CN109536366A - A kind of detection of nucleic acids micro-fluidic chip and nucleic acid detection system based on modified capillary - Google Patents
A kind of detection of nucleic acids micro-fluidic chip and nucleic acid detection system based on modified capillary Download PDFInfo
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Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/02—Adapting objects or devices to another
- B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
- B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Clinical Laboratory Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Dispersion Chemistry (AREA)
- Analytical Chemistry (AREA)
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Abstract
The present invention relates to a kind of detection of nucleic acids micro-fluidic chips and nucleic acid detection system based on modified capillary, include modified capillary inside the micro-fluidic chip, carrier for adsorbing the target nucleic acid in solution sample to be measured, as the automation implementation for realizing nucleic acid absorption, purifying, amplification and detection.The present invention realizes integrated sample cracking, nucleic acid extraction, nucleic acid amplification and detection of nucleic acids function, can be deployed in entry and exit port, the field Lab region Deng Fei with realization and detect to infectious agent.
Description
Technical field
The present invention relates to the technical fields of micro-fluidic foranalysis of nucleic acids chip, and in particular to a kind of core based on modified capillary
Acid detection micro-fluidic chip and nucleic acid detection system.
Background technique
In recent years, several extensive infectious diseases, such as H1N1, Ebola virus and stockaded village's card etc. have been broken out in the whole world, need to lead to
The detection of nucleic acids instrument for crossing profession, can just detect by nucleic acid extraction and the process of amplification.Typically this quasi-instrument has body
Product is huge, the disadvantages of being difficult to field deployment, involving great expense and special messenger is needed to safeguard.Micro-fluidic chip is because of its being easily integrated, automatic
Change, the feature that fluid controllable and required sample size are small, is widely used in biochemical analysis field, such as bacterium, virus and protein
Detection field etc. compensates for professional detection of nucleic acids equipment to a certain extent and is difficult to be deployed in the non-experiment such as entry and exit port, field
The defect of chamber region.
The core of nucleic acid detection method based on micro-fluidic chip first is that the method for nucleic acid extraction, it is usually following several
One of method: boiling method, paramagnetic particle method and solid phase extraction.In order to develop the type scene Rapid nucleic acid detection device, applicant
The research team at place has carried out certain research to related fields already and has had deep Research foundation, and Ru Zhaoshu more et al. exists
" the integrated nucleic acid extraction real-time fluorescence PCR micro-total analysis system " delivered on analytical chemistry, this paper is reported a kind of for detecting
The micro-fluidic chip of virus in blood and the fluorescent quantitative detection device of collocation, but the chip of paper report is to use to be based on
The solid phase extraction of pellosil carries out nucleic acid extraction, and the sample being directed to is blood lysis liquid, cannot be handled whole blood
Analysis.A kind of base is described in the patent " devices and methods therefors of field quick detection infectious disease pathogens " of the applications such as Liu Yingle
In the nucleic acid-extracting apparatus design of precipitating boiling method, but the nucleic acid amount of this method extraction is few and purity is low, it is difficult to RNA class disease
The high sample of the nucleic acid purifications quality requirement such as poison is purified.Qiu Xianbo etc. is in patent " nucleic acid automatically extracts micro fluidic device "
A kind of nucleic acid extraction chip and device based on paramagnetic particle method is described, and paramagnetic particle method itself, which is not, to be designed for micro-fluidic chip
Method, in contrast step is complicated and is difficult to carry out automation integrated.Thiago Wu Qingqing et al. exists
Delivered on Analytical chemistry < Integrated glass microdevice for nucleic acid
Purification, loop-mediated isothermal amplification, and the online text of detection > mono-
In report the chip of the collection nucleic acid extraction based on solid phase extraction method and amplification a kind of, need during nucleic acid extraction additional
Nucleic Acid Elution step increases the integrated difficulty of micro-fluidic chip.Hui Junhou et al. delivered in Lab on a Chip <
Multiplex sample-to-answer detection ofbacteria using a pipette-actuated
capillary array comb with integrated DNA extraction,isothermal amplification,
And smartphone detection > propose a kind of capillary array based on Chitosan modified glass Adsorption of Filter Paper nucleic acid
Nucleic acid original position amplification method, realizing nucleic acid and needing not move through elution step can be realized amplification, greatly reduce integrated difficulty, but
It is that the method proposed in the paper still needs to rely on manual operation, is not able to achieve nucleic acid " extracting --- amplification --- detection " certainly
The process of dynamicization.
Summary of the invention
The present invention solves technical problem: overcoming above-mentioned prior art defect, provides a kind of nucleic acid based on modified capillary
Micro-fluidic chip and nucleic acid detection system are detected, sample cracking, nucleic acid extraction, nucleic acid amplification and detection of nucleic acids function are integrated, with
Realization can be deployed in non-Lab region and detect to infectious agent.
To achieve the goals above, technical scheme is as follows:
Include a kind of modified capillary inside micro-fluidic chip, for adsorbing the target nucleic acid in solution sample to be measured, makees
For the carrier for automating implementation for realizing nucleic acid absorption, purifying, amplification and detection；
The chip is respectively substrate, fluid path layer substrate, elastic film, gas circuit layer substrate four-layer structure from bottom to top；Liquid
The upper surface of road floor substrate is embedded with fluid flow paths, liquid hybrid channel and separating liquid channel, and has through hole to lead to liquid
Road floor lower surface；Chamber is reserved embedded with cracking chamber and capillary in fluid path layer lower surface, and the latter is internally embedded modified capillary, realizes
The absorption and amplification of sample of nucleic acid；The cavity that the upper surface of gas circuit layer substrate has multiple protrusions hollow, for store detection sample,
Lysate or PCR reagent；The air valve of multiple recess is contained in lower surface, and is run through with upper surface by through-hole, fluid path layer substrate
Lower surface is bonded with bottom substrate, and wherein middle chamber part is chimeric with capillary, the upper surface of fluid path layer substrate and Thin Elastic
The bonding of film lower surface；Upper and lower surfaces connects with microchannel by microchannel so that liquid storage chamber, crack chamber, capillary with
And outlet；Gas circuit layer substrate lower surface is bonded with elastic film upper surface, constitutes one piece of complete micro-fluidic chip.
The substrate is glass or silicon wafer；Fluid path layer substrate and gas circuit layer substrate are PDMS, PC, PMMA or COC polymerization
Object material；Elastic membrane is the elastic material of PDMS or TPU；
In addition, according to one embodiment of present invention, the micro-fluidic chip gas circuit layer substrate upper surface further includes three
Liquid storage chamber, the liquid storage chamber are commonly connected to by penetrating the through-hole of gas circuit layer substrate upper and lower surface positioned at fluid path layer upper surface
Serpentine channel, the serpentine channel are connected to the cracking chamber for cracking nucleic acid, and the cracking chamber is connected to for being fitted into capillary
The cavity of pipe.
The technical solution that the modified capillary uses are as follows: make capillary surface carry positive electricity by process for modifying surface
Lotus, target nucleic acid of the sample after cracking in solution can be enriched with by the modified capillary, while the nucleic acid being enriched with is just
It can be dissociated with spontaneous into solution under the conditions of normal nucleic acid amplification；
In addition, according to an embodiment of the present invention, the modification capillary is glass, silica or thermoplastic
In any one hard transparent material be made；
In addition, according to one embodiment of present invention, modified capillary tube inner wall contains functional group of the end with amino structure
Or the compound of ion, including-NH2Or [NH4]+Structure, is based primarily upon hydrogen bond and ionic bond adsorbs nucleic acid；
In addition, according to an embodiment of the present invention, the modification capillary passes through interference fit, heat bonding or plasma
Cleaning bonding is fixed in micro-fluidic chip fluid path substrate lower surface channel；
In addition, according to an embodiment of the present invention, target nucleic acid of the sample after cracking in solution, which refers to, to be in
Nucleic acid in lysate environment changes lysate solution system without additional；
In addition, according to an embodiment of the present invention, referring mainly to need not move through under the conditions of the normal nucleic acid amplification additional
Means, including change temperature, pH value in capillary, in solution under the premise of the conditions such as other ion concentrations, the core being adsorbed
Acid is spontaneously decoupled capillary wall suction-operated in nucleic acid amplification reaction liquid system and participates in nucleic acid amplification reaction.
In order to realize the automatic control to above-mentioned micro-fluidic chip, the present invention provides a kind of the mating of micro-fluidic chip
Device includes fluid driving control system, air valve control system, temperature control system, optical system, control system and intelligent hand
Machine, for realizing above-mentioned micro-fluidic chip full-automatic operation and detection process.It is full-automatic for realizing above-mentioned micro-fluidic chip
Change operation and detection process, realizes the target of " sample into as a result go out ".
The technical solution that micro-fluidic chip uses in the present invention are as follows: simultaneously using the modified capillary attraction in insertion chip
Purifying nucleic acid, union at nucleic acid amplification and detection micro-fluidic chip；
The technical solution that the corollary apparatus of micro-fluidic chip uses in the present invention are as follows:
The micro-fluidic chip is by gas circuit layer substrate, elastic film, fluid path layer substrate, substrate, heating cracking chamber, the first storage
Sap cavity, the second liquid storage chamber, third liquid storage chamber, the first injection pump interface, the second injection pump interface, several elastic film micro-valves, snake
Shape liquid mixture flow road, several modified capillaries reserve chamber, the first modified capillary, the second modified capillary, the modified hair of third
Tubule, the 4th modified capillary are constituted；
Described device includes fluid driving control system, air valve control system, temperature control system, optical system, control system
And smart phone, fluid driving control system connect chip gas circuit layer substrate upper surface, temperature control system with air valve control system
It is close to chip base lower surface, optical system is placed in right above micro-fluidic chip, and smart phone is placed in right above optical system.
Fluid in the fluid driving control system control micro-fluidic chip, the air valve control system manipulate Thin Elastic
The opening and closing of film micro-valve, temperature needed for the temperature control system maintains nucleic acid amplification, the optical system provide the excitation of specific wavelength
Light and the transmitting light for obtaining specific wavelength, the control system are real to fluid driving control system, temperature control system and optical system
Control is applied, the image identification function of the smart phone carries out quantitative and semi-quantitative detection to sample；
In addition, according to an embodiment of the present invention, the fluid driving control system is by the first syringe pump, the second syringe pump
It constitutes, syringe pump is connected to the inlet and outlet on micro-fluidic chip, drives fluid by way of positive pressure and negative pressure；
In addition, according to an embodiment of the present invention, the air valve control system is made of gas source and several solenoid valves, gas
Source generates the positive pressure for continuing 0.2MPa and is connected to solenoid valve, and it is micro- that the solenoid valve other end is separately connected an at least elastic film
Valve is opened and closed by the opening and closing control elastic film micro-valve of solenoid valve；
In addition, according to an embodiment of the present invention, the temperature control system is close to the substrate of micro-fluidic chip, inside includes temperature
Sensor, Peltier, heat sink, radiating fin and fan are spent, temperature regulating range is 0-100 DEG C, and temperature degree of regulation is 0.1
DEG C, warming and cooling rate is 5 DEG C/s, for controlling the capillary temperature inside micro-fluidic chip, meets the temperature of nucleic acid amplification needs
Spend environment；
In addition, according to an embodiment of the present invention, the optical system includes an excitation light source, optical filter, one
A lens and a focus lamp are irradiated in micro flow control chip capillary surface mainly for generation of the exciting light of specific wavelength, and
The transmitting light of specific wavelength is obtained, realizes and capillary nucleic acid amplification situation quantitative and semi-quantitative is detected；
In addition, according to an embodiment of the present invention, the control system includes channel radio at a microprocessor and one
Communication interface, microprocessor is for operating fluid driving control system, optical system and temperature control system, and wireless communication interface is for micro-
Processor and smart phone communicate；
In addition, according to an embodiment of the present invention, the smart phone drives fluid by control microprocessor machine and controls
System, temperature control system and optical system processed issue an order and obtain feedback data, and pass through included camera detection fluorescence letter
Number, by the way that built-in image processing software is qualitative to nucleic acid amplification result or semi-quantitative analysis.
In addition, according to an embodiment of the present invention, steps are as follows for the implementation method that the micro-fluidic chip is analyzed:
(1) sample loads
Sample needed for foranalysis of nucleic acids, lysate and reagent sequentially add the first liquid storage chamber, the second liquid storage chamber, third liquid storage
Chamber；
(2) sample is mixed with lysate
Sample and lysate are mixed by the effect of serpentine fluid mixing runner under the driving for the negative pressure that syringe pump generates
It closes, and is finally uniformly mixed and imports cracking chamber；
(3) sample cracks
Discharge DNA into liquid in cracking intracavitary heating the mixed liquor of sample and lysate by temperature control system；
(4) nucleic acid absorption
Driving force size allows sample to pass through modified capillary, and removes lysate；
(5) Impurity elution
The modified capillary of eluent injection first, second are modified by syringe pump by being located at the through-hole above gas circuit layer substrate
Then the modified capillary of capillary, third removes eluent to get sample of nucleic acid；
(6) reaction solution loads
By external force driving effect, the reaction solution cavity above gas circuit layer substrate will be located at and pass through bifurcated flow-channel for reaction solution
It loads to the first modified capillary, the second modified capillary, the modified capillary of third, the 4th modified capillary；
(7) nucleic acid amplification
The the first modified capillary, the second modified capillary, the modified capillary of third of chip interior are heated by temperature control system
Pipe, the 4th are modified capillary, and temperature condition needed for specific nucleic acid amplification is determined by the reagent reaction system selected；
(8) detection of nucleic acids
It is taken pictures using smart phone to modified capillary timing and acquires fluorescent image, and pass through built-in image recognition software
Identification and the modified capillary fluorescence intensity change of detection, realize the qualitative and half-quantitative detection to sample of nucleic acid.
The principle of the invention lies in:
(1) micro-fluidic chip is constituted by 4 layers, by lower from upper respectively substrate, fluid path layer substrate, elastic film and gas circuit layer
Substrate makes the microchannel and microchannel disengaging of reagent and sample by customization by the cooperation of outer power drive and elastic film valve
Cavity；Furthermore chip interior is embedded in the capillary changed, and elecrtonegativity is presented in nucleic acid (pH > 7) in alkaline solution, thus by capillary
It is adsorbed to wall surface, the enrichment of sample to be tested is realized, and elute capillary using weakly alkaline solution, so that extra impurity be gone
It removes, and in contrast the nucleic acid for being attracted to modified capillary tube inner wall is difficult to be eluted since inside pipe wall face flow rate of liquid is lower,
To obtain more pure sample of nucleic acid.
(2) order is sent by the means of wireless telecommunications based on smart phone and controls each system, and pass through included camera shooting
Head obtains capillary fluorescent image, capillary fluorescence intensity change is detected by built-in image recognition software, thus to amplification
As a result quantitative and semi-quantitative judgement is carried out.
The present invention is a little compared with prior art:
(1) compared to traditional nucleic acid extraction and detection method, micro-fluidic chip and system in the present invention be may be implemented
Nucleic acid cleavage, nucleic acid absorption, Nucleic Acid Elution, nucleic acid amplification and the overall process of detection, operating procedure are simple, it is only necessary to add sample
, all processes realize in the chip, complete " sample into --- result goes out ".
(2) micro-fluidic chip of nucleic acid is extracted compared to traditional paramagnetic particle method and solid phase extraction, contains ammonia in the present invention
The micro-fluidic chip of the modified capillary of base, avoids the step of eluting nucleic acid, it is possible to reduce nucleic acid pre-treatment operating procedure simplifies
Process；
(3) compared to traditional micro-fluidic chip instrument, the present invention is based on smart phones to realize the detection to sample, reduces
The manufacturing cost of equipment, improves the portability and flexibility of instrument；
In short, the present invention provides a kind of more function with sample pre-treatments, nucleic acid enriching and purifying, nucleic acid amplification and detection
Can foranalysis of nucleic acids chip and corollary apparatus, realize automation, the totally enclosed type micro-fluidic chip of " sample into --- result goes out ", fit
Conjunction is applied in the scene for being difficult to dispose large-scale instrument and equipment.
Detailed description of the invention
Fig. 1 is the top view of micro-fluidic chip in the present invention；
Fig. 2 is the analysis diagram of micro-fluidic chip in the present invention；
Fig. 3 is the apparatus structure schematic diagram of micro-fluidic chip sample introduction, nucleic acid enriching and elution process in the present invention；
Fig. 4 is the apparatus structure schematic diagram of amplification and detection process in the present invention.
In figure, Greek alphabet number is respectively as follows: micro-fluidic chip I, fluid driving control system II, air valve control system
III, control system IV, smart phone V, optical system VI, temperature control system VII；Arabic numerals number is respectively as follows: gas circuit layer base
Piece 1, elastic film 2, fluid path layer substrate 3, substrate 4 crack chamber 5, the first liquid storage chamber 61, the second liquid storage chamber 62, third liquid storage chamber
63, the first injection pump interface 64, the second injection pump interface 65, the first elastic film micro-valve 71, the second elastic film micro-valve 72, the
Three elastic film micro-valves 73, the 4th elastic film micro-valve 74, the 5th elastic film micro-valve 75, the 6th elastic film micro-valve 76, the
Seven elastic film micro-valves 77, serpentine fluid mixing runner 8, the first modified capillary reserve chamber 81, and the second modified capillary is reserved
Chamber 82, the modified capillary of third reserve chamber 83, and the 4th modified capillary reserves chamber 84, and the first modified capillary 91, second is modified
Capillary 92, the modified capillary 93 of third, the 4th modified capillary 94, the first solenoid valve 101, second solenoid valve 102, third electricity
Magnet valve 103, the 4th solenoid valve 104, gas source 11, the first syringe pump 121, the second syringe pump 122, lens 13, optical filter 14, excitation
Light source 15, focus lamp 16, heat sink 17, radiating fin 18, radiator fan 19.
Specific embodiment
The present invention is further described below with reference to drawings and examples.These embodiments are merely to illustrate without limiting
Application range of the invention.In addition, it should also be understood that, those skilled in the art can be to this after reading the content that the present invention lectures
Any change and modification are made in invention, and such equivalent forms equally fall within the application scope of the appended claims.
As depicted in figs. 1 and 2, micro-fluidic chip I is gas circuit layer substrate 1, elastic film 2, fluid path layer substrate 3 from top to bottom
With substrate 4.4 capillaries are put into H2SO41h is impregnated in solution, it is ensured that capillary inner surface is completely capped, to remove capillary
The spot of pipe internal surface；Capillary is taken out again, with pure water rinsing 1h, to remove the H of capillary inner surface2SO4；Finally by hair
Tubule is placed in the alkali compounds of the functional group that amino structure is contained in end or ion, such as-NH2, [NH4]+etc. structures, this
When solution in compound can react with capillary tube inner wall, thus by corresponding chemical group be coated in capillary tube inner wall
On to get the modified capillary 92 of the first modified capillary 91, second, the modified capillary 94 of the modified capillary the 93, the 4th of third.With
The first modified capillary that 4 modified capillaries are horizontally placed on 3 bottom of fluid path layer substrate respectively is reserved into the modified hair of chamber 81, second afterwards
Tubule is reserved the modified capillary of chamber 82, third and is reserved in the reserved chamber 84 of the modified capillary of chamber the 83, the 4th.Then by micro-fluidic chip
I is successively bonded according to the sequence of gas circuit layer substrate 1, elastic film 2, fluid path layer substrate 3 and substrate 4, that is, forms Fig. 1 and Fig. 2 institute
The micro-fluidic chip I shown.
III air valve control system includes 4 solenoid valves in Fig. 3, wherein the first solenoid valve 101 is connected by gas circuit sealing pipeline
The first elastic film micro-valve 71 in map interlinking 1, second solenoid valve 102 connect the second elastic film micro-valve 72 and the 7th elastic film
Micro-valve 77, third solenoid valve 103 connect third elastic film micro-valve 73 and the 6th elastic film micro-valve 76, the 4th solenoid valve 104
Connect the 4th elastic film micro-valve 74 and the 5th elastic film micro-valve 75；The other end of solenoid valve is connect with gas source 11, Hou Zheti
The air pressure for being 0.2MPa for constant magnitude；Solenoid valve is implemented to control by control system IV, when solenoid valve is opened, gas source 11
It is passed through the corresponding elastic film micro-valve of the solenoid valve, runner is caused to be closed.
As shown in Fig. 3 and Fig. 1, fluid driving control system II includes the first syringe pump 121, the second syringe pump in Fig. 3
122, wherein the first syringe pump 121 connection the first injection pump intake 64, pump are provided with the deionized water that pH is 7.0；Second injection
122 connection the second injection pump intakes 65 of pump, without sample loading and residual volume inside syringe pump.
As shown in Fig. 4 and Fig. 1, micro-fluidic core I piece is placed on temperature control system VII in Fig. 4, and with heat sink 17 close phases
Even, heat sink 17 bottom is radiating fin 18 and fan 19.Heat sink heating and cooling are controlled by the pid algorithm built in temperature control system, and
Cooperate radiating fin 18 and fan 19, to maintain temperature needed for nucleic acid amplification reaction；Optical filter 14 in optical system VI
It is modified to be right against the modified capillary 92 of the modified capillary 91, second of micro-fluidic chip I first, the modified capillary the 93, the 4th of third
The top of capillary 94, excitation light source 15 irradiate 470nm exciting light, and it is modified to reflex to first by focus lamp 16 and lens 13
Capillary 91, second is modified the modified capillary 94 of the modified capillary the 93, the 4th of capillary 92, third, and obtains 590nm transmitting
Light；Smart phone camera is right against right above lens 13, is taken pictures and is acquired fluorescence signal, and passes through built-in image processing software
Analysis of fluorescence intensity.
Specific embodiment two, present embodiment are the nucleic acid inspection described in specific embodiment one based on modified capillary
Micrometer fluidic chip and nucleic acid detection system application process:
Step 1: sample load.The bottom micro-fluidic chip I is filled into heat-conducting silicone grease, and is close to temperature control system VII.It will
250 μ l blood sample to be measured and 500 μ l lysates are respectively placed in the first liquid storage chamber 61, in the second liquid storage chamber 62, by 150 μ l PCR
Mix is added in third liquid storage chamber 63；Solenoid valve and syringe pump are connected to miniflow by seal tube according in embodiment one
Control each elastic film micro-valve of chip I or injection pump interface.
Step 2: sample is mixed with lysate.Manipulation mobile phone control control system IV issues an order, starting gas source 11 and electricity
Magnet valve and syringe pump.By opening and the first solenoid valve 101 and the 4th solenoid valve 104, the first elastic film micro-valve 71, the are closed
Four elastic film micro-valves 74, the 5th elastic film micro-valve 75 make and the first liquid storage chamber 61, the second liquid storage chamber 62 and the second injection
Liquid flow path between pump intake 65 forms access；Make to generate negative pressure in channel by 122 stretching action of the second syringe pump, drives sample
This reconciliation cracking enters cracking chamber 5, wherein when the two passes through liquid mixture flow road 8, it can the spontaneous mixing in snake pipe；Work as sample
When originally filling up cracking chamber 5, second solenoid valve 102 is opened, keeps the second elastic film micro-valve 72 and the 7th elastic film micro-valve 77 close
Envelope.
Step 3: sample cracking.Temperature control system VII is manipulated by smart phone V, heats heat sink 17 to 95 DEG C, the time is
10min makes the blood sample cracked in chamber 5 crack and release determined nucleic acid.
Step 4: nucleic acid absorption.Second solenoid valve 102 is closed, while controlling the second syringe pump 122 and continuing to stretch, allows cracking
Sample afterwards, which is slow transitted through, is modified the modified capillary 93 of capillary 92, third, core to be measured by the first modified capillary 91, second
Acid is fixed on surface under the charge adsorption effect of capillary surface, and last waste liquid enters in the second syringe pump 122.
Step 5: Impurity elution.The first solenoid valve 101 is closed, and opens second solenoid valve 102, so that the first injection is pumped into
Channel between mouth 64 and the second injection pump intake 65 is connected to.5ml ionized water is gone to lead to it slowly by the driving of the first syringe pump 121
The modified capillary 92 of the first modified capillary 91, second, the modified capillary 93 of third are crossed, by remaining Impurity elution, and it is to be measured
Nucleic acid is difficult to be washed away under capillary attraction effect；The second syringe pump 122 is stretched simultaneously, and the liquid after elution finally enters the
In two syringe pumps 122.
Step 6: reaction solution load.Close the first solenoid valve 101, and open the 4th solenoid valve 104 so that reagent chamber 63 with
Channel connection between second injection pump intake 65.By stretching the second syringe pump 122, so that PCR mix enters the first modified hair
Tubule 91, second is modified the modified capillary 94 of the modified capillary the 93, the 4th of capillary 92, third, fills up 4 hairs simultaneously to liquid
When tubule, all solenoid valves are opened, are closed all elastic film micro-valve, to seal all modified capillaries.
Step 7: nucleic acid amplification.Temperature control system VII is opened, controls the amplification of sample nucleic acid, temperature by heat sink heating and cooling
Spend parameter are as follows: 95 DEG C first lasting 600s realize Taq enzyme initial denaturation；Untwist 95 DEG C of lasting 15s, and anneal 62 DEG C of lasting 20s, extends
72 DEG C of lasting 20s, continue 35 circulations.
Step 8: detection of nucleic acids.Coordinate excitation light source 15 and smart phone V using smart phone, in the extension of each circulation
Stage takes pictures, and identifies capillary edge contour using internal image processing software and obtain fluorescence signal, passes through built-in calculation
Method obtains fluorescence intensity curves, realizes and half-quantitative detection qualitative to target nucleic acid.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily
Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and legend shown and described herein.
Claims (9)
1. a kind of detection of nucleic acids micro-fluidic chip based on modified capillary, it is characterised in that: packet inside the micro-fluidic chip
Containing modified capillary, for adsorbing the target nucleic acid in solution sample to be measured, as realization nucleic acid absorption, purifying, amplification and inspection
The carrier of the automation implementation of survey.
2. the detection of nucleic acids micro-fluidic chip according to claim 1 based on modified capillary, it is characterised in that: described micro-
Fluidic chip is respectively substrate, fluid path layer substrate, elastic film and gas circuit layer substrate from bottom to top；The upper surface of fluid path layer substrate
Embedded with fluid flow paths, liquid hybrid channel and separating liquid channel, and there is through hole to lead to fluid path layer lower surface；Fluid path layer
Chamber is reserved embedded with cracking chamber and modified capillary in lower surface, and the latter is internally embedded modified capillary；The upper surface of gas circuit layer substrate
The liquid storage chamber for having multiple protrusions hollow；The elastic film micro-valve of multiple recess is contained in lower surface, and is passed through with upper surface by through-hole
It wears, the lower surface of fluid path layer substrate is bonded with bottom substrate, and wherein middle chamber part is chimeric with modified capillary, fluid path layer base
The upper surface of piece is bonded with elastic film lower surface；Upper and lower surfaces are connected by microchannel with microchannel, so that liquid storage
Chamber, cracking chamber, modified capillary and outlet；Gas circuit layer substrate lower surface is bonded with elastic film upper surface, constitutes one
The complete micro-fluidic chip of block.
3. the detection of nucleic acids micro-fluidic chip according to claim 2 based on modified capillary, it is characterised in that: the base
Bottom is glass or silicon wafer；Fluid path layer substrate and gas circuit layer substrate are PDMS, PC, PMMA or COC polymer material；Elastic membrane is
PDMS or TPU elastic material.
4. the detection of nucleic acids micro-fluidic chip according to claim 2 based on modified capillary, it is characterised in that: described to change
Property capillary by interference fit, heat bonding or plasma cleaning bonding be fixed on micro-fluidic chip fluid path substrate lower surface channel
It is interior.
5. the detection of nucleic acids micro-fluidic chip according to claim 1 or 2 based on modified capillary, it is characterised in that: change
Property capillary is made of hard transparent material, and the hard transparent material includes glass, silica or thermoplastic.
6. the detection of nucleic acids micro-fluidic chip according to claim 1 or 2 based on modified capillary, it is characterised in that: institute
The inner wall for stating modified capillary contains the compound of functional group of the end with amino structure or ion, including-NH2Or [NH4]+Knot
Structure.
7. a kind of detection of nucleic acids for realizing the detection of nucleic acids micro-fluidic chip based on modified capillary described in operational rights requirement 1
System, it is characterised in that: the nucleic acid detection system is the micro-fluidic chip and corollary apparatus, comprising: micro-fluidic chip, stream
Body driving control system, air valve control system, control system, smart phone, optical system, temperature control system；The micro-fluidic core
Piece is by gas circuit layer substrate, elastic film, fluid path layer substrate, substrate, heating cracking chamber, the first liquid storage chamber, the second liquid storage chamber, third
Liquid storage chamber, the first injection pump interface, the second injection pump interface, several elastic film micro-valves, serpentine fluid mixing runner, Ruo Gangai
Property capillary reserve chamber, the first modified capillary, the second modified capillary, the modified capillary of third, the 4th modified capillary structure
At；The fluid driving control system is made of the first syringe pump, the second syringe pump；The air valve control system by gas source and
Several solenoid valves are constituted；The optical system is made of lens, optical filter, excitation light source, focus lamp；The temperature control system is by warm
Heavy, radiating fin, radiator fan is constituted；One end of each solenoid valve is micro- by the corresponding elastic film of gas circuit sealing pipeline connection
Valve, the other end are connect with gas source；First syringe pump connection the first injection pump intake, pump are provided with the deionized water that pH is 7.0；The
Two syringe pumps connection the second injection pump intake, without sample loading and residual volume inside syringe pump；Micro-fluidic chip is placed in temperature
In control system, and with it is heat sink closely coupled, heat sink bottom be radiating fin and fan；Optical filter in optical system is right against micro-
Above fluidic chip capillary, excitation light source 15 irradiates 470nm exciting light, by focus lamp and reflection from lens to capillary,
And obtain 590nm transmitting light；Smart phone camera is right against right above lens, is taken pictures and is acquired fluorescence signal, and by built-in
Image processing software analysis of fluorescence intensity.
8. the nucleic acid detection system of the detection of nucleic acids micro-fluidic chip according to claim 7 based on modified capillary,
Be characterized in that: the smart phone assigns life to fluid driving control system, temperature control system and optical system by control system
Feedback data is enabled and obtained, and fluorescence signal is detected by smart phone, by built-in image processing software to nucleic acid amplification
As a result qualitative or semi-quantitative analysis.
9. the nucleic acid detection system of the detection of nucleic acids micro-fluidic chip according to claim 7 based on modified capillary, special
Sign is: including the following steps:
(1) sample loads
Sample needed for foranalysis of nucleic acids, lysate and reagent sequentially add the first liquid storage chamber, the second liquid storage chamber, third liquid storage chamber；
(2) sample is mixed with lysate
Sample and lysate mix under the driving for the negative pressure that syringe pump generates by the effect of serpentine fluid mixing runner, and
Final be uniformly mixed imports cracking chamber；
(3) sample cracks
Discharge DNA into liquid in cracking intracavitary heating the mixed liquor of sample and lysate by temperature control system；
(4) nucleic acid absorption
Driving force size allows sample to pass through modified capillary, and removes lysate；
(5) Impurity elution
Syringe pump is by being located at the through-hole above gas circuit layer substrate for the modified capillary of eluent injection first, the second modified capillary
Then the modified capillary of pipe, third removes eluent to get sample of nucleic acid；
(6) reaction solution loads
By external force driving effect, the reaction solution cavity being located above gas circuit layer substrate is loaded reaction solution by bifurcated flow-channel
To the first modified capillary, the second modified capillary, the modified capillary of third, the 4th modified capillary；
(7) nucleic acid amplification
The first modified capillary of chip interior, the second modified capillary, the modified capillary of third, the are heated by temperature control system
Four are modified capillaries, and temperature condition needed for specific nucleic acid amplification is determined by the reagent reaction system selected；
(8) detection of nucleic acids
It is taken pictures using smart phone to modified capillary timing and acquires fluorescent image, and identified by built-in image recognition software
And modified capillary fluorescence intensity change is detected, realize the qualitative and half-quantitative detection to sample of nucleic acid.
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