CN209923319U - Microfluidic chip structure and analysis device - Google Patents

Abstract

The application relates to a micro-fluidic chip structure and an analysis device, wherein the micro-fluidic chip structure comprises a chip substrate, a sample adding cavity, a gas outlet, a sample enrichment cavity, a first waste liquid cavity, a dilution and cracking cavity, a capillary valve, a second waste liquid cavity, a reagent distribution pipeline and a plurality of PCR amplification cavities; the sample enrichment cavity is communicated with the first waste liquid cavity and is also communicated with the dilution cracking cavity through a sample output pipeline; the sample enrichment cavity and the lysis cavity flow pipeline are provided with a first connecting position, the sample enrichment cavity and the sample output pipeline are provided with a second connecting position, and the distance between the first connecting position and the sample adding cavity is smaller than the distance between the second connecting position and the sample adding cavity; the chip substrate is provided with a positioning area. The design of the positioning area is favorable for conveniently and quickly positioning and fixing the structure of the microfluidic chip, and the PCR amplification technology is applied to the centrifugal microfluidic technology to realize molecular diagnosis based on PCR amplification, thereby realizing the requirement of rapid detection anytime and anywhere.

Description

Microfluidic chip structure and analysis device

Technical Field

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

SUMMERY OF THE UTILITY MODEL

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

A micro-fluidic chip structure comprises a chip substrate, and a sample adding cavity, a sample enrichment cavity, a first waste liquid cavity, a dilution cracking cavity, a capillary valve, a second waste liquid cavity, a sample adding cavity circulation pipeline, a cracking cavity circulation pipeline, a gas circulation pipeline, a sample output pipeline, a reagent distribution pipeline and a plurality of PCR amplification cavities which are arranged in the chip substrate; the microfluidic chip structure 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 circulation pipeline; 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; the sample enrichment cavity and the lysis cavity flow pipeline are provided with a first connecting position, the sample enrichment cavity and the sample output pipeline are provided with a second connecting position, and the distance between the first connecting position and the sample adding cavity is smaller than the distance between the second connecting position and the sample adding cavity; the dilution and lysis cavity is communicated with a reagent distribution pipeline through a capillary valve and is respectively communicated with each PCR amplification cavity and the second waste liquid cavity through the reagent distribution pipeline; the micro-fluidic chip structure is also provided with a gas outlet communicated with the outside in the chip substrate, and the first waste liquid cavity and the second waste liquid cavity are respectively communicated with the gas outlet through gas circulation pipelines; the chip substrate is provided with a positioning area.

The microfluidic chip structure is suitable for centrifugal microfluidic analysis, sample enrichment, cracking, dilution after cracking, equal distribution and PCR amplification of multiple chambers are sequentially realized, a nucleic acid purification-free molecular diagnosis function can be realized, the design of a positioning area is favorable for conveniently and quickly positioning and fixing the microfluidic chip structure, the PCR amplification technology is applied to the centrifugal microfluidic technology to realize molecular diagnosis based on PCR amplification, a large-scale molecular diagnosis laboratory is not required to be built, a large amount of manual operation is not required, the whole reaction process is in the closed microfluidic chip structure, the burden of operators and the possibility of pollution are reduced, the whole molecular diagnosis process does not depend on a molecular diagnosis laboratory any more and does not depend on professional operators any more, the requirement of rapid detection anytime and anywhere is met, and great help is brought to medical inspection and disease prevention and control.

In other embodiments, the full automation of the molecular diagnosis project free of nucleic acid purification steps can be realized by cooperating with a full-automatic nucleic acid analyzer.

In other embodiments, the microfluidic chip structure is provided with 8 PCR amplification chambers, and a nucleic acid analyzer corresponding to each amplification chamber has 5 fluorescence channels, so that detection of 40 indexes can be simultaneously 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 positioning area is a positioning hole and the number of the positioning holes 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 structure.

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 center of rotation of the microfluidic chip structure.

In one embodiment, the sample output pipeline is provided with a phase change valve, and the chip substrate is provided with a packaging hole for communicating the outside and the phase change valve.

In one embodiment, the microfluidic chip structure has a center of rotation and the center of rotation is located outside the chip substrate;

the distance from the rotating center is arranged in the order from small to large: the device comprises a sample adding cavity, a sample enrichment cavity, a first waste liquid cavity, a dilution cracking cavity and a second waste liquid cavity;

the phase change valve is arranged at the position of the sample output pipeline close to the dilution cracking cavity;

the positioning area is arranged between the air outlet and the packaging hole;

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;

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 cracking cavity circulation pipeline is bent;

the sample output pipeline is provided with at least one direction-changing area;

the packaging hole is arranged in a sealed mode or the micro-fluidic chip structure is provided with a sealing cover part at the packaging hole.

An assay device comprising any one of the microfluidic chip structures.

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 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 another schematic view of the embodiment of fig. 1.

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

Fig. 9 is another schematic view of the embodiment of fig. 8.

Fig. 10 is another schematic view of the embodiment of fig. 8.

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 microfluidic chip structure includes a chip substrate, and a sample application cavity, a sample enrichment cavity, a first waste liquid cavity, a dilution lysis cavity, a capillary valve, a second waste liquid cavity, a sample application cavity circulation pipeline, a lysis cavity circulation pipeline, a gas circulation pipeline, a sample output pipeline, a reagent distribution pipeline, and a plurality of PCR amplification cavities disposed in the chip substrate; the microfluidic chip structure 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 circulation pipeline; 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; the sample enrichment cavity and the lysis cavity flow pipeline are provided with a first connecting position, the sample enrichment cavity and the sample output pipeline are provided with a second connecting position, and the distance between the first connecting position and the sample adding cavity is smaller than the distance between the second connecting position and the sample adding cavity; the dilution and lysis cavity is communicated with a reagent distribution pipeline through a capillary valve and is respectively communicated with each PCR amplification cavity and the second waste liquid cavity through the reagent distribution pipeline; the micro-fluidic chip structure is also provided with a gas outlet communicated with the outside in the chip substrate, and the first waste liquid cavity and the second waste liquid cavity are respectively communicated with the gas outlet through gas circulation pipelines; the chip substrate is provided with a positioning area. The microfluidic chip structure is suitable for centrifugal microfluidic analysis, sample enrichment, cracking, dilution after cracking, equal distribution and PCR amplification of multiple chambers are sequentially realized, a nucleic acid purification-free molecular diagnosis function can be realized, the design of a positioning area is favorable for conveniently and quickly positioning and fixing the microfluidic chip structure, the PCR amplification technology is applied to the centrifugal microfluidic technology to realize molecular diagnosis based on PCR amplification, a large-scale molecular diagnosis laboratory is not required to be built, a large amount of manual operation is not required, the whole reaction process is in the closed microfluidic chip structure, the burden of operators and the possibility of pollution are reduced, the whole molecular diagnosis process does not depend on a molecular diagnosis laboratory any more and does not depend on professional operators any more, the requirement of rapid detection anytime and anywhere is met, and great help is brought to medical inspection and disease prevention and control.

In one embodiment, a microfluidic chip structure includes a part or all of the following embodiments; that is, the microfluidic chip structure includes some or all of the following technical features. In one embodiment, the microfluidic chip structure comprises a chip substrate, and a sample adding cavity, a sample enrichment cavity, a first waste liquid cavity, a dilution lysis cavity, a capillary valve, a second waste liquid cavity, a sample adding cavity circulation pipeline, a lysis cavity circulation pipeline, a gas circulation pipeline, a sample output pipeline, a reagent distribution pipeline and a plurality of PCR amplification cavities which are arranged in the chip substrate; it can be understood that the shape and size of the sample application cavity, the sample enrichment cavity, the first waste liquid cavity, the dilution lysis cavity, the capillary valve, the sample application cavity flow channel, the lysis cavity flow channel, the gas flow channel, the sample output channel, the reagent distribution channel, the PCR amplification cavity, the second waste liquid cavity, and the like can be designed according to actual requirements. Further, in one embodiment, the sample application cavity, the sample enrichment cavity, the first waste liquid cavity, the dilution lysis cavity, the capillary valve, the sample application cavity flow channel, the lysis cavity flow channel, the gas flow channel, the sample output channel, the reagent distribution channel, the PCR amplification cavity, and the second waste liquid cavity are disposed in the chip substrate and are communicated with the external environment only through the sample application hole and the gas outlet or other structures such as the packaging hole. 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 beneficial to the regular arrangement of a plurality of micro-fluidic chip structures 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 chip structures 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 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 substrate has a substrate portion and a cover plate portion, the cover plate portion is packaged on the substrate portion by hot pressing, ultrasonic welding, laser welding or gluing, and the sample-adding cavity, the gas outlet, the sample enrichment cavity, the first waste liquid cavity, the dilution and lysis cavity, the capillary valve, the PCR amplification cavity and the second waste liquid cavity are all disposed on or in the substrate portion; 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 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 substrate so as to position and fix the micro-fluidic chip structure. Further, in one embodiment, the fixing portion includes a convex portion and/or a concave portion. Therefore, the structure of 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 one embodiment, the microfluidic chip structure is further provided with a sample adding hole which is communicated with the outside and the sample adding cavity 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, 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; 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; 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 first connection position is located in the middle or the middle lower part of the sample enrichment cavity, during centrifugation, liquid in the sample enrichment cavity above the first connection position enters the first waste liquid cavity, and liquid in the sample enrichment cavity below the first connection position enters the dilution and lysis cavity through the sample output pipeline after the phase change valve is opened. The first attachment site determines the total volume of liquid that will participate in the subsequent reaction. Further, in one embodiment, the first connection position is set according to the total volume of the target liquid participating in the reaction. In one embodiment, as shown in FIG. 2, the first connection location 146 is located a distance from the sample application chamber 120 that is less than the distance from the second connection location 147 to the sample application chamber 120, and the sample enriched in the sample enrichment chamber enters the dilution lysis chamber 170 through the sample output channel 161. 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. Further, in one embodiment, the sample enrichment cavity is provided with an enrichment cavity and a lysis cavity; 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, 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 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, in one embodiment, the enrichment cavity is communicated with the sample output pipeline at the lower part or the bottom position of the enrichment cavity, and 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, the bottom of the enrichment cavity is spaced from 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 chamber flow pipe is arranged in a bent mode, in one embodiment, the cracking chamber flow pipe has an П -shaped structure, further, in one embodiment, the distance between the top position of the cracking chamber flow pipe and the rotation center is smaller than the distance between the top position of the sample enrichment chamber and the rotation center, the design is that the liquid level of liquid filled on one side, close to the sample enrichment chamber, of the cracking chamber flow pipe and the liquid level in the chamber of the sample enrichment chamber are on a centrifugal circumference during high-speed centrifugation, and therefore the control effect of a siphon valve is achieved.

In one embodiment, the dilution and lysis cavity is communicated with the reagent distribution pipeline through a capillary valve and is respectively communicated with each PCR amplification cavity and the second waste liquid cavity through the reagent distribution pipeline; 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. Further, in one embodiment, the PCR amplification chamber contains dry PCR reagent powder. 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 structure 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 structure is further provided with a plurality of measurement cavities, 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, and the reagent distribution pipeline is respectively communicated with each PCR amplification cavity through each measurement cavity; in one embodiment, the microfluidic chip structure 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 microfluidic chip structure is further provided with a gas outlet communicated with the outside in the chip substrate, and the first waste liquid cavity and the second waste liquid cavity are respectively communicated with the gas outlet through gas circulation pipelines; 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, in one embodiment, the first waste liquid chamber is connected with a gas circulation pipe at one end near the rotation center so as to prevent waste liquid from overflowing through the gas outlet.

In order to facilitate the rapid and accurate installation of the microfluidic chip structure during centrifugation, in one embodiment, the chip substrate is provided with a positioning area, and the positioning area is used for positioning and installing the microfluidic chip structure. 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 structure. 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 center of rotation of the microfluidic chip structure. In one embodiment, the positioning area is disposed between the air outlet and the packaging hole.

In one embodiment, the sample output pipeline is provided with a phase change valve, the chip substrate is provided with a packaging hole communicated with the outside and the phase change valve, and the phase change valve is used for closing or communicating the sample output pipeline; in such a design, the phase change material can be injected through the packaging hole to form the phase change valve, and then the packaging hole is sealed by forming the phase change valve. Further, in one embodiment, the packaging hole is closed; furthermore, the micro-fluidic chip structure is provided with a sealing cover part at the packaging hole. In one embodiment, the phase change valve is arranged at the position of the sample output pipeline close to the dilution and lysis cavity; further, in one embodiment, the phase change valve is made of a phase change material; further, in one embodiment, 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.

In one embodiment, the diversion area has an arc-shaped structure, and further, in one embodiment, the diversion area has a C-shape or S-shape, which is beneficial to reducing the impact force of the liquid flowing to the phase change valve or the dilution cracking chamber through the sample output pipeline, so that a certain slow flow effect is realized, and the effect of the centrifugal speed is better, and in the embodiment with the phase change valve, particularly the embodiments shown in FIGS. 8 to 10, the sample enrichment method is more direct, the enrichment method is suitable for a scene that the volume of the precipitated substances in the sample is larger while the sample flows into the waste liquid chamber under the centrifugal action, and the enrichment method is suitable for a scene that the volume of the precipitated substances in the sample is larger, and the application is improved, the enrichment of the sample is also based on a density difference mode, but the capillary flow of the sample is precipitated to the bottom of the cracking chamber under the centrifugal action of the centrifugal force, and the capillary flow of the sample is more stable, and the enrichment method is suitable for a case that the capillary flow of the sample is more stable, and the enrichment method is suitable for a more stable and the application is suitable for a more efficient and more suitable for a more efficient, and more convenient, and more rapid, and more convenient, and more rapid, and more convenient, and more convenient, more rapid, and more rapid.

In one embodiment, the microfluidic chip structure 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 enrichment cavity, a first waste liquid cavity, a dilution cracking cavity and a second waste liquid cavity; namely, the sample adding cavity, the sample enrichment cavity, the first waste liquid cavity, the dilution and lysis cavity and the reagent distribution pipeline are sequentially arranged in the centrifugal direction, and the position of the gas outlet is closer to the sample adding cavity relative to the first waste liquid cavity; in one embodiment, the distance between the liquid level in the dilution lysis chamber and the rotation center is greater than the distance between the capillary valve and the rotation center; thus, when centrifugal operation is carried out, a sample in the sample adding cavity enters the sample enrichment cavity through the sample adding cavity circulation pipeline, supernatant on the upper part of the sample enrichment cavity enters the first waste liquid cavity through the cracking cavity circulation 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 sample is diluted in the dilution cracking cavity and then sequentially enters each PCR amplification cavity through the capillary valve 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. 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 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. Further, in one embodiment, a diluent is disposed in the dilute lysis chamber. 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, as shown in fig. 1, a microfluidic chip structure comprises a chip substrate 100, and a sample application cavity 120, a sample enrichment cavity 140, a first waste liquid cavity 150, a dilution lysis cavity 170, a capillary valve 180, a second waste liquid cavity 200, a sample application cavity flow channel 121, a lysis cavity flow channel 141, a gas flow channel 151, a sample output channel 161, a reagent distribution channel 191 and a plurality of PCR amplification cavities 190, which are disposed in the chip substrate 100; the chip base 100 has a partial fan-shaped structure; the microfluidic chip structure has a rotation center, the rotation center is located outside the chip substrate 100, and the distance from the rotation center is arranged in the order from small to large: the microfluidic chip structure further comprises a gas outlet 130 communicated with the outside, which is arranged in the chip substrate 100, wherein in each embodiment, the gas outlet 130 is closer to the sample adding cavity 120 relative to the first waste liquid cavity 150; the microfluidic chip structure is further provided with a sample adding hole 110 communicating the outside and the sample adding cavity 120 in the chip substrate 100, and the sample adding cavity 120 is communicated with the sample enrichment cavity 140 through a sample adding cavity circulation pipeline 121; the sample enrichment chamber 140 is communicated with the first waste liquid chamber 150 through a lysis chamber circulation pipeline 141, and the sample enrichment chamber 140 is also communicated with the dilution lysis chamber 170 through a sample output pipeline 161; referring to fig. 2, fig. 5, fig. 6 and fig. 7, the sample enrichment chamber 140 and the lysis chamber flow channel 141 have a first connection position 146, the sample enrichment chamber 140 and the sample output channel 161 have a second connection position 147, and the distance between the first connection position 146 and the sample application chamber 120 is smaller than the distance between the second connection position 147 and the sample application chamber 120; the dilution and lysis cavity 170 is communicated with a reagent distribution pipeline 191 through a capillary valve 180, and is respectively communicated with each PCR amplification cavity 190 and the second waste liquid cavity 200 through the reagent distribution pipeline 191; the PCR amplification cavities 190 are arranged side by side, the micro-fluidic chip structure is also provided with a plurality of measurement cavities 192 and reagent conveying pipelines 193 thereof, the measurement cavities are arranged in one-to-one correspondence with the PCR amplification cavities 190, each measurement 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 measurement cavity 192 and is communicated with each PCR amplification cavity 190, and the measurement cavities 192 are communicated with the corresponding PCR amplification cavities 190 through the reagent conveying pipelines 193 thereof; 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; the gas circulation pipe 151 is provided with a branch channel 152, the first waste liquid cavity 150 is communicated with the gas circulation pipe 151 through the branch channel 152, and the position where the first waste liquid cavity 150 is communicated with the branch channel 152 is positioned at the position where the first waste liquid cavity 150 is closest to the sample adding cavity 120; the chip substrate 100 is provided with a positioning area 101, the sample output pipeline 161 is provided with a phase change valve 162, and the phase change valve 162 is arranged at the position of the sample output pipeline 161 close to the dilution cracking cavity 170; as shown in fig. 3 and 4, the chip substrate 100 is provided with a packaging hole 163 communicating with the outside and the phase change valve 162, the sample addition hole 110, the gas outlet 130 and the packaging hole 163 are respectively communicated with the outside, the positioning region 101 is a positioning groove, the positioning groove is arc-shaped, the center of the arc coincides with the rotation center of the microfluidic chip structure, and the positioning region 101 is disposed between the gas outlet 130 and the packaging hole 163. Furthermore, a positioning column 201 is convexly disposed at the second waste liquid cavity 200. The sample hole 110 and the gas outlet 130 are located at the same end of the chip substrate, i.e. the end near the rotation center; the chip substrate 100 has a partial fan-shaped structure and the partial fan-shaped structure has three straight sides. In each embodiment, the PCR amplification chambers are arranged side by side, that is, as shown in FIGS. 1 to 8, the mouth of each PCR amplification chamber is at the same distance from the center of rotation.

With the embodiment shown in fig. 1 to 7, in a specific application, when the microfluidic chip structure is stationary, a sample is loaded into the loading chamber 120 through the loading hole 110. Subsequently, the microfluidic chip structure is centrifuged at high speed, and the sample fills the sample enrichment chamber 140 through the sample application chamber flow channel 121. In various embodiments, the high speed centrifugation is performed at a speed of about 3000rpm to about 6000 rpm. Due to the existence of the phase change valve 162, the liquid of the sample enrichment chamber 140 cannot enter the subsequent reaction chamber through the sample output pipe 161. The lysis chamber flow channel 141 may be a normal liquid flow channel or may additionally have a valve 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. If the lysis chamber circulation line 141 is a normal liquid circulation line, when the sample fills the sample enrichment chamber 140, cells, tissues, pathogens, etc. in the sample will settle to the bottom of the sample enrichment chamber 140 due to the centrifugal field, and the supernatant will overflow into the first waste liquid chamber 150 through 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. If the lysis chamber circulation line 141 has a siphon valve or a phase change valve, then the sample can be centrifuged at a high speed at this time to fully enrich the sample in the sample enrichment chamber 140, and then if the lysis chamber circulation line 141 has a siphon valve, the rotation speed is reduced to make the supernatant in the sample enrichment chamber 140 break through the siphon valve of the lysis chamber circulation line 141 and enter the first waste liquid chamber 150, or if the lysis chamber circulation line 141 has a phase change valve, the phase change valve is opened by heating or other phase change methods to make the supernatant in the sample enrichment chamber 140 break through the phase change valve of the lysis chamber circulation line 141 and enter 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.

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. Heating the phase change valve 162 melts the phase change material of the phase change valve 162, so that the remaining liquid in the sample enrichment chamber 140 can flow into the dilution lysis chamber 170 via the sample output pipe 161. Phase change materials 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 capillary valve 180 and the centrifugal center, i.e., the liquid level in the dilution cracking cavity 170 is lower than the capillary valve 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 capillary valve 180, before the dilution, no matter how large the centrifugal force is, the liquid in the dilution cracking cavity 170 cannot break through the capillary valve 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 cracked liquid, and the mixing can be realized by rotating the microfluidic chip structure forward and backward or rotating the microfluidic chip structure at an accelerated speed and a decelerated speed.

Subsequently, upon centrifugation at medium speed, the diluted lysed liquid breaks through the capillary valve 180 and enters the reagent dispensing channel 191, and fills the measuring chambers 192 in sequence from left to right. After high speed centrifugation, the liquid in each measurement cavity 192 enters into the corresponding PCR amplification cavity 190 through the reagent delivery pipe 193. 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, 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, starting temperature cycle, starting 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 the PCR reaction process, the structure of the microfluidic 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, 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 microfluidic chip structure has a center of rotation and the center of rotation is located outside the chip substrate; the distance from the rotating center is arranged in the order from small to large: the device comprises a sample adding cavity, a sample enrichment cavity, a first waste liquid cavity, a dilution cracking cavity and a second waste liquid cavity; the phase change valve is arranged at the position of the sample output pipeline close to the dilution cracking cavity; the positioning area is arranged between the air outlet and the packaging hole; 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; 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 cracking cavity circulation pipeline is bent; the sample output pipeline is provided with at least one direction changing area.

In one embodiment, as shown in fig. 8, 9 and 10, the lysis chamber flow channel 141 is bent and has an П -shaped structure, the distance between the top position of the lysis chamber flow channel 141 and the rotation center is smaller than 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 smaller than the distance between the top position of the sample enrichment chamber 140 and the sample application chamber 120, and the sample output channel 161 has a first direction changing region 166 and a second direction changing region 167.

In one embodiment, the whole design of the microfluidic chip structure adopts a centrifugal force driving mode. The whole microfluidic chip structure is of a fan-shaped structure, the eight microfluidic chip structures form a circular 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 structure 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. Detection and typing of HPVDNA has important value for understanding disease condition, judging prevention and guiding treatment.

In the embodiments shown in fig. 1 to 7, the detection process using the structure of 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 centrifugal field, the cells, tissues and pathogens in the sample can be fully precipitated to the bottom of the chamber, and the supernatant can enter the first waste liquid chamber 150;

3. heating the sample enrichment cavity 140 at 95 ℃ or higher, and boiling at high temperature to fully crack cells, tissues and pathogens in the sample;

4.1500rpm, and then the phase change valve 162 is heated to melt the phase change material sealing the phase change valve, the liquid in the sample enrichment chamber 140 flows into the dilution lysis chamber 170 through the sample output line 161. In one embodiment, the phase change valve 162 is heated to 60 ℃ or higher, the paraffin melts, the sample output line 161 is connected, and the remaining liquid in the sample enrichment chamber 140 flows into the dilution lysis chamber 170 through the sample output line 161.

5. The centrifugal speed is reduced to 200rpm, because the diluting and cracking cavity 170 is pre-filled with diluent, the diluent is pre-filled in an aluminum foil with one end packaged by paraffin and the other end bonded by glue, the aluminum foil is bonded on the top of the diluting and cracking cavity 170 by the glue, the diluting and cracking cavity 170 is heated at 70 ℃ or higher, after heating, the paraffin is melted, and the diluent is released.

6. After the liquid cracked in the dilution cracking cavity 170 and the diluent are fully mixed, the centrifugal speed is increased to 1500rpm, the liquid in the dilution cracking cavity 170 above the inlet of the capillary valve 180 breaks through the capillary valve 180, enters the reagent distribution pipeline 191, fills all the measurement cavities 192 in sequence, and the redundant liquid enters the second waste liquid cavity 200;

7.3000rpm centrifugal microfluidic chip structure, 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, melting and floating the paraffin wax preset in the PCR amplification cavity 190 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 fluorescent signal in each PCR amplification chamber during the extension phase of each cycle to plot a PCR curve.

In the embodiments shown in fig. 8 to 10, the detection process using the structure of 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 centrifugal field, the cells, tissues and pathogens in the sample can be fully precipitated to the bottom of the chamber, and the supernatant can enter the first waste liquid chamber 150;

3. the centrifugal speed is reduced to 200rmm and then increased to 1500rpm, and the liquid above the inlet of the cracking cavity circulating pipeline 141 with siphon pipeline effect in the sample enrichment cavity 140 enters the first waste liquid cavity 150 along the cracking cavity circulating pipeline 141;

4. heating the sample enrichment cavity 140 at 95 ℃ or higher, and boiling at high temperature to fully crack cells, tissues and pathogens in the sample;

5. the centrifugal speed of 1500rpm is maintained, then the phase change valve 162 is heated to melt the phase change material sealing the phase change valve, and the liquid in the sample enrichment chamber 140 flows into the dilution lysis chamber 170 through the sample output pipe 161. In one embodiment, the phase change valve 162 is heated to 60 ℃ or higher, the paraffin melts, the sample output line 161 is connected, and the remaining liquid in the sample enrichment chamber 140 flows into the dilution lysis chamber 170 through the sample output line 161.

6. The centrifugal speed is reduced to 200rpm, because the diluting and cracking cavity 170 is pre-filled with diluent, the diluent is pre-filled in an aluminum foil with one end packaged by paraffin and the other end bonded by glue, the aluminum foil is bonded on the top of the diluting and cracking cavity 170 by the glue, the diluting and cracking cavity 170 is heated at 70 ℃ or higher, after heating, the paraffin is melted, and the diluent is released.

7. After the liquid cracked in the dilution cracking cavity 170 and the diluent are fully mixed, the centrifugal speed is increased to 1500rpm, the liquid in the dilution cracking cavity 170 above the inlet of the capillary valve 180 breaks through the capillary valve 180, enters the reagent distribution pipeline 191, fills all the measurement cavities 192 in sequence, and the redundant liquid enters the second waste liquid cavity 200;

8.3000rpm centrifugal microfluidic chip structure, 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 9.500rpm, starting hot start heating, melting and floating the paraffin wax preset in the PCR amplification cavity 190 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 fluorescent signal in each PCR amplification chamber during the extension phase of each cycle to plot a PCR curve.

Then, fluorescence detection, negative and positive judgment and other operations can be performed in a targeted manner, and data processing, writing into a database and printing a report can also be performed.

By the design, the 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 structure, 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 anytime and anywhere is met, and great help is brought to medical inspection and disease prevention and control. Meanwhile, the micro-fluidic chip structure 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.

In one embodiment, an assay device comprises any one of the microfluidic chip structures. Further, in one embodiment, the assay device is provided with at least three fluorescence channels; further, in one of the embodiments, the nucleic acid analyzing apparatus is provided with 5 kinds of fluorescence channels. In one embodiment, the analysis device is used for controlling the structure of the microfluidic chip to keep a low-speed centrifugation state during the PCR reaction; in one embodiment, the analysis device is further configured to scan and read out light intensity of each fluorescence channel in each PCR amplification cavity 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 device 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 device 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. Further, in one embodiment, the assay device is provided with at least three fluorescence channels; and/or the analysis device is used for controlling the structure of the microfluidic chip to keep a low-speed centrifugal state in the PCR reaction process; and/or the analysis device is also used for respectively scanning and reading 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 device 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 cell residues after cracking to be precipitated at the bottom of the sample enrichment cavity and enable the sample after cracking to enter the dilution cracking cavity, heating to melt paraffin in the dilution cracking cavity so as to release diluent to enable the sample after cracking 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 device further comprises a permanent magnet disposed below the ferromagnetic mass.

Further, in one embodiment, the nucleic acid analysis device uses the microfluidic chip structure of each embodiment, and is matched 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 structure of the microfluidic chip, sample enrichment, lysis, dilution after lysis and equal distribution are sequentially realized, and PCR amplification of multiple chambers is realized. In one embodiment, the 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 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 structure, 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 include a microfluidic chip structure and an analysis device, 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 (10)

1. A micro-fluidic chip structure is characterized by comprising a chip substrate, and a sample adding cavity, a sample enrichment cavity, a first waste liquid cavity, a dilution cracking cavity, a capillary valve, a second waste liquid cavity, a sample adding cavity circulation pipeline, a cracking cavity circulation pipeline, a gas circulation pipeline, a sample output pipeline, a reagent distribution pipeline and a plurality of PCR amplification cavities which are arranged in the chip substrate;

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 the sample adding cavity circulation pipeline;

the sample enrichment cavity is communicated with the first waste liquid cavity through the cracking cavity circulation pipeline, and the sample enrichment cavity is also communicated with the dilution cracking cavity through the sample output pipeline; the sample enrichment cavity and the lysis cavity flow channel are provided with a first connecting position, the sample enrichment cavity and the sample output channel are provided with a second connecting position, and the distance between the first connecting position and the sample adding cavity is smaller than the distance between the second connecting position and the sample adding cavity;

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

the micro-fluidic chip is also provided with a gas outlet communicated with the outside in the chip substrate, and the first waste liquid cavity and the second waste liquid cavity are respectively communicated with the gas outlet through the gas circulation pipeline;

the chip substrate is provided with a positioning area.

2. The microfluidic chip structure of claim 1, wherein the positioning region is a positioning hole and the number of positioning holes is at least one.

3. The microfluidic chip structure according to claim 2, wherein the number of the positioning holes is plural and is uniformly distributed with respect to a rotation center of the microfluidic chip structure.

4. The microfluidic chip structure of claim 1, wherein the positioning region is a positioning groove.

5. The microfluidic chip structure of claim 4, wherein the positioning grooves are linear.

6. The microfluidic chip structure of claim 4, wherein the positioning groove is arc-shaped.

7. The microfluidic chip structure of claim 6, wherein the center of the arc line coincides with the center of rotation of the microfluidic chip structure.

8. The microfluidic chip structure according to any one of claims 1 to 7, wherein the sample output pipeline is provided with a phase change valve, and the chip substrate is provided with a packaging hole for communicating the outside and the phase change valve.

9. The microfluidic chip structure according to claim 8, wherein the microfluidic chip structure has a center of rotation and the center of rotation is located outside the chip substrate;

the distances from the rotating center are arranged in the order from small to large: the sample application chamber, the sample enrichment chamber, the first waste fluid chamber, the dilution lysis chamber, and the second waste fluid chamber;

the phase change valve is arranged at the position of the sample output pipeline close to the dilution cracking cavity;

the positioning area is arranged between the air outlet and the packaging hole;

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 where the first waste liquid cavity is closest to the sample adding cavity;

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 cracking cavity circulation pipeline is arranged in a bending way;

the sample output pipeline is provided with at least one direction-changing area;

the packaging hole is arranged in a sealed mode or a sealing cover portion is arranged at the packaging hole of the micro-fluidic chip.

10. An analysis device comprising the microfluidic chip structure according to any one of claims 1 to 9.

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