CN209778828U

CN209778828U - centrifugal micro-fluidic chip structure and nucleic acid analysis device

Info

Publication number: CN209778828U
Application number: CN201920211548.9U
Authority: CN
Inventors: 汤明辉
Original assignee: Shenzhen City Of Gang Zhu Medical Science And Technology Co Ltd
Current assignee: Shenzhen Chenghui Medical Technology Co ltd
Priority date: 2019-02-19
Filing date: 2019-02-19
Publication date: 2019-12-13
Anticipated expiration: 2029-02-19

Abstract

The application relates to a centrifugal micro-fluidic chip structure and a nucleic acid analysis device, wherein the centrifugal micro-fluidic chip structure comprises a chip substrate, a sample adding cavity, a gas outlet, a sample enrichment and lysis cavity, a first waste liquid cavity, a first capillary valve, a dilution cavity, a second capillary valve, a PCR amplification cavity and a second waste liquid cavity; the sample adding cavity is provided with a sample adding hole and is communicated with the sample enrichment and cracking cavity through a sample adding cavity circulation pipeline; the sample enrichment cracking cavity is communicated with the first waste liquid cavity through a cracking cavity circulation pipeline, is also communicated with a cracking cavity mixing pipeline through a first capillary valve, and is communicated with the dilution cavity through a cracking cavity mixing pipeline; the dilution cavity is communicated with the PCR amplification cavity and the second waste liquid cavity through a second capillary valve, and the first waste liquid cavity and the second waste liquid cavity are communicated with the gas outlet through gas circulation pipelines respectively. The PCR amplification technology can be applied to the centrifugal microfluidic technology to realize molecular diagnosis based on PCR amplification, and a large molecular diagnosis laboratory is not required to be built, and a large amount of manual operation is not required to be adopted.

Description

Centrifugal micro-fluidic chip structure and nucleic acid analysis device

Technical Field

The application relates to the field of centrifugal microfluidics, in particular to a centrifugal microfluidic chip structure and a nucleic acid analysis device.

background

Microfluidics (Microfluidics) refers to the manipulation of liquids on a sub-millimeter scale, which is typically several microns to several hundred microns. The microfluidic technology integrates the basic operation units related to the biological and chemical fields, even the functions of the whole laboratory, including sampling, dilution, reaction, separation, detection, etc., on a 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 greatly 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 testing (POCT).

The molecular diagnosis based on PCR amplification is to detect the existence of endogenous (genetic or variant) or exogenous (pathogen) target gene by primer-mediated specific amplification of the target gene, and further provide information and decision basis for disease diagnosis and treatment. The main application scenes of the kit include diagnosis of infectious diseases, blood screening, tumor mutation site detection, diagnosis of genetic diseases, prenatal diagnosis, tissue typing and the like. 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. This has greatly increased 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.

SUMMERY OF THE UTILITY MODEL

In view of the above, there is a need for a centrifugal microfluidic chip structure and a nucleic acid analysis device.

A centrifugal micro-fluidic chip structure comprises a chip substrate, and a sample adding cavity, a gas outlet, a sample enrichment and lysis cavity, a first waste liquid cavity, a first capillary valve, a dilution cavity, a second capillary valve, a PCR amplification cavity and a second waste liquid cavity which are arranged on the chip substrate; the sample adding cavity is provided with a sample adding hole and is communicated with the sample enrichment and lysis cavity through a sample adding cavity circulation pipeline; the sample enrichment and cracking cavity is communicated with the first waste liquid cavity through a cracking cavity circulation pipeline, and is also communicated with a cracking cavity mixing pipeline through the first capillary valve and is communicated with the dilution cavity through the cracking cavity mixing pipeline; the dilution cavity is communicated with the PCR amplification cavity and the second waste liquid cavity through the second capillary valve, and the first waste liquid cavity and the second waste liquid cavity are communicated with the gas outlet through gas circulation pipelines respectively.

The centrifugal microfluidic chip structure can utilize the PCR amplification technology to be applied to the centrifugal microfluidic technology to realize molecular diagnosis based on PCR amplification through the skillfully designed chamber structure, a large-scale molecular diagnosis laboratory is not required to be built, a large amount of manual operation is not required to be adopted, and PCR amplification can be realized in the PCR amplification cavity through integration of the sample adding cavity, the sample enrichment and cracking cavity, the dilution cavity and the PCR amplification cavity.

In one embodiment, the centrifugal microfluidic chip structure further comprises the sample application cavity flow channel, the lysis cavity flow channel, the gas flow channel, and the lysis cavity mixing channel.

In one embodiment, the sample application hole and the gas outlet are located at the same end of the chip substrate.

in one embodiment, the chip substrate has a partial fan-shaped structure.

In one embodiment, the partial sector comprises a sector ring shape and a sector blade shape or the partial sector structure has three straight sides.

in one embodiment, the air outlet is provided in a convex shape.

In one embodiment, the second waste liquid cavity is convexly provided with an auxiliary air outlet or a positioning column.

In one embodiment, the number of the PCR amplification chambers is multiple, and the centrifugal microfluidic chip structure further includes a reagent distribution pipeline disposed on the chip base body, the reagent distribution pipeline respectively communicating each of the PCR amplification chambers and the second waste liquid chamber; the dilution cavity is communicated with the reagent distribution pipeline through the second capillary valve, and is respectively communicated with the PCR amplification cavity and the second waste liquid cavity through the reagent distribution pipeline.

In one embodiment, the number of the PCR amplification cavities is 8, the PCR amplification cavities are arranged side by side, the centers of the PCR amplification cavities are distributed on the same circumference, and the PCR amplification cavities have cylindrical structures; the sample enrichment and lysis cavity is internally 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 a cracking cavity mixing pipeline through the first capillary valve; according to the centrifugal direction, the sample adding cavity, the sample enrichment and lysis cavity, the first waste liquid cavity, the dilution cavity and the reagent distribution pipeline are arranged in sequence, and the position of the gas outlet is closer to the sample adding cavity relative to the first waste liquid cavity; the distance between the liquid level in the dilution cavity and the centrifugal circle center is larger than the distance between the second capillary valve and the centrifugal circle center; the sum of the volumes of the first waste liquid cavity and the second waste liquid cavity is not less than the volume of the sample adding cavity; a liquid storage container is arranged in the dilution cavity; the chip matrix is packaged by adopting a hot pressing, ultrasonic welding, laser welding or gluing mode; the chip substrate is provided with at least three fixing parts.

a nucleic acid analysis device comprising the centrifugal microfluidic chip structure according to any one of the above.

The nucleic acid analysis device can apply the PCR amplification technology to the centrifugal microfluidic technology to realize molecular diagnosis based on PCR amplification by utilizing the skillfully designed centrifugal microfluidic chip structure, does not need to build a large molecular diagnosis laboratory and also does not need to adopt a large amount of manual operation, and can realize PCR amplification in the PCR amplification cavity by integrating the sample adding cavity, the sample enrichment and cracking cavity, the dilution cavity and the PCR amplification cavity, wherein the whole reaction process is in the closed microfluidic chip structure, so that the burden and the pollution possibility of operators are reduced, the whole molecular diagnosis process does not depend on the 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.

Drawings

Fig. 1 is a schematic structural diagram according to an embodiment of the present application.

Fig. 2 is another schematic view of the embodiment shown in fig. 1.

Fig. 3 is a schematic structural diagram of another embodiment of the present application.

fig. 4 is a schematic structural diagram of another embodiment of the present application.

Fig. 5 is a schematic structural diagram of another embodiment of the present application.

Fig. 6 is a schematic structural diagram of another embodiment of the present application.

fig. 7 is a schematic structural diagram of another embodiment of the present application.

Fig. 8 is a schematic structural diagram of another embodiment of the present application.

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.

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

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.

In one embodiment of the present application, a centrifugal microfluidic chip structure includes a chip substrate, and a sample loading cavity, a gas outlet, a sample enrichment and lysis cavity, a first waste liquid cavity, a first capillary valve, a dilution cavity, a second capillary valve, a PCR amplification cavity, and a second waste liquid cavity, which are disposed on the chip substrate; the sample adding cavity is provided with a sample adding hole and is communicated with the sample enrichment and lysis cavity through a sample adding cavity circulation pipeline; the sample enrichment and cracking cavity is communicated with the first waste liquid cavity through a cracking cavity circulation pipeline, and is also communicated with a cracking cavity mixing pipeline through the first capillary valve and is communicated with the dilution cavity through the cracking cavity mixing pipeline; the dilution cavity is communicated with the PCR amplification cavity and the second waste liquid cavity through the second capillary valve, and the first waste liquid cavity and the second waste liquid cavity are communicated with the gas outlet through gas circulation pipelines respectively. The centrifugal microfluidic chip structure can utilize the PCR amplification technology to be applied to the centrifugal microfluidic technology to realize molecular diagnosis based on PCR amplification through the skillfully designed chamber structure, a large-scale molecular diagnosis laboratory is not required to be built, a large amount of manual operation is not required to be adopted, and PCR amplification can be realized in the PCR amplification cavity through integration of the sample adding cavity, the sample enrichment and cracking cavity, the dilution cavity and the PCR amplification cavity.

In one embodiment, a centrifugal microfluidic chip structure comprises a part of or the whole structure of the following embodiments; namely, the centrifugal microfluidic chip structure comprises the following technical features in part or all. In one embodiment, the centrifugal microfluidic chip structure comprises a chip substrate, and a sample adding cavity, a gas outlet, a sample enrichment and lysis cavity, a first waste liquid cavity, a first capillary valve, a dilution cavity, a second capillary valve, a PCR amplification cavity and a second waste liquid cavity which are arranged on the chip substrate. It can be understood that the shapes and sizes of the sample application cavity, the gas outlet, the sample enrichment and lysis cavity, the first waste liquid cavity, the first capillary valve, the dilution cavity, the second capillary valve, the PCR amplification cavity and the second waste liquid cavity can be designed according to actual requirements. Further, in one embodiment, the sample application cavity, the gas outlet, the sample enrichment and lysis cavity, the first waste liquid cavity, the first capillary valve, the dilution cavity, the second capillary valve, the PCR amplification cavity, and the second waste liquid cavity are disposed on or in the chip substrate. In one embodiment, the centrifugal microfluidic chip structure further comprises the sample application cavity flow channel, the lysis cavity flow channel, the gas flow channel, and the lysis cavity mixing channel. Further, in one embodiment, the sample application chamber flow channel, the lysis chamber flow channel, the gas flow channel, and the lysis chamber mixing channel are disposed on or in the chip substrate.

In one embodiment, the sample adding cavity is provided with a sample adding hole, and the sample adding cavity is communicated with the sample enrichment lysis cavity through a sample adding cavity circulation pipeline; in one embodiment, the sample application hole and the gas outlet are located at the same end of the chip substrate. In one embodiment, the sample addition hole and the gas outlet are both located at one end of the chip substrate close to the centrifugal center.

In one embodiment, the sample enrichment lysis chamber is provided with lysate dry powder, glass grinding beads and/or quartz sand. And/or, in one embodiment, a ferromagnet is arranged in the sample enrichment lysis cavity. In one embodiment, the sample enrichment lysis chamber is communicated with the first waste liquid chamber through a lysis chamber flow pipeline, the sample enrichment lysis chamber is also communicated with a lysis chamber mixing pipeline through the first capillary valve, and is communicated with the dilution chamber through the lysis chamber mixing pipeline; in one embodiment, the sample enrichment lysis cavity has two functions of enrichment and lysis, and a ferromagnetic substance block is preset in the cavity. In one embodiment, the sample enrichment lysis chamber is provided with an enrichment chamber and a lysis chamber, and the enrichment chamber and the lysis chamber are separated by a filter membrane; the cracking cavity is communicated with the first waste liquid cavity through the cracking cavity circulation pipeline, and the enrichment cavity is communicated with the cracking cavity mixing pipeline through the first capillary valve. The filter membrane can intercept large granular substances such as cell tissues and the like, thereby realizing sample enrichment and separating an enrichment cavity and a cracking cavity. Further, in one embodiment, the enrichment chamber is communicated with the first capillary valve at a middle or middle-lower position, that is, the bottom of the enrichment chamber is spaced from the first capillary valve, so as to prevent the sediment of the cell debris from blocking the first capillary valve or entering the PCR amplification chamber through the first capillary valve 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 centrifugal circle center is greater than that between the neck and the centrifugal circle center, and the enrichment cavity is communicated with the first capillary valve at the position above the neck. Wherein the neck has a narrowed design relative to other regions of the enrichment chamber. In one embodiment, the sample enrichment lysis chamber is provided with an enrichment chamber and a lysis chamber, and the enrichment chamber and the lysis chamber 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 a cracking cavity mixing pipeline through the first capillary valve, and cracking liquid dry powder is arranged in the cracking cavity and/or ferromagnetism is arranged in the enrichment cavity. Further, in one embodiment, the lysis chamber is filled with dry powder of lysis solution, glass grinding beads and/or quartz sand. Further, in one embodiment, the enrichment chamber is used for enriching the sample by centrifugation, and the lysis chamber is used for overflowing the supernatant obtained by centrifugation to the first waste liquid chamber through the lysis chamber circulation pipeline.

In one embodiment, the dilution cavity is communicated with the PCR amplification cavity and the second waste liquid cavity through the second capillary valve; in one embodiment, the second waste liquid cavity is convexly provided with an auxiliary air outlet or a positioning column. In one embodiment, the number of the PCR amplification chambers is multiple, and the centrifugal microfluidic chip structure further includes a reagent distribution pipeline disposed on the chip base body, the reagent distribution pipeline respectively communicating each of the PCR amplification chambers and the second waste liquid chamber; the dilution cavity is communicated with the reagent distribution pipeline through the second capillary valve, and is respectively communicated with the PCR amplification cavity and the second waste liquid cavity through the reagent distribution pipeline. In one embodiment, the PCR amplification cavities are arranged side by side, and the centers of the PCR amplification cavities are distributed on the same circumference. In one embodiment, the number of the PCR amplification cavities is 8, the PCR amplification cavities are arranged side by side, the centers of the PCR amplification cavities are distributed on the same circumference, and the PCR amplification cavities have cylindrical structures; in one embodiment, the reagent distribution pipeline is respectively communicated with each PCR amplification cavity at the same distance position relative to the center of the centrifugal circle.

In one embodiment, the first waste liquid chamber and the second waste liquid chamber are respectively communicated with the gas outlet through gas circulation pipelines. 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 gas flow conduit has a gas branch conduit, wherein the first waste chamber communicates with the gas outlet via one of the gas branch conduits, and the second waste chamber communicates with the gas outlet via the other gas branch conduit. In one embodiment, the air outlet is provided in a convex shape. Thus, the waste liquid can be prevented from overflowing while the exhaust is convenient. Further, in one embodiment, the first waste liquid chamber is connected with a gas circulation pipeline at one end close to the center of the centrifugal circle, so that waste liquid is prevented from overflowing through the gas outlet.

In one embodiment, the chip substrate is a PMMA, PDMS, PC, ABS, COC, or COP part, or a mixed part of multiple materials. In one embodiment, the chip substrate has a partial fan-shaped structure. In one embodiment, the partial sector comprises a sector ring shape and a sector blade shape or the partial sector structure has three straight sides. The design is favorable for the regular arrangement of the centrifugal microfluidic chip structures to form a structure similar to a circular ring, so that the centrifugal effect is reasonably utilized, the processing efficiency is improved, and the centrifugal microfluidic chip structures can simultaneously carry out centrifugal processing and PCR amplification to complete the whole molecular diagnosis process.

in one embodiment, the sample application chamber, the sample enrichment lysis chamber, the first waste chamber, the dilution chamber, and the reagent distribution conduit are arranged in order in the centrifugal direction, and the gas outlet is positioned closer to the sample application chamber than to the first waste chamber; in one embodiment, the distance between the liquid level in the dilution cavity and the center of a centrifugal circle is larger than the distance between the second capillary valve and the center of the centrifugal circle; thus, when centrifugal operation is carried out, a sample in the sample adding cavity enters the sample enrichment and lysis cavity through the sample adding cavity circulation pipeline, supernatant on the upper part of the sample enrichment and lysis cavity enters the first waste liquid cavity through the sample adding cavity circulation pipeline, the sample on the lower part of the sample enrichment and lysis cavity enters the lysis cavity mixing pipeline through the first capillary valve after being enriched, then the sample enters the dilution cavity, the sample is diluted in the dilution cavity and then sequentially enters each PCR amplification cavity through the second capillary valve and the reagent distribution pipeline, and the sample is redundant to enter the second waste liquid cavity.

In one embodiment, a liquid storage container is arranged in the dilution cavity; the liquid storage container is used for containing diluent. Or the diluting cavity is used for containing a diluting liquid. Thus, PCR amplification can be used directly for molecular diagnosis without the need for a diluent. Or in one embodiment, the diluting cavity is provided with a liquid injection hole for injecting the diluting liquid. In one embodiment, the liquid injection hole is a one-way hole. Or in one embodiment, the diluting cavity is provided with a liquid injection cover for closing the liquid injection hole. Further, in one embodiment, the liquid storage container is provided with diluent. In one embodiment, the reservoir is provided with an opening closed with a heat-fusible layer. In one embodiment, the liquid storage container is adhered to the dilution cavity. 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 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 cavity, and the puncturing piece is used for matching with the elastic piece to generate displacement to puncture the sealing film during centrifugation. Further, in one embodiment, the dilution chamber is filled with a dilution liquid. In one embodiment, the diluent is disposed in a heat-fusible wrapping layer disposed in the dilution chamber. In one embodiment, the diluent is disposed in a wrapping layer disposed in the dilution chamber and the wrapping layer is provided with an opening closed by a hot melt layer.

In one embodiment, the chip substrate is packaged by hot pressing, ultrasonic welding, laser welding or gluing; therefore, a relatively sealed centrifugal micro-fluidic chip structure can be formed, and only the sample adding hole is communicated with the air outlet and the outside. Further, in one embodiment, the chip base body has a base body portion and a cover plate portion, the cover plate portion is packaged on the base body portion by using a hot pressing, ultrasonic welding, laser welding or gluing manner, and the sample adding cavity, the gas outlet, the sample enrichment and lysis cavity, the first waste liquid cavity, the first capillary valve, the dilution cavity, the second capillary valve, the PCR amplification cavity and the second waste liquid cavity are all disposed on the base body portion; further, the sample-adding cavity circulation pipeline, the lysis cavity circulation pipeline, the gas circulation pipeline and the lysis cavity mixing pipeline are all arranged on the base body part; this allows the formation of a reusable centrifugal microfluidic chip structure. Further, in one embodiment, the cover plate portion is a hot-pressed 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 centrifugal microfluidic chip structure. Further, in one embodiment, the fixing portion includes a convex portion and/or a concave portion. Therefore, the centrifugal microfluidic chip structure can be conveniently fixed, and operations such as centrifugation, PCR amplification and subsequent nucleic acid analysis and molecular diagnosis can be conveniently carried out after sample addition.

in one embodiment, as shown in fig. 1 and fig. 2, the centrifugal microfluidic chip structure includes a chip substrate 100, and a sample application cavity flow channel 121, a lysis cavity flow channel 141, a gas flow channel 151, a lysis cavity mixing channel 161, a reagent distribution channel 191, a sample application cavity 120, a gas outlet 130, a sample enrichment lysis cavity 140, a first waste liquid cavity 150, a first capillary valve 160, a dilution cavity 170, a second capillary valve 180, 8 PCR amplification cavities 190 and a second waste liquid cavity 200, which are disposed on the chip substrate 100; the sample adding cavity 120 is provided with a sample adding hole 110, and the sample adding cavity 120 is communicated with the sample enrichment lysis cavity 140 through a sample adding cavity circulation pipeline 121; the sample enrichment lysis chamber 140 is communicated with the first waste liquid chamber 150 through a lysis chamber flow line 141, the sample enrichment lysis chamber 140 is further communicated with a lysis chamber mixing line 161 through the first capillary valve 160, and is communicated with the dilution chamber 170 through the lysis chamber mixing line 161; the dilution chamber 170 is communicated with a reagent distribution pipeline 191 through the second capillary valve 180, and is respectively communicated with the PCR amplification chambers 190 and the second waste liquid chamber 200 through the reagent distribution pipeline 191, the PCR amplification chambers 190 are arranged side by side, and the first waste liquid chamber 150 and the second waste liquid chamber 200 are respectively communicated with the gas outlet 130 through a gas circulation pipeline 151. Wherein, according to the centrifugal direction, i.e. the distance relative to the centrifugal center, the sample loading cavity 120, the sample enrichment and lysis cavity 140, the first waste liquid cavity 150, the dilution cavity 170 and the reagent distribution pipeline 190 are sequentially arranged, and the position of the air outlet is closer to the sample loading cavity relative to the first waste liquid cavity. A positioning column 201 is convexly arranged at the second waste liquid cavity 200. The gas outlet 130 is arranged in a protruding manner, and the sample adding hole 110 and the gas outlet 130 are located at the same end of the chip substrate, namely, at one end close to the center of the centrifugal circle; the chip substrate 100 has a partial sector structure having three straight sides. In each embodiment, the PCR amplification chambers are arranged side by side and the centers of the PCR amplification chambers are distributed on the same circumference, that is, as shown in fig. 1 to 8, the distances between the mouths of the PCR amplification chambers and the centers of the centrifugal circles are the same.

In other embodiments, as shown in fig. 3, fig. 4, fig. 5, fig. 6, fig. 7 and fig. 8, the centrifugal microfluidic chip structure includes a chip substrate 100, and a sample application cavity flow channel 121, a lysis cavity flow channel 141, a gas flow channel 151, a lysis cavity mixing channel 161, a reagent distribution channel 191, a sample application cavity 120, a gas outlet 130, a sample enrichment lysis cavity 140, a first waste liquid cavity 150, a first capillary valve 160, a dilution cavity 170, a second capillary valve 180, 8 PCR amplification cavities 190 and a second waste liquid cavity 200, which are disposed on the chip substrate 100, and are not repeated herein.

In one embodiment, a nucleic acid analysis device comprises the centrifugal microfluidic chip structure of any one of the embodiments. The nucleic acid analysis device can apply the PCR amplification technology to the centrifugal microfluidic technology to realize molecular diagnosis based on PCR amplification by utilizing the skillfully designed centrifugal microfluidic chip structure, does not need to build a large molecular diagnosis laboratory and also does not need to adopt a large amount of manual operation, and can realize PCR amplification in the PCR amplification cavity by integrating the sample adding cavity, the sample enrichment and cracking cavity, the dilution cavity and the PCR amplification cavity, wherein the whole reaction process is in the closed microfluidic chip structure, so that the burden and the pollution possibility of operators are reduced, the whole molecular diagnosis process does not depend on the 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. In one embodiment, the nucleic acid analysis device further comprises a permanent magnet disposed below the ferromagnetic mass, and the permanent magnet is used for driving the ferromagnetic mass to move or rotate in the sample enrichment lysis cavity.

further, in one embodiment, the nucleic acid analysis apparatus uses the centrifugal microfluidic chip structure described in each embodiment, and is matched with a full-automatic nucleic acid analysis instrument to realize full automation of a molecular diagnosis project free from a nucleic acid purification step. 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 centrifugal microfluidic chip structure is provided with 8 PCR amplification cavities, and a nucleic acid analysis instrument corresponding to each amplification cavity is provided with 5 fluorescent channels, so that detection of 40 indexes 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.

in one embodiment, the centrifugal microfluidic chip structure is integrally designed in a centrifugal force driving mode. The whole microfluidic chip is of a fan-like structure, the eight microfluidic chips form a ring, and the centrifugal rotation center is located at the center of the fan-like structure. Continuing with an example of a specific application, as shown in fig. 1-8, a sample is loaded into the loading chamber 120 through the loading hole 110 while the centrifugal microfluidic chip structure is stationary. Subsequently, the microfluidic chip structure is centrifuged at a medium speed, and the sample fills the sample enrichment lysis chamber 140, i.e., the sample enrichment/lysis chamber. When the sample fills the sample enrichment lysis chamber, due to the centrifugal field, cells, tissues and/or pathogens, etc. in the sample may settle to the bottom of the sample enrichment lysis chamber 140, and the supernatant may overflow into the first waste liquid chamber 150. Thus, the function of sample enrichment is realized. In some embodiments of specific applications, a filter membrane may be further added to the sample enrichment lysis chamber 140, and effective enrichment of cells, tissues, and/or pathogens, etc. in the sample may be achieved by filtering through the filter membrane. Lysis of the cells, tissues, and/or pathogens, etc. within the sample is then achieved in the sample enrichment lysis chamber 140. The cracking method includes but is not limited to ultrasonic cracking, boiling cracking at high temperature or magnetic stirring cracking, etc. Then, high speed centrifugation is performed to make the cracked liquid break through the first capillary valve 160 and then enter the dilution chamber 170 along the mixing pipe 161 of the cracking chamber. In certain embodiments for specific applications, centrifugation at high and medium speeds may also be used to allow the centrifuged cell debris to settle sufficiently below the first capillary valve 160 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 lysis chamber 140 and to allow the lysed liquid to enter the dilution chamber 170 through the first capillary valve 160. Because the volume of the liquid after cracking is limited, at this time, the distance between the liquid level in the dilution cavity 170 and the center of the centrifugal circle is greater than the distance between the second capillary valve 180 and the center of the centrifugal circle, that is, the liquid level in the dilution cavity 170 is lower than the second capillary valve 180, that is, the liquid level in the dilution cavity 170 is lower than the communication position between the dilution cavity 170 and the second capillary valve 180, so that before dilution, no matter how large the centrifugal force is, the liquid in the dilution cavity 170 cannot break through the second capillary valve 180 and enter the subsequent reaction cavity. Further, the diluting cavity 170 is pre-filled with a diluent, and in some embodiments of specific applications, 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 to the top of the diluting cavity 170 by glue, and after heating, the paraffin melts and the diluent is released. Alternatively, in some embodiments for specific applications, the diluent in the dilution chamber 170 is pre-placed in a sealed aluminum foil placed over the spike, the spike is fixed to the top of the dilution 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 embodiments of specific applications, the diluent is released and mixed with the cracked 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. Subsequently, after high speed centrifugation, the diluted lysed liquid breaks through the second capillary valve 180 and enters the sample distribution channel, and then fills 8 PCR amplification chambers 190 from left to right at a time. PCR reaction reagent dry powder and paraffin are preset in the PCR amplification cavity 190, and in one embodiment, the PCR reaction reagent dry powder comprises enzymes, dNTPs, primers and the like required by PCR reaction. In particular, each amplification chamber in the 8 PCR amplification chambers 190 can be preset with a plurality of primers, and each amplification chamber can realize multiplex PCR. The amplification products are distinguished by different fluorescent dyes. Then, temperature cycling is started, PCR reaction starts in the PCR amplification cavity 190, and paraffin in the amplification cavity starts to melt. 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 the PCR reaction process, the microfluidic chip is always kept in a low-speed centrifugation state, so that an optical system in a full-automatic nucleic acid analyzer which is matched with the centrifugal microfluidic chip structure and arranged in the nucleic acid analysis device can respectively read the light intensity of each fluorescence channel in each PCR amplification cavity in a scanning mode, a QPCR curve is drawn, the Ct value is calculated, and a negative and positive report is given. In particular, in order to match the multiplex PCR in the PCR amplification chamber, the optical system in the fully automatic nucleic acid analyzer also has a plurality of sets of optical and photoelectric detector systems to read the fluorescent signals with different wavelengths.

In one embodiment, the optical system of the fully automatic nucleic acid analyzer can read 5-channel fluorescence signals respectively, and the specific parameters are shown in the following table:

Fluorescent dyes	Excitation light wavelength (nm)	Emission wavelength (nm)
			5-FAM	494±10	522
VIC/HEX	538±10	554
			TAMRA	560±10	582
ROX	588±10	608
			CY5	649±10	670

the following takes the typing of Human Papilloma Virus (HPV) as an example to illustrate the specific implementation of the centrifugal microfluidic chip structure. 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. The detection flow applying the centrifugal microfluidic chip structure is explained as follows.

1. Taking 1ml of cervical brushing washing liquid as a sample, and adding the sample into the sample adding cavity through the sample adding hole;

2.1000 rpm for 1 minute, after the sample is filtered and enriched by the filter membrane, 900 mul of supernatant fluid flows into the first waste liquid cavity, and the residual 100 mul of sample is left in the sample enrichment lysis cavity; alternatively, the first and second electrodes may be,

Centrifuging at 1000rpm for 1 minute, precipitating the sample at the bottom of the sample enrichment lysis cavity under the action of a centrifugal field, allowing 900 μ l of supernatant to flow into the first waste liquid cavity, and leaving the remaining 100 μ l of sample in the sample enrichment lysis cavity;

Centrifuging at 3.200 rpm for 4 minutes, driving the ferromagnetic block to move up and down in the cracking cavity by the permanent magnet fixed below the chip, accelerating the melting of the dry powder of the cracking solution and tearing the filter membrane, and stirring to accelerate the cell cracking process;

4.1000 rpm for 1 minute, fully precipitating impurities in the solution after cracking in the cracking cavity;

5.3000 rpm for 2 minutes, the supernatant in the cracking cavity breaks through the capillary valve and enters the mixing cavity and fills the bottom of the mixing cavity;

6.1000 rpm for 1 minute, heating the temperature control zone to 60 ℃, opening the end of the liquid storage container sealed by paraffin, releasing the diluent in the liquid storage container and mixing with the supernatant from the lysis cavity, and heating at 95 ℃ to lyse the HPV virus;

7.3000 rpm for 1 minute, the mixed liquid on the capillary valve in the mixing cavity breaks through the capillary valve and enters the liquid distribution pipeline to fill the amplification cavity one by one, and the redundant mixed liquid is filled in the waste liquid cavity;

8.200 rpm, and starting hot start heating, the paraffin in the PCR amplification cavity is melted and floats to the inlet of the PCR amplification cavity, the PCR amplification cavity is sealed, and then the temperature cycle of the PCR reaction is started.

Then, the operations such as fluorescence detection and negative and positive judgment can be performed in a targeted manner.

By the design, the centrifugal microfluidic chip structure is matched with a full-automatic nucleic acid analysis instrument, so that a nucleic acid analysis device is realized, and the full automation of a molecular diagnosis project without a nucleic acid purification step can be realized by adopting the nucleic acid analysis device. In the centrifugal microfluidic chip structure, sample enrichment, lysis, dilution after lysis and equal distribution are sequentially realized, and PCR amplification of multiple chambers is realized. In one embodiment, 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. Meanwhile, the micro-fluidic chip can realize the detection of multiple indexes of a single sample by matching with a full-automatic nucleic acid analyzer, and provides possibility for molecular diagnosis to realize pathogen screening facing diseases.

Other embodiments of the present application include a centrifugal microfluidic chip structure and a nucleic acid analysis device, which are capable of being implemented by combining technical features of the above embodiments.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments 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 centrifugal micro-fluidic chip structure is characterized by comprising a chip substrate, and a sample adding cavity, a gas outlet, a sample enrichment and lysis cavity, a first waste liquid cavity, a first capillary valve, a dilution cavity, a second capillary valve, a PCR amplification cavity and a second waste liquid cavity which are arranged on the chip substrate;

The sample adding cavity is provided with a sample adding hole and is communicated with the sample enrichment and lysis cavity through a sample adding cavity circulation pipeline;

The sample enrichment and cracking cavity is communicated with the first waste liquid cavity through a cracking cavity circulation pipeline, and is also communicated with a cracking cavity mixing pipeline through the first capillary valve and is communicated with the dilution cavity through the cracking cavity mixing pipeline;

The dilution cavity is communicated with the PCR amplification cavity and the second waste liquid cavity through the second capillary valve, and the first waste liquid cavity and the second waste liquid cavity are communicated with the gas outlet through gas circulation pipelines respectively.

2. the centrifugal microfluidic chip structure of claim 1, further comprising the sample application chamber flow channel, the lysis chamber flow channel, the gas flow channel, and the lysis chamber mixing channel.

3. the centrifugal microfluidic chip structure of claim 1, wherein the sample well and the gas outlet are located at the same end of the chip substrate.

4. The centrifugal microfluidic chip structure of claim 1, wherein the chip substrate has a partial fan-shaped structure.

5. the centrifugal microfluidic chip structure of claim 4, wherein the partial sector comprises a sector ring shape and a sector blade shape or the partial sector structure has three straight sides.

6. The centrifugal microfluidic chip structure of claim 1, wherein the gas outlet is convexly disposed.

7. The centrifugal microfluidic chip structure of claim 1, wherein the second waste liquid chamber is convexly provided with an auxiliary gas outlet or a positioning column.

8. The centrifugal microfluidic chip structure according to any one of claims 1 to 7, wherein the number of the PCR amplification chambers is plural, and the centrifugal microfluidic chip structure further comprises a reagent distribution pipeline disposed on the chip base body, the reagent distribution pipeline respectively communicating each of the PCR amplification chambers and the second waste liquid chamber;

the dilution cavity is communicated with the reagent distribution pipeline through the second capillary valve, and is respectively communicated with the PCR amplification cavity and the second waste liquid cavity through the reagent distribution pipeline.

9. The centrifugal microfluidic chip structure of claim 8, wherein the number of the PCR amplification cavities is 8, the PCR amplification cavities are arranged side by side, the centers of the PCR amplification cavities are distributed on the same circumference, and the PCR amplification cavities have a cylindrical structure;

The sample enrichment and lysis cavity is internally 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 a cracking cavity mixing pipeline through the first capillary valve;

According to the centrifugal direction, the sample adding cavity, the sample enrichment and lysis cavity, the first waste liquid cavity, the dilution cavity and the reagent distribution pipeline are arranged in sequence, and the position of the gas outlet is closer to the sample adding cavity relative to the first waste liquid cavity;

The distance between the liquid level in the dilution cavity and the centrifugal circle center is larger than the distance between the second capillary valve and the centrifugal circle center;

the sum of the volumes of the first waste liquid cavity and the second waste liquid cavity is not less than the volume of the sample adding cavity;

A liquid storage container is arranged in the dilution cavity;

The chip matrix is packaged by adopting a hot pressing, ultrasonic welding, laser welding or gluing mode;

The chip substrate is provided with at least three fixing parts.

10. A nucleic acid analysis device comprising the centrifugal microfluidic chip structure according to any one of claims 1 to 9.