CN110029052A

CN110029052A - Micro-fluidic chip and analysis system

Info

Publication number: CN110029052A
Application number: CN201910312447.5A
Authority: CN
Inventors: 汤明辉
Original assignee: Shenzhen City Of Gang Zhu Medical Science And Technology Co Ltd
Current assignee: Shenzhen City Of Gang Zhu Medical Science And Technology Co Ltd
Priority date: 2019-04-18
Filing date: 2019-04-18
Publication date: 2019-07-19

Abstract

This application involves micro-fluidic chip and analysis system, micro-fluidic chip includes chip basal body, feeding chamber, gas outlet, sample enrichment cracking chamber, the first waste liquid chamber, dilution chamber, capillary valves, the second waste liquid chamber, feeding chamber circulation duct, cracking chamber circulation duct, gas flow pipe road, sample output channel, reagent distribution pipeline and multiple PCR amplification chambers；Sample output channel is equipped with phase transformation valve, and chip basal body is equipped with the encapsulated holes of connection phase transformation valve；Chip basal body offers positioning area.It is analyzed suitable for centrifugal microfluidic control, it can be realized and exempt from nucleic acid purification molecular diagnosis function, phase transformation valve can be set in sample output channel conveniently by encapsulated holes enters dilution chamber to control the sample in sample enrichment cracking chamber under certain condition, the design of positioning area is conducive to conveniently and efficiently positioning and fixing micro-fluidic chip, realize that the molecular diagnosis of based on PCR amplification, entire reaction process are in closed microfluidic chip structure applied to centrifugal microfluidic control technology.

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.Microflow control technique is by basic operation unit involved in biological and chemical field, even entire The function in laboratory, including sampling, dilution, reaction, separation, detection etc. are integrated on a small chip, therefore also known as chip is real Test room (Lab-on-a-Chip).This chip is usually to be made of various liquid storage tanks and microchannel network interconnected, can be very Big degree shortens the sample process time, and controls liquid flowing by accurate, realizes the maximum utilization efficiency of reagent consumptive material.

Microfluidic system refers to the device of the handling liquids on submillimeter scale.Centrifugal microfluidic control is under the jurisdiction of micro-fluidic one A branch refers in particular to drive the flowing of liquid by rotation centrifugal microfluidic control chip, to realize using centrifugal force in submillimeter Handling liquids on scale.Basic operation unit involved in biological and chemical field is integrated in a small-sized dish by centrifugal microfluidic control On (disc-shaped) chip of formula.Other than micro-fluidic specific advantage, since centrifugal microfluidic control only needs a motor Power required for liquid manipulation is provided, so whole equipment more concision and compact.And it is ubiquitous on disc-type chip Centrifugal field can make liquid driven more effective, it is ensured that without residual liquid in pipeline, and can effectively realize based on density The sample of difference separates, and also parallel processing can be allowed more simple.Therefore, centrifugal microfluidic control is also more and more applied instant It diagnoses in (Point-of-care testing, POCT).

The molecular diagnosis of based on PCR amplification is that specific amplification target gene is mediated (to lose to detect endogenous by primer Pass or variation) or exogenous (pathogen) target gene presence or absence, and then to the diagnosing and treating of disease provide information and Decision-making foundation.Its main application scenarios has the diagnosis of infectious disease, blood sieve, Tumor mutations site primer, the diagnosis of hereditary disease, production Preceding diagnosis, tissue typing etc..The molecular diagnosis of based on PCR amplification generally comprises following steps: sample cracking, nucleic acid purification, core Acid expands under specific primer constraint, the acquisition and analysis of fluorescence signal.In the project of certain molecular diagnosises, due to sample ratio It is relatively simple, usually can directly it be expanded after sample cracking；On the other hand, increasingly mature one-step method DNA now The appearance of amplification kit is extracted but also directly amplification is possibly realized after sample cracking, avoiding nucleic acid purification, this is more multiple Miscellaneous step.

But based on PCR amplification molecular diagnosis system in, due to having Aerosol Pollution when PCR amplification, also for The cross contamination between sample is avoided, to set up a subregion laboratory under normal circumstances.This laboratory will realize sample Processing, nucleic acid extraction, the division operation of PCR amplification, and must have good ventilating system, laboratory build it is at high cost, it is past Just there are the financial resources built toward only larger medical mechanism.On the other hand, laboratory operation personnel will take appointment with certificate, and also greatly increase Cost of labor.At the same time, excessive artificial intervention certainly will also bring along artificial operation error.These problems greatly mention The technology of the high molecular diagnosis of based on 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 comprising chip basal body, and be set in chip basal body feeding chamber, sample enrichment split Solve chamber, the first waste liquid chamber, dilution chamber, capillary valves, the second waste liquid chamber, feeding chamber circulation duct, cracking chamber circulation duct, gas stream It threads a pipe, sample output channel, reagent distribute pipeline and multiple PCR amplification chambers；Micro-fluidic chip is also equipped in chip basal body The well of connection outside and feeding chamber, and feeding chamber is connected to sample enrichment cracking chamber by feeding chamber circulation duct；Sample is rich Collection cracking chamber is connected to the first waste liquid chamber by cracking chamber circulation duct, and sample enrichment cracking chamber also passes through sample output channel and is connected to Dilution chamber；Dilution chamber is connected to reagent by capillary valves and distributes pipeline, and distributes pipeline by reagent and be respectively communicated with each PCR amplification chamber And the second waste liquid chamber；Micro-fluidic chip is equipped with the gas outlet outside connection also in chip basal body, and the first waste liquid chamber and second are given up Sap cavity passes through gas flow pipe road connection gas outlet respectively；Sample output channel is equipped with phase transformation valve, and chip basal body is equipped with connection External and phase transformation valve encapsulated holes；Chip basal body offers positioning area.

Above-mentioned micro-fluidic chip is suitable for centrifugal microfluidic control and analyzes, can be realized and exempt from nucleic acid purification molecular diagnosis function, phase Becoming valve can be set in sample output channel to control the sample in sample enrichment cracking chamber one conveniently by encapsulated holes Enter dilution chamber under fixed condition, the design of positioning area is conducive to conveniently and efficiently positioning and fixing micro-fluidic chip, is expanded using PCR Increasing technology is applied to the molecular diagnosis that centrifugal microfluidic control technology realizes based on PCR amplification, without building larger molecule diagnostic test Room need not also use a large amount of manual operations, and entire reaction process is in closed microfluidic chip structure, reduce operator A possibility that burden and pollution of member, but also entire molecular diagnostic procedure is no longer dependent on molecular diagnostic laboratories, also no longer Dependent on professional operator, the demand quickly detected whenever and wherever possible is realized, is brought for medical inspection and control and prevention of disease huge Big help.

Micro-fluidic chip is additionally provided with multiple measurement chambers and its reagent conveyance conduit in one of the embodiments, each to measure Chamber is arranged in a one-to-one correspondence with each PCR amplification chamber, and each measurement chamber is set between reagent distribution pipeline and a PCR amplification chamber, examination Agent distribution pipeline is respectively communicated with each measurement chamber and is connected to each PCR amplification chamber, and measures chamber and corresponded to by the connection of its reagent conveyance conduit PCR amplification chamber.

Cracking chamber circulation duct bending setting in one of the embodiments,.

Cracking chamber circulation duct has П character form structure in one of the embodiments,.

Sample output channel has an at least direction-changing area in one of the embodiments,.

Direction-changing area has arcuate structure in one of the embodiments,.

Sample enrichment cracking chamber is equipped with filter membrane in one of the embodiments,.

Cracking chamber circulation duct is equipped with valve arrangement in one of the embodiments,.

Sample enrichment cracking chamber and cracking chamber circulation duct have the first link position, sample in one of the embodiments, This enrichment, which cracks chamber and sample output channel, has the second link position, and the first link position is at a distance from feeding chamber less than second Link position is at a distance from feeding chamber；It cracks chamber circulation duct and is equipped with valve arrangement；Further, valve arrangement be siphon valve arrangement or Person's phase transformation valve arrangement；Positioning area is set between gas outlet and encapsulated holes；Positioning area be positioning convex portion, location hole or locating slot, The quantity of positioning area be one, two or more；Locating slot be linear or locating slot be arc line shaped and the center of circle of its camber line with The rotation center of micro-fluidic chip coincides；Further, the quantity of location hole is multiple and relative to micro-fluidic chip rotations Turn center to be uniformly distributed；The encapsulated holes closing setting；Further, the micro-fluidic chip is equipped with envelope at the encapsulated holes Close cover；Micro-fluidic chip has rotation center and rotation center is located at outside chip basal body, presses by a distance from rotation center Sequence arranges from small to large are as follows: feeding chamber, sample enrichment cracking chamber, the first waste liquid chamber, dilution chamber, the second waste liquid chamber；Gas stream It threads a pipe and is equipped with branched bottom, the first waste liquid chamber is connected to gas flow pipe road, and the first waste liquid chamber and branch by branched bottom The position that channel is connected is located at the first closest feeding chamber of waste liquid chamber.

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

Further, analysis system is equipped at least three kinds of fluorescence channels；And/or analysis system is used in PCR reaction process In, control micro-fluidic chip keeps the state of low-speed centrifugal；And/or analysis system is also used to using full-automatic nucleic acids instrument Optical system scan the light intensity for reading the intracavitary each fluorescence channel of each PCR amplification respectively, draw QPCR curve, calculate Ct Value, and provide yin and yang attribute report；And/or analysis system, for sequentially carrying out static sample-adding, medium-speed centrifuge is so that sample fills sample This enrichment cracking chamber simultaneously realizes lytic cell after sample enrichment function, and high speed is centrifuged so that the cell residue after cracking is deposited in Sample enrichment cracking chamber bottom and make cracking after sample enter in dilution chamber, heating with melt the paraffin in dilution chamber to Release dilution enable cracking after sample dilute, high speed centrifugation so that dilution after sample enter each PCR amplification it is intracavitary, add Heat is to melt the intracavitary paraffin of each PCR amplification, and low-speed centrifugal is to carry out PCR amplification.

Detailed description of the invention

Fig. 1 is the structural schematic diagram of one embodiment of the application.

Fig. 2 is the schematic diagram of another direction of embodiment illustrated in fig. 1.

Fig. 3 is 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 the schematic diagram of another direction of embodiment illustrated in fig. 1.

Fig. 6 is the schematic diagram of another direction of embodiment illustrated in fig. 1.

Fig. 7 is the schematic diagram of another direction of embodiment illustrated in fig. 1.

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

Fig. 9 is the schematic diagram of another direction of embodiment illustrated in fig. 8.

Figure 10 is the schematic diagram of another direction of embodiment illustrated in fig. 8.

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.

It should be noted that it can be directly another when element is referred to as " being fixed on " or " being set to " another element On one element or there may also be elements placed in the middle.When an element is considered as " connection " another element, it can be with It is directly to another element or may be simultaneously present centering elements.Term as used herein " vertically ", " level ", "left", "right" and similar statement for illustrative purposes only, be not meant to be the only embodiment.

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.

In the application one embodiment, a kind of micro-fluidic chip comprising chip basal body, and it is set to chip basal body In feeding chamber, sample enrichment cracking chamber, the first waste liquid chamber, dilution chamber, capillary valves, the second waste liquid chamber, feeding chamber circulation duct, Crack chamber circulation duct, gas flow pipe road, sample output channel, reagent distribution pipeline and multiple PCR amplification chambers；Micro-fluidic core Piece is equipped with the well of connection outside and feeding chamber also in chip basal body, and feeding chamber is connected to sample by feeding chamber circulation duct This enrichment cracks chamber；Sample enrichment cracking chamber is connected to the first waste liquid chamber by cracking chamber circulation duct, and sample enrichment cracking chamber is also Dilution chamber is connected to by sample output channel；Dilution chamber is connected to reagent by capillary valves and distributes pipeline, and passes through reagent dispensing tube Road is respectively communicated with each PCR amplification chamber and the second waste liquid chamber；Micro-fluidic chip is equipped with the outlet outside connection also in chip basal body Mouthful, the first waste liquid chamber and the second waste liquid chamber pass through gas flow pipe road connection gas outlet respectively；Sample output channel is equipped with phase transformation Valve, and chip basal body is equipped with the encapsulated holes of connection outside and phase transformation valve；Chip basal body offers positioning area.Above-mentioned micro-fluidic core Piece is suitable for centrifugal microfluidic control and analyzes, can be realized and exempt from nucleic acid purification molecular diagnosis function, phase transformation valve can be conveniently by envelope Dress hole is set in sample output channel enters dilution chamber to control the sample in sample enrichment cracking chamber under certain condition, fixed The design in position area is conducive to conveniently and efficiently positioning and fixing micro-fluidic chip, is applied to centrifugal microfluidic using PCR amplification Control technology realizes the molecular diagnosis of based on PCR amplification, also need not be using a large amount of artificial without larger molecule diagnostic test room is built Operation, entire reaction process are in closed microfluidic chip structure, reduce the burden of operator and the possibility of pollution Property, but also entire molecular diagnostic procedure is no longer dependent on molecular diagnostic laboratories, it is also no longer dependent on professional operator, The demand quickly detected whenever and wherever possible is realized, brings huge help for medical inspection and control and prevention of disease.

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 be set in chip basal body feeding chamber, sample enrichment chamber, the first waste liquid chamber, Dilution cracking chamber, capillary valves, the second waste liquid chamber, feeding chamber circulation duct, cracking chamber circulation duct, gas flow pipe road, sample Output channel, reagent distribution pipeline and multiple PCR amplification chambers；It is understood that feeding chamber, sample are enriched with chamber, the first waste liquid Chamber, dilution cracking chamber, capillary valves, feeding chamber circulation duct, cracking chamber circulation duct, gas flow pipe road, sample output channel, The shapes and sizes of reagent distribution pipeline, PCR amplification chamber and the second waste liquid chamber etc. design according to actual needs.Further Ground, feeding chamber, sample enrichment chamber, the first waste liquid chamber, dilution crack chamber, capillary valves, feeding chamber stream in one of the embodiments, It threads a pipe, crack chamber circulation duct, gas flow pipe road, sample output channel, reagent distribution pipeline, PCR amplification chamber and second Waste liquid chamber is opened in chip basal body, and only external by such as encapsulated holes connection of well and gas outlet or other structures is external rings Border.Chip basal body is PMMA, PDMS, PC, ABS, COC or COP product in one of the embodiments,.An implementation wherein In example, chip basal body has partial sector structure.In one of the embodiments, partial sector include fan annular and flaabellum shape or Partial sector structure has three straight flanges.Such design is conducive to regularly arranged formed of multiple micro-fluidic chips and is similar to circle Annular structure, thus rationally utilize centrifugal action, improve treatment effeciency, allow multiple micro-fluidic chips carry out simultaneously from The heart handles and carries out PCR amplification and then complete entire molecular diagnostic procedure.Chip basal body is using heat in one of the embodiments, Pressure, ultrasonic bonding, laser welding or adhesive means encapsulation；The micro-fluidic chip of an opposing seal can be formed in this way, only There is well to be in communication with the outside with gas outlet.Further, chip basal body has matrix part and lid in one of the embodiments, Plate portion, ceiling plate portion are packaged on matrix part using hot pressing, ultrasonic bonding, laser welding or adhesive means, feeding chamber, outlet Mouth, sample enrichment chamber, the first waste liquid chamber, dilution cracking chamber, capillary valves, PCR amplification chamber and the second waste liquid chamber are all set in matrix In portion or in；Further, feeding chamber circulation duct, cracking chamber circulation duct, gas flow pipe road and sample output channel are equal It is set on matrix part；Reusable centrifugal microfluidic control chip can be formed in this way.Further, an implementation wherein In example, ceiling plate portion is hot pressing film layer；Reusable matrix part can be formed in this way.Chip in one of the embodiments, Matrix is equipped at least three fixed parts.Fixed part positions fixed micro-fluidic chip for positioning fixed chip matrix.Further Ground, fixed part includes protrusion and/or recess portion in one of the embodiments,.In this way, fix micro-fluidic chip with can be convenient, Convenient for being centrifuged after sample-adding, PCR amplification and subsequent foranalysis of nucleic acids and molecular diagnosis etc. operate.Also, such design, has Conducive to full-automatic foranalysis of nucleic acids instrument is cooperated, the full-automation for exempting from the molecular diagnosis project of nucleic acid purification step can be realized.

Micro-fluidic chip is equipped with the sample-adding of connection outside and feeding chamber also in chip basal body in one of the embodiments, Hole, and feeding chamber is connected to sample by feeding chamber circulation duct and is enriched with chamber；It is set in chip basal body in one of the embodiments, There is connection external and the well of feeding chamber, and feeding chamber is connected to sample by feeding chamber circulation duct and is enriched with chamber；Wherein one In a embodiment, well and gas outlet are located at the same end of chip basal body.Well and outlet in one of the embodiments, Mouth is respectively positioned on one end of rotation center of the chip basal body close to centrifugation when.Well and gas outlet in one of the embodiments, Positioned at 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 Collection chamber also passes through sample output channel connection dilution cracking chamber；Sample enrichment chamber passes through cracking chamber in one of the embodiments, Circulation duct is connected to the first waste liquid chamber, and sample enrichment chamber also passes through sample output channel connection dilution cracking chamber；One wherein In embodiment, sample, which is enriched with chamber and cracking chamber circulation duct, has the first link position, and sample is enriched with chamber and sample output channel With 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；? In one embodiment, the first link position is located at the middle part or middle and lower part of sample enrichment chamber, and in centrifugation, sample is enriched with chamber Liquid more than the first link position will enter the first waste liquid chamber, and sample enrichment chamber will in the first link position liquid below After phase transformation valve opening, it is intracavitary to enter dilution cracking via sample output channel.So after the first link position determines participation The total liquid volume of continuous reaction.Further, it is set in one of the embodiments, according to the target liq total volume for participating in reaction Set the first link position.In one of the embodiments, as shown in Fig. 2, the first link position 146 is at a distance from feeding chamber 120 Less than the second link position 147 at a distance from feeding chamber 120, the sample being enriched in sample enrichment chamber passes through sample output channel 161 enter in dilution cracking chamber 170.Sample enrichment chamber is equipped with filter membrane in one of the embodiments, to separate sample enrichment Chamber, so that supernatant is preferably separated with pregnant solution, to reach better sample concentration effect.Further, wherein In one embodiment, sample, which is enriched in chamber, is equipped with enrichment chamber and cracking chamber；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 used to pass through centrifugation in one of the embodiments, It is enriched with sample, cracking chamber is used to the supernatant that centrifugation obtains spilling into the first waste liquid chamber by cracking chamber circulation duct.At it In middle one embodiment, sample, which is enriched in chamber, is equipped with enrichment chamber and cracking chamber, is enriched with chamber and cracking chamber passes through a filter membrane phase point Every；It cracks chamber and the first waste liquid chamber is connected to by cracking chamber circulation duct, enrichment chamber is connected to sample output channel.Further, exist In one embodiment, enrichment chamber is connected to sample output channel in its underpart or bottom position, wherein one embodiment In, enrichment chamber is connected to sample output channel, the i.e. bottom of enrichment chamber and sample efferent duct at its middle part or middle and lower part position Between road have a certain distance, in order to avoid the precipitating blocked sample output channel of cell residue or by sample output channel into Enter PCR amplification chamber and subsequent PCR is inhibited to react；Further, the bottom for being enriched with chamber is equipped with neck and below neck Settling zone, i.e. settling zone are greater than distance of the neck relative to rotation center relative to the distance of rotation center, and are enriched with chamber in it Sample output channel is connected at neck top position.Wherein, neck has relative to other regions of enrichment chamber narrows design.? In one embodiment, it is equipped with enrichment chamber and cracking chamber in sample enrichment chamber, is enriched with chamber and cracking chamber passes through a filter membrane phase Separate；It cracks chamber and the first waste liquid chamber is connected to by cracking chamber circulation duct, enrichment chamber is connected to sample output channel, cracks and set in chamber Have in lysate dry powder and/or enrichment chamber and is equipped with paramagnet.

Cracking chamber circulation duct bending setting in one of the embodiments,；Chamber stream is cracked in one of the embodiments, It threads a pipe with П character form structure.Further, top position and the rotation of chamber circulation duct are cracked in one of the embodiments, The distance for turning center, less than sample enrichment chamber top position at a distance from rotation center, such design, in high speed centrifugation When, cracking chamber circulation duct is enriched with liquid in the chamber of chamber with sample close to the liquid level of the filled liquid in side of sample enrichment chamber Face is on a centrifugation circumference, thus the control effect with siphon valve.Chamber circulation duct is cracked in one of the embodiments, Equipped with valve arrangement.Valve arrangement is siphon valve arrangement or phase transformation valve arrangement in one of the embodiments,；An implementation wherein In example, П character form structure can reach the effect of siphon valve arrangement.Such design is advantageously implemented the liquid of sample enrichment chamber From cracking chamber circulation duct outflow control.

Dilution cracking chamber is connected to reagent distribution pipeline by capillary valves in one of the embodiments, and passes through reagent point Hair pipeline is respectively communicated with each PCR amplification chamber and the second waste liquid chamber；It is outer to be equipped with connection for the second waste liquid chamber in one of the embodiments, The auxiliary gas outlet in portion is convexly equipped with positioning column.Each PCR amplification chamber is arranged side by side in one of the embodiments,.Wherein one In a embodiment, the quantity of PCR amplification chamber is 8, and each PCR amplification chamber is arranged side by side, and PCR expands in one of the embodiments, Increasing chamber has cylindrical structure；In one of the embodiments, reagent distribution pipeline respectively at relative rotation center it is identical away from The place of setting of offing normal is connected to each PCR amplification chamber.Further, PCR reaction examination is accommodated in PCR amplification chamber in one of the embodiments, Agent dry powder.Paraffin is also housed in PCR amplification chamber in one of the embodiments,.PCR amplification chamber in one of the embodiments, In paraffin dosage be used for match close PCR amplification chamber accent.Also hold in PCR amplification chamber in one of the embodiments, Set fluorescent dye.PCR reaction reagent dry powder in one of the embodiments, in each PCR amplification chamber is identical or different setting.? In one embodiment, the fluorescent dye accommodated in each PCR amplification chamber is identical or different setting.In this manner it is achieved that various Identical or different fluorescence detection.Also, such design, micro-fluidic chip are equipped with 8 PCR amplification chambers, each amplification chamber pair The foranalysis of nucleic acids instrument answered has 5 kinds of fluorescence channels, and the detection of 40 indexs simultaneously at most can be achieved.This single sample multi objective Mode, also realized for molecular diagnosis and provide possibility in face of more pathogen screenings of illness.

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 micro-fluidic chip is equipped with the gas outlet outside connection also in chip basal body in one of the embodiments, First waste liquid chamber and the second waste liquid chamber pass through gas flow pipe road connection gas outlet respectively；Gas in one of the embodiments, Circulation duct be equipped with branched bottom, the first waste liquid chamber by branched bottom be connected to gas flow pipe road, and the first waste liquid chamber with divide The position that subchannel is connected is located at the first closest feeding chamber of waste liquid chamber；First waste liquid chamber in one of the embodiments, It is not less than the volume of feeding chamber with the sum of the volume of the second waste liquid chamber；It in this way can be to avoid overload.An implementation wherein In example, gas outlet has the terminal part of protrusion setting.Waste liquid can be avoided to overflow while easy to exhaust in this way.Further Ground, the first waste liquid chamber connects gas flow pipe road close to one end of rotation center at it in one of the embodiments, so as not to it is useless Liquid is overflowed by gas outlet.

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, and 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.

Sample output channel is equipped with phase transformation valve in one of the embodiments, and chip basal body is equipped with connection outside and phase Become the encapsulated holes of valve, phase transformation valve is for closing or being connected sample output channel；Such design can inject phase by encapsulated holes Become material to form the phase transformation valve, then forms encapsulated holes described in the phase transformation valve closure.Further, wherein one In a embodiment, encapsulated holes closing setting；Further, micro-fluidic chip is equipped with closing cover at encapsulated holes.Wherein one In a embodiment, phase transformation valve is set to sample output channel close to the position of dilution cracking chamber；Further, a reality wherein It applies in example, phase transformation valve is prepared using phase-change material；Further, in one of the embodiments, phase transformation valve have paraffin or Phase transformation valve is paraffin, and in each embodiment, phase-change material can be wax, for example, wax can be paraffin, microwax, synthetic wax or day Right wax.Alternatively, phase-change material is also possible to gel or thermoplastic resin.Gel can be polyacrylamide, and polyacrylate gathers Methacrylate or polyvinylamine.Thermoplastic resin can be cricoid olefin copolymer (COC), poly-methyl methacrylate Ester (PMMA), polycarbonate (PC), polystyrene (PS), polyformaldehyde (POM), perfluoro alkoxy (PFA), polyvinyl chloride (PVC), Polypropylene (PP), polyethylene terephthalate (PET), polyether-ether-ketone (PEEK), polyacrylate (PA), polysulfones (PSU) or Polyethylene diene fluoride (PVDF) etc..

Such design, in the initial stage, phase transformation valve closure sample output channel；When sample enrichment chamber completes example enrichment When cracking, by heating or other processing modes, so that phase transformation valve is undergone phase transition, so that sample output channel is connected, sample warp Sample output channel is crossed to enter in dilution cracking chamber.Sample output channel has an at least deflecting in one of the embodiments, Area.Direction-changing area has arcuate structure in one of the embodiments,.Further, direction-changing area has in one of the embodiments, There are C-shaped or S-shaped.Such design advantageously reduces liquid and flows to phase transformation valve through sample output channel or dilute rushing for cracking chamber Power is hit, certain unhurried current effect is realized, cooperates centrifugal speed better effect.Embodiment with phase transformation valve, especially Fig. 8 are to 10 Illustrated embodiment, sample enrichment mode is more direct, and the sample of applicant's previous design, which exists to be centrifuged while flowing into waste liquid chamber, to be made Cracking cavity bottom is deposited under, such enrichment method is often suitable for the biggish scene of deposit volume in sample.And The application has carried out Curve guide impeller to this, and sample is enriched with the enrichment mode for being again based on density contrast, but since sample is enriched with Chamber all liq outlet be in off state, therefore can with high speed centrifugation it is to be precipitated sufficiently after again low speed open cracking chamber circulation The siphon valve of pipeline П character form structure for example therein designs, therefore sample enrichment universality is wider, and enrichment is more abundant.And And the cracking of the application is to crack intracavitary realization in dilution, cracking mode is high-temperature boiling cracking.Further, wherein one In a embodiment, chip basal body using water wetted material prepare, or cracking chamber circulation duct inner surface be equipped with hydrophilic material or Inner surface carries out hydrophilic treated；Such П character form structure design in centrifugal force very little (low-speed centrifugal) or does not have centrifugal force When (micro-fluidic chip stops operating), sample is enriched with intracavity liquid and does not cross siphon piping near centrifugation by capillary force traction It is hydraulically full in the center point to siphon piping；Then increase centrifugal speed, under the influence of centrifugal force, siphon inside siphon piping Flowing occurs, and it is intracavitary that liquid all flows into subsequent first waste liquid.

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, the second waste liquid chamber；That is, press centrifugal direction, feeding chamber, sample enrichment chamber, the first waste liquid chamber, Dilution cracking chamber, reagent distribution pipeline sequence are arranged, and the position of gas outlet relative to the first waste liquid chamber closer to feeding chamber；? In one embodiment, dilution crack the distance between intracavitary liquid level and rotation center be greater than capillary valves and rotation center it Between distance；In this way, the sample in feeding chamber enters sample enrichment by feeding chamber circulation duct when carrying out centrifugally operated Chamber, the supernatant that sample is enriched with chamber top enter the first waste liquid chamber by cracking chamber circulation duct, and sample is enriched with the sample of chamber lower part Enter sample output channel after this enrichment, crack chamber subsequently into dilution, passes through capillary valves after dilution cracking chamber is diluted And reagent distribution pipeline sequence enters each PCR amplification chamber, extra part enters the second waste liquid chamber.One embodiment wherein In, liquid storage container is equipped in dilution cracking chamber；Liquid storage container is for accommodating dilution.Alternatively, dilution cracking chamber is dilute for accommodating Release liquid.In this way, can directly be used when carrying out PCR amplification or even molecular diagnosis, without reassembling dilution.Alternatively, In one embodiment, dilution cracking chamber is equipped with liquid injection hole, and liquid injection hole is for injecting dilution.One embodiment wherein In, liquid injection hole is unidirectional hole.Alternatively, dilution cracking chamber is equipped with the fill cap of closing liquid injection hole in one of the embodiments,.Into One step, dilution is equipped in liquid storage container in one of the embodiments,.Liquid storage container is set in one of the embodiments, Have using the closed opening of hot melt layer.Liquid storage container is viscous in one of the embodiments, is set in dilution cracking chamber.Wherein one In a embodiment, liquid storage container has aluminium foil layer.Liquid storage container is equipped with opening, thorn part, elastic component in one of the embodiments, With sealer, sealer is fixed in dilution cracking chamber for closing opening, elastic component one end connection thorn part, the other end, and thorn part is used for In centrifugation, cooperation elastic component generates displacement to puncture sealer.Further, in one of the embodiments, in dilution cracking chamber Equipped with dilution.Dilution is set in hot melt wrapping layer in one of the embodiments, and hot melt wrapping layer is set to dilution and splits It solves in chamber.Dilution is set in wrapping layer in one of the embodiments, and wrapping layer is set in dilution cracking chamber and wraps up Layer, which is equipped with, uses the closed opening of hot melt layer.

In one of the embodiments, as shown in Figure 1, a kind of micro-fluidic chip comprising chip basal body 100, Yi Jishe Feeding chamber 120, sample enrichment chamber 140, the first waste liquid chamber 150, dilution the cracking chamber 170, capillary valves being placed in chip basal body 100 180,200 feeding chamber circulation duct 121 of the second waste liquid chamber, cracking chamber circulation duct 141, gas flow pipe road 151, sample output Pipeline 161, reagent distribution pipeline 191 and multiple PCR amplification chambers 190；Chip basal body 100 has partial sector structure；It is micro-fluidic Chip has rotation center and rotation center is located at outside chip basal body 100, suitable from small to large by pressing at a distance from rotation center Sequence arrangement are as follows: feeding chamber 120, sample enrichment chamber 140, the first waste liquid chamber 150, dilution cracking chamber 170, the second waste liquid chamber 200, it is micro- Fluidic chip is equipped with the gas outlet 130 outside connection, in each embodiment, the position phase of gas outlet 130 also in chip basal body 100 For the first waste liquid chamber 150 closer to feeding chamber 120；Micro-fluidic chip is equipped with connection outside and is added also in chip basal body 100 The well 110 of sample chamber 120, and feeding chamber 120 is connected to sample by feeding chamber circulation duct 121 and is enriched with chamber 140；Sample enrichment Chamber 140 is connected to the first waste liquid chamber 150 by cracking chamber circulation duct 141, and sample is enriched with chamber 140 and also passes through sample output channel 161 connection dilution cracking chambers 170；Referring to Figure 2 together, Fig. 5, Fig. 6 and Fig. 7, sample are enriched with chamber 140 and cracking chamber circulation duct 141 have the first link position 146, and sample, which is enriched with chamber 140 and sample output channel 161, has the second link position 147, and first Link position 146 is at a distance from feeding chamber 120 less than the second link position 147 at a distance from feeding chamber 120；Dilution cracking chamber 170, which are connected to reagent by capillary valves 180, distributes pipeline 191, and distributes pipeline 191 by reagent and be respectively communicated with each PCR amplification chamber 190 and the second waste liquid chamber 200；Each PCR amplification chamber 190 be arranged side by side, and micro-fluidic chip be additionally provided with multiple measurement chambers 192 and its Reagent conveyance conduit 193, each chamber that measures are arranged in a one-to-one correspondence with each PCR amplification chamber 190, and each measurement chamber 192 is set to reagent Distribute between pipeline 191 and a PCR amplification chamber 190, reagent distribution pipeline 191 is respectively communicated with each measurement chamber 192 and is connected to each PCR expansion Increase chamber 190, and measures chamber 192 and corresponding PCR amplification chamber 190 is connected to by its reagent conveyance conduit 193；First waste liquid chamber 150 And second waste liquid chamber 200 respectively by gas flow pipe road 151 be connected to gas outlet 130；Gas flow pipe road 151 is logical equipped with branch Road 152, the first waste liquid chamber 150 is connected to gas flow pipe road 151 by branched bottom 152, and the first waste liquid chamber 150 and branch are logical The position that road 152 is connected is located at the closest feeding chamber 120 of the first waste liquid chamber 150；Chip basal body 100 offers positioning area 101, sample output channel 161 is equipped with phase transformation valve 162, and phase transformation valve 162 is set to sample output channel 161 close to dilution cracking chamber 170 position；As shown in Figures 3 and 4, chip basal body 100 is equipped with the encapsulated holes 163 of connection outside and phase transformation valve 162, well 110, gas outlet 130 and encapsulated holes 163 are respectively communicated with outside, and positioning area 101 is locating slot, and locating slot is arc line shaped, arc line shaped The center of circle of camber line and the rotation center of micro-fluidic chip coincide, positioning area 101 is set to gas outlet 130 and encapsulated holes 163 Between.Further, positioning column 201 is convexly equipped at the second waste liquid chamber 200.Well 110 and gas outlet 130 are located at chip basal body The same end, i.e., close to one end of rotation center；Chip basal body 100 is with partial sector structure and partial sector structure has three Straight flange.In each embodiment, each PCR amplification chamber is arranged side by side, i.e., as shown in Figures 1 to 8, the accent of each PCR amplification chamber and rotation The distance for turning center is identical.

Using Fig. 1 to embodiment illustrated in fig. 7, in a particular application, when micro-fluidic chip is static, sample is by adding Sample hole 110 is added in feeding chamber 120.Then, high speed centrifugation micro-fluidic chip, sample passes through feeding chamber circulation duct at this time 121 filling samples are enriched with chamber 140.In each embodiment, ultracentrifugal revolving speed is about 3000rpm-6000rpm.Due to phase transformation The presence of valve 162, so that the liquid of sample enrichment chamber 140 can not enter subsequent reactions chamber via sample output channel 161.It splits Solution chamber circulation duct 141, which can be a normal liquid communication pipeline, can also additionally have a valve arrangement such as phase transformation valve Structure or siphon valve arrangement etc., phase transformation valve arrangement are also referred to as phase transformation valve, and siphon valve arrangement is also referred to as siphon valve.If cracking Chamber circulation duct 141 is normal liquid communication pipeline, then in sample filling sample enrichment chamber 140, due to centrifugal field In the presence of cell, tissue, pathogen in sample etc. can be deposited to the bottom of sample enrichment chamber 140, and supernatant can be via cracking Chamber circulation duct 141 overflows into the first waste liquid chamber 150, and the function of sample enrichment has been achieved；In other embodiments, may be used One layer of filter membrane is added in sample enrichment chamber 140, cell in sample, tissue, disease are realized in such a way that filter membrane filters The enrichment of substance etc..If cracking chamber circulation duct 141 has siphon valve or phase transformation valve, can be allowed at this time with high speed centrifugation Sample is sufficiently enriched in sample enrichment chamber 140, later if having siphon valve at cracking chamber circulation duct 141, passes through drop The slow-speed of revolution 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 if there is phase transformation valve at cracking chamber circulation duct 141, open phase transformation valve by heating or other phase transformation modes and make The phase transformation valve that the supernatant in sample enrichment chamber 140 is broken through at cracking chamber circulation duct 141 is obtained to enter in the first waste liquid chamber 150. In some embodiments of concrete application, can also first high speed centrifugation allow the cell residue after centrifugation to be sufficiently deposited to sample enrichment The bottom of chamber 140 inhibits subsequent PCR reaction to avoid cell residue.In some embodiments of concrete application, can also it take The mode of high speed is slowly accelerated to, the bottom for being deposited to sample enrichment chamber 140 of cell residue is realized on one side, realizes on one side Liquid after cracking enters in dilution cracking chamber 170 via sample output channel 161.

In the present embodiment, the cracking of cell, tissue, pathogen in sample etc. is realized in sample enrichment chamber 140.Cracking Mode includes but is not limited to ultrasonic treatment, high-temperature boiling cracking, magnetic stirring cracking etc..It is heated at phase transformation valve 162, phase transformation valve 162 phase-change material melts, and remaining liq in sample enrichment chamber 140 is diluted via the inflow of sample output channel 161 It cracks in chamber 170.Phase-change material includes but is not limited to paraffin.Since the liquid volume after cracking is limited, the chamber of dilution cracking at this time The distance between liquid level and the centrifugation center of circle, that is, rotation center in 170 are greater than the distance between capillary valves 180 and the centrifugation center of circle, i.e., Liquid level in dilution cracking chamber 170 is lower than capillary valves 180, that is, dilutes the liquid level in cracking chamber 170 lower than dilution cracking chamber 170 With the communicating position of capillary valves 180, so no matter centrifugal force is much before dilution, dilution cracks the liquid in chamber 170 also not Capillary valves 180 can be broken through to enter in subsequent reaction chamber.

Further, in each embodiment, dilution cracking chamber 170 presets dilution；In some instances, dilution is preset Among at one end by the paraffin encapsulation other end by the aluminium foil of glue bond, aluminium foil is glued at the top of dilution cracking chamber 170, is added Paraffin melting after heat, dilution can just release.In other examples, the dilution in dilution cracking chamber 170 is preset Among the aluminium foil for the sealing being placed on above spine, spine is fixed on the top of dilution cracking chamber 170, between aluminium foil and spine It is fixed by spring.In this way, aluminium foil is punctured by spine when high speed centrifugation, to discharge dilution.In some instances, it dilutes It is mixed after liquid release with the liquid after cracking, mixing can rotate micro-fluidic core by positive and negative rotation micro-fluidic chip or acceleration and deceleration Piece is realized.

Then, medium-speed centrifuge, the liquid after the cracking after being diluted break through capillary valves 180 and enter reagent distribution pipeline 191, And from left to right successively fill up each measurement chamber 192.Subsequent high speed centrifugation, each liquid for measuring chamber 192 are conveyed by reagent respectively Pipeline 193 enters in corresponding PCR amplification chamber 190.Further, it is anti-that PCR is preset in each embodiment, in PCR amplification chamber 190 Answer reagent dry powder and paraffin, in one of the embodiments, PCR reaction reagent dry powder include PCR reaction needed for enzyme, dNTPs, Primer etc..Particularly, each amplification chamber can be with preset a variety of primers, and each amplification is intracavitary to may be implemented multiplex PCR.Wherein In one embodiment, amplified production is done by different fluorescent dyes to distinguish.Then, opening temperature recycles, and expands intracavitary Start to realize PCR reaction, expands intracavitary paraffin and start to melt.Since paraffin density is less than water, in centrifugal field, paraffin can float To PCR amplification chamber entrance and seal PCR amplification chamber, to avoid the Aerosol Pollution that easily occurs in PCR reaction.It is reacted in PCR In the process, micro-fluidic chip remains the state of low-speed centrifugal, and in each embodiment, the revolving speed of low-speed centrifugal is about 0- 300rpm；The each PCR amplification of reading respectively of optical system energy scan-type inside full-automatic nucleic acids instrument matched in this way The light intensity of intracavitary each fluorescence channel, to calculate Ct value, provides yin and yang attribute report to draw QPCR curve.Particularly, it is The multiplex PCR for cooperating PCR amplification intracavitary, full-automatic nucleic acids instrument the inside optical system also need the optics more covered and photoelectricity to visit Device system is surveyed, to read the fluorescence signal of different wave length.

Micro-fluidic chip has rotation center in one of the embodiments, and rotation center is located at outside chip basal body； It is arranged from small to large ord by a distance from rotation center are as follows: feeding chamber, sample enrichment chamber, the first waste liquid chamber, dilution cracking Chamber, the second waste liquid chamber；Phase transformation valve is set to sample output channel close to the position of dilution cracking chamber；Positioning area is set to gas outlet Between encapsulated holes；Gas flow pipe road is equipped with branched bottom, and the first waste liquid chamber is connected to gas flow pipe road by branched bottom, And first the position that is connected with branched bottom of waste liquid chamber be located at the first closest feeding chamber of waste liquid chamber；Micro-fluidic chip is also set Have multiple measurement chambers, each chamber that measures is arranged in a one-to-one correspondence with each PCR amplification chamber, each measurement chamber be set to reagent distribute pipeline and Between one PCR amplification chamber and reagent distribution pipeline is connected to each PCR amplification chamber by each measurement chamber respectively；It is curved to crack chamber circulation duct Folding setting；Sample output channel has an at least direction-changing area.

In one of the embodiments, as shown in Fig. 8, Fig. 9 and Figure 10, the cracking bending of chamber circulation duct 141 setting and tool There is П character form structure, crack the top position of chamber circulation duct 141 at a distance from rotation center, less than sample enrichment chamber 140 Top position is at a distance from rotation center, i.e., the top position of cracking chamber circulation duct 141 is less than at a distance from feeding chamber 120 Sample is enriched with the top position of chamber 140 at a distance from feeding chamber 120；Sample output channel 161 has the first direction-changing area 166 and the Two direction-changing areas 167.

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 implementation of micro-fluidic chip.Modern times doctor Research confirmation is learned, it is cervix caused by human papilloma virus infection that women, which suffers from cervical carcinoma, and HPV is the general name of one group of virus, It is mainly made of DNA core and protein coat.The HPV type classification about more than 80 having determined at present, different genotype HPV have different pathogenic risks, low risk, middle danger type and high-risk-type three classes can be divided by its carcinogenicity size.HPV The detection of DNA and parting have important value to the state of an illness, judgement prevention and guiding treatment is understood.

In embodiment as shown in Figures 1 to 7, it is described as follows using the testing process of micro-fluidic chip.

1. scrubbing liquid as sample using 1ml Uterine neck bush, it is added in feeding chamber 120 by well 110；

2.4000rpm is centrifuged 5 minutes, and sample flows into sample enrichment chamber 140 through feeding chamber circulation duct 121；In sample It is enriched in chamber 140, due to the presence of centrifugal field, cell, tissue, pathogen in sample can sufficiently be deposited to cavity bottom, and And supernatant can enter in the first waste liquid chamber 150；

3. carrying out 95 DEG C at pair sample enrichment chamber 140 or higher temperature heating, high-temperature boiling making sample inner cell, group It knits, pathogen sufficiently cracks；

4.1500rpm centrifugation, then heats phase transformation valve 162, so that the phase-change material of sealing phase transformation valve is melted, sample Liquid in this enrichment chamber 140 flows into dilution cracking chamber 170 through sample output channel 161.In an application example, to phase Become valve 162 and carry out 60 DEG C or higher temperature heating, paraffin melting at this time, sample output channel 161 is connected, and sample is enriched with chamber 140 Interior remaining liq flows into dilution cracking chamber 170 through sample output channel 161.

5. centrifugal speed is down to 200rpm, since dilution cracking chamber 170 presets dilution, dilution it is preset at one end by Paraffin encapsulate the other end by the aluminium foil of glue bond among, aluminium foil be glued dilution cracking chamber 170 top, to dilution crack Chamber 170 carries out 70 DEG C or higher temperature heating, and paraffin melting after heating, dilution can just release.

6. centrifugal speed rises to after the liquid and dilution in cracking chamber 170 to be diluted after cracking are sufficiently mixed 1500rpm, the liquid more than 180 inlet of capillary valves that dilution cracks in chamber 170 can break through capillary valves 180 and enter reagent point It sends out in pipeline 191, and successively fills up each measurement chamber 192, surplus liquid enters in the second waste liquid chamber 200；

7.3000rpm centrifugal microfluidic control chip, each liquid measured in chamber 192 pass through respectively reagent conveyance conduit 193 into Enter in PCR amplification chamber 190, preset reagent dry powder melts again in PCR amplification chamber 190.

8.500rpm centrifugation, and start thermal starting heating, preset paraffin melting in PCR amplification chamber 190 simultaneously float to The entrance of PCR amplification chamber 190 seals PCR amplification chamber 190, subsequently enters the temperature cycles of PCR reaction.And in each circulation Read each PCR amplification intracavitary fluorescence signal extension stage scan-type to draw PCR curve.

In such as Fig. 8 into embodiment illustrated in fig. 10, it is described as follows using the testing process of micro-fluidic chip.

3. centrifugal speed is reduced to 200rmm and rises to 1500rpm again, sample enrichment chamber 140 is in siphon piping effect The liquid for cracking 141 entrance of chamber circulation duct or more enters the first waste liquid chamber 150 along cracking chamber circulation duct 141；

4. carrying out 95 DEG C at pair sample enrichment chamber 140 or higher temperature heating, high-temperature boiling making sample inner cell, group It knits, pathogen sufficiently cracks；

5. maintaining 1500rpm centrifugal speed, then phase transformation valve 162 is heated, so that the phase-change material of sealing phase transformation valve obtains To melt, sample is enriched with the liquid in chamber 140 and flows into dilution cracking chamber 170 through sample output channel 161.It is real in an application In example, 60 DEG C are carried out to phase transformation valve 162 or higher temperature heats, paraffin melting at this time, sample output channel 161 is connected, sample Remaining liq in chamber 140 is enriched with to flow into dilution cracking chamber 170 through sample output channel 161.

6. centrifugal speed is down to 200rpm, since dilution cracking chamber 170 presets dilution, dilution it is preset at one end by Paraffin encapsulate the other end by the aluminium foil of glue bond among, aluminium foil be glued dilution cracking chamber 170 top, to dilution crack Chamber 170 carries out 70 DEG C or higher temperature heating, and paraffin melting after heating, dilution can just release.

7. centrifugal speed rises to after the liquid and dilution in cracking chamber 170 to be diluted after cracking are sufficiently mixed 1500rpm, the liquid more than 180 inlet of capillary valves that dilution cracks in chamber 170 can break through capillary valves 180 and enter reagent point It sends out in pipeline 191, and successively fills up each measurement chamber 192, surplus liquid enters in the second waste liquid chamber 200；

8.3000rpm centrifugal microfluidic control chip, each liquid measured in chamber 192 pass through respectively reagent conveyance conduit 193 into Enter in PCR amplification chamber 190, preset reagent dry powder melts again in PCR amplification chamber 190.

9.500rpm centrifugation, and start thermal starting heating, preset paraffin melting in PCR amplification chamber 190 simultaneously float to The entrance of PCR amplification chamber 190 seals PCR amplification chamber 190, subsequently enters the temperature cycles of PCR reaction.And in each circulation Read each PCR amplification intracavitary fluorescence signal extension stage scan-type to draw PCR curve.

The operation such as fluorescence detection or even yin and yang attribute judgement can be targetedly carried out later, can also carry out data Database, printed report is written in processing.

Such design, micro-fluidic chip cooperate full-automatic foranalysis of nucleic acids instrument, foranalysis of nucleic acids system are realized, using this Foranalysis of nucleic acids system can realize the full-automation for exempting from the molecular diagnosis project of nucleic acid purification step.In such centrifugal microfluidic control core In piece, the enrichment of sample, cracking, dilution and equivalent distribution, the PCR amplification of multi-chamber are all able to sequence and realize after cracking.? In one embodiment, entire reaction process is in closed micro-fluidic chip, reduces the burden and dirt of operator A possibility that dye, is also no longer dependent on profession but also 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.With this Meanwhile this micro-fluidic chip, cooperate full-automatic nucleic acids instrument to be able to achieve the detection of single sample multi objective, is that molecular diagnosis is real Now possibility is provided in face of the pathogen screening of illness.

A kind of analysis system in one of the embodiments, comprising any one micro-fluidic chip.Further, at it In middle one embodiment, analysis system is equipped at least three kinds of fluorescence channels；Further, nucleic acid in one of the embodiments, Analysis system is equipped with 5 kinds of fluorescence channels.Analysis system is used in PCR reaction process in one of the embodiments, is controlled micro- The state of fluidic chip holding low-speed centrifugal；Analysis system is also used to using full-automatic nucleic acid point in one of the embodiments, The optical system of analyzer scans the light intensity for reading the intracavitary each fluorescence channel of each PCR amplification respectively, draws QPCR curve, meter Ct value is calculated, and provides yin and yang attribute report；Analysis system is for sequentially carrying out static sample-adding, middling speed in one of the embodiments, It is centrifuged so that sample filling sample is enriched with chamber and realizes lytic cell after sample enrichment function, high speed is centrifuged so that after cracking Cell residue be deposited in sample enrichment chamber bottom and make cracking after sample enter dilution cracking it is intracavitary, heating with melt dilution Crack intracavitary paraffin to discharge dilution enable cracking after sample dilute, high speed centrifugation so that dilution after sample into Enter that each PCR amplification is intracavitary, heats to melt the intracavitary paraffin of each PCR amplification, low-speed centrifugal is to carry out PCR amplification；Wherein one In a embodiment, nucleic acid analyzer further includes the permanent magnet being arranged in below ferromagnetic block, and permanent magnet is for driving paramagnet Block is enriched with intracavitary movement or rotation in sample, and such design cooperates the embodiment with paramagnet, may be implemented preferably to split Solve effect.Further, analysis system is equipped at least three kinds of fluorescence channels in one of the embodiments,；And/or analysis system System is in PCR reaction process, control micro-fluidic chip to keep the state of low-speed centrifugal；And/or analysis system is also used to adopt Scan the light intensity for reading the intracavitary each fluorescence channel of each PCR amplification respectively with the optical system of full-automatic nucleic acids instrument, QPCR curve is drawn, Ct value is calculated, and provides yin and yang attribute report；And/or analysis system is used to sequentially carry out static sample-adding, in Speed is centrifuged so that sample filling sample is enriched with chamber and realizes lytic cell after sample enrichment function, and high speed is centrifuged so that after cracking Cell residue be deposited in sample enrichment chamber bottom and make cracking after sample enter dilution cracking it is intracavitary, heat it is dilute to melt It releases and cracks intracavitary paraffin and so that the sample after cracking is diluted to discharge dilution, high speed centrifugation is so that sample after dilution It is intracavitary into each PCR amplification, it heats to melt the intracavitary paraffin of each PCR amplification, low-speed centrifugal is to carry out PCR amplification；And/or Nucleic acid analyzer further includes the permanent magnet being arranged in below ferromagnetic block.

Further, foranalysis of nucleic acids system uses each embodiment micro-fluidic chip in one of the embodiments, and cooperation is complete Automatic foranalysis of nucleic acids instrument, realizes the full-automation for exempting from the molecular diagnosis project of nucleic acid purification step.In this micro-fluidic chip In, the enrichment of sample, cracking, dilution and equivalent distribution, the PCR amplification of multi-chamber are all able to sequence and realize after cracking.At it In in an embodiment, micro-fluidic chip is equipped with 8 PCR amplification chambers, and the corresponding foranalysis of nucleic acids instrument of each amplification chamber is glimmering equipped with 5 kinds The detection of 40 indexs simultaneously at most can be achieved in optical channel.The mode of this single sample multi objective is also molecular diagnosis realization More pathogen screenings in face of illness provide possibility.Further, entire reaction process is in closed micro-fluidic chip, A possibility that reducing the burden and pollution of operator, but also entire molecular diagnostic procedure is no longer dependent on molecular diagnosis reality Room is tested, professional operator is also no longer dependent on, realizes the demand quickly detected whenever and wherever possible, is medical inspection and disease Prevention and control bring huge help.

It should be noted that the other embodiments of the application further include, the mutually group of the technical characteristic in the various embodiments described above Close be formed by, the micro-fluidic chip that can implement and analysis system.

Each technical characteristic of above embodiments can be combined arbitrarily, for simplicity of description, not to above-described embodiment In each technical characteristic it is all possible combination be all described, as long as however, the combination of these technical characteristics be not present lance Shield all should be considered as described in this specification.

Above embodiments only express the several embodiments of the application, and the description thereof is more specific and detailed, but can not Therefore it is interpreted as the limitation to claim.It should be pointed out that for those of ordinary skill in the art, Under the premise of not departing from the application design, various modifications and improvements can be made, these belong to the protection scope of the application. Therefore, the scope of patent protection of the application should be determined by the appended claims.

Claims

1. a kind of micro-fluidic chip, which is characterized in that including chip basal body, and the sample-adding being set in the chip basal body Chamber, sample enrichment cracking chamber, the first waste liquid chamber, dilution chamber, capillary valves, the second waste liquid chamber feeding chamber circulation duct, cracking chamber stream It threads a pipe, gas flow pipe road, sample output channel, reagent distribute pipeline and multiple PCR amplification chambers；

The micro-fluidic chip is equipped with that connection is external and the well of the feeding chamber also in the chip basal body, and it is described plus Sample chamber is connected to the sample enrichment cracking chamber by the feeding chamber circulation duct；

The sample enrichment cracking chamber is connected to the first waste liquid chamber by the cracking chamber circulation duct, and the sample enrichment is split It solves chamber and the dilution chamber is also connected to by the sample output channel；

The dilution chamber is connected to the reagent by the capillary valves and distributes pipeline, and distributes pipeline by the reagent and connect respectively Lead to each PCR amplification chamber and the second waste liquid chamber；

The micro-fluidic chip is equipped with the gas outlet outside connection, the first waste liquid chamber and described also in the chip basal body Second waste liquid chamber is connected to the gas outlet by the gas flow pipe road respectively；

The sample output channel is equipped with phase transformation valve, and the chip basal body is equipped with the encapsulation of connection outside and the phase transformation valve Hole；

The chip basal body offers positioning area.

2. micro-fluidic chip according to claim 1, which is characterized in that the micro-fluidic chip be additionally provided with multiple measurement chambers and Its reagent conveyance conduit, each measurement chamber are arranged in a one-to-one correspondence with each PCR amplification chamber, and each measurement chamber is set to Between the reagent distribution pipeline and a PCR amplification chamber, the reagent distribution pipeline is respectively communicated with each measurement chamber and connects Lead to each PCR amplification chamber, and the measurement chamber is connected to the corresponding PCR amplification chamber by its described reagent conveyance conduit.

3. micro-fluidic chip according to claim 1, which is characterized in that the cracking chamber circulation duct bending setting.

4. micro-fluidic chip according to claim 3, which is characterized in that the cracking chamber circulation duct has П font knot Structure.

5. micro-fluidic chip according to claim 1, which is characterized in that the sample output channel has an at least deflecting Area.

6. micro-fluidic chip according to claim 5, which is characterized in that the direction-changing area has arcuate structure.

7. micro-fluidic chip according to claim 1, which is characterized in that the sample enrichment cracking chamber is equipped with filter membrane.

8. micro-fluidic chip according to claim 1, which is characterized in that the cracking chamber circulation duct is equipped with valve arrangement.

9. according to claim 1 to micro-fluidic chip described in any one of 8, which is characterized in that sample enrichment cracking chamber with The cracking chamber circulation duct has the first link position, and sample enrichment cracking chamber and the sample output channel have the Two link positions, first link position are less than second link position and the feeding chamber at a distance from the feeding chamber Distance；

The cracking chamber circulation duct is equipped with valve arrangement；Further, the valve arrangement is siphon valve arrangement or phase transformation valve knot Structure；

The positioning area is set between the gas outlet and the encapsulated holes；

The positioning area is positioning convex portion, location hole or locating slot, and the quantity of the positioning area is one, two or more；Institute State that locating slot is linear or the locating slot is the rotation center of arc line shaped and the center of circle of its camber line and the micro-fluidic chip It coincides；Further, the quantity of the location hole is that multiple and rotation centers relative to the micro-fluidic chip uniformly divides Cloth；

The encapsulated holes closing setting；Further, the micro-fluidic chip is equipped with closing cover at the encapsulated holes；

The micro-fluidic chip has rotation center and the rotation center is located at outside the chip basal body, presses and the rotation The distance at center arranges from small to large ord are as follows: the feeding chamber, the sample enrichment cracking chamber, the first waste liquid chamber, The dilution chamber, the second waste liquid chamber；

The gas flow pipe road is equipped with branched bottom, and the first waste liquid chamber is connected to the gas stream by the branched bottom It threads a pipe, and to be located at the first waste liquid chamber closest described for the position that is connected with the branched bottom of the first waste liquid chamber At feeding 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；Into one Step ground, the analysis system are equipped at least three kinds of fluorescence channels；And/or the analysis system is used in PCR reaction process, Control the state that the micro-fluidic chip keeps low-speed centrifugal；And/or the analysis system is also used to using full-automatic nucleic acid point The optical system of analyzer scans the light intensity for reading the intracavitary each fluorescence channel of each PCR amplification respectively, draws QPCR curve, meter Ct value is calculated, and provides yin and yang attribute report；And/or the analysis system, for sequentially carrying out static sample-adding, medium-speed centrifuge is so that sample This filling sample enrichment cracking chamber simultaneously realizes lytic cell after sample enrichment function, and high speed is centrifuged so that the cell after cracking is residual Slag is deposited in the bottom of sample enrichment cracking chamber and enters the sample after cracking in dilution chamber, heats to melt in dilution chamber Paraffin enables the sample after cracking to dilute to discharge dilution, and high speed centrifugation is so that the sample after dilution expands into each PCR Increase it is intracavitary, heat to melt the intracavitary paraffin of each PCR amplification, low-speed centrifugal is to carry out PCR amplification.