CN110142066B

CN110142066B - Microfluidic chip and analysis system

Info

Publication number: CN110142066B
Application number: CN201910339790.9A
Authority: CN
Inventors: 汤明辉
Original assignee: Shenzhen Gangzhu Medical Technology Co ltd
Current assignee: Shenzhen Chenghui Medical Technology Co ltd
Priority date: 2019-04-25
Filing date: 2019-04-25
Publication date: 2021-09-03
Anticipated expiration: 2039-04-25
Also published as: CN110142066A

Abstract

The microfluidic chip comprises a sample adding hole, a sample adding cavity, a gas outlet, a sample enrichment cavity, a waste liquid cavity, a dilution cracking cavity, a siphon pipeline, a sample adding cavity circulating pipeline, a cracking cavity circulating pipeline, a gas circulating pipeline, a sample output pipeline, a reagent distributing pipeline, a gas outlet pipeline and a PCR amplification cavity; the dilution cracking cavity is sequentially communicated with each PCR amplification cavity and the second waste liquid cavity through siphon pipelines; the gas outlet is also communicated with a waste liquid cavity through a gas outlet pipeline, and the position where the waste liquid cavity is communicated with the gas outlet is farther away from the rotating center than the gas outlet. Enrichment, cracking, dilution after cracking, equal distribution and PCR amplification of multiple cavities of samples are sequentially realized, the function of nucleic acid purification-free molecular diagnosis can be realized, on one hand, the position of the air outlet can be adjusted according to requirements, on the other hand, the relative relation between capillary force and centrifugal force is skillfully utilized by designing a siphon pipeline, and a regulating valve is formed to control liquid to flow into a reagent distribution pipeline.

Description

Microfluidic chip and analysis system

Technical Field

The application relates to the field of centrifugal microfluidics, in particular to a microfluidic chip and an analysis system.

Background

Microfluidics (Microfluidics) refers to the manipulation of liquids on a sub-millimeter scale, which is typically several microns to several hundred microns. Microfluidic technology integrates the functions of the entire laboratory, including sampling, dilution, reaction, separation, detection, etc., into a single small Chip, so it is also called Lab-on-a-Chip. The chip generally comprises various liquid storage tanks and a micro-channel network which is connected with each other, can shorten the sample processing time, and realizes the maximum utilization efficiency of reagent consumables by precisely controlling the liquid flow. Microfluidic systems refer to devices that manipulate liquids on a sub-millimeter scale. Centrifugal microfluidics belongs to a branch of microfluidics, and particularly relates to a method for driving liquid to flow by rotating a centrifugal microfluidic chip, so that the liquid is controlled on a sub-millimeter scale by using centrifugal force. Centrifugal microfluidics integrates basic operational units involved in biological and chemical fields into a small disc-shaped (disc-shaped) chip. In addition to the advantages specific to microfluidics, the overall device is more compact since only one motor is required for centrifugal microfluidics to provide the force required for liquid manipulation. And the ubiquitous centrifugal field on the disc chip can not only make liquid drive more effective and ensure that no liquid remains in the pipeline, but also can effectively realize sample separation based on density difference and make parallel processing simpler. Therefore, centrifugal microfluidics is also increasingly used in point-of-care diagnostics.

Molecular diagnostics based on PCR amplification generally comprise the following steps: sample cracking, nucleic acid purification, nucleic acid amplification under the constraint of specific primers, and fluorescent signal acquisition and analysis. In some molecular diagnostic projects, because the sample is relatively simple, amplification can be performed directly after sample lysis; on the other hand, the emergence of the one-step DNA extraction amplification kit which is mature day by day also makes direct amplification of the sample after lysis possible, and avoids the complex step of nucleic acid purification. However, in the molecular diagnostic system based on PCR amplification, a partitioned laboratory is generally constructed because aerosol contamination occurs during PCR amplification and cross-contamination between samples is avoided. The laboratory needs to realize the partition operation of sample treatment, nucleic acid extraction and PCR amplification, and has a good ventilation system, the building cost of the laboratory is high, and the laboratory is often built only by large-scale medical institutions. On the other hand, laboratory operators need to be certified and on duty, and labor cost is greatly increased. Meanwhile, too much manual intervention will bring about manual operation errors. These problems greatly raise the technical use threshold for PCR-based molecular diagnostics.

In addition, the current molecular diagnosis laboratory mode finishes operations of multiple samples and multiple detection projects in a centralized experiment field, and the requirement on process quality control is high. In addition, the current molecular diagnosis laboratory mode is generally a multi-sample single-index detection mode, the detection indexes are limited, and the screening of single-sample multi-index infectious pathogens cannot be realized. In addition, although the advantages of molecular diagnosis technology are obvious, the steps are complicated, the process is time-consuming, professional operation is required, and the construction cost of a clinical molecular diagnosis laboratory is generally high, so that the molecular diagnosis is expensive.

Disclosure of Invention

In view of the above, there is a need for a microfluidic chip and an analysis system.

A micro-fluidic chip is provided with a rotation center and comprises a chip substrate, and a sample adding hole, a sample adding cavity, a gas outlet, a sample enrichment cavity, a waste liquid cavity, a dilution cracking cavity, a siphon pipeline, a sample adding cavity circulating pipeline, a cracking cavity circulating pipeline, a gas circulating pipeline, a sample output pipeline, a reagent distributing pipeline, a gas outlet pipeline and a plurality of PCR amplification cavities which are arranged in the chip substrate; the waste liquid cavity comprises a first waste liquid cavity and a second waste liquid cavity; the sample adding hole is respectively communicated with the outside and the sample adding cavity, and the sample adding cavity is communicated with the sample enrichment cavity through a sample adding cavity circulation pipeline; the sample enrichment cavity is communicated with the first waste liquid cavity through a cracking cavity circulation pipeline, and the bottom position of the sample enrichment cavity, which is far away from the rotation center, is communicated with the dilution cracking cavity through a sample output pipeline; the dilution and cracking cavity is sequentially communicated with each PCR amplification cavity and the second waste liquid cavity through a siphon pipeline and a reagent distribution pipeline; the first waste liquid cavity is communicated with the second waste liquid cavity through a gas circulation pipeline; the gas outlet communicates with the outside, and the gas outlet still communicates a waste liquid chamber through the pipeline of giving vent to anger, and the position that is linked together with the gas outlet in waste liquid chamber is more far away from the rotation center than the gas outlet. The microfluidic chip is suitable for centrifugal microfluidic analysis, enrichment, cracking, dilution after cracking, equal distribution and PCR amplification of multiple chambers can be sequentially realized, a nucleic acid purification-free molecular diagnosis function can be realized, on one hand, the gas outlet is communicated with one waste liquid chamber and is communicated with the other waste liquid chamber through the waste liquid chamber, the position of the gas outlet can be adjusted according to requirements, on the other hand, the relative relation between capillary force and centrifugal force is ingeniously utilized through designing a siphon pipeline, a regulating valve is formed to control liquid to flow into a reagent distribution pipeline, the PCR amplification technology is applied to the centrifugal microfluidic technology to realize molecular diagnosis based on PCR amplification, the whole reaction process is in the closed microfluidic chip, and the molecular diagnosis effect of rapid detection at any time and any place is realized.

In one embodiment, the chip substrate is provided with a positioning region. In one embodiment, the air outlet is communicated with the second waste liquid cavity through an air outlet pipeline. In one embodiment, the air outlet is communicated with the first waste liquid cavity through an air outlet pipeline. In one embodiment, the siphon pipe has a bent structure. Further, the bending structure is

A glyph configuration or an arch configuration. In one embodiment, the minimum distance between the bending structure and the rotation center is smaller than or equal to the minimum distance between the dilution and lysis chamber and the rotation center. In one embodiment, the chip substrate is made of a hydrophilic material. In one embodiment, the inner surface of the siphon pipe is provided with a hydrophilic material layer or the inner surface is subjected to hydrophilic treatment. In one embodiment, the sample output pipeline is provided with a first phase change valve, the chip substrate is provided with a first packaging hole for communicating the outside with the first phase change valve, and the microfluidic chip is provided with a first sealing cover part at the first packaging hole; the sample enrichment cavity is provided with a top position close to the rotation center, the top position is provided with a vent hole, the gas outlet is communicated with the second waste liquid cavity through a gas outlet pipeline, the gas circulation pipeline is provided with a second phase change valve, the chip substrate is provided with a second packaging hole communicated with the second phase change valve, and the microfluidic chip is provided with a second sealing cover part at the second packaging hole; the micro-fluidic chip structure is also provided with a plurality of measuring cavities, each measuring cavity is arranged in one-to-one correspondence with each PCR amplification cavity, each measuring cavity is arranged between a reagent distribution pipeline and one PCR amplification cavity, and the reagent distribution pipeline is respectively communicated with each PCR amplification cavity through each measuring cavity; the chip substrate has a partial fan-shaped structure; the chip substrate is provided with a positioning area which is arranged between the gas outlet and the second waste liquid cavity; the positioning areas are positioning convex parts, positioning holes or positioning grooves, and the number of the positioning areas is one, two or more; the positioning groove is in a linear shape or an arc shape, and the circle center of the arc line of the positioning groove is coincided with the rotation center of the microfluidic chip; the number of the positioning holes is multiple and is uniformly distributed relative to the rotation center of the microfluidic chip; the center of rotation being located on the chip baseThe external part of the body is arranged from small to large according to the distance from the rotating center: the device comprises a sample adding cavity, a sample enriching cavity, a first waste liquid cavity, a dilution cracking cavity, a reagent distributing pipeline, a second waste liquid cavity and a PCR amplification cavity; a liquid storage container is arranged in the dilution cracking cavity and used for containing diluent; the PCR amplification cavity is filled with PCR reaction reagent dry powder, fluorescent dye and a sealing body.

An analysis system comprising any one of the microfluidic chips.

Drawings

Fig. 1 is a schematic structural diagram according to an embodiment of the present application. Fig. 2 is an enlarged schematic view of the embodiment shown in fig. 1 at a. Fig. 3 is another schematic view of the embodiment shown in fig. 1. Fig. 4 is another schematic view of the embodiment shown in fig. 1. Fig. 5 is another schematic view of the embodiment shown in fig. 1. Fig. 6 is another schematic view of the embodiment shown in fig. 1. Fig. 7 is an enlarged schematic view of the embodiment shown in fig. 6 at B. Fig. 8 is another schematic view of the embodiment of fig. 1. Fig. 9 is a schematic structural diagram of another embodiment of the present application. Fig. 10 is another schematic view of the embodiment of fig. 9. Fig. 11 is another schematic view of the embodiment of fig. 9. Fig. 12 is a schematic structural diagram of another embodiment of the present application. Fig. 13 is another schematic view of the embodiment of fig. 12. Fig. 14 is another schematic view of the embodiment of fig. 12. Fig. 15 is another schematic view of the embodiment of fig. 12.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present application more comprehensible, embodiments accompanying the present application are described in detail below with reference to the accompanying drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. This application is capable of embodiments in many different forms than those described herein and that modifications may be made by one skilled in the art without departing from the spirit and scope of the application and it is therefore not intended to be limited to the specific embodiments disclosed below.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Although the traditional molecular diagnosis technology has obvious advantages, the steps are complicated, the process is time-consuming, the operation of professional personnel is needed, and the construction cost of a clinical molecular diagnosis laboratory is generally high, so the molecular diagnosis is expensive. In one embodiment of the present application, a microfluidic chip having a rotation center includes a chip substrate, and a sample application hole, a sample application cavity, a gas outlet, a sample enrichment cavity, a waste liquid cavity, a dilution cleavage cavity, a siphon pipeline, a sample application cavity circulation pipeline, a cleavage cavity circulation pipeline, a gas circulation pipeline, a sample output pipeline, a reagent distribution pipeline, a gas outlet pipeline, and a plurality of PCR amplification cavities disposed in the chip substrate; the waste liquid cavity comprises a first waste liquid cavity and a second waste liquid cavity; the sample adding hole is respectively communicated with the outside and the sample adding cavity, and the sample adding cavity is communicated with the sample enrichment cavity through a sample adding cavity circulation pipeline; the sample enrichment cavity is communicated with the first waste liquid cavity through a cracking cavity circulation pipeline, and the bottom position of the sample enrichment cavity, which is far away from the rotation center, is communicated with the dilution cracking cavity through a sample output pipeline; the dilution and cracking cavity is sequentially communicated with each PCR amplification cavity and the second waste liquid cavity through a siphon pipeline and a reagent distribution pipeline; the first waste liquid cavity is communicated with the second waste liquid cavity through a gas circulation pipeline; the gas outlet communicates with the outside, and the gas outlet still communicates a waste liquid chamber through the pipeline of giving vent to anger, and the position that is linked together with the gas outlet in waste liquid chamber is more far away from the rotation center than the gas outlet. The microfluidic chip is suitable for centrifugal microfluidic analysis, enrichment, cracking, dilution after cracking, equal distribution and PCR amplification of multiple chambers can be sequentially realized, a nucleic acid purification-free molecular diagnosis function can be realized, on one hand, the gas outlet is communicated with one waste liquid chamber and is communicated with the other waste liquid chamber through the waste liquid chamber, the position of the gas outlet can be adjusted according to requirements, on the other hand, the relative relation between capillary force and centrifugal force is ingeniously utilized through designing a siphon pipeline, a regulating valve is formed to control liquid to flow into a reagent distribution pipeline, the PCR amplification technology is applied to the centrifugal microfluidic technology to realize molecular diagnosis based on PCR amplification, the whole reaction process is in the closed microfluidic chip, and the molecular diagnosis effect of rapid detection at any time and any place is realized.

In one embodiment, a microfluidic chip includes a part or all of the following embodiments; that is, the microfluidic chip includes some or all of the following technical features. In one embodiment, the microfluidic chip comprises a chip substrate, and a sample adding hole, a sample adding cavity, a gas outlet, a sample enrichment cavity, a waste liquid cavity, a dilution cracking cavity, a siphon pipeline, a sample adding cavity circulation pipeline, a cracking cavity circulation pipeline, a gas circulation pipeline, a sample output pipeline, a reagent distribution pipeline, a gas outlet pipeline and a plurality of PCR amplification cavities which are arranged in the chip substrate; it can be understood that the shapes and sizes of the sample adding hole, the sample adding cavity, the gas outlet, the sample enrichment cavity, the waste liquid cavity, the dilution cracking cavity, the siphon pipeline, the sample adding cavity circulating pipeline, the cracking cavity circulating pipeline, the gas circulating pipeline, the sample output pipeline, the reagent distribution pipeline, the gas outlet pipeline, the PCR amplification cavity and the like can be designed according to actual requirements. Furthermore, the sample adding cavity, the sample enriching cavity, the waste liquid cavity, the dilution cracking cavity, the siphon pipeline, the sample adding cavity circulating pipeline, the cracking cavity circulating pipeline, the gas circulating pipeline, the sample output pipeline, the reagent distribution pipeline, the gas outlet pipeline and the plurality of PCR amplification cavities are arranged in the chip substrate and are communicated with the outside, namely the external environment, only through the sample adding hole and the gas outlet or other structures such as the packaging hole.

In one embodiment, the microfluidic chip has a center of rotation and the center of rotation is located outside the chip substrate; in one embodiment, the distances from the rotation center are arranged in order from small to large: the device comprises a sample adding cavity, a sample enriching cavity, a first waste liquid cavity, a dilution cracking cavity, a reagent distributing pipeline, a second waste liquid cavity and a PCR amplification cavity; (ii) a The sample adding cavity, the sample enrichment cavity, the first waste liquid cavity, the dilution and cracking cavity and the reagent distribution pipeline are sequentially arranged at the top of the sample adding cavity, namely the position of the sample adding cavity closest to the rotation center, namely the position of the gas outlet is closer to the sample adding cavity relative to the first waste liquid cavity according to the centrifugal direction; further, the minimum distance from the rotation center is arranged in order from small to large: the device comprises a sample adding cavity, a sample enriching cavity, a first waste liquid cavity, a dilution cracking cavity, a second waste liquid cavity, a reagent distributing pipeline and a second waste liquid cavity; the minimum distance between the sample adding hole of each PCR amplification cavity and the rotation center is equal to or less than the minimum distance between the second waste liquid cavity and the rotation center. In one embodiment, the distance between the liquid level in the dilution cracking cavity and the rotation center is larger than the minimum distance between the siphon pipe and the rotation center; namely, the minimum distance between the siphon pipeline and the rotation center is less than or equal to the distance between the liquid level in the dilution cracking cavity and the rotation center. In various embodiments, the dilute lysis chamber communicates with the siphon channel at a bottom location thereof remote from the center of rotation. Thus, when centrifugal operation is carried out, a sample in the sample adding cavity enters the sample enrichment cavity through the sample adding cavity circulating pipeline, supernatant on the upper part of the sample enrichment cavity enters the first waste liquid cavity through the cracking cavity circulating pipeline, the sample on the lower part of the sample enrichment cavity enters the sample output pipeline after being enriched, then the sample enters the dilution cracking cavity, the diluted cracking cavity is diluted and then sequentially enters each PCR amplification cavity through the siphon pipeline and the reagent distribution pipeline, and redundant parts enter the second waste liquid cavity. In one embodiment, a liquid storage container is arranged in the dilution and lysis cavity; the liquid storage container is used for containing diluent. Alternatively, the dilute lysis chamber is used to contain a diluent. Thus, PCR amplification can be used directly for molecular diagnosis without the need for a diluent. Alternatively, in one embodiment, the dilute lysis chamber is provided with a liquid injection port for injecting a diluent. In one embodiment, the liquid injection hole is a one-way hole. Alternatively, in one embodiment, the diluting lysis chamber is provided with a filling cap closing the filling hole. Furthermore, a diluent is arranged in the liquid storage container. In one embodiment, the reservoir is provided with an opening closed with a heat-fusible layer. In one embodiment, the reservoir is affixed to the dilution lysis chamber. In one embodiment, the reservoir has an aluminum foil layer. In one embodiment, the liquid storage container is provided with an opening, a puncturing piece, an elastic piece and a sealing membrane, wherein the sealing membrane is used for sealing the opening, one end of the elastic piece is connected with the puncturing piece, the other end of the elastic piece is fixed in the dilution and lysis cavity, and the puncturing piece is used for being matched with the elastic piece to generate displacement to puncture the sealing membrane during centrifugation. Furthermore, a diluent is arranged in the dilution cracking cavity. In one embodiment, the diluent is disposed in a heat-fusible wrapping disposed in the dilute lysis chamber. In one embodiment, the diluent is disposed in a casing disposed in the dilute lysis chamber and the casing is provided with an opening closed with a hot melt layer.

In one embodiment, the chip substrate is made of a hydrophilic material, or the inner surface of the flow channel of the lysis chamber is provided with a hydrophilic material layer or is subjected to hydrophilic treatment. In one embodiment, the inner surface of the siphon pipe is provided with a hydrophilic material layer or the inner surface is subjected to hydrophilic treatment. In one embodiment, the chip substrate is a PMMA, PDMS, PC, ABS, COC or COP product. In one embodiment, the chip substrate has a partial fan-shaped structure. In one embodiment, the partial sector shape includes a sector ring shape and a fan blade shape or partial sector shape structure has three straight sides. The design is favorable for the regular arrangement of the plurality of micro-fluidic chips to form a structure similar to a circular ring, thereby reasonably utilizing the centrifugal action, improving the processing efficiency, and simultaneously carrying out centrifugal processing and PCR amplification on the plurality of micro-fluidic chips so as to complete the whole molecular diagnosis process. In one embodiment, the chip substrate is packaged by hot pressing, ultrasonic welding, laser welding or gluing; therefore, a relatively sealed micro-fluidic chip can be formed, and only the sample adding hole is communicated with the air outlet and the outside. Furthermore, the chip base body is provided with a base body part and a cover plate part, the cover plate part is packaged on the base body part in a hot pressing, ultrasonic welding, laser welding or gluing mode, and the sample adding cavity, the gas outlet, the sample enrichment cavity, the waste liquid cavity comprising the first waste liquid cavity and the second waste liquid cavity, the dilution cracking cavity, the gas outlet pipeline, the PCR amplification cavity and the like are all arranged on or in the base body part; furthermore, the sample-adding cavity circulation pipeline, the cracking cavity circulation pipeline, the gas circulation pipeline and the sample output pipeline are all arranged on the base body part; this allows the formation of a reusable centrifugal microfluidic chip. Further, the cover plate part is a hot-pressing film layer; this allows the formation of a reusable base portion. In one embodiment, the chip base body is provided with at least three fixing parts. The fixing part is used for positioning and fixing the chip matrix so as to position and fix the microfluidic chip. Further, the fixing portion includes a convex portion and/or a concave portion. Therefore, the microfluidic chip can be conveniently fixed, and operations such as centrifugation, PCR amplification, subsequent nucleic acid analysis and molecular diagnosis and the like can be conveniently carried out after sample addition. Moreover, the design is favorable for matching with a full-automatic nucleic acid analysis instrument, and can realize full automation of a molecular diagnosis project without a nucleic acid purification step.

In order to conveniently and quickly and accurately install the microfluidic chip during centrifugation, in one embodiment, the chip substrate is provided with a positioning area; the positioning area is used for positioning and mounting the microfluidic chip. In order to better realize the positioning function, one or more positioning structures can be designed, and in one embodiment, the number of the positioning areas is one, two or more; in one embodiment, the positioning area is a positioning hole and the number of the positioning holes is at least one. In one embodiment, the positioning area is a positioning protrusion and the number of positioning protrusions is at least one. In one embodiment, the number of the positioning holes is multiple and is uniformly distributed relative to the rotation center of the microfluidic chip. In one embodiment, the positioning area is a positioning slot. In one embodiment, the detents are linear. In one embodiment, the positioning slot is arcuate. In one embodiment, the center of the arc line coincides with the rotation center of the microfluidic chip. In one embodiment, the positioning area is disposed between the air outlet and the packaging hole.

In one embodiment, the waste liquid chamber comprises a first waste liquid chamber and a second waste liquid chamber; in one embodiment, the first waste liquid chamber is further away from the center of rotation than the second waste liquid chamber and/or the liquid inlet of the first waste liquid chamber is further away from the center of rotation than the liquid inlet of the second waste liquid chamber, i.e. the two waste liquid chambers are at different distances from the center of rotation. In one embodiment, the first waste liquid cavity is communicated with the second waste liquid cavity through a gas circulation pipeline; the design enables the first waste liquid cavity and the second waste liquid cavity to share one air outlet.

In one embodiment, the sample adding hole is respectively communicated with the outside and the sample adding cavity, and the sample adding cavity is communicated with the sample enrichment cavity through a sample adding cavity circulation pipeline; namely, the micro-fluidic chip is also provided with a sample adding hole which is communicated with the outside and the sample adding cavity in the chip matrix, and the sample adding cavity is communicated with the sample enrichment cavity through a sample adding cavity circulating pipeline; in one embodiment, a sample adding hole for communicating the outside and the sample adding cavity is arranged in the chip substrate, and the sample adding cavity is communicated with the sample enrichment cavity through a sample adding cavity circulation pipeline; in one embodiment, the well and the gas outlet are located at the same end of the chip substrate. In one embodiment, the sample well and the gas outlet are located at one end of the chip substrate near the rotation center during centrifugation. In one embodiment, the well and the gas outlet are located at the same end of the chip substrate.

In one embodiment, the sample enrichment cavity is communicated with the first waste liquid cavity through a cracking cavity circulation pipeline, and the sample enrichment cavity is also communicated with the dilution cracking cavity through a sample output pipeline at the bottom position far away from the rotation center; in one embodiment, the sample enrichment chamber and the lysis chamber flow conduit have a first connection position, the sample enrichment chamber and the sample output conduit have a second connection position, and the first connection position is less than the second connection position; in one embodiment, the second connection position is located at the bottom of the sample enrichment chamber, the first connection position is located at the middle or middle lower part of the sample enrichment chamber, during centrifugation, liquid in the sample enrichment chamber above the first connection position enters the first waste liquid chamber, and liquid in the sample enrichment chamber below the first connection position enters the dilution lysis chamber through the sample output pipeline after the first phase change valve is opened. The first attachment site determines the total volume of liquid that will participate in the subsequent reaction. Further, the first connection position is set according to the total volume of the target liquid participating in the reaction. In one embodiment, the sample enrichment cavity is provided with a filter membrane to separate the sample enrichment cavity, so that the supernatant and the enrichment liquid are better separated, and a better sample enrichment effect is achieved. Furthermore, an enrichment cavity and a cracking cavity are arranged in the sample enrichment cavity; the cracking cavity passes through the cracking cavityThe circulation pipeline is communicated with the first waste liquid cavity, and the enrichment cavity is communicated with the sample output pipeline. Further, the enrichment cavity is used for enriching the sample through centrifugation, and the cracking cavity is used for overflowing the supernatant obtained through centrifugation to the first waste liquid cavity through a flow pipeline of the cracking cavity. In one embodiment, the sample enrichment cavity is provided with an enrichment cavity and a lysis cavity which are separated by a filter membrane; the cracking cavity is communicated with the first waste liquid cavity through a cracking cavity circulation pipeline, and the enrichment cavity is communicated with the sample output pipeline. Further, the enrichment cavity is communicated with the sample output pipeline at the lower part or the bottom part of the enrichment cavity, in one embodiment, the enrichment cavity is communicated with the sample output pipeline at the middle part or the middle-lower part of the enrichment cavity, namely, a certain distance is reserved between the bottom of the enrichment cavity and the sample output pipeline, so that the sediment of cell residues can not block the sample output pipeline or enter the PCR amplification cavity through the sample output pipeline to inhibit the subsequent PCR reaction; furthermore, the bottom of the enrichment cavity is provided with a neck and a settling zone positioned below the neck, namely, the distance between the settling zone and the rotation center is greater than that between the neck and the rotation center, and the enrichment cavity is communicated with the sample output pipeline at a position above the neck. Wherein the neck has a narrowed design relative to other regions of the accumulation chamber. In one embodiment, the sample enrichment cavity is provided with an enrichment cavity and a lysis cavity which are separated by a filter membrane; the cracking cavity is communicated with the first waste liquid cavity through a cracking cavity circulation pipeline, the enrichment cavity is communicated with the sample output pipeline, and cracking liquid dry powder is arranged in the cracking cavity and/or paramagnetic substances are arranged in the enrichment cavity. In one embodiment, the cracking cavity flow pipeline is arranged in a bent mode; in one embodiment, the lysis chamber flow conduit has

A font structure. Furthermore, the distance between the top position of the circulation pipeline of the cracking cavity and the rotation center is smaller than the distance between the top position of the circulation pipeline of the sample enrichment cavity and the rotation center, and the design is that the liquid level of the liquid filled at one side of the circulation pipeline of the cracking cavity, which is close to the sample enrichment cavity, and the liquid level in the cavity of the sample enrichment cavity are on a centrifugal circumference during high-speed centrifugation, so that the control of a siphon valve is realizedAnd (5) preparing an effect. In one embodiment, the lysis chamber flow conduit is provided with a valve structure. In one embodiment, the valve structure is a siphon valve structure or a phase change valve structure; in one of the embodiments, the first and second electrodes are,

the font structure can reach siphon valve structure's effect. The design is favorable for realizing the control of the liquid of the sample enrichment cavity flowing out of the flow pipeline of the lysis cavity. According to the II-shaped structural design, when the centrifugal force is very small (low-speed centrifugation) or no centrifugal force is generated (the micro-fluidic chip stops rotating), liquid in the sample enrichment cavity is drawn by capillary force to submerge the cracking cavity circulation pipeline with the siphoning effect and is closest to the center of the centrifugal circle until the cracking cavity circulation pipeline is filled with liquid; then the centrifugal speed is increased, under the action of centrifugal force, siphon flow occurs in the inner part of the circulation pipeline of the cracking cavity, and all liquid flows into the first waste liquid cavity behind.

In one embodiment, the dilution and lysis chamber is communicated with the reagent distribution pipeline through a siphon pipeline, and is respectively communicated with each PCR amplification chamber and the second waste liquid chamber through the reagent distribution pipeline; namely, the dilution cracking cavity is communicated with each PCR amplification cavity and the second waste liquid cavity respectively through a siphon pipeline and a reagent distribution pipeline in sequence. Furthermore, the depth of the reagent conveying pipeline is shallower than that of the measurement cavity, the depth of the reagent conveying pipeline is shallower than that of the PCR amplification cavity, the depth of the reagent distribution pipeline is shallower than that of the reagent conveying pipeline, and the depth of the gas circulation pipeline, the depth of the gas outlet pipeline and the depth of the reagent distribution pipeline are shallower than that of the second waste liquid cavity. In one embodiment, the second waste liquid cavity is provided with an auxiliary air outlet communicated with the outside or a positioning column is convexly arranged. In one embodiment, the PCR amplification chambers are arranged side by side. In one embodiment, the number of the PCR amplification cavities is 8, the PCR amplification cavities are arranged side by side, and in one embodiment, the PCR amplification cavities have a cylindrical structure; in one embodiment, the reagent distribution pipelines are respectively communicated with each PCR amplification cavity at the same distance position relative to the rotation center. Furthermore, PCR reaction reagent dry powder is accommodated in the PCR amplification cavity. In one embodiment, the PCR amplification cavity contains PCR reaction reagent dry powder, fluorescent dye and a sealing body. In one embodiment, the seal is wax. In one embodiment, paraffin is also contained in the PCR amplification chamber. In one embodiment, the amount of paraffin wax in the PCR amplification chamber is used to match the mouth of the closed PCR amplification chamber. In one embodiment, the PCR amplification chamber also contains a fluorescent dye. In one embodiment, the dry PCR reagent powders in the PCR amplification chambers are arranged in the same or different ways. In one embodiment, the fluorescent dyes contained in the PCR amplification chambers are arranged in the same or different ways. In this way, a variety of identical or different fluorescence detections can be achieved. In addition, in the design, the microfluidic chip is provided with 8 PCR amplification cavities, and a nucleic acid analyzer corresponding to each amplification cavity is provided with 5 fluorescence channels, so that detection of 40 indexes can be realized at most. The single-sample multi-index mode also provides possibility for molecular diagnosis to realize multi-pathogen screening facing diseases.

In one embodiment, the microfluidic chip is further provided with a plurality of measurement cavities, each measurement cavity corresponds to each PCR amplification cavity one by one, each measurement cavity is arranged between the reagent distribution pipeline and one PCR amplification cavity, and the reagent distribution pipeline is communicated with each PCR amplification cavity through each measurement cavity; in one embodiment, the microfluidic chip is further provided with a plurality of measurement cavities and reagent conveying pipelines thereof, each measurement cavity is arranged in one-to-one correspondence with each PCR amplification cavity, each measurement cavity is arranged between the reagent distribution pipeline and one PCR amplification cavity, the reagent distribution pipeline is respectively communicated with each measurement cavity and each PCR amplification cavity, and the measurement cavities are communicated with the corresponding PCR amplification cavities through the reagent conveying pipelines of the measurement cavities. The design of measuring the chamber is favorable to realizing even branch liquid, avoids mutual interference or pollution, if do not measure the chamber, liquid fills up PCR amplification chamber in proper order, because prestore has reagent dry powder in the PCR amplification chamber, can lead to, after liquid fills up a PCR amplification chamber, brings out the reagent dry powder of the inside and gets into next PCR amplification chamber through reagent distribution pipeline, and then has stopped this kind of phenomenon through the design of measuring the chamber. In one embodiment, the number of the PCR amplification cavities is 8, the number of the measurement cavities is 8, the PCR amplification cavities are arranged side by side, and each measurement cavity corresponds to one PCR amplification cavity; the measuring cavities are communicated with the PCR amplification cavities, and the measuring cavities are communicated with the corresponding PCR amplification cavities through reagent conveying pipelines. By the design, liquid can enter the measurement cavity first, and then enter the PCR amplification cavity in a high-speed centrifugal mode after all the measurement cavities are full, so that the quantity consistency of the liquid in the PCR amplification cavity is ensured.

In one embodiment, the air outlet is communicated with the outside, and the air outlet is also communicated with a waste liquid cavity through an air outlet pipeline, namely the air outlet is communicated with the first waste liquid cavity or the second waste liquid cavity through the air outlet pipeline, and the position of the waste liquid cavity communicated with the air outlet is far away from the rotation center than the air outlet; that is, the outlet is closer to the rotation center, which is designed to make the liquid close to the waste liquid chamber during centrifugation, facilitating communication with the outside environment through the outlet. In one embodiment, the air outlet is communicated with the first waste liquid cavity through an air outlet pipeline; or the air outlet is communicated with the second waste liquid cavity through an air outlet pipeline. The design of the air outlets at different positions is favorable for realizing the main exhaust effect of the first waste liquid cavity or the second waste liquid cavity, and the micro-fluidic chip is suitable for micro-fluidic chips with different reaction requirements. Further, the gas outlet is communicated with a gas circulation pipeline through a gas outlet pipeline, and the first waste liquid cavity and the second waste liquid cavity are respectively communicated with the gas outlet through the gas circulation pipeline; in one embodiment, the gas circulation pipeline is provided with a branch channel, the first waste liquid cavity is communicated with the gas circulation pipeline through the branch channel, and the position where the first waste liquid cavity is communicated with the branch channel is positioned at the position, closest to the sample adding cavity, of the first waste liquid cavity; in one embodiment, the sum of the volumes of the first waste liquid chamber and the second waste liquid chamber is not less than the volume of the sample adding chamber; this avoids excessive sample application. In one embodiment, the air outlet has a convexly disposed distal end portion. Thus, the waste liquid can be prevented from overflowing while the exhaust is convenient. Further, the first waste liquid chamber is connected with a gas circulation pipeline at one end close to the rotation center so as to prevent waste liquid from overflowing through the gas outlet.

In one embodiment, the siphon pipe is bent. In one embodiment, the siphon pipe has a bent structure. Further, the bending structure is

A glyph configuration or an arch configuration. The arch can be plane or solid, and the arch can be a part of a circle, a cylinder or a sphere, or a part of a parabola or an ellipse. In one embodiment, the minimum distance between the bending structure and the rotation center is less than or equal to the minimum distance between the dilution and lysis chamber and the rotation center; i.e. the top of the meander is closer to the centre of rotation or equally closer to the centre of rotation than the top of the dilute lysis chamber. In one embodiment, the siphon pipe has a bent structure. The bending structure is

A glyph configuration or an arch configuration. In one embodiment, the siphon pipeline has a bent structure which is

The minimum distance between the bending structure and the rotation center is less than or equal to the minimum distance between the dilution cracking cavity and the rotation center. Furthermore, the chip substrate is provided with a positioning area; further, the inner surface of the siphon pipeline is provided with a hydrophilic material layer. Such that

The liquid in the dilution cracking cavity is drawn by capillary force to flow through the siphon pipeline at the position closest to the center of the centrifugal circle to fill the siphon pipeline with the liquid when the centrifugal force is very small (low-speed centrifugation) or no centrifugal force (the microfluidic chip stops rotating); and then, the centrifugal speed is increased, siphon flow occurs in the siphon pipeline under the action of centrifugal force, liquid flows into the reagent distribution pipeline behind and is sequentially distributed to each measuring cavity and the corresponding PCR amplification cavity through the reagent distribution pipeline, and all redundant liquid flows into the second waste liquid cavity.

In one embodiment, the sample output pipeline is provided with a first phase change valve, and the chip substrate is provided with a first packaging hole for communicating the outside and the first phase change valve. First phase change valveSealing or connecting the sample output pipeline; by adopting the design, the phase change material can be injected through the first packaging hole to form the first phase change valve, and then the first packaging hole is closed after the first phase change valve is formed. Further, the first packaging hole is arranged in a closed mode; in one embodiment, the microfluidic chip is provided with a first sealing cover part at the first packaging hole. In one embodiment, the sample output pipeline is provided with a first phase change valve, the chip substrate is provided with a first packaging hole for communicating the outside with the first phase change valve, and the microfluidic chip is provided with a first sealing cover part at the first packaging hole. Further, the first phase change valve is arranged at a position, close to the dilution cracking cavity, of the sample output pipeline; furthermore, the phase change valve comprises a first phase change valve and a second phase change valve, and is prepared by adopting a phase change material; further, the phase change valve has paraffin or the phase change valve is paraffin, and in various embodiments, the phase change material may be wax, for example, the wax may be paraffin, microcrystalline wax, synthetic wax or natural wax. Alternatively, the phase change material may be a gel or a thermoplastic resin. The gel may be polyacrylamide, polyacrylate, polymethacrylate, or polyvinylamine. The thermoplastic resin may be Cyclic Olefin Copolymer (COC), Polymethylmethacrylate (PMMA), Polycarbonate (PC), Polystyrene (PS), Polyoxymethylene (POM), Perfluoroalkoxy (PFA), polyvinyl chloride (PVC), polypropylene (PP), polyethylene terephthalate (PET), Polyetheretherketone (PEEK), Polyacrylate (PA), Polysulfone (PSU), or polyethylenediene fluoride (PVDF), and the like. With the design, in the initial stage, the first phase change valve closes the sample output pipeline; when the sample enrichment cavity finishes sample enrichment cracking, the first phase change valve is subjected to phase change through heating or other processing modes, so that the sample output pipeline is conducted, and a sample enters the dilution cracking cavity through the sample output pipeline. In one embodiment, the sample output conduit has at least one redirecting area. In one embodiment, the redirecting area has an arcuate configuration. Further, the redirecting area has a C-shape or an S-shape. Such design is favorable to reducing the impact force that liquid flowed to first phase change valve or dilution schizolysis chamber through sample output tube, realizes certain slow-flow effect, and the cooperation centrifugal speed effect is better. Thus, the sample enrichment baseIn the concentration mode of density difference, all liquid outlets of the sample enrichment cavity are in a closed state, so that the sample enrichment cavity can be centrifuged at a high speed, fully precipitated and then cracked at a low speed to form a cavity cracking flow pipeline such as the one in the cavity cracking flow pipeline

The siphon valve with the character-shaped structure is designed, so that the sample enrichment universality is wider, and the enrichment effect is more sufficient. And the cracking of this application is realized in diluting the schizolysis intracavity, and the schizolysis mode is the pyrolysis of high temperature boiling.

In one embodiment, the sample enrichment chamber is provided with a vent. The vent is used for communicating with the external environment, namely the external atmospheric pressure. In one embodiment, the sample enrichment chamber has a top position adjacent the center of rotation and a vent is provided at the top position. In one embodiment, the air outlet is communicated with the second waste liquid cavity through an air outlet pipeline. In one embodiment, the gas flow conduit is provided with a second phase change valve and the chip substrate is provided with a second package hole communicating with the second phase change valve. In one embodiment, the microfluidic chip is provided with a second sealing cover part at the second packaging hole. In one embodiment, the sample enrichment chamber has a top position adjacent to the rotation center, the top position is provided with a vent, the gas outlet is communicated with the second waste liquid chamber through a gas outlet pipeline, the gas outlet pipeline is provided with a second phase change valve, the chip substrate is provided with a second packaging hole communicated with the second phase change valve, and the microfluidic chip is provided with a second sealing cover part at the second packaging hole. By adopting the design, the second packaging hole is closed after the second phase change valve is realized, so that the liquid in the sample adding cavity can enter the sample enrichment cavity through the sample adding cavity flow pipeline, and particularly when the gas flow pipeline is provided with the second phase change valve, the liquid is difficult to flow down or even does not flow down if the vent is not arranged.

In one embodiment, the chip substrate is provided with a positioning area; the siphon pipeline has a bending structure which is

Of a letter-shaped or arched structure, curvedThe minimum distance between the folding structure and the rotation center is less than or equal to the minimum distance between the dilution cracking cavity and the rotation center; the inner surface of the siphon pipeline is provided with a hydrophilic material layer. In one embodiment, the sample output pipeline is provided with a first phase change valve, the chip substrate is provided with a first packaging hole for communicating the outside with the first phase change valve, and the microfluidic chip is provided with a first sealing cover part at the first packaging hole; the sample enrichment cavity is provided with a top position close to the rotation center, the top position is provided with a vent hole, the gas outlet is communicated with the second waste liquid cavity through a gas outlet pipeline, the gas circulation pipeline is provided with a second phase change valve, the chip substrate is provided with a second packaging hole communicated with the second phase change valve, and the microfluidic chip is provided with a second sealing cover part at the second packaging hole; the micro-fluidic chip structure is also provided with a plurality of measuring cavities, each measuring cavity is arranged in one-to-one correspondence with each PCR amplification cavity, each measuring cavity is arranged between a reagent distribution pipeline and one PCR amplification cavity, and the reagent distribution pipeline is respectively communicated with each PCR amplification cavity through each measuring cavity; the chip substrate has a partial fan-shaped structure; the chip substrate is provided with a positioning area which is arranged between the gas outlet and the second waste liquid cavity; the positioning areas are positioning convex parts, positioning holes or positioning grooves, and the number of the positioning areas is one, two or more; the positioning groove is in a linear shape or an arc shape, and the circle center of the arc line of the positioning groove is coincided with the rotation center of the microfluidic chip; the number of the positioning holes is multiple and is uniformly distributed relative to the rotation center of the microfluidic chip; the rotation center is positioned outside the chip substrate, and the distances from the rotation center to the rotation center are arranged in the order from small to large: the device comprises a sample adding cavity, a sample enriching cavity, a first waste liquid cavity, a dilution cracking cavity, a reagent distributing pipeline, a second waste liquid cavity and a PCR amplification cavity; a liquid storage container is arranged in the dilution cracking cavity and used for containing diluent; the PCR amplification cavity is filled with PCR reaction reagent dry powder, fluorescent dye and a sealing body.

In one embodiment, as shown in fig. 1, a microfluidic chip includes a chip substrate 100, and a sample application hole 110, a sample application chamber 120, a gas outlet 220, a sample enrichment chamber 140, a waste solution chamber, a dilution lysis chamber 170, a siphon tube 180, a sample application chamber flow tube 121, a lysis chamber flow tube 141, a gas flow tube disposed in the chip substrate 100The through pipe 151, the sample output pipe 161, the reagent distribution pipe 191, the air outlet pipe 210 and the plurality of PCR amplification chambers 190; the waste liquid chamber comprises a first waste liquid chamber 150 and a second waste liquid chamber 200; the chip substrate 100 has a partial fan-shaped structure and the partial fan-shaped structure has three straight sides. The micro-fluidic chip is provided with a rotation center which is positioned outside the chip substrate 100, the micro-fluidic chip is also provided with an air outlet 220 communicated with the outside in the chip substrate 100, the sample adding holes 110 are respectively communicated with the outside and the sample adding cavity 120, and the sample adding cavity 120 is communicated with the sample enrichment cavity 140 through a sample adding cavity circulation pipeline 121; the distance from the rotating center is arranged in the order from small to large: a sample addition well 110, a sample addition chamber 120, a sample enrichment chamber 140, a first waste liquid chamber 150, a dilution lysis chamber 170, a gas outlet 220, a second waste liquid chamber 200, and a PCR amplification chamber 190; the sample enrichment cavity 140 is communicated with the first waste liquid cavity 150 through a lysis cavity circulation pipeline 141, and the sample enrichment cavity 140 is also communicated with a dilution lysis cavity 170 through a sample output pipeline 161 at the bottom position 143 away from the rotation center; the dilution and lysis chamber 170 is communicated with a reagent distribution pipeline 191 through a siphon pipeline 180, and is respectively communicated with each PCR amplification chamber 190 and the second waste liquid chamber 200 through the reagent distribution pipeline 191; the first waste liquid chamber 150 is communicated with the second waste liquid chamber 200 through a gas circulation pipeline 151; the air outlet 220 is communicated with the outside, the air outlet 220 is also communicated with the second waste liquid cavity 200 through an air outlet pipeline 210, and the position of the second waste liquid cavity 200 communicated with the air outlet 220 is far away from the rotation center than the air outlet 220; for convenience of description and understanding, different reference numerals are used for gas outlets connecting different waste liquid chambers, in one embodiment, as shown in fig. 12 to 15, a gas circulation pipeline 151 and a gas outlet pipeline are combined, and the gas outlet 130 is respectively communicated with the first waste liquid chamber 150 and the second waste liquid chamber 200 through the gas circulation pipeline 151, that is, the gas outlet 130 is communicated with the first waste liquid chamber 150 through the gas circulation pipeline 151 and is communicated with the second waste liquid chamber 200 through the gas circulation pipeline 151. The siphon pipe 180 is bent to form a

The term "zigzag" is also understood to mean a zigzag structure, i.e. a combination of two symmetrical L-shaped structures and a transverse line, as shown in the figures, a siphonOf conduits 180

The minimum distance between the glyph structure and the center of rotation is less than or equal to the minimum distance between the dilute lysis chamber 170 and the center of rotation. Referring to fig. 2, the siphon channel 180 is sequentially provided with a horizontal inlet 181, a vertical inlet 182, a horizontal part 183, a vertical outlet 184 and a horizontal outlet 185, which are communicated with each other, wherein the horizontal inlet 181 is communicated with the bottom of the dilution and lysis chamber 170, i.e. a position away from the rotation center, and the horizontal outlet 185 is communicated with the reagent dispensing channel 191. The sample enrichment chamber 140 has a top position adjacent to the center of rotation and a vent 142 is provided at the top position; referring to fig. 4 and 5 together, the chip substrate 100 is provided with a first packaging hole 163 for communicating the outside and the first phase change valve 162; the sample adding hole 110, the air vent 142, the air outlet 220 and the packaging hole 163 are respectively communicated with the outside, the positioning area 101 is a positioning groove which is arc-shaped, the circle center of the arc is coincident with the rotation center of the microfluidic chip, the positioning area 101 is arranged between the air outlet 220 and the packaging hole 163, or the positioning area 101 is arranged between the air vent 142 and the air outlet 220, or the packaging hole 163 is positioned in the positioning area 101. In each embodiment, the PCR amplification chambers 190 are arranged side by side, and as shown in fig. 1, 3, 6 and 8, the distance between the mouth and the rotation center of each PCR amplification chamber is the same. The micro-fluidic chip is also provided with a plurality of measuring cavities 192 and reagent conveying pipelines 193 thereof, each measuring cavity 192 and each PCR amplification cavity 190 are arranged in a one-to-one correspondence manner, each measuring cavity 192 is arranged between a reagent distribution pipeline 191 and one PCR amplification cavity 190, the reagent distribution pipeline 191 is respectively communicated with each PCR amplification cavity 190 through each measuring cavity 192, and the measuring cavity 192 is communicated with the corresponding PCR amplification cavity 190 through the reagent conveying pipeline 193 thereof; the first waste liquid chamber 150 is communicated with the second waste liquid chamber 200 through a gas circulation pipe 151, and the second waste liquid chamber 200 is communicated with a gas outlet 220 through a gas outlet pipe 210. As shown in FIG. 7, the depth of the reagent delivery conduit 193 is shallower than the depth of the measurement chamber 192, and the depth of the reagent delivery conduit 193 is shallower than the depth of the PCR amplification chamber 190,the depth of reagent dispensing channel 191 is shallower than the depth of reagent delivery channel 193, and the depth of gas flow channel 151, gas outlet channel 210, and reagent dispensing channel 191 are shallower than the depth of second waste chamber 200. In one embodiment, as shown in fig. 9 to 11, the gas flow pipe 151 is provided with a second phase change valve 230, and the chip substrate 100 is provided with a second package hole communicating with the second phase change valve 230. In one embodiment, the lysis chamber flow conduit 141 is bent and has

The distance between the top position of the lysis chamber flow channel 141 and the rotation center is less than or equal to the distance between the top position of the sample enrichment chamber 140 and the rotation center, that is, the distance between the top position of the lysis chamber flow channel 141 and the sample application chamber 120 is less than the distance between the top position of the sample enrichment chamber 140 and the sample application chamber 120; the sample output conduit 161 has a first direction change region and a second direction change region. With the embodiment shown in fig. 1 to 8, in a specific application, a sample is loaded into the loading chamber 120 through the loading hole 110 when the microfluidic chip is at rest. Subsequently, the microfluidic chip is centrifuged at high speed, and the sample fills the sample enrichment chamber 140 through the sample chamber flow channel 121. In various embodiments, the high speed centrifugation is performed at a speed of about 3000rpm to about 6000 rpm. When the sample fills the sample enrichment chamber, the liquid in the sample enrichment chamber 140 cannot enter the subsequent reaction chamber through the sample output pipe 161 due to the presence of the first phase change valve 162. The presence of a control structure, such as a phase change valve, in the lysis chamber flow conduit 141 or the gas flow conduit 151 results in the first waste chamber 150 being in a sealed state such that liquid in the sample enrichment chamber cannot flow into the first waste chamber 150 via the lysis chamber flow conduit 141. The lysis chamber flow channel 141 may be a normal liquid flow channel or may additionally have a control structure such as a phase change valve structure, which may also be referred to as a phase change valve, or a siphon valve structure, which may also be referred to as a siphon valve. For example, the lysis chamber flow channel 141 is a normal fluid flow channel, and then the high-speed centrifugation is continued while the sample fills the sample enrichment chamber 140, and the cells, tissues, pathogens in the sample due to the centrifugal fieldThe supernatant will be deposited to the bottom of the sample enrichment chamber 140, and the supernatant will overflow into the first waste liquid chamber 150 via the lysis chamber circulation line 141, thus realizing the function of sample enrichment; in other embodiments, a filter membrane may be added to the sample enrichment chamber 140, and the enrichment of cells, tissues, pathogens, etc. in the sample may be achieved by filtering through the filter membrane. Or, the lysis chamber circulation line 141 has a siphon valve or a phase change valve, and then the sample can be sufficiently enriched in the sample enrichment chamber 140 by high-speed centrifugation, and then when the lysis chamber circulation line 141 has a siphon valve formed by a siphon line, the rotation speed is reduced so that the supernatant in the sample enrichment chamber 140 breaks through the siphon valve of the lysis chamber circulation line 141 and enters the first waste liquid chamber 150, or when the lysis chamber circulation line 141 has a third phase change valve, the third phase change valve is opened by heating or other phase change methods so that the supernatant in the sample enrichment chamber 140 breaks through the third phase change valve of the lysis chamber circulation line 141 and enters the first waste liquid chamber 150. In some embodiments for specific applications, the centrifuged cell debris can be sufficiently settled to the bottom of the sample enrichment chamber 140 by first performing medium-high speed centrifugation to prevent the cell debris from inhibiting the subsequent PCR reaction. In some embodiments of specific applications, slow acceleration to high speed may also be used to deposit the cell debris on the bottom of the sample enrichment chamber 140 and to introduce the lysed liquid into the dilution lysis chamber 170 via the sample output tube 161. Further, when a control structure is present in the lysis chamber flow conduit 141, the vent 220 may be located at the top of either the first waste chamber 150 or the second waste chamber 200; however, when a control structure such as a phase change valve is present somewhere on the gas flow conduit 151, the gas outlet 220 may be located only at the top of the second waste liquid chamber 200. After the cells, tissues and pathogens in the sample are fully precipitated, the siphon valve breakthrough at the flow pipeline 141 of the lysis chamber is realized by changing the centrifugal rotating speed and adopting a low-speed centrifugal mode, or the phase change valve breakthrough at a certain part of the flow pipeline 141 of the lysis chamber or a certain part of the gas flow pipeline 151 is realized by heating. At this time, the supernatant in the sample enrichment chamber 140 will flow into the first waste liquid chamber 150 until the liquid level in the sample enrichment chamber 140 is lower than the lysis chamber flow line 141 for sample enrichmentAn inlet in the cavity 140. In this embodiment, lysis of cells, tissues, pathogens, etc. within the sample is achieved in the sample enrichment chamber 140. The lysis method includes but is not limited to ultrasonic lysis, boiling lysis, magnetic stirring lysis, etc. The phase change material sealing the first phase change valve 162 is then melted by heating the first phase change valve 162, so that the remaining liquid in the sample enrichment chamber 140 can flow into the dilution lysis chamber 170 through the sample output tube 161. Phase change materials used to seal the phase change valve include, but are not limited to, paraffin. Because the volume of the liquid after the cracking is limited, the distance between the liquid level in the dilution cracking cavity 170 and the centrifugal center, i.e., the rotation center, is larger than the distance between the siphon pipe 180 and the centrifugal center, i.e., the liquid level in the dilution cracking cavity 170 is lower than the siphon pipe 180, i.e., the liquid level in the dilution cracking cavity 170 is lower than the communication position between the dilution cracking cavity 170 and the siphon pipe 180, before the dilution, no matter how large the centrifugal force is, the liquid in the dilution cracking cavity 170 cannot break through the siphon pipe 180 and enter the subsequent reaction cavity. Further, in various embodiments, the dilute lysis chamber 170 is pre-loaded with a diluent; in some embodiments, the diluent is pre-placed in a paraffin-encapsulated aluminum foil with one end and an adhesive bonded to the top of the dilute lysis chamber 170, and the diluent is released when the paraffin melts after heating. In other examples, the diluent in the dilute lysis chamber 170 is pre-loaded in a sealed aluminum foil placed over a spike, the spike is fixed to the top of the dilute lysis chamber 170, and the aluminum foil and the spike are fixed by a spring. Thus, upon high speed centrifugation, the aluminum foil is punctured by the tip, thereby releasing the diluent. In some examples, the diluent is released and mixed with the lysed liquid, and the mixing can be achieved by rotating the microfluidic chip forward and backward or rotating the microfluidic chip at an accelerated speed and a decelerated speed. Thereafter, the dilution and lysis chamber 170 is heated at a high temperature, and the tissue, cells, and pathogens in the dilution and lysis chamber 170 are boiled and lysed. After full cracking, the centrifugal speed is reduced, low-speed centrifugation is carried out, then intermediate-speed centrifugation is carried out, the liquid in the dilution cracking cavity 170 passes through the siphon pipeline 180 with hydrophilic treatment on the inner surface, during low-speed centrifugation, the capillary force is greater than the centrifugal force, and the liquid can submerge the siphon pipeline under the driving of the capillary force to be closest to the separation cavityThe center of the circle reaches the junction of the siphon pipeline 180 and the reagent distribution pipeline 191, then the liquid is centrifuged at medium speed, the distance from the junction of the siphon pipeline 180 and the reagent distribution pipeline 191 to the center of the circle is greater than the distance from the junction of the siphon pipeline 180 and the dilution cracking cavity 170 to the center of the circle, so that a siphon effect is generated, the diluted cracked liquid breaks through the siphon pipeline 180 and enters the reagent distribution pipeline 191, and the measurement cavities 192 are filled in sequence from left to right; that is, the liquid in the diluting and lysing chamber 170 above the junction of the siphon pipe 180 and the diluting and lysing chamber 170 flows into the reagent distributing pipe 191 through the siphon pipe 180, enters the reagent distributing pipe 191, and fills 8 measuring chambers 192, and the excess liquid enters the second waste liquid chamber 200. Subsequently, by high-speed centrifugation, the liquids in the measurement chambers 192 enter the corresponding PCR amplification chambers 190 through the reagent delivery pipes 193, respectively. Further, in each embodiment, the PCR amplification chamber 190 is pre-filled with PCR reaction reagent dry powder and paraffin, and in one embodiment, the PCR reaction reagent dry powder includes enzymes, dNTPs, fluorescent dyes, primers, and the like required for PCR reaction. In particular, each amplification cavity can be preset with a plurality of primers, and multiple PCR can be realized in each amplification cavity. In one embodiment, the amplification products are distinguished by different fluorescent dyes. And then, carrying out temperature cycle, starting to realize PCR reaction in the amplification cavity, and melting paraffin in the amplification cavity. Because the density of the paraffin is less than that of water, the paraffin floats to the inlet of the PCR amplification cavity and seals the PCR amplification cavity in a centrifugal field, so that aerosol pollution which is easy to occur in PCR reaction is avoided. In one embodiment, the substance used to seal the PCR amplification chamber is mineral oil. In particular, the positioning region 101 forms a bayonet structure for mounting the microfluidic chip or its chip substrate. In the PCR reaction process, the micro-fluidic chip is always kept in a low-speed centrifugation state, and in each embodiment, the rotation speed of the low-speed centrifugation is about 0-300 rpm; the optical system in the matched full-automatic nucleic acid analyzer can respectively read the light intensity of each fluorescence channel in each PCR amplification cavity in a scanning mode, so that a QPCR curve is drawn, the Ct value is calculated, and a positive and negative report is given. In particular, for matching multiple PCR in PCR amplification chamber, in a fully automatic nucleic acid analyzerThe area optical system also has multiple sets of optical and photodetector systems to read the fluorescent signals at different wavelengths. The design is suitable for centrifugal microfluidic analysis, enrichment, cracking, dilution after cracking, equal distribution and PCR amplification of multiple chambers can be sequentially realized, a nucleic acid purification-free molecular diagnosis function can be realized, on one hand, the gas outlet is communicated with one waste liquid chamber and is communicated with the other waste liquid chamber through the waste liquid chamber, the position of the gas outlet can be adjusted according to requirements, on the other hand, the bent siphon pipeline ingeniously utilizes the relative relation between capillary force and centrifugal force to form a regulating valve to control liquid to flow into a reagent distribution pipeline, the PCR amplification technology is applied to the centrifugal microfluidic technology to realize molecular diagnosis based on PCR amplification, the whole reaction process is in a closed microfluidic chip, and the molecular diagnosis effect of rapid detection at any time and any place is realized.

In one embodiment, the microfluidic chip is integrally designed in a centrifugal force driving mode. The whole microfluidic chip is of a fan-shaped structure, the eight microfluidic chips form a ring, and the centrifugal rotation center, namely the rotation center, is positioned at the center of the circle of the fan-shaped structure. The following takes the typing of Human Papilloma Virus (HPV) as an example to illustrate the specific implementation of the microfluidic chip. Modern medical research confirms that cervical cancer in women is caused by infection of the cervix by human papilloma virus, HPV is a generic term for a group of viruses, which is composed mainly of a DNA core and a protein capsid. The types of HPV which are determined at present are about more than 80, and the HPV with different genotypes have different pathogenic risks and can be divided into three types of low-risk type, medium-risk type and high-risk type according to the carcinogenicity of the HPV. The detection and typing of HPV DNA has important value for understanding the state of illness, judging prevention and guiding treatment.

In the embodiments shown in fig. 9, 10 and 11, the detection process using the microfluidic chip is described as follows. 1. Taking 1ml of cervical brushing washing liquid as a sample, and adding the sample into the sample adding cavity 120 through the sample adding hole 110; centrifuging at 2.4000rpm for 5 minutes, and allowing the sample to flow into the sample enrichment chamber 140 through the sample application chamber flow pipeline 121; in the sample enrichment chamber 140, due to the existence of the centrifugal field, cells, tissues and pathogens in the sample can be fully precipitated to the bottom of the chamber, and when the first waste liquid chamber 150 is communicated with the external environment, the supernatant can enter the first waste liquid chamber 150; 3. after the cells, tissues and pathogens in the sample are sufficiently precipitated, the second phase change valve 230 of the gas circulation pipe 151 is heated to melt the phase change material for sealing the phase change valve, so that the first waste liquid chamber 150 is communicated with the gas outlet 220 via the gas circulation pipe 151, and the first waste liquid chamber 150 is communicated with the external atmosphere, at this time, the supernatant in the sample enrichment chamber 140 flows into the first waste liquid chamber 150 until the liquid level in the sample enrichment chamber 140 is lower than the inlet of the lysis chamber circulation pipe 141 in the sample enrichment chamber 140. 4. The centrifugal speed of 4000rpm is maintained, and then the first phase change valve 162 of the sample output pipe 161 is heated, so that the phase change material for sealing the phase change valve is melted, and the liquid in the sample enrichment chamber 140 flows into the dilution cracking chamber 170 through the sample output pipe 161. The paraffin-forming first phase change valve 162 is heated to 70 ℃, the paraffin melts, the sample output pipe 161 is opened, and the liquid in the sample enrichment chamber 140 below the inlet of the lysis chamber circulation pipe 141 in the sample enrichment chamber 140 wraps the tissue, the cells and the pathogen sediment and flows into the dilution lysis chamber 170 through the sample output pipe 161. In one embodiment, the first phase change valve 162 is heated to 60 ℃ or higher, the paraffin melts, the sample output line 161 is conducted, and the remaining liquid in the sample enrichment chamber 140 flows into the dilution and lysis chamber 170 through the sample output line 161.

5.3000rpm, and the diluted lysis chamber 170 is heated at 100 ℃ for 5 minutes to boil and lyse the tissue, cells, pathogens, etc. in the diluted lysis chamber 170. 6. The centrifugal speed is reduced to 200rpm and then increased to 1000rpm, the liquid after the dilution cracking cavity 170 cracks through the siphon pipe 180, flows into the reagent distribution pipe 191 through the siphon pipe 180 and sequentially fills 8 measuring cavities 192, and the redundant liquid enters the second waste liquid cavity 200; in one embodiment, the diluting and cracking chamber 170 is pre-filled with a diluent, the diluent is pre-filled in an aluminum foil with one end encapsulated by paraffin and the other end bonded by glue, the aluminum foil is bonded on the top of the diluting and cracking chamber 170 by glue, the diluting and cracking chamber 170 is heated at 70 ℃ or higher, and after heating, the paraffin is melted and the diluent is released. 7.3000rpm centrifugal microfluidic chip, the liquid in each measurement cavity 192 enters the PCR amplification cavity 190 through the reagent conveying pipeline 193 respectively, and the reagent dry powder preset in the PCR amplification cavity 190 is re-melted. Centrifuging at 8.500rpm, starting hot start heating control, melting paraffin in the PCR amplification cavity 190, floating the paraffin to the inlet of the PCR amplification cavity 190, sealing the PCR amplification cavity 190, and then entering the temperature cycle of PCR reaction. And scanningly reading the fluorescence signal in each PCR amplification cavity in the extension phase of each cycle to draw a PCR curve.

In the embodiments shown in fig. 12 to 15, the detection process using the microfluidic chip is described as follows. 1.1ml of cervical brushing solution as a sample is added to the sample addition chamber 120 through the sample addition hole 110. 2.4000rpm for 5 minutes, the sample flows through the sample application chamber flow channel 121 into the sample enrichment chamber 140. In the sample enrichment cavity 140, due to the existence of the centrifugal field, cells, tissues and pathogens in the sample can be fully precipitated to the bottom of the cavity; 3. the centrifugal speed is reduced to 200rpm, at this time, due to the siphon tube design of the lysis chamber circulation tube 141, the inner surface of the lysis chamber circulation tube 141 is coated with a hydrophilic material or is subjected to hydrophilic treatment or the chip substrate 100 is made of a hydrophilic material, the lysis chamber circulation tube 141 has a strong capillary force to the liquid, at this time, the capillary force is greater than the centrifugal force, the liquid in the sample enrichment chamber 140 above the inlet of the lysis chamber circulation tube 141 having the siphon tube effect flows through the position closest to the center of the centrifugal circle along the lysis chamber circulation tube 141, flows to the junction of the lysis chamber circulation tube 141 and the first waste liquid chamber 150, and at this time, the flow is stopped due to the breakthrough of a liquid contact angle. 4. The centrifugation speed is increased to 2000rpm, and due to the siphon action, the supernatant in the sample enrichment chamber 140 above the junction of the lysis chamber flow channel 141 and the inlet of the sample enrichment chamber 140 flows into the first waste fluid chamber 150 along the lysis chamber flow channel 141. 5. The paraffin-formed first phase change valve 162 is heated to 70 ℃, the paraffin is melted at the moment, the sample output pipeline 161 is opened, and the liquid below the junction of the lysis cavity circulation pipeline 141 and the sample enrichment cavity 140 in the sample enrichment cavity 140 wraps tissues, cells and pathogen sediments and flows into the dilution lysis cavity 170 through the sample output pipeline 161. 6. The diluted lysis chamber 170 is heated at 100 ℃ for 5 minutes, and the tissue, cells, pathogens, etc. in the diluted lysis chamber 170 are boiled and lysed. 7. The centrifugal speed is reduced to 200rpm and then increased to 1000rpm, the liquid which is cracked in the dilution cracking cavity 170 and is above the inlet of the siphon pipe 180 flows into the reagent distributing pipe 191 through the siphon pipe 180, 8 measuring cavities 192 are filled in sequence, and the redundant liquid enters the second waste liquid cavity 200; 8.3000rpm centrifugal microfluidic chip, the liquid in each measurement cavity 192 enters the PCR amplification cavity 190 through the reagent conveying pipeline 193 respectively, and the reagent dry powder preset in the PCR amplification cavity 190 is re-melted. 9.200rpm, and starting hot start heating control, the paraffin wax in the PCR amplification cavity 190 melts and floats to the entrance of the PCR amplification cavity 190, the PCR amplification cavity 190 is sealed, and then the temperature cycle of the PCR reaction is started. And scanningly reading the fluorescent signal in each PCR amplification chamber during the extension phase of each cycle to plot a PCR curve. Then, the operations such as fluorescence detection, negative and positive judgment and the like can be performed in a targeted manner, and data processing, writing into a database and printing a report can also be performed.

In one embodiment, an analysis system comprises the microfluidic chip of any one of the embodiments, and further, the analysis system is provided with at least three fluorescence channels; further, the assay system was provided with 5 fluorescence channels. In one embodiment, the analysis system is used for controlling the microfluidic chip to keep a low-speed centrifugation state during the PCR reaction; in one embodiment, the analysis system is further configured to scan and read out light intensity of each fluorescence channel in each PCR amplification chamber by using an optical system of the full-automatic nucleic acid analyzer, draw a QPCR curve, calculate a Ct value, and give a positive and negative report; in one embodiment, the analysis system is used for sequentially carrying out static sample adding, medium-speed centrifugation to enable a sample to fill a sample enrichment cavity and realize a sample enrichment function and then lyse cells, medium-speed centrifugation to enable lysed cell residues to be precipitated at the bottom of the sample enrichment cavity and enable the lysed sample to enter a dilution lysis cavity, heating to melt paraffin in the dilution lysis cavity so as to release a diluent to enable the lysed sample to be diluted, high-speed centrifugation to enable the diluted sample to enter each PCR amplification cavity, heating to melt paraffin in each PCR amplification cavity, and low-speed centrifugation to carry out PCR amplification; in one embodiment, the nucleic acid analysis system further comprises a permanent magnet arranged below the ferromagnetic substance block, and the permanent magnet is used for driving the paramagnetic substance block to move or rotate in the sample enrichment cavity, so that a better cracking effect can be realized by matching with the embodiment with the paramagnetic substance. Further, the analysis system is provided with at least three fluorescence channels; and/or the analysis system is used for controlling the microfluidic chip to keep a low-speed centrifugal state in the PCR reaction process; and/or the analysis system is also used for respectively scanning and reading out the light intensity of each fluorescence channel in each PCR amplification cavity by adopting an optical system of the full-automatic nucleic acid analyzer, drawing a QPCR curve, calculating a Ct value and giving a positive and negative report; and/or the analysis system is used for carrying out static sample adding in sequence, carrying out medium-speed centrifugation to enable the sample to fill the sample enrichment cavity and realize the function of sample enrichment and then cracking cells, carrying out medium-high-speed centrifugation to enable the cracked cell residues to be precipitated at the bottom of the sample enrichment cavity and enable the cracked sample to enter the dilution cracking cavity, heating to melt paraffin in the dilution cracking cavity so as to release diluent to enable the cracked sample to be diluted, carrying out high-speed centrifugation to enable the diluted sample to enter each PCR amplification cavity, heating to melt paraffin in each PCR amplification cavity, and carrying out low-speed centrifugation to carry out PCR amplification; and/or, the nucleic acid analysis system further comprises a permanent magnet disposed below the ferromagnetic mass.

Furthermore, the analysis system uses the microfluidic chip of each embodiment to match with a full-automatic nucleic acid analysis instrument, so that the full automation of the molecular diagnosis project without a nucleic acid purification step is realized. In the microfluidic chip, sample enrichment, lysis, dilution after lysis and equal distribution are sequentially realized, and PCR amplification in multiple chambers is realized. In one embodiment, the microfluidic chip is provided with 8 PCR amplification cavities, and a nucleic acid analysis instrument corresponding to each amplification cavity is provided with 5 fluorescence channels, so that detection of 40 indexes can be realized at most simultaneously. The single-sample multi-index mode also provides possibility for molecular diagnosis to realize multi-pathogen screening facing diseases. Furthermore, the whole reaction process is in a closed microfluidic chip, so that the burden and the pollution possibility of operators are reduced, the whole molecular diagnosis process does not depend on a molecular diagnosis laboratory or professional operators, the requirement of rapid detection at any time and any place is met, and great help is brought to medical inspection and disease prevention and control. Other embodiments of the present application further include a microfluidic chip and an analysis system, which are formed by combining technical features of the above embodiments. The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, but should be considered as the scope of the present specification as long as there is no contradiction between the combinations of the technical features. The above examples only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the claims. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present application shall be subject to the appended claims.

Claims

1. A micro-fluidic chip is provided with a rotation center and is characterized by comprising a chip substrate, and a sample adding hole, a sample adding cavity, a gas outlet, a sample enrichment cavity, a waste liquid cavity, a dilution cracking cavity, a siphon pipeline, a sample adding cavity circulating pipeline, a cracking cavity circulating pipeline, a gas circulating pipeline, a sample output pipeline, a reagent distribution pipeline, a gas outlet pipeline and a plurality of PCR amplification cavities which are arranged in the chip substrate;

the waste liquid cavity comprises a first waste liquid cavity and a second waste liquid cavity;

the sample adding hole is respectively communicated with the outside and the sample adding cavity, and the sample adding cavity is communicated with the sample enrichment cavity through the sample adding cavity circulation pipeline;

the sample enrichment cavity is communicated with the first waste liquid cavity through the pyrolysis cavity circulation pipeline, and the sample enrichment cavity is also communicated with the dilution pyrolysis cavity through the sample output pipeline at the bottom position far away from the rotation center; wherein the sample enrichment chamber and the lysis chamber flow channel have a first connection location, the sample enrichment chamber and the sample output channel have a second connection location, and the first connection location is less distant from the sample application chamber than the second connection location is distant from the sample application chamber;

the dilution and lysis cavity is sequentially communicated with each PCR amplification cavity and the second waste liquid cavity through the siphon pipeline and the reagent distribution pipeline respectively;

the first waste liquid cavity is communicated with the second waste liquid cavity through the gas circulation pipeline;

the gas outlet is communicated with the outside, the gas outlet is communicated with the waste liquid cavity through the gas outlet pipeline, and the position of the waste liquid cavity communicated with the gas outlet is far away from the rotating center compared with the position of the gas outlet.

2. The microfluidic chip according to claim 1, wherein the chip substrate is provided with a positioning region.

3. The microfluidic chip according to claim 1, wherein the gas outlet is communicated with the second waste liquid chamber through the gas outlet pipeline.

4. The microfluidic chip according to claim 1, wherein the gas outlet is communicated with the first waste liquid chamber through the gas outlet pipeline.

5. The microfluidic chip according to claim 1, wherein the siphon channel has a bent structure.

6. The microfluidic chip according to claim 5, wherein the minimum distance between the bending structure and the rotation center is smaller than or equal to the minimum distance between the diluted lysis chamber and the rotation center.

7. The microfluidic chip according to claim 1, wherein the chip substrate is made of a hydrophilic material.

8. The microfluidic chip according to claim 1, wherein the inner surface of the siphon channel is provided with a hydrophilic material layer.

9. The microfluidic chip according to claim 1, wherein the inner surface of the siphon tube is subjected to hydrophilic treatment.

10. The microfluidic chip according to any one of claims 1 to 9, wherein the sample output channel is provided with a first phase change valve, the chip substrate is provided with a first packaging hole for communicating the outside with the first phase change valve, and the microfluidic chip is provided with a first sealing cover at the first packaging hole;

setting the first connection position according to the total volume of the target liquid participating in the reaction;

an enrichment cavity and a cracking cavity are arranged in the sample enrichment cavity; the lysis cavity is communicated with the first waste liquid cavity through the lysis cavity circulation pipeline, the enrichment cavity is communicated with the sample output pipeline, the enrichment cavity is used for enriching a sample through centrifugation, the lysis cavity is used for overflowing supernatant obtained through centrifugation to the first waste liquid cavity through the lysis cavity circulation pipeline, and the enrichment cavity and the lysis cavity are separated through a filter membrane;

the sample enrichment cavity is provided with a top position close to the rotation center, the top position is provided with a vent hole, the gas outlet is communicated with the second waste liquid cavity through the gas outlet pipeline, the gas circulation pipeline is provided with a second phase change valve, the chip substrate is provided with a second packaging hole communicated with the second phase change valve, and the microfluidic chip is provided with a second sealing cover part at the second packaging hole;

the microfluidic chip structure is also provided with a plurality of measuring cavities, each measuring cavity is in one-to-one correspondence with each PCR amplification cavity, each measuring cavity is arranged between the reagent distribution pipeline and one PCR amplification cavity, and the reagent distribution pipeline is respectively communicated with each PCR amplification cavity through each measuring cavity;

the chip substrate is provided with a partial fan-shaped structure;

the chip substrate is provided with a positioning area, and the positioning area is arranged between the gas outlet and the second waste liquid cavity; the positioning areas are positioning convex parts, positioning holes or positioning grooves, and the number of the positioning areas is one, two or more; the positioning groove is linear or is arc-shaped, and the circle center of the arc line of the positioning groove is coincided with the rotation center of the microfluidic chip; the number of the positioning holes is multiple and is uniformly distributed relative to the rotation center of the microfluidic chip;

the rotation center is positioned outside the chip substrate, and the distance from the rotation center to the rotation center is arranged in the order from small to large: the sample application cavity, the sample enrichment cavity, the first waste liquid cavity, the dilution and lysis cavity, the reagent distribution pipeline, the second waste liquid cavity and the PCR amplification cavity;

a liquid storage container is arranged in the dilution cracking cavity and used for containing diluent; the liquid storage container is provided with an opening, a puncturing piece, an elastic piece and a sealing film, the sealing film is used for sealing the opening, one end of the elastic piece is connected with the puncturing piece, the other end of the elastic piece is fixed in the dilution cracking cavity, and the puncturing piece is used for being matched with the elastic piece to generate displacement to puncture the sealing film during centrifugation; or, diluent is arranged in the dilution cracking cavity; wherein, the diluent is arranged in a hot-melt wrapping layer which is arranged in the diluting and cracking cavity; or the diluent is arranged in the wrapping layer which is arranged in the dilution cracking cavity and provided with an opening which is sealed by a hot melt layer;

the PCR amplification cavity is internally provided with PCR reaction reagent dry powder, fluorescent dye and a sealing body.

11. An analysis system comprising the microfluidic chip according to any one of claims 1 to 10.