CN110331092A - A kind of nucleic acid complete analysis micro-fluidic chip system and its application method - Google Patents
A kind of nucleic acid complete analysis micro-fluidic chip system and its application method Download PDFInfo
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- CN110331092A CN110331092A CN201910712073.6A CN201910712073A CN110331092A CN 110331092 A CN110331092 A CN 110331092A CN 201910712073 A CN201910712073 A CN 201910712073A CN 110331092 A CN110331092 A CN 110331092A
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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
- 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
- B01L3/50273—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 characterised by the means or forces applied to move the fluids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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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
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
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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
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
- B01L2400/043—Moving fluids with specific forces or mechanical means specific forces magnetic forces
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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
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
Abstract
The invention discloses a kind of nucleic acid complete analysis micro-fluidic chip system and its application methods, belong to micro-fluidic chip nucleic acid analysis technique field.Including nucleic acid complete analysis micro-fluidic chip and portability device, the micro-fluidic chip, comprising: the foranalysis of nucleic acids channel of several parallel arrangements, the channel contain three-dimensional hybrid pond and several reaction tanks respectively, contain isolation tank between the adjacent reaction tank；The portability device, comprising: rotated three dimensional magnetic field control module, one-dimensional movement magnetic field control module, temperature control modules and signal detection module.Nucleic acid complete analysis micro-fluidic chip and portability device of the present invention can realize multistep magnetic bead efficiently mix, reaction process, and it is easily integrated other function unit, automation, low cost, highly sensitive nucleic acid micro-total analysis process are completed, is had wide practical use.
Description
Technical field
The present invention relates to a kind of nucleic acid complete analysis micro-fluidic chip system and its application methods, belong to micro-fluidic chip nucleic acid
Analysis technical field.
Background technique
Foranalysis of nucleic acids biology, medicine, study of pharmacy and disease prevention, diagnosis, in terms of play important work
With.PCR amplification of the conventional nucleic acid analysis method majority based on target fragment, whole process further include the sample treatment before amplification
The many more manipulations such as product detection and analysis after (such as cell cracking, nucleic acid purification and extraction), amplification, relate generally to more instruments
Equipment, and professional is needed to operate, various sample handling processes and product detection process often separately carry out, it is difficult to realize certainly
Dynamicization, low consumption cause analysis efficiency and detection accuracy low, are not suitable for field quick detection demand.Needed for foranalysis of nucleic acids
Monotechnics can be single or be integrally transferred on micro-fluidic chip and completes, finally by a complete foranalysis of nucleic acids process
Related basic operation unit is integrated to same micro-fluidic chip system, forms fully integrated micro-fluidic chip nucleic acid point
Analysis system, it will thoroughly change the feature that traditional method for nucleic acid analysis is time-consuming, laborious, automaticity is poor, accuracy is low, can effectively drop
The loss of low sample and pollution, hence it is evident that reduce analysis time and cost, complete fast and automatically to change analysis, with important theory and
Practical significance.Research in terms of nucleic acid complete analysis micro-fluidic chip system both at home and abroad it has been reported that but so far, have method
Have a single function mostly, " micro- without complete ", be not able to achieve automation, although integrated level it is higher, chip, apparatus structure are multiple
Miscellaneous, high expensive, efficiency are relatively low, flux is insufficient, cause testing result precision relatively low, less reproducible, not very practical.
Summary of the invention
To solve the above problems, the present invention provides a kind of nucleic acid complete analysis micro-fluidic chip system and its application method,
Specifically provide a kind of nucleic acid complete analysis micro-fluidic chip based on paramagnetic particle method and mancarried device and its application method, this method
The automation for being particularly suitable for nucleic acid samples quickly detects, and can be applied to particular surroundings.
To achieve the goals above, the present invention provides a kind of nucleic acid complete analysis micro-fluidic chip systems, including rotation three
Tie up magnetic field control module 1, one-dimensional movement magnetic field control module 2, temperature control modules 3, signal detection module 4 and micro-fluidic chip
5, in which:
The rotated three dimensional magnetic field control module 1 includes upper plectane 11, upper plectane magnet 12, lower plectane 13, lower plectane
Magnet 14, driving motor 15, shaft 16 and shaft base 17；The upper plectane 11 and lower plectane 13 are that corresponding horizontal parallel is set
It sets, the upper plectane 11 and lower plectane 13 are equipped with shaft 16, and the shaft 16 is set to the position for deviateing the center of circle and extends vertically through
In upper plectane 11 and lower plectane 13；16 top of shaft is equipped with driving motor 15, and 16 bottom end of shaft is equipped with shaft base
17, the upper plectane 11 and lower plectane 13 are driven by shaft 16 by driving motor 15；The upper plectane 11 and lower plectane 13
Edge is successively respectively equipped with identical shape, evenly arranged multiple upper plectane magnets 12 and lower plectane magnet 14, the upper plectane
The long side of magnet 12 and lower plectane magnet 14 is vertical with shaft 16, and the upper plectane magnet 12 and lower plectane magnet 14 are hanging down
It is interlaced on straight position；
The one-dimensional movement magnetic field control module 2 includes micro-fluidic chip 5, rectangle permanent magnet fixation kit 21 and micro-
Fluidic chip moves control assembly 22；One end of the micro-fluidic chip 5 is equipped with three-dimensional hybrid pond 51, the three-dimensional hybrid pond 51
In field region between upper plectane 11 and lower plectane 13, the side of the other end of the micro-fluidic chip 5 is equipped with miniflow
The mobile control assembly 22 of chip is controlled, the lower end of the micro-fluidic chip 5 is close in mobile 22 side of control assembly of micro-fluidic chip
Equipped with rectangle permanent magnet fixation kit 21, the other side of the rectangle permanent magnet fixation kit 21 is equipped with temperature control modules
3, the temperature control modules 3 are close to 5 lower end of micro-fluidic chip, and 5 upper end of micro-fluidic chip is equipped with signal detection module 4,
The signal detection module 4 is located at 3 top of temperature control modules.
Further, in above-mentioned technical proposal, at least two channels, the channel are flat on the micro-fluidic chip 5
Row is arranged, and is successively arranged a three-dimensional hybrid pond 51 and at least two reaction tanks 52, the three-dimensional hybrid on each channel
It is equipped with isolation tank 54 between pond 51 and reaction tank 52, isolation tank 54 is equipped between the adjacent reaction tank 52, it is described three-dimensional mixed
It closes and is equipped with liquid filling hole 53 on pond 51, reaction tank 52 and isolation tank 54, the side endpoint in the channel is three-dimensional hybrid pond 51, institute
The other side endpoint for stating channel is isolation tank 54.
Further, in above-mentioned technical proposal, the diameter of the upper plectane 1 and lower plectane 3 is 45mm, and thickness is
3mm, the eccentricity of the shaft 6 are 7.5mm, and the upper plectane magnet 2 and lower plectane magnet 4 are Nd-Fe-B permanent magnet, described
Upper plectane magnet 2 and the long 8.5mm of lower plectane magnet 4, width 1.5mm, a height of 5mm.
Further, in above-mentioned technical proposal, the three-dimensional hybrid pond 51 is enclosed construction, depth > 1mm；The reaction
Pond 52 is platypelloid type, depth < 0.5mm, the volume ratio > 4:1 in the three-dimensional hybrid pond 51 and reaction tank 52.
Further, in above-mentioned technical proposal, the temperature control region overlay reaction tank 52 of the temperature control modules 3 is described
Temperature control modules 3 include temperature control component and control circuit.
Further, in above-mentioned technical proposal, the detection zone of the signal detection module 4 covers reaction tank 52, described
Signal detection module 4 includes signal acquisition unit and data processing unit.
The present invention also provides a kind of application methods of micro-fluidic chip nucleic acid analysis system comprising following steps:
1. the modified magnetic bead in surface and nucleic acid samples to be detected are added in three-dimensional hybrid pond 51, it is added in reaction tank 52 corresponding
Reagent, blank medium is added in isolation tank 54, the liquid filling hole of each liquid pool is then closed using sealed membrane；
2. three-dimensional hybrid pond 51 is fully seated at rotated three dimensional magnetic field control area, starting rotated three dimensional magnetic field controls mould
Block 1 drives upper plectane 11 and lower plectane 13 to rotate, magnetic bead is made to come into full contact with, mix with nucleic acid samples by drive shaft 16；
3. step 2. after the completion of, close rotated three dimensional magnetic field control module 1, the mobile control assembly of starting micro-fluidic chip
22, so that magnetic bead is left three-dimensional hybrid pond and is entered in next reaction tank by isolation tank；
4. step 3. after the completion of, the mobile control assembly 22 of control micro-fluidic chip carries out magnetic bead in reaction tank one-dimensional
It moves back and forth, realizes magnetic bead and being sufficiently mixed of reagent in reaction tank, reacts；
5. step 4. after the completion of, the mobile control assembly 22 of control micro-fluidic chip makes magnetic bead sequentially enter subsequent reactions pond,
Subsequent mixing, reaction process are sequentially completed, and is reacted and is detected in last reaction tank, to realize nucleic acid complete analysis
Process.
Further, in above-mentioned technical proposal, the step 1. in the modified magnetic bead in surface include versatility magnetism silica bead,
Specificity DNA probing needle is modified magnetic bead, immunomagnetic beads etc..
Further, in above-mentioned technical proposal, the step 1. in the reagent that is added into reaction tank include cleaning buffering
Liquid, elution buffer, nucleic acid amplification agents, detection reagent.
Further, in above-mentioned technical proposal, the step 1. in spacer medium be another flowing incompatible with water
Medium, another flow media incompatible with water include air, mineral oil etc., realize different aqueous solutions in adjacent reaction pond
Separation.
The invention has the following advantages:
1, the present invention rotated three dimensional magnetic field microflow control technique is used for foranalysis of nucleic acids for the first time, may be implemented bulk sample and
The rapidly and efficiently mixing of magnetic bead, so as to obtain higher detection spirit by improving sampling volume come enriching low-concentration sample
Sensitivity；
2, the integrated one-dimensional movement magnetic field control module of the present invention and platypelloid type reaction tank, it is only necessary to one-dimensional reciprocating motion
It realizes the efficient mixing of magnetic bead and reagent, react, chip structure and control mode relative ease, suitable for multistep magnetic bead operation stream
Journey, and it is easily integrated other function unit, construct the nucleic acid micro-total analysis system of automation, portability, low cost.
Detailed description of the invention
Fig. 1 is nucleic acid complete analysis micro-fluidic chip system structural schematic diagram；
Fig. 2 is microfluidic chip structure schematic diagram；
Fig. 3 is rotated three dimensional magnetic field magnetic bead mixing principle schematic diagram；
Fig. 4 is used for triple channel/tetra- liquid pool microfluidic chip structure schematic diagrames of nucleic acid complete analysis；
Fig. 5 is used for two channels/seven liquid pool microfluidic chip structure schematic diagrames of nucleic acid complete analysis；
Fig. 6 nucleic acid extraction three-dimensional hybrid time is to real-time fluorescent PCR amplification curve CTThe influence of value.
In figure: 1, rotated three dimensional magnetic field control module, 11, upper plectane, 12, upper plectane magnet, 13, lower plectane, 14, lower circle
Plate magnet, 15, driving motor, 16, shaft, 17, shaft base；2, magnetic field control module, 21, rectangle permanent magnet are moved one-dimensionally
Fixation kit, 22, the mobile control assembly of micro-fluidic chip；3, temperature control modules；4, signal detection module；5, micro-fluidic core
Piece, 51, three-dimensional hybrid pond, 52, reaction tank, 53, liquid filling hole, 54, isolation tank.
Specific embodiment
Following non-limiting embodiments can with a person of ordinary skill in the art will more fully understand the present invention, but not with
Any mode limits the present invention.In the schematic diagram of the microfluidic chip of the invention and device, structure size is not marked, mainly
The structure for embodying this micro-fluidic chip and device constructs, and in actual production use process, can according to need adjustment structure proportion
And size.
Embodiment 1
The invention will be further described below in conjunction with the accompanying drawings.Fig. 1 is nucleic acid complete analysis micro-fluidic chip system of the invention
System structural block diagram, Fig. 2 microfluidic chip structure figure, Fig. 3 are rotated three dimensional magnetic field magnetic bead mixing principle schematic diagram；As seen from the figure,
The present invention is made of nucleic acid complete analysis micro-fluidic chip 5 and portability device, wherein the portability device, by rotation three
Magnetic field control module 1, one-dimensional movement magnetic field control module 2, temperature control modules 3 and signal detection module 4 is tieed up to constitute.
Wherein, rotated three dimensional magnetic field control module 1 of the present invention by upper plectane 11, upper plectane magnet 12, lower plectane 13,
Lower plectane magnet 14, driving motor 15, shaft 16 and shaft base 17 form.Specifically, the upper plectane 11 and lower plectane 13
For the setting of corresponding horizontal parallel, the upper plectane 11 and lower plectane 13 are equipped with shaft 16, and the shaft 16 is set to stray circle
It the position of the heart and extends vertically through in two plectanes；16 top of shaft is equipped with driving motor 15, and 16 bottom end of shaft is equipped with
Shaft base 17, the upper plectane 11 and lower plectane 13 are driven by shaft 16 by driving motor 15.Upper circle used in the present embodiment
The diameter of plate 11 and lower plectane 13 is 45mm, and thickness is 3mm, and the eccentricity of the shaft 16 is 7.5mm；The upper plectane
Plectane magnetic on 11 edges are 6 identical, evenly arranged equipped with shape, 12, it is identical, uniform that lower 13 edge of plectane is equipped with shape
The long side of 6 lower plectane magnets 14 of arrangement, the upper plectane magnet 12 and lower plectane magnet 14 is vertical with shaft 6, it is described on
Plectane magnet 12 and the long 8.5mm of lower plectane magnet 14, width 1.5mm, a height of 5mm, the upper plectane magnet 12 and lower plectane magnet
14 be Nd-Fe-B permanent magnet, and the upper plectane magnet 12 and lower plectane magnet 14 are interlaced on upright position.This hair
Micro-fluidic chip 5 used in bright embodiment, three-dimensional mixing pit 51 are located at one end of micro-fluidic chip 5, are enclosed construction,
Shape, length are unlimited, depth > 1mm, and position is in the field region between upper plectane 11, lower plectane 13.
The one-dimensional movement magnetic field control module 2 includes micro-fluidic chip 5, rectangle permanent magnet fixation kit 21 and micro-
Fluidic chip moves control assembly 22, and one end of the micro-fluidic chip 5 is equipped with three-dimensional hybrid pond 51, the three-dimensional hybrid pond 51
On the lower plectane magnet 14 of lower plectane 13, the other end of the micro-fluidic chip 5 is equipped with the mobile control group of micro-fluidic chip
The lower end of part 22, the micro-fluidic chip 5 equipped with mobile 22 one end of control assembly of micro-fluidic chip is close in micro-fluidic chip
Mobile 22 side of control assembly is equipped with rectangle permanent magnet fixation kit 21, the rectangle permanent magnet fixation kit 21 it is another
Side is equipped with temperature control modules 3, and the temperature control modules 3 are located at 5 lower end of micro-fluidic chip, and are close to micro-fluidic chip bottom
Plate, 5 upper end of micro-fluidic chip are equipped with signal detection module 4, and the signal detection module 4 is located on temperature control modules 3
Side.
As shown in Fig. 2, at least two channels on the micro-fluidic chip 5, the channel is parallel arrangement, described each
Be successively arranged a three-dimensional hybrid pond 51 and at least two reaction tanks 52 on channel, the three-dimensional hybrid pond 51 and reaction tank 52 it
Between be equipped with isolation tank 54, isolation tank 54, the three-dimensional hybrid pond 51,52 and of reaction tank are equipped between the adjacent reaction tank 52
Liquid filling hole 53 is equipped on isolation tank 54, the side in the channel is three-dimensional hybrid pond 51, and the other side in the channel is isolation
Pond 54.The depth in three-dimensional hybrid pond 51 described in the present embodiment is 2mm, and the reaction tank 52 is platypelloid type, depth 0.4mm, institute
The volume ratio for stating three-dimensional hybrid pond 51 and reaction tank 52 is 10:1.
Reaction tank position needed for the temperature control region overlay of the temperature control modules 3, the temperature control modules 3 include temperature
Spend control element and control circuit.
Reaction tank position needed for the detection zone of the signal detection module 4 covers, the signal detection module 4 include letter
Number acquisition unit and data processing unit.
Below in conjunction with Fig. 3 schematic diagram, rotated three dimensional magnetic field magnetic bead mixing principle is described in detail:
Three-dimensional hybrid pond 51 equipped with nucleic acid samples and magnetic bead is placed in control mould in rotated three dimensional magnetic field of the present invention
In field region between the upper plectane 11 of block 1, lower plectane 13, after starting driving motor 15, upper and lower plectane simultaneously around the shaft 16
It starts turning；When rotating plectane, it is embedded in the Three-Dimensional Magnetic that the magnet in plectane generates periodical gradual change between two layers of plectane
, so that the magnetic bead that control is located in this region in three-dimensional hybrid pond 51 does the movement of three-dimensional S type, and then realize magnetic bead and nucleic acid sample
Product come into full contact with efficiently mix, fast reaction, significantly improve magnetic bead and nucleic acid molecules Percentage bound.By 1/2 period, magnetic
Pearl from the one end in three-dimensional hybrid pond 51 along space S type move to the other end (see Fig. 3, a), cycle movement track such as Fig. 3
(b) shown in.
Embodiment 2
Micro-fluidic chip described in the present embodiment is illustrated in Fig. 4, is triple channel/tetra- liquid pool micro-fluidic chips, wherein containing
There are three foranalysis of nucleic acids channels, and each channel is containing there are four reaction tank, a respectively three-dimensional hybrid ponds, two service sinks and one
A elution/amplification pond is isolation tank between reaction tank；Wherein, three-dimensional hybrid tank depth is 2mm, width 4.5mm, service sink
It is 0.4mm, width 2.25mm with elution/amplification pond depth.
As shown in Figure 4, the method for carrying out nucleic acid complete analysis using chip described in the embodiment of the present invention and device, including it is following
Step:
1) micro-fluidic chip prepares: universal magnetic silica bead and its buffer (such as 6M hydrochloric acid being added in three-dimensional hybrid pond
Guanidine, 10mM Tris-1mM edta buffer liquid, pH 6.1) and nucleic acid samples, be added in service sink cleaning buffer solution (such as 75~
80% ethyl alcohol), nucleic acid amplification/detection reagent (the real-time fluorescence PCR reaction solution outside such as removing template) is added in elution/amplification pond,
It is spaced in pond and mineral oil is added as blank medium, with the liquid filling hole of each liquid pool of rear enclosed；
2) based on the nucleic acid extraction of micro-fluidic chip: three-dimensional hybrid pond (51) are fully seated at the control of rotated three dimensional magnetic field
Region starts driving motor 15, and the magnetic bead in three-dimensional hybrid pond is driven to carry out three-dimensional motion, realizes that magnetic bead sufficiently connects with sample
Touching mixes, the nucleic acid molecules efficient absorption in solution to magnetic bead surfaces；Then, driving motor 15 is closed, micro-fluidic chip is started
Mobile control assembly 22 makes magnetic bead leave three-dimensional hybrid pond, enters in first service sink and carry out one-dimensional past by isolation tank
Multiple movement, sufficiently to clean the impurity of magnetic bead surfaces absorption；The mobile control assembly 22 of micro-fluidic chip is controlled again, enters magnetic bead
In second service sink and one-dimensional reciprocating motion is carried out, further to clean the impurity of magnetic bead surfaces absorption.
3) nucleic acid amplification based on micro-fluidic chip and detection: the mobile control assembly 22 of control micro-fluidic chip makes magnetic bead
Into in elution/amplification pond and one-dimensional reciprocating motion is carried out, it is anti-that the nucleic acid molecules of magnetic bead surfaces absorption are sufficiently eluted to amplification
It answers in liquid；Finally, elution/amplification pond is moved to 3 region of temperature control modules, start-up temperature control module 3 and signal detection
Module 4 starts nucleic acid amplification and detection process, completes automatic nucleic acid complete analysis process.
Embodiment 3
Micro-fluidic chip described in the present embodiment is illustrated in Fig. 5, is two channels/seven liquid pool micro-fluidic chips, wherein containing
There are two foranalysis of nucleic acids channels, and seven reaction tanks, a respectively three-dimensional hybrid pond, two service sinks, one are contained in each channel
A amplification pond, another two service sink and a detection cell are isolation tank between reaction tank；Wherein, service sink, amplification pond and detection
The depth < 0.5mm in pond, width are less than the 1/2 of three-dimensional hybrid pond width.
As shown in Figure 5, the method for carrying out nucleic acid complete analysis using chip described in the embodiment of the present invention and device, including it is following
Step:
1) micro-fluidic chip prepares: the modified magnetic bead of DNA capture probe and its buffer and core being added in three-dimensional hybrid pond
Cleaning buffer solution (such as 75~80% ethyl alcohol) are added in service sink in sour sample, expand and nucleic acid amplification agents are added in pond (as removed mould
PCR reaction solution outside plate), blank medium is added in isolation tank, then in fixed dna detection probe microarray in advance in detection cell
Close the liquid filling hole of each liquid pool；
2) based on the nucleic acid purification of micro-fluidic chip: micro-fluidic chip (5) being fixed on bracket and is adjusted mixed to three-dimensional
Close pond (51) and be fully located at rotated three dimensional magnetic field control area, start driving motor 15, drive magnetic bead in three-dimensional hybrid pond into
Row three-dimensional motion realizes that magnetic bead comes into full contact with sample, mixes, the nucleic acid molecules and magnetic bead surfaces in solution containing particular sequence
DNA probe efficiently combines；Then, driving motor 15 is closed, the mobile control assembly 22 of starting micro-fluidic chip makes magnetic bead leave three
Dimension mixing pit enters in first service sink by isolation tank and carries out one-dimensional reciprocating motion, sufficiently to clean magnetic bead surfaces
The nucleic acid molecules of impurity and non-specific binding；The mobile control assembly 22 of micro-fluidic chip is controlled again, and magnetic bead is made to enter second
In service sink and one-dimensional reciprocating motion is carried out, further to clean the impurity of magnetic bead surfaces and the nucleic acid point of non-specific binding
Son.
3) based on the nucleic acid amplification of micro-fluidic chip: the mobile control assembly 22 of control micro-fluidic chip makes magnetic bead enter expansion
Increase in pond and amplification pond is moved to 3 region of temperature control modules, start-up temperature control module 3 starts amplification process.
4) based on the nucleic acid multiple determination of micro flow controlling chip DNA microarray: after amplification, control micro-fluidic chip is moved
Dynamic control assembly 22, combines magnetic bead efficiently with the reaction product expanded in pond, then magnetic bead is made to sequentially enter subsequent two clearly
Wash pool, sufficiently to clean the nucleic acid molecules of magnetic bead surfaces remaining impurity and non-specific binding；Finally, magnetic bead is made to enter detection
Pond simultaneously carries out one-dimensional reciprocating motion, while start-up temperature control module 3 starts nucleic acid hybridization reaction process；After hybridization reaction
Enabling signal detection module 4 obtains nucleic acid multiple determination as a result, completing automatic nucleic acid complete analysis process.
Embodiment 4
Micro-fluidic chip described in the present embodiment is illustrated in Fig. 4, is mentioned using the paramagnetic particle method bacterial genomes DNA of commercialization
Kit (AU2001, hundred Tyke Biotechnology Co., Ltd of Beijing) is taken, is examined using chip described in the embodiment of the present invention and device
Examine influence of the three-dimensional hybrid time to nucleic acid extraction effect, comprising the following steps:
1) nucleic acid samples prepare: 1.5mL Vibrio harveyi bacterium solution being centrifuged 3min in 12000r/min, removes supernatant；1mL is added
TE buffer and mix, the bacterium solution of resuspension is boiled into 5~10min at 100 DEG C, then 12000r/min be centrifuged 3min, take
Supernatant is saved in -20 DEG C or is used immediately；
2) micro-fluidic chip prepares: magnetic bead combination liquid CB, the magnetic bead solution of mixing and cracking being added in three-dimensional hybrid pond
Bacterium solution, rinsing liquid WB is added in service sink, elution buffer TE is added in elution/amplification pond, is spaced in pond and mineral oil is added；
3) based on the nucleic acid extraction of micro-fluidic chip: micro-fluidic chip 5 being fixed on bracket and is adjusted to three-dimensional hybrid
Pond 51 is fully located at rotated three dimensional magnetic field control area, and experiment parameter, starting portability device is then arranged, starts chip nucleic acid
Extract experiment.To investigate influence of the three-dimensional hybrid condition to magnetic bead and DNA molecular joint efficiency, when different three-dimensional hybrids are set
Between (respectively 0,1,5,9min).
4) chip nucleic acid extracts result detection: after chip nucleic acid extracts process, drawing institute from the liquid filling hole of elution pool
There is solution into centrifuge tube, carries out real-time fluorescence PCR detection, the different three-dimensional hybrid times are to amplified production PCR amplification CTValue
Fig. 6 is shown in influence, and the DNA sample for showing that chip extracts can be directly used for PCR amplification, and in 0~9min, the three-dimensional hybrid time
Longer, the DNA concentration of extraction is higher, it was demonstrated that the three-dimensional hybrid of magnetic bead and sample helps to improve DNA molecular and magnetic bead in sample
Joint efficiency.
Claims (9)
1. a kind of nucleic acid complete analysis micro-fluidic chip system, it is characterised in that: including rotated three dimensional magnetic field control module (1), one
Tie up shifting magnetic field control module (2), temperature control modules (3), signal detection module (4) and micro-fluidic chip (5), in which:
The rotated three dimensional magnetic field control module (1) include upper plectane (11), upper plectane magnet (12), lower plectane (13), under
Plectane magnet (14), driving motor (15), shaft (16) and shaft base (17)；The upper plectane (11) and lower plectane (13) are
Corresponding horizontal parallel setting, the upper plectane (11) and lower plectane (13) are equipped with shaft (16), and the shaft (16) is set to
Deviate the position in the center of circle and extends vertically through in upper plectane (11) and lower plectane (13)；Shaft (16) top is equipped with driving motor
(15), shaft (16) bottom end is equipped with shaft base (17), and the upper plectane (11) and lower plectane (13) pass through shaft (16)
It is driven by driving motor (15)；It is identical, uniform that the upper plectane (11) with the edge of lower plectane (13) is successively respectively equipped with shape
The multiple upper plectane magnets (12) and lower plectane magnet (14) of arrangement, the upper plectane magnet (12) and lower plectane magnet (14)
Long side is vertical with shaft (16), and the upper plectane magnet (12) and lower plectane magnet (14) are mutually handed on upright position
It is wrong；
The one-dimensional movement magnetic field control module (2) include micro-fluidic chip (5), rectangle permanent magnet fixation kit (21) and
Micro-fluidic chip is mobile control assembly (22)；One end of the micro-fluidic chip (5) is equipped with three-dimensional hybrid pond (51), the three-dimensional
Mixing pit (51) is in the field region between upper plectane (11) and lower plectane (13), the micro-fluidic chip (5) it is another
The side at end is equipped with micro-fluidic chip movement control assembly (22), and the lower end of the micro-fluidic chip (5) is close in micro-fluidic core
Mobile control assembly (22) side of piece is equipped with rectangle permanent magnet fixation kit (21), the rectangle permanent magnet fixation kit
(21) the other side is equipped with temperature control modules (3), and the temperature control modules (3) are close to micro-fluidic chip (5) lower end, described
Micro-fluidic chip (5) upper end is equipped with signal detection module (4), and the signal detection module (4) is located on temperature control modules (3)
Side.
2. nucleic acid complete analysis micro-fluidic chip system according to claim 1, it is characterised in that: the micro-fluidic chip
(5) at least two channels on, the channel are parallel arrangement, are successively arranged a three-dimensional hybrid pond on each channel
(51) and at least two reaction tanks (52) isolation tank (54), are equipped between the three-dimensional hybrid pond (51) and reaction tank (52), institute
It states and is equipped with isolation tank (54) between adjacent reaction tank (52), the three-dimensional hybrid pond (51), reaction tank (52) and isolation tank
(54) it is equipped on liquid filling hole (53), the side endpoint in the channel is three-dimensional hybrid pond (51), another side in the channel
Point is isolation tank (54).
3. nucleic acid complete analysis micro-fluidic chip system according to claim 1, it is characterised in that: the three-dimensional hybrid pond
It (51) is enclosed construction, depth > 1mm, the reaction tank (52) is platypelloid type, depth < 0.5mm, the three-dimensional hybrid pond
(51) with the volume ratio > 4:1 of reaction tank (52).
4. nucleic acid complete analysis micro-fluidic chip system according to claim 1, it is characterised in that: the temperature control modules
(3) temperature control region overlay reaction tank (52), the temperature control modules (3) include temperature control component and control circuit.
5. nucleic acid complete analysis micro-fluidic chip system according to claim 1, it is characterised in that: the signal detection module
(4) detection zone covers reaction tank (52), and the signal detection module (4) includes signal acquisition unit and data processing list
Member.
6. the application method of nucleic acid complete analysis micro-fluidic chip system according to claim 1, it is characterised in that: including following
Step:
1. the modified magnetic bead in surface and nucleic acid samples to be detected are added in three-dimensional hybrid pond (51), it is added in reaction tank (52) corresponding
Reagent, blank medium is added in isolation tank (54), the liquid filling hole of each liquid pool is then closed using sealed membrane；
2. three-dimensional hybrid pond (51) are fully seated at rotated three dimensional magnetic field control area, start rotated three dimensional magnetic field control module
(1), it is rotated by plectane (11) in drive shaft (16) drive and lower plectane (13), come into full contact with magnetic bead and nucleic acid samples,
Mixing；
3. step 2. after the completion of, close rotated three dimensional magnetic field control module (1), the mobile control assembly of starting micro-fluidic chip
(22), magnetic bead is made to leave three-dimensional hybrid pond and enter in next reaction tank by isolation tank；
4. step 3. after the completion of, control micro-fluidic chip is mobile control assembly (22), carries out magnetic bead in reaction tank one-dimensional past
Multiple movement is realized magnetic bead and being sufficiently mixed of reagent in reaction tank, is reacted；
5. step 4. after the completion of, control micro-fluidic chip is mobile control assembly (22), and magnetic bead is made to sequentially enter subsequent reactions pond, suitable
It is secondary to complete subsequent mixing, reaction process, and reacted and detected in last reaction tank, to realize nucleic acid complete analysis stream
Journey.
7. application method according to claim 6, it is characterised in that: the step 1. in the modified magnetic bead in surface include logical
With property magnetism silica bead, specificity DNA probing needle modified magnetic bead, immunomagnetic beads.
8. application method according to claim 6, it is characterised in that: the step 1. in the reagent that is added into reaction tank
Including cleaning buffer solution, elution buffer, nucleic acid amplification agents, detection reagent.
9. application method according to claim 6, it is characterised in that: the step 1. in spacer medium be and water not phase
Another flow media held, another flow media incompatible with water includes air, mineral oil.
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