conduits, each measurement chamber communicating with a corresponding one of the PCR amplification chambers via a corresponding one of the reagent delivery conduits; alternatively, the end of the sample distribution conduit communicates with the second waste chamber through a drain conduit.

7. The all-extracted-molecule-diagnostic microfluidic chip according to any one of claims 1 to 6, wherein the all-extracted-molecule-diagnostic microfluidic chip has a body provided with a part or all of a storage chamber, a first capillary valve, a sample chamber, a drain chamber, a first siphon conduit, a second siphon conduit, a first liquid-adding chamber, a second liquid-adding chamber, a first buffer chamber, a third siphon conduit, a pooling conduit, a first controlled liquid-adding chamber, a second controlled liquid-adding chamber, a first gas conduit, a second gas conduit, a first liquid-adding conduit, a second liquid-adding conduit, an elution conduit, a pooling chamber, a second capillary valve, a dispensing chamber, a waste conduit, a collection conduit, a second buffer chamber, a fourth siphon conduit, a first waste chamber, a sample dispensing conduit, measurement chambers, PCR amplification chambers, a second waste chamber, and a reagent delivery conduit; the first sample adding hole, the second sample adding hole, the first liquid adding hole, the first buffer hole, the second liquid adding hole, the waste liquid vent hole and the air outlet hole respectively penetrate through the body and are communicated with the external environment;

A magnetic piece or a grinding piece is arranged in the sample cavity;

paraffin is preset in the PCR amplification cavity.

8. The fully extracted molecular diagnostic microfluidic chip of claim 7, wherein the first sample addition well, the second sample addition well, the first liquid addition well, the first buffer well, the second liquid addition well, the waste liquid vent hole, and the air outlet hole respectively pass through the same side of the body and are in communication with an external environment.

9. A microfluidic system comprising a full extraction molecular diagnostic microfluidic chip according to any one of claims 1 to 8.