CN110142066A

CN110142066A - Micro-fluidic chip and analysis system

Info

Publication number: CN110142066A
Application number: CN201910339790.9A
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: 2019-04-25
Filing date: 2019-04-25
Publication date: 2019-08-20
Anticipated expiration: 2039-04-25
Also published as: CN110142066B

Abstract

Micro-fluidic chip and analysis system, micro-fluidic chip include well, feeding chamber, gas outlet, sample enrichment chamber, waste liquid chamber, dilution cracking chamber, siphon piping, feeding chamber circulation duct, cracking chamber circulation duct, gas flow pipe road, sample output channel, reagent distribution pipeline, outlet pipe and PCR amplification chamber；Dilution cracking chamber sequence passes through siphon piping and is respectively communicated with each PCR amplification chamber and the second waste liquid chamber；Gas outlet is also connected to position that a waste liquid chamber and waste liquid chamber are connected to gas outlet compared with gas outlet further from rotation center by outlet pipe.It is diluted after the enrichment of sample, cracking, cracking and the PCR amplification of equivalent distribution, multi-chamber is all able to sequence and realizes, it can be realized and exempt from nucleic acid purification molecular diagnosis function, on the one hand the position of gas outlet can be adjusted according to demand, on the other hand the relativeness for capillary force and centrifugal force being dexterously utilized by designing siphon piping forms controlling valve to control liquid and flow into reagent distribution pipeline.

Description

Micro-fluidic chip and analysis system

Technical field

This application involves centrifugal microfluidic control fields, more particularly to micro-fluidic chip and analysis system.

Background technique

Micro-fluidic (Microfluidics) refers to the handling liquids on submillimeter scale, wherein submillimeter scale is generally Several microns are arrived several hundred microns.Function, including sampling, dilution, reaction, separation, detection etc. of the microflow control technique entire laboratory It is integrated on a small chip, therefore also known as chip lab (Lab-on-a-Chip).This chip is usually by various liquid storages Pond and microchannel network interconnected composition can be shortened the sample process time, and control liquid flowing by accurate, realize examination The maximum utilization efficiency of agent consumptive material.Microfluidic system refers to the device of the handling liquids on submillimeter scale.Centrifugal microfluidic control is subordinate to Belong to a micro-fluidic branch, refers in particular to drive the flowing of liquid by rotation centrifugal microfluidic control chip, to realize use Centrifugal force handling liquids on submillimeter scale.Centrifugal microfluidic control is by basic operation unit collection involved in biological and chemical field On (disc-shaped) chip of Cheng Yi small-sized dish-style.Other than micro-fluidic specific advantage, due to centrifugal microfluidic control A motor is only needed to provide power required for liquid manipulation, so whole equipment more concision and compact.And disc-type chip On ubiquitous centrifugal field liquid driven can be made more effective, it is ensured that without residual liquid in pipeline, and can be effective Realization based on density variation sample separate, also parallel processing can be allowed more simple.Therefore, centrifugal microfluidic control is also by increasingly More applies in care diagnostic.

The molecular diagnosis of based on PCR amplification generally comprises following steps: sample cracking, nucleic acid purification, nucleic acid draw specific The lower amplification of object constraint, the acquisition and analysis of fluorescence signal.In the project of certain molecular diagnosises, since sample is fairly simple, often Often can directly it be expanded after sample cracking；On the other hand, increasingly the one-step method DNA of maturation extracts amplification examination now The appearance of agent box is possibly realized but also directly expanding after sample cracking, avoids this more complicated step of nucleic acid purification. But in the molecular diagnosis system of based on PCR amplification, due to having Aerosol Pollution when PCR amplification, also for avoiding sample Cross contamination between this, will set up a subregion laboratory under normal circumstances.This laboratory will realize sample process, nucleic acid It extracts, the division operation of PCR amplification, and must have good ventilating system, at high cost, often only large size is built in laboratory Just there are the financial resources built in medical institutions.On the other hand, laboratory operation personnel will take appointment with certificate, also considerably increase manually at This.At the same time, excessive artificial intervention certainly will also bring along artificial operation error.These problems, which greatly increase, to be based on The technology of the molecular diagnosis of PCR uses threshold.

And current molecular diagnostic laboratories mode, concentrating experimental site to complete multisample and more detection projects behaviour Make, process quality control requires high.Also, current molecular diagnostic laboratories mode, generally multisample single index detect mould Formula, Testing index is limited, cannot achieve the screening of single sample multi objective pathogen infection.In addition, although molecular diagnostic techniques are excellent For gesture it is obvious that still due to its complex steps, process is time-consuming, and professional is needed to operate, and clinical molecular diagnosis laboratory To build cost generally higher, so molecular diagnosis is also expensive.

Summary of the invention

Based on this, it is necessary to provide a kind of micro-fluidic chip and analysis system.

A kind of micro-fluidic chip, micro-fluidic chip have rotation center, and micro-fluidic chip includes chip basal body, and setting Well, feeding chamber, gas outlet, sample enrichment chamber, waste liquid chamber, dilution cracking chamber, siphon piping, sample-adding in chip basal body Chamber circulation duct, cracking chamber circulation duct, gas flow pipe road, sample output channel, reagent distribution pipeline, outlet pipe and more A PCR amplification chamber；Waste liquid chamber includes the first waste liquid chamber and the second waste liquid chamber；Well is respectively communicated with external and feeding chamber, and adds Sample chamber is connected to sample by feeding chamber circulation duct and is enriched with chamber；Sample is enriched with chamber and is connected to the first waste liquid by cracking chamber circulation duct Chamber, sample are enriched with chamber and also pass through sample output channel connection dilution cracking chamber in its bottom position far from rotation center；It is dilute Release cracking chamber sequence pass through siphon piping and reagent distribution pipeline be respectively communicated with each PCR amplification chamber and the second waste liquid chamber；First is useless Sap cavity is connected to the second waste liquid chamber by gas flow pipe road；The external setting of gas outlet connection, gas outlet is also connected by outlet pipe A logical waste liquid chamber, and the position of waste liquid chamber being connected with gas outlet compared with gas outlet more far from rotation center.It is above-mentioned micro-fluidic Chip, is suitable for centrifugal microfluidic control and analyzes, and dilutes after the enrichment of sample, cracking, cracking and the PCR of equivalent distribution, multi-chamber Amplification be all able to sequence realize, can be realized and exempt from nucleic acid purification molecular diagnosis function, one side gas outlet be connected to a waste liquid chamber and It is connected to another waste liquid chamber by the waste liquid chamber, the position of gas outlet can be adjusted according to demand, on the other hand passes through design siphon pipe The relativeness of capillary force and centrifugal force is dexterously utilized in road, forms controlling valve to control liquid and flow into reagent dispensing tube Road is applied to the molecular diagnosis that centrifugal microfluidic control technology realizes based on PCR amplification, entire reaction process using PCR amplification In closed micro-fluidic chip, the molecular diagnosis effect quickly detected whenever and wherever possible is realized.

Chip basal body offers positioning area in one of the embodiments,.Gas outlet passes through in one of the embodiments, Outlet pipe is connected to the second waste liquid chamber.Gas outlet is connected to the first waste liquid chamber by outlet pipe in one of the embodiments,.? In one embodiment, siphon piping has bending structure.Further, bending structure isCharacter form structure or arch knot Structure.The minimum range of bending structure and rotation center is less than or equal in dilution cracking chamber and rotation in one of the embodiments, The minimum range of the heart.Chip basal body is prepared using water wetted material in one of the embodiments,.In one of the embodiments, Siphon piping inner surface is equipped with hydrophilic material or inner surface carries out hydrophilic treated.Sample is defeated in one of the embodiments, Pipeline is equipped with the first phase transformation valve out, and chip basal body is equipped with the first encapsulated holes of connection outside and the first phase transformation valve, micro-fluidic core Piece is equipped with the first closing cover at the first encapsulated holes；Sample, which is enriched with chamber, has the top position of neighbouring rotation center and in top Blow vent is equipped at position, and gas outlet is connected to the second waste liquid chamber by outlet pipe, gas flow pipe road is equipped with the second phase transformation Valve, and chip basal body is equipped with the second encapsulated holes of the second phase transformation valve of connection, micro-fluidic chip is equipped with second at the second encapsulated holes Close cover；Microfluidic chip structure is additionally provided with multiple measurement chambers, and each chamber that measures is arranged in a one-to-one correspondence with each PCR amplification chamber, often One measurement chamber is set between reagent distribution pipeline and a PCR amplification chamber and reagent distribution pipeline passes through each measurement chamber respectively and is connected to Each PCR amplification chamber；Chip basal body has partial sector structure；Chip basal body offers positioning area and positioning area is set to gas outlet Between the second waste liquid chamber；Positioning area is positioning convex portion, location hole or locating slot, and the quantity of positioning area is one, two or more It is a；Locating slot is linear or locating slot is that the center of circle of arc line shaped and its camber line and the rotation center of micro-fluidic chip coincide； The quantity of location hole is that multiple and relative to micro-fluidic chip rotation centers are uniformly distributed；Rotation center is located at outside chip basal body Portion is arranged from small to large ord as at a distance from rotation center are as follows: feeding chamber, sample enrichment chamber, the first waste liquid chamber, dilution are split Solve chamber, reagent distributes pipeline, the second waste liquid chamber, PCR amplification chamber；It is equipped with liquid storage container in dilution cracking chamber, liquid storage container is used for Accommodate dilution；PCR reaction reagent dry powder, fluorescent dye and seal are equipped in PCR amplification chamber.

A kind of analysis system comprising any one micro-fluidic chip.

Detailed description of the invention

Fig. 1 is the structural schematic diagram of one embodiment of the application.Fig. 2 is enlarged diagram at the A of embodiment illustrated in fig. 1.Fig. 3 For the schematic diagram of another direction of embodiment illustrated in fig. 1.Fig. 4 is the schematic diagram of another direction of embodiment illustrated in fig. 1.Fig. 5 is Fig. 1 institute Show the schematic diagram of another direction of embodiment.Fig. 6 is the schematic diagram of another direction of embodiment illustrated in fig. 1.Fig. 7 is to implement shown in Fig. 6 Enlarged diagram at the B of example.Fig. 8 is the schematic diagram of another direction of embodiment illustrated in fig. 1.Fig. 9 is another embodiment of the application Structural schematic diagram.Figure 10 is the schematic diagram of another direction of embodiment illustrated in fig. 9.Figure 11 is the other direction of embodiment illustrated in fig. 9 Schematic diagram.Figure 12 is the structural schematic diagram of another embodiment of the application.Figure 13 is that the other direction of embodiment illustrated in fig. 12 is illustrated Figure.Figure 14 is the schematic diagram of another direction of embodiment illustrated in fig. 12.Figure 15 is the schematic diagram of another direction of embodiment illustrated in fig. 12.

Specific embodiment

In order to make the above objects, features, and advantages of the present application more apparent, with reference to the accompanying drawing to the application Specific embodiment be described in detail.Many details are explained in the following description in order to fully understand this Shen Please.But the application can be implemented with being much different from other way described herein, those skilled in the art can be not Similar improvement is done in the case where violating the application intension, therefore the application is not limited by the specific embodiments disclosed below.

Unless otherwise defined, all technical and scientific terms used herein and the technical field for belonging to the application The normally understood meaning of technical staff is identical.The term used in the description of the present application is intended merely to description tool herein The purpose of the embodiment of body, it is not intended that in limitation the application.Term " and or " used herein includes one or more Any and all combinations of relevant listed item.

Although traditional molecular diagnostic techniques advantage is it is obvious that still due to its complex steps, process is time-consuming, needs profession Personnel's operation, and clinical molecular diagnosis laboratory to build cost generally higher, so molecular diagnosis is also expensive.At this Apply in one embodiment, a kind of micro-fluidic chip, micro-fluidic chip has rotation center, and micro-fluidic chip includes chip base Body, and the well, feeding chamber, gas outlet, sample enrichment chamber, waste liquid chamber, dilution the cracking chamber, rainbow that are set in chip basal body Suction pipe road, feeding chamber circulation duct, cracking chamber circulation duct, gas flow pipe road, sample output channel, reagent distribution pipeline, Outlet pipe and multiple PCR amplification chambers；Waste liquid chamber includes the first waste liquid chamber and the second waste liquid chamber；Well be respectively communicated with it is external and Feeding chamber, and feeding chamber is connected to sample by feeding chamber circulation duct and is enriched with chamber；Sample is enriched with chamber and passes through cracking chamber circulation duct It is connected to the first waste liquid chamber, it is also dilute by the connection of sample output channel in its bottom position far from rotation center that sample is enriched with chamber Release cracking chamber；Dilution cracking chamber sequence passes through siphon piping and reagent distribution pipeline is respectively communicated with each PCR amplification chamber and second useless Sap cavity；First waste liquid chamber is connected to the second waste liquid chamber by gas flow pipe road；The external setting of gas outlet connection, gas outlet also passes through Outlet pipe be connected to a waste liquid chamber, and the position of waste liquid chamber being connected with gas outlet compared with gas outlet more far from rotation center. Above-mentioned micro-fluidic chip, is suitable for centrifugal microfluidic control and analyzes, and dilutes after the enrichment of sample, cracking, cracking and equivalent distribution, more The PCR amplification of chamber is all able to sequence and realizes, can be realized and exempts from nucleic acid purification molecular diagnosis function, one side gas outlet connection one Waste liquid chamber and another waste liquid chamber is connected to by the waste liquid chamber, the position of gas outlet can be adjusted according to demand, on the other hand by setting The relativeness of capillary force and centrifugal force is dexterously utilized in meter siphon piping, forms controlling valve to control liquid and flow into reagent Distribute pipeline, is applied to the molecular diagnosis that centrifugal microfluidic control technology realizes based on PCR amplification using PCR amplification, it is entire anti- It answers process to be in closed micro-fluidic chip, realizes the molecular diagnosis effect quickly detected whenever and wherever possible.

A kind of micro-fluidic chip in one of the embodiments, comprising the part-structure of following embodiment or all knot Structure；That is, micro-fluidic chip includes some technical characteristics or all technical features below.It is a kind of in one of the embodiments, Micro-fluidic chip comprising chip basal body, and the well, the feeding chamber, gas outlet, sample enrichment that are set in chip basal body Chamber, waste liquid chamber, dilution cracking chamber, siphon piping, feeding chamber circulation duct, cracking chamber circulation duct, gas flow pipe road, sample Output channel, reagent distribution pipeline, outlet pipe and multiple PCR amplification chambers；It is understood that well, feeding chamber, outlet Mouth, sample enrichment chamber, waste liquid chamber, dilution cracking chamber, siphon piping, feeding chamber circulation duct, cracking chamber circulation duct, gas stream It threads a pipe, sample output channel, reagent distribute the shapes and sizes of pipeline, outlet pipe and PCR amplification chamber etc. according to practical need Seek design.Further, feeding chamber, sample enrichment chamber, waste liquid chamber, dilution cracking chamber, siphon piping, feeding chamber runner pipe Road, cracking chamber circulation duct, gas flow pipe road, sample output channel, reagent distribution pipeline, outlet pipe and multiple PCR expand Increase chamber to be opened in chip basal body, only external by such as encapsulated holes connection of well and gas outlet or other structures is external rings Border.

Micro-fluidic chip has rotation center in one of the embodiments, and rotation center is located at outside chip basal body； Arranged from small to large ord by a distance from rotation center in one of the embodiments, are as follows: feeding chamber, sample enrichment chamber, First waste liquid chamber, dilution cracking chamber, reagent distribute pipeline, the second waste liquid chamber, PCR amplification chamber；；Positioned at the top position of feeding chamber Place is feeding chamber closest at the position of rotation center, and also that is, pressing centrifugal direction, feeding chamber, sample are enriched with chamber, the first waste liquid Chamber dilutes cracking chamber, reagent distribution pipeline sequence setting, and the position of gas outlet is relative to the closer sample-adding of the first waste liquid chamber Chamber；Further, it is arranged from small to large ord by the minimum range with rotation center are as follows: feeding chamber, sample are enriched with chamber, first Waste liquid chamber, dilution cracking chamber, the second waste liquid chamber, reagent distribute pipeline, the second waste liquid chamber；Each PCR amplification chamber well and rotation The minimum range at center is equal to or less than the minimum range of the second waste liquid chamber and rotation center.It is dilute in one of the embodiments, It releases and cracks the minimum range that the distance between intracavitary liquid level and rotation center are greater than between siphon piping and rotation center；That is rainbow The minimum range of suction pipe road and rotation center is less than or equal to dilution and cracks intracavitary the distance between liquid level and rotation center.Each reality It applies in example, dilution cracking chamber is connected to siphon piping in its bottom position far from rotation center.In this way, carrying out centrifugally operated When, the sample in feeding chamber enters sample by feeding chamber circulation duct and is enriched with chamber, and the supernatant on sample enrichment chamber top passes through It cracks chamber circulation duct and enters the first waste liquid chamber, sample enters sample output channel after being enriched with the sample enrichment of chamber lower part, then Into dilution cracking chamber, pipeline is distributed sequentially into each by siphon piping and reagent after diluting cracking chamber and being diluted PCR amplification chamber, extra part enter the second waste liquid chamber.Liquid storage is equipped in dilution cracking chamber in one of the embodiments, to hold Device；Liquid storage container is for accommodating dilution.Alternatively, dilution cracking chamber is for accommodating dilution.In this way, being carrying out PCR amplification It can directly be used when as molecular diagnosis, without reassembling dilution.Alternatively, dilution cracking in one of the embodiments, Chamber is equipped with liquid injection hole, and liquid injection hole is for injecting dilution.Liquid injection hole is unidirectional hole in one of the embodiments,.Alternatively, at it In middle one embodiment, dilution cracking chamber is equipped with the fill cap of closing liquid injection hole.Further, dilution is equipped in liquid storage container Liquid.Liquid storage container, which is equipped with, in one of the embodiments, uses the closed opening of hot melt layer.It stores up in one of the embodiments, Liquid container is viscous to be set in dilution cracking chamber.Liquid storage container has aluminium foil layer in one of the embodiments,.An implementation wherein In example, liquid storage container is equipped with opening, pierces part, elastic component and sealer, and sealer pierces part for closing opening, the connection of elastic component one end, The other end is fixed in dilution cracking chamber, and thorn part is used for the cooperation elastic component in centrifugation and generates displacement to puncture sealer.Further Ground, dilution, which cracks, is equipped with dilution in chamber.Dilution is set in hot melt wrapping layer in one of the embodiments, hot melt packet Covering layer is set in dilution cracking chamber.Dilution is set in wrapping layer in one of the embodiments, and wrapping layer is set to dilute It releases in cracking chamber and wrapping layer is equipped with using the closed opening of hot melt layer.

Chip basal body is prepared using water wetted material in one of the embodiments, or cracking chamber circulation duct inner surface is set There are hydrophilic material or inner surface to carry out hydrophilic treated.Siphon piping inner surface is equipped with hydrophilic in one of the embodiments, Material layer or inner surface carry out hydrophilic treated.In one of the embodiments, chip basal body be PMMA, PDMS, PC, ABS, COC or COP product.Chip basal body has partial sector structure in one of the embodiments,.In one of the embodiments, Partial sector includes that fan annular and flaabellum shape or partial sector structure have three straight flanges.Such design is conducive to multiple micro- Fluidic chip is regularly arranged to form the structure for being similar to circular ring shape, to rationally utilize centrifugal action, improves treatment effeciency, so that Multiple micro-fluidic chips can carry out centrifugal treating simultaneously and carry out PCR amplification and then complete entire molecular diagnostic procedure.At it In middle one embodiment, chip basal body is using hot pressing, ultrasonic bonding, laser welding or adhesive means encapsulation；It in this way can be with shape At the micro-fluidic chip of an opposing seal, only well is in communication with the outside with gas outlet.Further, chip basal body has Matrix part and ceiling plate portion, ceiling plate portion are packaged on matrix part using hot pressing, ultrasonic bonding, laser welding or adhesive means, are added Sample chamber, gas outlet, sample enrichment chamber, the waste liquid chamber including the first waste liquid chamber and the second waste liquid chamber, dilution cracking chamber, outlet pipe And PCR amplification chamber etc. be all set on matrix part or in；Further, feeding chamber circulation duct, cracking chamber circulation duct, gas Circulation duct and sample output channel are all set on matrix part；Reusable centrifugal microfluidic control core can be formed in this way Piece.Further, ceiling plate portion is hot pressing film layer；Reusable matrix part can be formed in this way.One embodiment wherein In, chip basal body is equipped at least three fixed parts.Fixed part positions fixed micro-fluidic chip for positioning fixed chip matrix. Further, fixed part includes protrusion and/or recess portion.In this way, micro-fluidic chip is fixed with can be convenient, convenient for carrying out after sample-adding Centrifugation, PCR amplification and the operation such as subsequent foranalysis of nucleic acids and molecular diagnosis.Also, it is full-automatic to be conducive to cooperation for such design Foranalysis of nucleic acids instrument can be realized the full-automation for exempting from the molecular diagnosis project of nucleic acid purification step.

For the ease of it is faster in centrifugation, micro-fluidic chip is accurately installed, chip base in one of the embodiments, Body offers positioning area；Positioning area is used for location and installation micro-fluidic chip.In order to which positioning function is better achieved, one can be designed A or multiple location structures, in one of the embodiments, the quantity of positioning area be one, two or more；One wherein In embodiment, positioning area is that the quantity of location hole and location hole is at least one.Positioning area is fixed in one of the embodiments, The quantity of position protrusion and positioning convex portion is at least one.The quantity of location hole is multiple and opposite in one of the embodiments, It is uniformly distributed in the rotation center of micro-fluidic chip.Positioning area is locating slot in one of the embodiments,.A reality wherein It applies in example, locating slot is linear.Locating slot is arc line shaped in one of the embodiments,.Arc in one of the embodiments, The center of circle of linear camber line and the rotation center of micro-fluidic chip coincide.Positioning area is set in one of the embodiments, Between gas outlet and encapsulated holes.

Waste liquid chamber includes the first waste liquid chamber and the second waste liquid chamber in one of the embodiments,；One embodiment wherein In, the first waste liquid chamber is useless compared with second compared with the inlet of the more separate close rotation center of the second waste liquid chamber and/or the first waste liquid chamber The more separate close rotation center of the inlet of sap cavity, also that is, two waste liquid chambers are different at a distance from rotation center.? In one embodiment, the first waste liquid chamber is connected to the second waste liquid chamber by gas flow pipe road；Such design, so that first Waste liquid chamber and the second waste liquid chamber can share a gas outlet.

Well is respectively communicated with external and feeding chamber in one of the embodiments, and feeding chamber is circulated by feeding chamber Pipeline is connected to sample and is enriched with chamber；I.e. micro-fluidic chip is equipped with the well for being connected to external and feeding chamber also in chip basal body, and Feeding chamber is connected to sample by feeding chamber circulation duct and is enriched with chamber；The company of being equipped in chip basal body in one of the embodiments, Logical external and feeding chamber well, and feeding chamber is connected to sample by feeding chamber circulation duct and is enriched with chamber；A reality wherein It applies in example, well and gas outlet are located at the same end of chip basal body.Well and gas outlet are equal in one of the embodiments, One end of rotation center when positioned at chip basal body close to centrifugation.Well and gas outlet are located in one of the embodiments, The same end of chip basal body.

Sample enrichment chamber is connected to the first waste liquid chamber by cracking chamber circulation duct in one of the embodiments, and sample is rich Collect chamber and also passes through sample output channel connection dilution cracking chamber in its bottom position far from rotation center；A reality wherein It applies in example, sample, which is enriched with chamber and cracking chamber circulation duct, has the first link position, and sample is enriched with chamber and sample efferent duct stage property There is the second link position, the first link position is at a distance from feeding chamber less than the second link position at a distance from feeding chamber；At it In middle one embodiment, the second link position is located at the bottom of sample enrichment chamber, and the first link position is located at sample enrichment chamber Middle part or middle and lower part, in centrifugation, sample, which is enriched with liquid of the chamber more than the first link position, will enter the first waste liquid chamber, sample Dilution will be entered via sample output channel and split in the first link position liquid below after the first phase transformation valve opening by being enriched with chamber It solves intracavitary.So the first link position determines the total liquid volume for participating in subsequent reactions.Further, according to participation reaction The first link position is arranged in target liq total volume.Sample enrichment chamber is equipped with filter membrane in one of the embodiments, to separate Sample is enriched with chamber, so that supernatant is preferably separated with pregnant solution, to reach better sample concentration effect.Further Ground, sample, which is enriched in chamber, is equipped with enrichment chamber and cracking chamber；It cracks chamber and the first waste liquid chamber, enrichment is connected to by cracking chamber circulation duct Chamber is connected to sample output channel.Further, enrichment chamber is used for through centrifugal enrichment sample, and cracking chamber, which is used to being centrifuged, to be obtained Supernatant spills into the first waste liquid chamber by cracking chamber circulation duct.It is equipped in sample enrichment chamber in one of the embodiments, It is enriched with chamber and cracking chamber, chamber is enriched with and cracking chamber is mutually separated by a filter membrane；It cracks chamber and passes through cracking chamber circulation duct connection First waste liquid chamber, enrichment chamber are connected to sample output channel.Further, enrichment chamber is connected to sample in its underpart or bottom position Output channel is enriched with chamber in one of the embodiments, and is connected to sample output channel at its middle part or middle and lower part position, i.e., Being enriched between the bottom and sample output channel of chamber has a certain distance, in order to avoid the precipitating blocked sample efferent duct of cell residue Road enters PCR amplification chamber by sample output channel subsequent PCR is inhibited to react；Further, it is enriched with the bottom of chamber Settling zone equipped with neck and below neck, i.e. settling zone are greater than neck relative to rotation relative to the distance of rotation center The distance at center, and be enriched with chamber and be connected to sample output channel at its neck top position.Wherein, neck is relative to enrichment chamber Other regions, which have, narrows design.Enrichment chamber and cracking chamber are equipped in sample enrichment chamber in one of the embodiments, is enriched with chamber And cracking chamber is mutually separated by a filter membrane；It cracks chamber and the first waste liquid chamber, enrichment chamber connection is connected to by cracking chamber circulation duct Sample output channel is cracked and is equipped in lysate dry powder and/or enrichment chamber in chamber equipped with paramagnet.One embodiment wherein In, cracking chamber circulation duct bending setting；Cracking chamber circulation duct has in one of the embodiments,Character form structure.Into One step, the top position of chamber circulation duct is cracked at a distance from rotation center, top position and rotation less than sample enrichment chamber Turn the distance at center, such design, in high speed centrifugation, cracking chamber circulation duct is filled close to the side of sample enrichment chamber Liquid level is on a centrifugation circumference in the liquid level of liquid, with the chamber of sample enrichment chamber, thus the control effect with siphon valve. Cracking chamber circulation duct is equipped with valve arrangement in one of the embodiments,.Valve arrangement is siphon valve in one of the embodiments, Structure or phase transformation valve arrangement；In one of the embodiments,Character form structure can reach the effect of siphon valve arrangement.In this way Design, be advantageously implemented sample enrichment chamber liquid from cracking chamber circulation duct outflow control.Such П character form structure is set Meter, centrifugal force very little (low-speed centrifugal) or do not have centrifugal force (micro-fluidic chip stops operating) when, sample be enriched with chamber Interior liquid is drawn by capillary force does not cross the cracking chamber circulation duct with siphonage near centrifugation the center point, until cracking chamber It is hydraulically full in circulation duct；Then increase centrifugal speed, under the influence of centrifugal force, siphon jet inside cracking chamber circulation duct Dynamic to occur, it is intracavitary that liquid all flows into subsequent first waste liquid.

Dilution cracking chamber is connected to reagent distribution pipeline by siphon piping in one of the embodiments, and passes through reagent Distribution pipeline is respectively communicated with each PCR amplification chamber and the second waste liquid chamber；That is dilution cracking chamber sequence passes through siphon piping and reagent point Hair pipeline is respectively communicated with each PCR amplification chamber and the second waste liquid chamber.Further, the depth of reagent conveyance conduit is shallower than measurement chamber Depth and the depth of reagent conveyance conduit are shallower than the depth of PCR amplification chamber, and the depth of reagent distribution pipeline is shallower than reagent delivery pipe The depth of the depth in road, the depth in gas flow pipe road, the depth of outlet pipe and reagent distribution pipeline is shallower than the second waste liquid The depth of chamber.The second waste liquid chamber is equipped with the auxiliary gas outlet outside connection or is convexly equipped with positioning column in one of the embodiments,. Each PCR amplification chamber is arranged side by side in one of the embodiments,.The quantity of PCR amplification chamber is 8 in one of the embodiments, A, each PCR amplification chamber is arranged side by side, and PCR amplification chamber has cylindrical structure in one of the embodiments,；One wherein In embodiment, reagent distribution pipeline is connected to each PCR amplification chamber at the same distance position of relative rotation center.Further Ground accommodates PCR reaction reagent dry powder in PCR amplification chamber.PCR reaction is equipped in PCR amplification chamber in one of the embodiments, Reagent dry powder, fluorescent dye and seal.Seal is wax in one of the embodiments,.In one of the embodiments, Paraffin is also housed in PCR amplification chamber.The dosage of the paraffin in PCR amplification chamber is for matching closing in one of the embodiments, The accent of PCR amplification chamber.Fluorescent dye is also housed in PCR amplification chamber in one of the embodiments,.One embodiment wherein In, the PCR reaction reagent dry powder in each PCR amplification chamber is identical or different setting.Each PCR amplification in one of the embodiments, The fluorescent dye accommodated in chamber is identical or different setting.In this manner it is achieved that various identical or different fluorescence detections.Also, Such design, micro-fluidic chip are equipped with 8 PCR amplification chambers, and the corresponding foranalysis of nucleic acids instrument of each amplification chamber has 5 kinds of fluorescence to lead to The detection of 40 indexs simultaneously at most can be achieved in road.The mode of this single sample multi objective also faces for molecular diagnosis realization More pathogen screenings of illness provide possibility.

Micro-fluidic chip is additionally provided with multiple measurement chambers in one of the embodiments, each to measure chamber and each PCR amplification chamber one One is correspondingly arranged, and each measurement chamber is set between reagent distribution pipeline and a PCR amplification chamber and reagent distribution pipeline leads to respectively It crosses each measurement chamber and is connected to each PCR amplification chamber；Micro-fluidic chip is additionally provided with multiple measurement chambers and its examination in one of the embodiments, Agent conveyance conduit, each chamber that measures are arranged in a one-to-one correspondence with each PCR amplification chamber, and each measurement chamber is set to reagent distribution pipeline and one Between PCR amplification chamber, reagent distribution pipeline is respectively communicated with each measurement chamber and is connected to each PCR amplification chamber, and measures chamber and pass through its reagent Conveyance conduit is connected to corresponding PCR amplification chamber.The design of measurement chamber is advantageously implemented uniform liquid separation, avoids interfering with each other or dirty Dye, if not measuring chamber, liquid successively fills up PCR amplification chamber, due to being pre-stored in reagent dry powder in PCR amplification chamber, will lead to, After liquid fills up a PCR amplification chamber, the reagent dry powder of the inside is brought out and enters next PCR amplification through reagent distribution pipeline Chamber, and such phenomenon has then been prevented by the design of measurement chamber.The quantity of PCR amplification chamber is 8 in one of the embodiments, A, the quantity for measuring chamber is also 8, and each PCR amplification chamber is arranged side by side, the corresponding PCR amplification chamber of each measurement chamber；Chamber is measured to connect Lead to each PCR amplification chamber, and measures chamber and corresponding PCR amplification chamber is connected to by its reagent conveyance conduit.Such design, Ke Yishi Existing liquid is introduced into measurement chamber, and all measurement chamber has all been expired and then high speed centrifugation enters PCR amplification chamber, it is ensured that PCR amplification chamber In liquid quantity consistency.

The external setting of gas outlet connection, gas outlet are also connected to a waste liquid by outlet pipe in one of the embodiments, Chamber, i.e. gas outlet be connected to the first waste liquid chamber or the second waste liquid chamber by outlet pipe, and waste liquid chamber is connected with gas outlet Position is compared with gas outlet more far from rotation center；Also that is, gas outlet is closer to rotation center, such design be in order to enable In centrifugation, liquid is conducive to realize connection external environment by gas outlet close to waste liquid chamber.Go out in one of the embodiments, Port is connected to the first waste liquid chamber by outlet pipe；Alternatively, gas outlet is connected to the second waste liquid chamber by outlet pipe.Different location Gas outlet design, be advantageously implemented the main exhaust effect of the first waste liquid chamber or the second waste liquid chamber, be suitable for differential responses demand Micro-fluidic chip.Further, gas outlet is connected to gas flow pipe road, the first waste liquid chamber and the second waste liquid by outlet pipe Chamber passes through gas flow pipe road connection gas outlet respectively；Gas flow pipe road is equipped with branched bottom in one of the embodiments, First waste liquid chamber is connected to gas flow pipe road by branched bottom, and the position that the first waste liquid chamber is connected with branched bottom is located at At the first closest feeding chamber of waste liquid chamber；The sum of the volume of first waste liquid chamber and the second waste liquid chamber in one of the embodiments, Not less than the volume of feeding chamber；It in this way can be to avoid overload.Gas outlet has protrusion setting in one of the embodiments, Terminal part.Waste liquid can be avoided to overflow while easy to exhaust in this way.Further, the first waste liquid chamber is at it close to rotation The one end at center connects gas flow pipe road, in case waste liquid is overflowed by gas outlet.

Siphon piping bending setting in one of the embodiments,.Siphon piping has curved in one of the embodiments, Folded structure.Further, bending structure isCharacter form structure or domes.What arch can be plane is also possible to solid , arch can be round or cylindrical body or spherical shape a part, be also possible to parabola, elliptical a part.One wherein In embodiment, the minimum range of bending structure and rotation center is less than or equal to the minimum range of dilution cracking chamber and rotation center； I.e. the top of bending structure is than the top of dilution cracking chamber closer to rotation center or similarly close to rotation center.Wherein In one embodiment, siphon piping has bending structure.Bending structure isCharacter form structure or domes.A reality wherein It applies in example, siphon piping has bending structure, and bending structure isCharacter form structure or domes, bending structure and rotation center Minimum range be less than or equal to dilution cracking chamber and rotation center minimum range.Further, chip basal body offers positioning Area；Further, siphon piping inner surface is equipped with hydrophilic material.It is suchCharacter form structure design is (low in centrifugal force very little Speed centrifugation) or do not have centrifugal force (micro-fluidic chip stops operating) when, dilution cracking intracavity liquid drawn by capillary force It is hydraulically full in centrifugation the center point to siphon piping siphon piping is not crossed；Then increase centrifugal speed, in centrifugal force Under effect, siphon flowing occurs inside siphon piping, and liquid flows into subsequent reagent distribution pipeline and distributes pipeline by reagent Sequence is distributed to each measurement chamber and corresponding PCR amplification chamber, and extra liquid all flows into the second waste liquid chamber.

Sample output channel is equipped with the first phase transformation valve in one of the embodiments, and chip basal body is equipped with connection outside And first phase transformation valve the first encapsulated holes.First phase transformation valve is for closing or being connected sample output channel；Such design, can be with Phase-change material is injected by the first encapsulated holes to form the first phase transformation valve, then is encapsulated in the rear enclosed first for forming the first phase transformation valve Hole.Further, the first encapsulated holes closing setting；Micro-fluidic chip is in the first encapsulated holes in one of the embodiments, Place is equipped with the first closing cover.Sample output channel is equipped with the first phase transformation valve in one of the embodiments, and chip basal body is set There is connection external and the first encapsulated holes of the first phase transformation valve, micro-fluidic chip is equipped with the first closing cover at the first encapsulated holes. Further, the first phase transformation valve is set to sample output channel close to the position of dilution cracking chamber；Further, phase transformation valve, packet The first phase transformation valve and the second phase transformation valve are included, is prepared using phase-change material；Further, phase transformation valve has paraffin or phase transformation valve is Paraffin, in each embodiment, phase-change material can be wax, for example, wax can be paraffin, microwax, synthetic wax or native paraffin.Or Person, phase-change material are also possible to gel or thermoplastic resin.Gel can be polyacrylamide, polyacrylate, poly- methyl-prop Olefin(e) acid ester or polyvinylamine.Thermoplastic resin can be cricoid olefin copolymer (COC), polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), polyformaldehyde (POM), perfluoro alkoxy (PFA), polyvinyl chloride (PVC) gather Propylene (PP), polyethylene terephthalate (PET), polyether-ether-ketone (PEEK), polyacrylate (PA), polysulfones (PSU) or poly- Ethylene diene fluoride (PVDF) etc..Such design, in the initial stage, the first phase transformation valve closure sample output channel；Work as sample When this enrichment chamber completes example enrichment cracking, by heating or other processing modes, so that the first phase transformation valve is undergone phase transition, thus Sample output channel is connected, sample enters in dilution cracking chamber by sample output channel.Sample in one of the embodiments, Output channel has an at least direction-changing area.Direction-changing area has arcuate structure in one of the embodiments,.Further, deflecting Area has C-shaped or S-shaped.Such design advantageously reduces liquid and flows to the first phase transformation valve through sample output channel or dilute and splits The impact force for solving chamber realizes certain unhurried current effect, cooperates centrifugal speed better effect.In this way, sample enrichment is based on density contrast Enrichment mode, but due to sample enrichment chamber all liq outlet be in off state, can wait sinking with high speed centrifugation Low speed opening cracking chamber circulation duct is for example therein again after forming sediment sufficientlyThe siphon valve of character form structure designs, therefore sample is enriched with Universality is wider, and enrichment is more abundant.And the cracking of the application is to crack intracavitary realization in dilution, and cracking mode is high Warm boiling lysis.

Sample enrichment chamber is equipped with blow vent in one of the embodiments,.Blow vent is connected to for being connected to external environment External atmosphere pressure.Sample enrichment chamber has the top position of neighbouring rotation center and in top position in one of the embodiments, It sets place and is equipped with blow vent.Gas outlet is connected to the second waste liquid chamber by outlet pipe in one of the embodiments,.One wherein In embodiment, gas flow pipe road is equipped with the second phase transformation valve, and chip basal body is equipped with the second encapsulated holes of the second phase transformation valve of connection. Micro-fluidic chip is equipped with the second closing cover at the second encapsulated holes in one of the embodiments,.One embodiment wherein In, sample, which is enriched with chamber, to be had the top position of neighbouring rotation center and is equipped with blow vent at top position, and gas outlet passes through Outlet pipe is connected to the second waste liquid chamber, and gas flow pipe road is equipped with the second phase transformation valve, and chip basal body is equipped with the second phase transformation of connection Second encapsulated holes of valve, micro-fluidic chip are equipped with the second closing cover at the second encapsulated holes.Such design is realizing second The second encapsulated holes of rear enclosed of phase transformation valve, it is ensured that the liquid in feeding chamber can enter sample enrichment by feeding chamber circulation duct In chamber, especially when gas flow pipe road is equipped with the second phase transformation valve, if being not provided with blow vent, liquid is difficult to flow down Do not flow down even.

Chip basal body offers positioning area in one of the embodiments,；Siphon piping has bending structure, bending structure ForThe minimum range of character form structure or domes, bending structure and rotation center is less than or equal in dilution cracking chamber and rotation The minimum range of the heart；Siphon piping inner surface is equipped with hydrophilic material.Sample output channel is equipped in one of the embodiments, First phase transformation valve, and chip basal body is equipped with the first encapsulated holes of connection outside and the first phase transformation valve, micro-fluidic chip is in the first envelope It fills and is equipped with the first closing cover at hole；Sample enrichment chamber has the top position of neighbouring rotation center and is equipped at top position Blow vent, and gas outlet is connected to the second waste liquid chamber by outlet pipe, gas flow pipe road is equipped with the second phase transformation valve, and chip base Body is equipped with the second encapsulated holes of the second phase transformation valve of connection, and micro-fluidic chip is equipped with the second closing cover at the second encapsulated holes；It is micro- Fluidic chip structure is additionally provided with multiple measurement chambers, and each chamber that measures is arranged in a one-to-one correspondence with each PCR amplification chamber, each measurement chamber setting Distribute between pipeline and a PCR amplification chamber in reagent and reagent distribution pipeline is connected to each PCR amplification chamber by each measurement chamber respectively； Chip basal body has partial sector structure；Chip basal body offers positioning area and positioning area is set to gas outlet and the second waste liquid chamber Between；Positioning area is positioning convex portion, location hole or locating slot, and the quantity of positioning area is one, two or more；Locating slot is straight Linear or locating slot is arc line shaped and the center of circle of its camber line and the rotation center of micro-fluidic chip coincide；The quantity of location hole is Multiple and relative to micro-fluidic chip rotation center is uniformly distributed；Rotation center is located at outside chip basal body, by in rotation The distance of the heart arranges from small to large ord are as follows: feeding chamber, sample enrichment chamber, the first waste liquid chamber, dilution cracking chamber, reagent distribution Pipeline, the second waste liquid chamber, PCR amplification chamber；It is equipped with liquid storage container in dilution cracking chamber, liquid storage container is for accommodating dilution；PCR PCR reaction reagent dry powder, fluorescent dye and seal are equipped in amplification chamber.

In one of the embodiments, as shown in Figure 1, a kind of micro-fluidic chip, micro-fluidic chip include chip basal body 100, and the well 110, feeding chamber 120, gas outlet 220, sample the enrichment chamber 140, waste liquid that are set in chip basal body 100 Chamber, dilution cracking chamber 170, siphon piping 180, feeding chamber circulation duct 121, cracking chamber circulation duct 141, gas flow pipe road 151, sample output channel 161, reagent distribution pipeline 191, outlet pipe 210 and multiple PCR amplification chambers 190；Waste liquid chamber includes First waste liquid chamber 150 and the second waste liquid chamber 200；Chip basal body 100 is with partial sector structure and partial sector structure has three Straight flange.Micro-fluidic chip has rotation center and rotation center is located at outside chip basal body 100, and micro-fluidic chip is also in chip The gas outlet 220 outside connection is equipped in matrix 100, well 110 is respectively communicated with external and feeding chamber 120, and feeding chamber 120 Sample, which is connected to, by feeding chamber circulation duct 121 is enriched with chamber 140；It is arranged from small to large ord by a distance from rotation center Are as follows: well 110, feeding chamber 120, sample enrichment chamber 140, the first waste liquid chamber 150, dilution cracking chamber 170, gas outlet 220, the Two waste liquid chambers 200, PCR amplification chamber 190；Sample is enriched with chamber 140 and is connected to the first waste liquid chamber 150 by cracking chamber circulation duct 141, Sample is enriched at bottom position 143 of the chamber 140 also in it far from rotation center through the connection dilution cracking of sample output channel 161 Chamber 170；Dilution cracking chamber 170 is connected to reagent by siphon piping 180 and distributes pipeline 191, and distributes pipeline 191 by reagent and divide It is not connected to each PCR amplification chamber 190 and the second waste liquid chamber 200；First waste liquid chamber 150 passes through the connection of gas flow pipe road 151 second Waste liquid chamber 200；Gas outlet 220 is connected to external setting, and gas outlet 220 is also connected to the second waste liquid chamber 200 by outlet pipe 210, And second waste liquid chamber 200 the position being connected with gas outlet 220 compared with gas outlet 220 more far from rotation center；For the ease of Description and understanding, the gas outlet for connecting different waste liquid chambers uses different label, in one of the embodiments, as schemed 12 to shown in Figure 15, and gas flow pipe road 151 merges setting with outlet pipe, and gas outlet 130 is divided by gas flow pipe road 151 It is not connected to the first waste liquid chamber 150 and the second waste liquid chamber 200, i.e. gas outlet 130 is connected to the first waste liquid by gas flow pipe road 151 Chamber 150 and by gas flow pipe road 151 be connected to the second waste liquid chamber 200.The bending setting of siphon piping 180 forms oneFont Structure also can be regarded as zigzag shape structure, that is, the combination of two symmetrical L-shaped structures and horizontal line as shown in each figures, siphon Pipeline 180The minimum range of character form structure and rotation center is less than or equal to the minimum of dilution cracking chamber 170 and rotation center Distance.Referring to Figure 2 together, siphon piping 180 is sequentially with interconnected Heng Ping import department 181, erects ascending part 182, is horizontal flat Portion 183, perpendicular drop portion 184 and horizontal oral area 185 of clearing, the bottom of the connection dilution cracking chamber 170 of Heng Ping import department 181 is i.e. far from rotation The position at center, horizontal oral area 185 of clearing are connected to reagent distribution pipeline 191.Sample is enriched with the top that chamber 140 has neighbouring rotation center Portion position and at top position be equipped with blow vent 142；Chip basal body 100 offers positioning area 101, sample output channel 161 Equipped with the first phase transformation valve 162, the first phase transformation valve 162 is set to the position that sample output channel 161 cracks chamber 170 close to dilution, Referring to Figure 4 together and Fig. 5, chip basal body 100 are equipped with the first encapsulated holes 163 of connection outside and the first phase transformation valve 162；Sample-adding Hole 110, blow vent 142, gas outlet 220 and encapsulated holes 163 are respectively communicated with outside, and positioning area 101 is locating slot, and locating slot is arc Linear, the center of circle of the camber line of arc line shaped and the rotation center of micro-fluidic chip coincide, and positioning area 101 is set to gas outlet 220 Perhaps positioning area 101 is set between blow vent 142 and gas outlet 220 between encapsulated holes 163 or encapsulated holes 163 are located at Positioning area 101.In each embodiment, each PCR amplification chamber 190 is arranged side by side, as shown in Fig. 1, Fig. 3, Fig. 6 and Fig. 8, each PCR amplification The accent of chamber is identical at a distance from rotation center.And micro-fluidic chip is additionally provided with multiple measurement chambers 192 and its reagent conveyance conduit 193, each chamber 192 that measures is arranged in a one-to-one correspondence with each PCR amplification chamber 190, and each measurement chamber 192 is set to reagent distribution pipeline 191 and one between PCR amplification chamber 190, and reagent distribution pipeline 191 is connected to each PCR amplification chamber 190 by each measurement chamber 192 respectively, And measurement chamber 192 is connected to corresponding PCR amplification chamber 190 by its reagent conveyance conduit 193；First waste liquid chamber 150 passes through gas Circulation duct 151 is connected to the second waste liquid chamber 200, and the second waste liquid chamber 200 is connected to gas outlet 220 by outlet pipe 210.Such as Fig. 7 Shown, the depth of reagent conveyance conduit 193 is shallower than the depth of measurement chamber 192 and the depth of reagent conveyance conduit 193 is shallower than PCR The depth of chamber 190 is expanded, the depth of reagent distribution pipeline 191 is shallower than the depth of reagent conveyance conduit 193, gas flow pipe road The depth of 151 depth, the depth of outlet pipe 210 and reagent distribution pipeline 191 is shallower than the depth of the second waste liquid chamber 200. In one of the embodiments, as shown in Figures 9 to 11, gas flow pipe road 151 is equipped with the second phase transformation valve 230, and chip base Body 100 is equipped with the second encapsulated holes of the second phase transformation valve 230 of connection.Cracking chamber circulation duct 141 is curved in one of the embodiments, Folding is arranged and hasCharacter form structure cracks the top position of chamber circulation duct 141 at a distance from rotation center, is less than or equal to sample The top position of this enrichment chamber 140 cracks the top position and feeding chamber of chamber circulation duct 141 at a distance from rotation center 120 distance is enriched with the top position of chamber 140 at a distance from feeding chamber 120 less than sample；Sample output channel 161 has the One direction-changing area and the second direction-changing area.Using Fig. 1 to embodiment illustrated in fig. 8, in a particular application, when micro-fluidic chip is static It waits, sample is added in feeding chamber 120 by well 110.Then, high speed centrifugation micro-fluidic chip, sample is by adding at this time Sample chamber circulation duct 121 fills sample and is enriched with chamber 140.In each embodiment, ultracentrifugal revolving speed is about 3000rpm- 6000rpm.In sample filling sample enrichment chamber, due to the presence of the first phase transformation valve 162, so that the liquid of sample enrichment chamber 140 Body can not enter subsequent reactions chamber via sample output channel 161.Crack chamber circulation duct 141 or gas flow pipe road 151 There are control structure such as phase transformation valves, and the first waste liquid chamber 150 to be caused to be in sealing state so that sample be enriched with intracavitary liquid without Method flows into the first waste liquid chamber 150 through cracking chamber circulation duct 141.Cracking chamber circulation duct 141 can be a normal liquid Circulation duct can also additionally have a control structure such as phase transformation valve arrangement or siphon valve arrangement etc., and phase transformation valve arrangement also may be used Referred to as phase transformation valve, siphon valve arrangement are also referred to as siphon valve.Such as cracking chamber circulation duct 141 is normal liquid flow siphunculus Road, then continue high speed centrifugation in sample filling sample enrichment chamber 140, due to the presence of centrifugal field, cell in sample, Tissue, pathogen etc. can be deposited to the bottom of sample enrichment chamber 140, and supernatant can be overflowed via cracking chamber circulation duct 141 Into the first waste liquid chamber 150, the function of sample enrichment has been achieved；In other embodiments, chamber 140 can be enriched in sample One layer of filter membrane of interior addition realizes the enrichment of cell in sample, tissue, pathogen etc. in such a way that filter membrane filters.Alternatively, Cracking chamber circulation duct 141 has siphon valve or phase transformation valve, then sample can be allowed to be enriched with chamber in sample with high speed centrifugation at this time It is sufficiently enriched in 140, later when cracking the siphon valve with siphon piping formation at chamber circulation duct 141, then passes through reduction Revolving speed makes the siphon valve that supernatant breaks through cracking chamber circulation duct 141 in sample enrichment chamber 140 enter the first waste liquid chamber 150 In, or when cracking has third phase transformation valve at chamber circulation duct 141, then third is opened by heating or other phase transformation modes The third phase transformation valve that phase transformation valve breaks through the supernatant in sample enrichment chamber 140 at cracking chamber circulation duct 141 enters first In waste liquid chamber 150.In some embodiments of concrete application, can also first high speed centrifugation make the cell residue after centrifugation sufficiently heavy It forms sediment to the bottom of sample enrichment chamber 140, inhibits subsequent PCR reaction to avoid cell residue.In certain implementations of concrete application In example, the mode for slowly accelerating to high speed can be also taken, realizes that the sample that is deposited to of cell residue is enriched with chamber 140 on one side Bottom, the liquid after realizing cracking on one side enter in dilution cracking chamber 170 via sample output channel 161.Further, when splitting Chamber circulation duct 141 is solved there are when control structure, gas outlet 220 can be located at the first waste liquid chamber 150 or the second waste liquid chamber 200 Top；But when there are when control structure such as phase transformation valve, gas outlet 220 may only position in somewhere on gas flow pipe road 151 In the top of the second waste liquid chamber 200.After sample inner cell, tissue, pathogen sufficiently precipitate, by changing centrifugal rotational speed with low Fast centrifugation realizes that cracking chamber circulates to realize that the siphon valve at cracking chamber circulation duct 141 is broken through, or by heating The phase transformation valve in 151 somewhere of 141 somewhere of pipeline or gas flow pipe road is broken through.At this point, the supernatant in sample enrichment chamber 140 will It can flow into the first waste liquid chamber 150, until liquid level is lower than cracking chamber circulation duct 141 in sample enrichment in sample enrichment chamber 140 Entrance in chamber 140.In the present embodiment, splitting for cell, tissue, pathogen in sample etc. is realized in sample enrichment chamber 140 Solution.Cracking mode includes but is not limited to ultrasonic treatment, high-temperature boiling cracking, magnetic stirring cracking etc..Then to the first phase transformation valve Heated at 162, the phase-change material to seal phase transformation valve melted, allow in sample enrichment chamber 140 remaining liq through It is flowed into dilution cracking chamber 170 by sample output channel 161.Phase-change material to seal phase transformation valve includes but is not limited to stone Wax.Since the liquid volume after cracking is limited, between the liquid level and the centrifugation center of circle, that is, rotation center in the chamber 170 of dilution cracking at this time Distance be greater than siphon piping 180 and centrifugation the distance between center of circle, i.e. liquid level in dilution cracking chamber 170 is lower than siphon piping 180, that is, communicating position of the liquid level in cracking chamber 170 lower than dilution cracking chamber 170 and siphon piping 180 is diluted, so Before dilution, no matter centrifugal force is much, dilution cracking chamber 170 in liquid will not break through siphon piping 180 enter it is subsequent Reaction chamber in.Further, in each embodiment, dilution cracking chamber 170 presets dilution；In some instances, it dilutes Liquid it is preset at one end by the paraffin encapsulation other end by the aluminium foil of glue bond among, aluminium foil is glued in dilution cracking chamber 170 Top, paraffin melting after heating, dilution can just release.It is dilute in dilution cracking chamber 170 in other examples Liquid is released to be preset among the aluminium foil for the sealing being placed on above spine, spine be fixed on dilution cracking chamber 170 top, aluminium foil and It is fixed between spine by spring.In this way, aluminium foil is punctured by spine when high speed centrifugation, to discharge dilution.In certain examples In, it is mixed after dilution release with the liquid after cracking, mixing can be rotated by positive and negative rotation micro-fluidic chip or acceleration and deceleration Micro-fluidic chip is realized.Hereafter, dilution cracking chamber 170 is heated at high temperature, to the tissue diluted in cracking chamber 170, carefully Born of the same parents, pathogen just boiling lysis.After cracking sufficiently, centrifugal speed is reduced, medium-speed centrifuge again after low-speed centrifugal, dilution cracking Liquid does the siphon piping 180 of hydrophily processing through inner surface in chamber 170, and in low-speed centrifugal, capillary force is greater than centrifugal force, Liquid can not have siphon piping then to reach siphon piping 180 and reagent point near centrifugation the center point under the drive of capillary force The intersection of pipeline 191 is sent out, at this time medium-speed centrifuge again, due to the intersection distance of siphon piping 180 and reagent distribution pipeline 191 The distance for being centrifuged the center of circle is greater than siphon piping 180 and dilutes the distance in the intersection distance centrifugation center of circle of cracking chamber 170, so meeting Generate siphonic effect, liquid after the cracking after being diluted, which breaks through siphon piping 180 and enters reagent, distributes pipeline 191, and from a left side Each measurement chamber 192 is successively filled up to the right side；It is as in siphon piping 180 in dilution cracking chamber 170 and dilution cracking chamber 170 has a common boundary Liquid more than place can flow into reagent through siphon piping 180 and distribute pipeline 191, into reagent distribution pipeline 191, and with this 8 measurement chambers 192 are filled up, surplus liquid enters in the second waste liquid chamber 200.Then, high speed centrifugation measures the liquid in chamber 192 Entered in corresponding PCR amplification chamber 190 by reagent conveyance conduit 193 respectively.Further, in each embodiment, PCR amplification chamber PCR reaction reagent dry powder and paraffin are preset in 190, PCR reaction reagent dry powder includes PCR anti-in one of the embodiments, Answer required enzyme, dNTPs, fluorescent dye, primer etc..Particularly, each amplification chamber can be with preset a variety of primers, each amplification chamber Multiplex PCR inside may be implemented.Amplified production is done by different fluorescent dyes to distinguish in one of the embodiments,. Then, temperature cycles are carried out, the intracavitary realization PCR that starts is expanded and reacts, expand intracavitary paraffin and start to melt.Since paraffin density is small Yu Shui, in centrifugal field, paraffin can float to the entrance of PCR amplification chamber and seal PCR amplification chamber, to avoid easy in PCR reaction The Aerosol Pollution of appearance.The substance for sealing PCR amplification chamber is mineral oil in one of the embodiments,.Particularly, fixed Position area 101 forms bayonet arrangement for installing micro-fluidic chip or its chip basal body.In PCR reaction process, micro-fluidic chip Remain the state of low-speed centrifugal, in each embodiment, the revolving speed of low-speed centrifugal is about 0-300rpm；It is matched complete in this way Optical system energy scan-type inside automatic nucleic acids instrument reads the intracavitary each fluorescence channel of each PCR amplification respectively Light intensity, to calculate Ct value, provides yin and yang attribute report to draw QPCR curve.It particularly, is intracavitary more of cooperation PCR amplification Weight PCR, full-automatic nucleic acids instrument the inside optical system also needs the optics more covered and photoelectric sensor system, to read difference The fluorescence signal of wavelength.Such design, is suitable for centrifugal microfluidic control and analyzes, diluted after the enrichment of sample, cracking, cracking and Equivalent is distributed, the PCR amplification of multi-chamber is all able to sequence and realizes, be can be realized and is exempted from nucleic acid purification molecular diagnosis function, on the one hand Gas outlet is connected to a waste liquid chamber and is connected to another waste liquid chamber by the waste liquid chamber, can adjust the position of gas outlet according to demand, separately On the one hand the relativeness of capillary force and centrifugal force is dexterously utilized in the siphon piping of bending setting, forms controlling valve to control Controlling flow enters reagent distribution pipeline, is applied to point that centrifugal microfluidic control technology realizes based on PCR amplification using PCR amplification Son diagnosis, entire reaction process are in closed micro-fluidic chip, realize the molecular diagnosis effect quickly detected whenever and wherever possible Fruit.

Micro-fluidic chip whole design is by the way of being centrifuged power drive in one of the embodiments,.It is entire micro-fluidic Chip is similar sector structure, and eight micro-fluidic chips form an annulus, and centrifugation center of rotation, that is, rotation center is located at similar The center of circle of sector structure.Below by taking the parting of human papilloma virus (HPV) as an example, to illustrate the specific reality of micro-fluidic chip It applies.Modern medicine study confirmation, it is cervix caused by human papilloma virus infection that women, which suffers from cervical carcinoma, and HPV is one group of disease The general name of poison, is mainly made of DNA core and protein coat.The HPV type classification about more than 80 having determined at present, no The HPV of homogenic type has different pathogenic risks, can be divided into low risk, middle danger type and high-risk-type three by its carcinogenicity size Class.The detection of HPV DNA and parting have important value to the state of an illness, judgement prevention and guiding treatment is understood.

In such as Fig. 9, Figure 10 and embodiment illustrated in fig. 11, it is described as follows using the testing process of micro-fluidic chip.1. with 1ml Uterine neck bush scrubs liquid as sample, is added in feeding chamber 120 by well 110；2.4000rpm is centrifuged 5 minutes, sample This is flowed into sample enrichment chamber 140 through feeding chamber circulation duct 121；In sample enrichment chamber 140, due to the presence of centrifugal field, Cell, tissue, pathogen in sample can sufficiently be deposited to cavity bottom, when the first waste liquid chamber 150 is connected to external environment, on Clear liquid can enter in the first waste liquid chamber 150；3. 4000rpm centrifugation is maintained, it is sufficiently heavy to sample inner cell, tissue, pathogen Behind shallow lake, heated at the second phase transformation valve 230 in gas flow pipe road 151, so that being able to the phase-change material for sealing phase transformation valve Melt, so that the first waste liquid chamber 150 is connected to via gas flow pipe road 151 with gas outlet 220, so that the first waste liquid Chamber 150 is connected to atmospheric environment, at this point, the supernatant in sample enrichment chamber 140 will flow into the first waste liquid chamber 150, Until entrance of the liquid level lower than cracking chamber circulation duct 141 in sample enrichment chamber 140 in sample enrichment chamber 140.4. maintaining 4000rpm centrifugal speed is then heated at the first phase transformation valve 162 of sample output channel 161, to seal the phase of phase transformation valve Become material to be melted, sample is enriched with the liquid in chamber 140 and flows into dilution cracking chamber 170 through sample output channel 161.To stone The first phase transformation valve 162 that wax is formed carries out 70 DEG C of heating, and paraffin melting at this time, sample output channel 161 is got through, and sample is enriched with chamber It is in entrance below liquid of the cracking chamber circulation duct 141 in sample enrichment chamber 140 in 140 and sweeps along tissue, cell, disease Substance precipitating flows into dilution cracking chamber 170 through sample output channel 161.In an application example, to the first phase transformation valve 162 60 DEG C or higher temperature heating are carried out, paraffin melting at this time, sample output channel 161 is connected, and sample is enriched with raffinate in chamber 140 Body flows into dilution cracking chamber 170 through sample output channel 161.

5.3000rpm centrifugation, and 100 DEG C are carried out to dilution cracking chamber 170 and is heated 5 minutes, in dilution cracking chamber 170 The boiling lysis such as tissue, cell, pathogen.6. centrifugal speed is reduced to 200rpm, 1000rpm, dilution cracking chamber 170 are then risen to Liquid after interior cracking breaks through siphon piping 180 and flows into reagent distribution pipeline 191 through siphon piping 180 and successively fill up 8 Chamber 192 is measured, surplus liquid enters in the second waste liquid chamber 200；In an application example, dilution cracking chamber 170 presets dilute Release liquid, dilution it is preset at one end by the paraffin encapsulation other end by the aluminium foil of glue bond among, aluminium foil is glued to be split in dilution The top for solving chamber 170 carries out 70 DEG C to dilution cracking chamber 170 or higher temperature heats, paraffin melting after heating, dilution It can just release.7.3000rpm centrifugal microfluidic control chip, each liquid measured in chamber 192 pass through reagent conveyance conduit respectively 193 enter in PCR amplification chamber 190, and preset reagent dry powder melts again in PCR amplification chamber 190.8.500rpm centrifugation, and start heat Start computer heating control, the entrance that is paraffin melting and floating to PCR amplification chamber 190 in PCR amplification chamber 190 seals PCR amplification chamber 190, subsequently enter the temperature cycles of PCR reaction.And each PCR amplification chamber of reading of the extension stage scan-type in each circulation Interior fluorescence signal is to draw PCR curve.

In such as Figure 12 into embodiment illustrated in fig. 15, it is described as follows using the testing process of micro-fluidic chip.1.1ml uterine neck Brush liquid is scrubbed as sample, is added in feeding chamber 120 by well 110.2.4000rpm is centrifuged 5 minutes, and sample is through being loaded Chamber circulation duct 121 flows into sample enrichment chamber 140.In sample enrichment chamber 140, due to the presence of centrifugal field, in sample Cell, tissue, pathogen can sufficiently be deposited to cavity bottom；3. centrifugal speed is reduced to 200rpm, at this time due to cracking chamber circulation The siphon piping of pipeline 141 designs, and inner surface is coated with water wetted material or has done hydrophilic treated or chip basal body 100 is Water wetted material, cracking chamber circulation duct 141 have stronger capillary force to liquid, and capillary force is greater than centrifugal force, sample enrichment at this time Chamber 140 is in the liquid of cracking 141 entrance of chamber circulation duct with siphon piping effect or more along cracking chamber circulation duct 141 flow through its near centrifugation the center point, flow to cracking chamber circulation duct 141 and the first waste liquid chamber 150 intersection, at this time by It is broken through in Liquid contact angle and stops flowing.4. centrifugal speed is upgraded to 2000rpm, due to the effect of siphon, sample is enriched at this time The supernatant that cracking chamber circulation duct 141 and sample enrichment 140 entrance intersection of chamber or more are in chamber 140 can be along cracking Chamber circulation duct 141 flows into the first waste liquid chamber 150.5. the first phase transformation valve 162 that pair paraffin is formed carries out 70 DEG C of heating, at this time Paraffin melting, sample output channel 161 is got through, and is in cracking chamber circulation duct 141 in sample enrichment chamber 140 and sample is enriched with chamber 140 entrance intersection liquid below sweep along tissue, cell, pathogen precipitating to split through the inflow dilution of sample output channel 161 It solves in chamber 170.6. a pair dilution cracking chamber 170 carries out 100 DEG C and heats 5 minutes, to tissue, the cell, disease in dilution cracking chamber 170 The boiling lysis such as substance.7. centrifugal speed is reduced to 200rpm, then rise to 1000rpm, in dilution cracking chamber 170 after cracking Liquid more than 180 inlet of siphon piping flows into reagent distribution pipeline 191 through siphon piping 180, and successively fills up 8 surveys Chamber 192 is measured, surplus liquid enters the second waste liquid chamber 200；8.3000rpm centrifugal microfluidic control chip, each liquid measured in chamber 192 Entered in PCR amplification chamber 190 by reagent conveyance conduit 193 respectively, preset reagent dry powder melts again in PCR amplification chamber 190. 9.200rpm centrifugation, and starts thermal starting computer heating control, and preset paraffin melting in PCR amplification chamber 190 simultaneously floats to PCR expansion Increase the entrance of chamber 190, seal PCR amplification chamber 190, subsequently enters the temperature cycles of PCR reaction.And in the extension rank of each circulation Section scan-type the intracavitary fluorescence signal of each PCR amplification is read to draw PCR curve.It can targetedly carry out later glimmering The operation such as light detection or even yin and yang attribute judgement, can also carry out data processing, database, printed report is written.

A kind of analysis system in one of the embodiments, comprising any embodiment micro-fluidic chip further, divides Analysis system is equipped at least three kinds of fluorescence channels；Further, analysis system is equipped with 5 kinds of fluorescence channels.One embodiment wherein In, analysis system is used in PCR reaction process, and control micro-fluidic chip keeps the state of low-speed centrifugal；A reality wherein It applies in example, analysis system, which is also used to scan respectively using the optical system of full-automatic nucleic acids instrument, reads each PCR amplification chamber The light intensity of interior each fluorescence channel draws QPCR curve, calculates Ct value, and provides yin and yang attribute report；An implementation wherein In example, analysis system is for sequentially carrying out static sample-adding, and medium-speed centrifuge is so that sample filling sample is enriched with chamber and realizes sample richness Lytic cell after collection function, high speed is centrifuged so that the cell residue after cracking is deposited in the bottom of sample enrichment chamber and makes to crack It is intracavitary that sample afterwards enters dilution cracking, and heating cracks intracavitary paraffin to melt dilution to discharge after dilution makes cracking Sample is diluted, and each PCR amplification is intracavitary, and heating is to melt each PCR amplification chamber so that the sample after dilution enters for high speed centrifugation Interior paraffin, low-speed centrifugal is to carry out PCR amplification；Foranalysis of nucleic acids system further includes being arranged in iron in one of the embodiments, Permanent magnet below magnetic object block, permanent magnet is for driving paramagnet block to be enriched with intracavitary movement or rotation in sample, in this way, cooperation tool There is the embodiment of paramagnet, preferable lytic effect may be implemented.Further, it is logical to be equipped at least three kinds of fluorescence for analysis system Road；And/or analysis system is used in PCR reaction process, control micro-fluidic chip keeps the state of low-speed centrifugal；And/or Analysis system is also used to the optical system using full-automatic nucleic acids instrument and scan intracavitary each of each PCR amplification of reading respectively The light intensity of fluorescence channel draws QPCR curve, calculates Ct value, and provides yin and yang attribute report；And/or analysis system is for sequence Static sample-adding is carried out, medium-speed centrifuge is so that sample filling sample is enriched with chamber and realizes lytic cell after sample enrichment function, middle height Speed is centrifuged so that the cell residue after cracking is deposited in the bottom of sample enrichment chamber and the sample after cracking is made to enter dilution cracking Intracavitary, heating cracks intracavitary paraffin to melt dilution enables the sample after cracking to dilute to discharge dilution, at a high speed from The heart so that the sample after dilution to enter each PCR amplification intracavitary, heating to melt the intracavitary paraffin of each PCR amplification, low-speed centrifugal with Carry out PCR amplification；And/or foranalysis of nucleic acids system further includes the permanent magnet being arranged in below ferromagnetic block.

Further, analysis system uses each embodiment micro-fluidic chip, cooperates full-automatic foranalysis of nucleic acids instrument, realization is exempted from The full-automation of the molecular diagnosis project of nucleic acid purification step.In this micro-fluidic chip, the enrichment of sample is cracked, cracking Dilution and equivalent distribution, the PCR amplification of multi-chamber are all able to sequence and realize afterwards.In one embodiment, micro-fluidic chip Equipped with 8 PCR amplification chambers, the corresponding foranalysis of nucleic acids instrument of each amplification chamber is equipped with 5 kinds of fluorescence channels, at most can be achieved simultaneously 40 The detection of a index.The mode of this single sample multi objective realizes the more pathogen screenings for facing illness also for molecular diagnosis Provide possibility.Further, entire reaction process is in closed micro-fluidic chip, reduce operator burden and A possibility that pollution, is also no longer dependent on profession so that entire molecular diagnostic procedure is no longer dependent on molecular diagnostic laboratories Operator realizes the demand quickly detected whenever and wherever possible, brings huge help for medical inspection and control and prevention of disease.It needs Illustrate, the application other embodiments further include, and the technical characteristic intercombination in the various embodiments described above is formed by, can The micro-fluidic chip and analysis system of implementation.Each technical characteristic of above embodiments can be combined arbitrarily, to make to describe Succinctly, combination not all possible to each technical characteristic in above-described embodiment is all described, as long as however, these technologies Contradiction is not present in the combination of feature, all should be considered as described in this specification.Above embodiments only express the application Several embodiments, the description thereof is more specific and detailed, and but it cannot be understood as the limitations to claim. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of this application, can also do Several modifications and improvements out, these belong to the protection scope of the application.Therefore, the scope of patent protection of the application should be with appended Subject to claim.

Claims

1. a kind of micro-fluidic chip, the micro-fluidic chip has rotation center, which is characterized in that the micro-fluidic chip includes Chip basal body, and the well, feeding chamber, gas outlet, sample enrichment chamber, waste liquid chamber, dilute being set in the chip basal body Release cracking chamber, siphon piping, feeding chamber circulation duct, cracking chamber circulation duct, gas flow pipe road, sample output channel, examination Pipeline, outlet pipe and multiple PCR amplification chambers are distributed in agent；

The waste liquid chamber includes the first waste liquid chamber and the second waste liquid chamber；

The well is respectively communicated with the external and described feeding chamber, and the feeding chamber is connected to by the feeding chamber circulation duct The sample is enriched with chamber；

Sample enrichment chamber is connected to the first waste liquid chamber by the cracking chamber circulation duct, the sample be enriched with chamber also in Its bottom position far from the rotation center is connected to the dilution by the sample output channel and cracks chamber；

The dilution cracking chamber sequence distributes pipeline by the siphon piping and the reagent and is respectively communicated with each PCR amplification Chamber and the second waste liquid chamber；

The first waste liquid chamber is connected to the second waste liquid chamber by the gas flow pipe road；

The external setting of gas outlet connection, the gas outlet also pass through the outlet pipe and are connected to a waste liquid chamber, and institute The position gas outlet of waste liquid chamber being connected with the gas outlet is stated more far from the rotation center.

2. micro-fluidic chip according to claim 1, which is characterized in that the chip basal body offers positioning area.

3. micro-fluidic chip according to claim 1, which is characterized in that the gas outlet is connected to institute by the outlet pipe State the second waste liquid chamber.

4. micro-fluidic chip according to claim 1, which is characterized in that the gas outlet is connected to institute by the outlet pipe State the first waste liquid chamber.

5. micro-fluidic chip according to claim 1, which is characterized in that the siphon piping has bending structure.

6. micro-fluidic chip according to claim 5, which is characterized in that the minimum of the bending structure and the rotation center Distance is less than or equal to the minimum range of dilution the cracking chamber and the rotation center.

7. micro-fluidic chip according to claim 1, which is characterized in that the chip basal body is prepared using water wetted material.

8. micro-fluidic chip according to claim 1, which is characterized in that the siphon piping inner surface is equipped with hydrophilic material Or inner surface carries out hydrophilic treated.

9. according to claim 1 to micro-fluidic chip described in any one of 8, which is characterized in that the sample output channel is equipped with First phase transformation valve, and the chip basal body is equipped with the first encapsulated holes of connection outside and the first phase transformation valve, it is described micro-fluidic Chip is equipped with the first closing cover at first encapsulated holes；

The sample enrichment chamber has the top position of the neighbouring rotation center and is equipped with blow vent at the top position, And the gas outlet is connected to the second waste liquid chamber by the outlet pipe, the gas flow pipe road is equipped with the second phase transformation Valve, and the chip basal body is equipped with the second encapsulated holes for being connected to the second phase transformation valve, the micro-fluidic chip is in described second The second closing cover is equipped at encapsulated holes；

The microfluidic chip structure is additionally provided with multiple measurement chambers, and each measurement chamber and each PCR amplification chamber correspond Setting, each measurement chamber is set between the reagent distribution pipeline and a PCR amplification chamber and the reagent is distributed Pipeline is connected to each PCR amplification chamber by each measurement chamber respectively；

The chip basal body has partial sector structure；

The chip basal body offers positioning area and the positioning area is set between the gas outlet and the second waste liquid chamber； The positioning area is positioning convex portion, location hole or locating slot, and the quantity of the positioning area is one, two or more；It is described fixed Position slot is linear or the locating slot is arc line shaped and the center of circle of its camber line is mutually be overlapped with the rotation center of the micro-fluidic chip It closes；The quantity of the location hole is that multiple and relative to the micro-fluidic chip rotation centers are uniformly distributed；

The rotation center is located at outside the chip basal body, arranges from small to large ord by a distance from the rotation center Are as follows: the feeding chamber, the sample enrichment chamber, the first waste liquid chamber, the dilution cracking chamber, the reagent distribute pipeline, The second waste liquid chamber, the PCR amplification chamber；

It is equipped with liquid storage container in the dilution cracking chamber, the liquid storage container is for accommodating dilution；

PCR reaction reagent dry powder, fluorescent dye and seal are equipped in the PCR amplification chamber.

10. a kind of analysis system, which is characterized in that including the micro-fluidic chip as described in any one of claims 1 to 9.