CN110317728A - Single cell whole genome amplification system and method based on micro-fluidic electron spray - Google Patents
Single cell whole genome amplification system and method based on micro-fluidic electron spray Download PDFInfo
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- CN110317728A CN110317728A CN201910628637.8A CN201910628637A CN110317728A CN 110317728 A CN110317728 A CN 110317728A CN 201910628637 A CN201910628637 A CN 201910628637A CN 110317728 A CN110317728 A CN 110317728A
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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
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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
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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
Abstract
The present invention provides a kind of single cell whole genome amplification system and method based on micro-fluidic electron spray.The system includes micro-fluidic chip and AC power supply module, wherein the micro-fluidic chip is equipped with the sample flow channel for injecting discrete phase and continuous phase, the AC power supply module is used to apply alternating current to the sample solution in sample flow channel, sample solution in sample flow channel generates drop under the action of electric field force and hydraulic power, and stretching of the drop in a distance Jing Guo AC field is cracked into extra small drop.The present invention can generate extra small drop by electrospray mode using micro-fluidic chip, improve the accuracy of sequencing.
Description
Technical field
The present invention relates to microfluidic art more particularly to a kind of unicellular full-length genomes based on micro-fluidic electron spray
Amplification system and method.
Background technique
The emulsion droplet generation technique of Water-In-Oil is the important directions of current life science and industrial production research, even more state
Inside and outside mainstream Bioexperiment, mechanism detection and industrial production in key link, such as digital pcr, it is unicellular sequencing, pharmacy and
Household chemicals production etc..Micro-fluidic technologies based on droplet have reagent consumption as the important branch of microflow control technique
Volume needed for operating far below traditional experiment executes the advantages such as big quantitative response while holding meanss miniaturization, therefore has
Broader prospect.The micro-fluidic technologies carry out the only of single drop by the way that discrete droplets are generated and manipulated inside micro element
Vertical control, to generate the microreactor that can individually transport, mix combined analysis.In addition, the technology can be formed in a short time
Multiple identical microreactor units effectively simplify subsequent processing and experimentation, and then promote the collection efficiency of big data.It should
The technology more increase of drop flux and drop scalability provide support.
Micro-fluidic field at home and abroad, drop formation method traditional at present are to rely on channel geometries control drop life
At, with T junction (T-junction) and flow focusing (flow-focusing) for representative, and the general character disadvantage of traditional technology
It is that debugging is slow, unstable, flux is low.Emerging technology then uses electrohydrodynamics (EHD) method, by being integrated into micro element
In electrode realize electric control to drop, representative two examples are dielectrophoresis (DEP) and dielectric electrowetting (EWOD),
And at present there is the problems such as control is complicated, limited to size droplet diameter modification scope in such methods.
Therefore, it is necessary to improve to the prior art, novel drop formation method and drop adjustment mechanism are provided.
Summary of the invention
It is an object of the invention to overcome the defect of the above-mentioned prior art, provide a kind of based on the slender of micro-fluidic electron spray
Born of the same parents' whole genome amplification system and method.
According to the first aspect of the invention, a kind of single cell whole genome amplification system based on micro-fluidic electron spray is provided
System.The system includes micro-fluidic chip and AC power supply module, wherein the micro-fluidic chip be equipped with for inject discrete phase and
The sample flow channel of continuous phase, the AC power supply module are used to apply alternating current, sample flow to the sample solution in sample flow channel
Sample solution in road generates drop under the action of electric field force and hydraulic power, and drop is in a distance by AC field
Stretching is cracked into extra small drop.
In one embodiment, first electrode and second electrode, the first electrode are fixed on the micro-fluidic chip
One end be electrically connected the sample flow channel of continuous phase, for the other end for accessing alternating voltage, one end of the second electrode is electrical
Connection generates the sample flow channel of drop, and the other end is for accessing alternating voltage.
In one embodiment, the sample flow channel is shaped to the point that drop formation betides Taylor cone
The discrete phase at end, injection with ion and has the conductivity set, and the continuous phase internal mix of injection has surfactant.
In one embodiment, according to the hydraulic power control in the electric field force and sample flow channel applied on the micro-fluidic chip
The subtended angle of Taylor cone processed, and then control the partial size and frequency of generated drop.
In one embodiment, by controlling one or more generations to control extra small drop in lower list: sample
The distance between temperature, the shape of electrode, first electrode and second electrode of solution, the conductivity of sample solution, sample solution
Sticking property, the shape of sample flow channel, the size of sample flow channel, the flow velocity of continuous phase, the flow velocity of discrete phase, application alternating current
Voltage and frequency.
In one embodiment, system of the invention further includes the extra small drop for generation, is triggered using ultraviolet light solid
Change mode is solidified into solid globules, and is carried out Analysis of Surface Topography with scanning electron microscope or atomic force microscope characteristic manner and obtained
The grain diameter characteristic of extra small drop is obtained, and carries out constant temperature nucleic acid amplification reaction, extra small drop internal is judged by fluorescence signal
Bioactivity.
In one embodiment, the bioactivity pair inside grain diameter characteristic and extra small liquid based on extra small drop obtained
The influence of amplified reaction is to control the generation for optimizing extra small drop.
In one embodiment, the partial size of extra small drop generated is less than 5 microns.
According to the second aspect of the invention, a kind of single cell whole genome amplification side based on micro-fluidic electron spray is provided
Method.This method comprises: utilizing the single cell whole genome amplification system provided in an embodiment of the present invention based on micro-fluidic electron spray
Generate extra small drop；Determine extra small size droplet diameter, the incidence relation between nucleic acid fragment size and amplification efficiency；To unicellular complete
Genome amplification result carries out gene sequencing, and the characteristic of the extra small drop of generation is controlled to adjust according to sequencing result.
In one embodiment, it is controlled by controlling voltage and the frequency of the alternating current being applied on the micro-fluidic chip
Make the partial size and frequency of extra small drop formation.
Compared with the prior art, the advantages of the present invention are as follows: by developing based on the exchange method of electrospraying in oily phase
Microfluidic system, realize the generation of high efficiency, high throughput, small volume drops, by observing experimental phenomena, study multi-parameter
To the synergic adjustment rule of drop formation, and then grasp the adjusting method for realizing that the fine droplet of ultra-high throughput generates；And it utilizes
Drop monodisperse generated and high-throughput feature, are applied to the foundation of single cell whole genome amplification and sequencing library
Aspect helps to eliminate amplification prejudice, realizes rare nucleic acid amplification more evenly.
Detailed description of the invention
The following drawings only makees schematical description and interpretation to the present invention, is not intended to limit the scope of the present invention, in which:
Fig. 1 is the single cell whole genome amplification system according to an embodiment of the invention based on micro-fluidic electron spray
Schematic diagram；
Fig. 2 is extra small drop formation process schematic according to an embodiment of the invention；
Fig. 3 shows the single cell whole genome amplification side according to an embodiment of the invention based on micro-fluidic electron spray
The flow chart of method.
Specific embodiment
It is logical below in conjunction with attached drawing in order to keep the purpose of the present invention, technical solution, design method and advantage more clear
Crossing specific embodiment, the present invention is described in more detail.It should be appreciated that specific embodiment described herein is only used for explaining
The present invention is not intended to limit the present invention.
It is as shown herein and discuss all examples in, any occurrence should be construed as merely illustratively, without
It is as limitation.Therefore, other examples of exemplary embodiment can have different values.
Technology, method and apparatus known to person of ordinary skill in the relevant may be not discussed in detail, but suitable
In the case of, the technology, method and apparatus should be considered as part of specification.
According to one embodiment of present invention, a kind of single cell whole genome amplification system based on micro-fluidic electron spray is provided
It unites and experiment porch occurs as the extra small drop of Highgrade integration, shown in Figure 1, which includes 110 He of micro-fluidic chip
AC power source 120, AC power source 120 are electrically connected by electrode 121 and 121 and micro-fluidic chip 110.The system is wrapped on the whole
Fluid sample injection process is included, target grain size drop process is generated using micro-fluidic chip, microlayer model collects process and sequencing is examined
Process is tested, wherein micro-fluidic chip 120 integrates multiple functional units, including fluid sample injection, liquid sheath stream generate, microlayer model is split
Solution, microlayer model collection etc., can complete the function that fluid sample is cracked into target grain size microlayer model.
It is shown in Figure 1, within the system, the fluid sample a extra small drop for being passed into the embodiment of the present invention is occurred micro-
In fluidic chip b (being also labeled as micro-fluidic chip 110)；Hundreds of thousands of to millions of a liquid are automatically completed on micro-fluidic chip
The cracking of drop obtains extra small drop；Extra small droplet sample after collecting cracking；The extra small droplet sample being collected into is carried out slender
Born of the same parents' whole genome amplification；Finally, being sent to machine on sequenator c carries out sequencing inspection.
It hereafter will specifically introduce micro-fluidic chip, drop adjustment mechanism and the unicellular full-length genome of the embodiment of the present invention
The content of amplification and sequencing.
1), about micro-fluidic chip
In embodiments of the present invention, using microlayer model that chip (or for micro-fluidic chip) occurs will by electrospray mode
Sample solution is effectively cracked into target grain size, such as is cracked into submicron droplets (less than 5 microns).
Shown in Figure 2, in micro-fluidic chip, drop formation mechanism is: in drop generating unit, flow focusing knot
Structure generates the sheath stream of fluid sample, and by flow dynamics analysis, Flow Field Distribution in calculating simulation micro-fluidic chip, optimization is set
Family planning makes the characteristic values such as its flow path and flow velocity be suitable for subsequent drop cleavage step at the shape and characteristic of sheath stream.Entire
After sheath flow structure passes through electrode position, by power drives AC field, cracking operation is carried out to sample liquids, and this is operated
It is subsequent to be expected to carry out high-throughput parallel-expansion.In the micro-fluidic chip, controlled in conjunction with flow velocity and field strength, it can be in lysate
The adjusting that extra small size droplet diameter is carried out while drop, allows to be oriented application and development.
Specifically, referring also to shown in Fig. 2, micro-fluidic chip is equipped with the sample channel for injecting discrete phase and continuous phase,
For applying the AC power supply module of two-phase alternating current, in the sample flow channel sample solution apply alternating voltage, two kinds
Immiscible liquid crosses at the fluidic structures of sample channel, due to the liquid surface of " oily phase " sample and " water phase " sample
Shearing force caused by power difference and impressed pressure, " water phase " sample intersection are divided by " oily phase " sample from continuous phase discrete
Drop, the sample solution in sample flow channel generate drop under the action of electric field force and hydraulic power, and drop is cone cell (i.e. Taylor
Cone), the high-speed stretch that drop (tip of taylor cone) in a relatively short distance passes through electric field force generates extra small drop.
In micro-fluidic chip of the invention, the process of extra small drop formation is the dynamic equilibrium of hydraulic power and electric field force,
It can be by adjusting such as temperature, humidity, electrode shape, to the distance of electrode, the conductivity of liquid, sticking property, micro-fluidic chip
Flow channel shape and size, two-phase flow velocity and the generation of the state modulators such as electric field strength and frequency meet desired extra small drop.Liquid
Drop cracking phenomenon only occurs under specific parameter combination, determines the effect that different parameters crack drop, facilitates preferably
Control drop formation.
In one embodiment, suitable ion concentration discrete phase and the continuous phase for being mixed with surfactant are selected, is studied
Influence of the different ratio to the stability of generated drop.Drop cracking betides the tip of Taylor cone, it is desirable that discrete phase (water
Phase) there must be ion so as to conduction, the concentration of ion determines conductivity, therefore has a direct impact to the stabilization of drop formation；And
The surfactant of continuous phase (oily phase) internal mix, and directly affect stabilization of the drop after generation and in constant-temperature amplification
Property.The influence for studying ion concentration and surfactant concentration helps to optimize drop stability, to carry out subsequent biological
Experiment.
In one embodiment, according to drop extra small in micro-fluidic chip cracking and regulation rule, Taylor cone angle is measured
Changing rule under the conditions of AC field carries out theory analysis based on Taylor cone formula, is controlled by founding mathematical models
System generates size and frequency of drop etc..The subtended angle of Taylor cone is directly affected subsequent life by the double action of electric field and flow field
Help to analyze and understand water-oil phase by founding mathematical models and Binding experiment phenomenon at the size and frequency of drop
Alternating current is sprayed mechanism.
In one embodiment, flow velocity, field when determining that the extra small drop of generation cracks according to the empirical parameter of drop cracking
By force, the parameter thresholds such as frequency need small scWGA to control exchange electron spray and can produce 1~100 micron of drop
In 10 microns of extra small drop.By experiment, it is concluded that the condition rule of extra small drop is stably generated, to be conducive to area
Divide key parameter and time key parameter.
To sum up, micro-fluidic chip of the invention is determined and liquid occurs by adjusting microchannel parameter and electrical parameter
The parameter working range of drop cracking phenomenon, establishes the coordinated regulation model of drop size Yu ac electric field strength and frequency, can
Obtain under the conditions of AC field, the conspiracy relation of multiple parameters and drop size, thus control stably generate it is highly homogeneous
Extra small drop cracking.In addition, it is spraying that two-phase alternating current can be informed in by establishing the changing rule model bored based on Taylor
Under the conditions of drop crack physical mechanism, be conducive to the adjusting of size droplet diameter.
2), about drop adjustment mechanism
In practical applications, after extra small drop being generated in micro-fluidic chip initial characterization, in order to further to drop
Partial size be adjusted to obtain accurate sequencing result, need each important key parameter to carry out quantitative test one by one, will
The parameter combination of the particle size adjustment of corresponding sample solution is advanced optimized.
For drop adjustment mechanism research can by characterize drop grain diameter characteristic and verifying drop bioactivity into
Row.For example, in order to characterize the thickness characteristics of generated drop cured method can be triggered using ultraviolet light, by extra small drop
Solid globules are solidified into, and carry out Analysis of Surface Topography with characteristic manners such as scanning electron microscope or atomic force microscope.Meanwhile to test
The bioactivity (still maintaining activity by the biomolecule after strong electrical field in it) for demonstrate,proving extra small drop, needs to carry out constant temperature
The reaction of nucleic acid amplification, such as LAMP and MDA, its bioactivity is judged by fluorescence signal, by digital pcr and in real time it is glimmering
Whether Fluorescent Quantitative PCR occurs to examine to expand, and assesses the quality of amplified production.
Specifically, ultra-small volume drop includes: to the effect of the single cell whole genome amplification uniformity
A), using common such as absorption spectrum, the methods of fluorescence lifetime spectrum, hybridization measure enzyme, nucleic acid in extra small drop
Activity.
Since drop cracking betides the tip of Taylor cone, electric field herein be it is extremely strong because all electric field lines are equal
Across this region.The biomolecule activity for characterizing extra small drop internal helps to understand that strong electrical field advises the effect of biomolecule
Rule.The extra small drop that the exchange electron spray of the embodiment of the present application generates can successfully carry out nucleic acid amplification, by characterizing extra small liquid
Biomolecule activity inside drop, can further determine that influence of the AC field to drop internal nucleic acids activity.
B), the condition for realizing constant temperature nucleic acid amplification (MDA) in extra small drop is explored, and carries out unicellular full-length genome expansion
Increase reaction.
Determine droplet size, the incidence relation between nucleic acid fragment size and amplification efficiency.Since nucleic acid amplification raw material has
Limitation, is not that smaller drop is better to reach certain amplification ratio；It is in the same size in view of drop, but fragmentation
DNA fragmentation size be not quite similar, usually have a distribution, the molecular amounts of such amplified production also have a degree of
Difference；Along with too small segment due to can not cyclization, MDA can not be carried out, therefore segment also has a lower limit.These are all determined
Determine to need to rake about an efficiency optimum interval during reducing droplet size and increasing amount of droplets.
3), about single cell whole genome amplification and sequencing experiment
Extra small drop is generated by above-mentioned micro-fluidic chip, and research ultra-small volume drop expands unicellular full-length genome
The effect for increasing the uniformity, on the one hand can stably generate drop, and accuracy controlling droplet size；On the other hand, it can be ensured that liquid
Drop carries out constant temperature MDA nucleic acid amplification.
After sample completes pre- amplification, further, gene sequencing is carried out to single cell whole genome amplification result, point
The uniformity of genome amplification is analysed, the inhibitory effect to amplification prejudice is assessed.For example, being carried out conventional after completing pre- amplification
Library is built, and delivers sequencing, sequencing result is analyzed, is compared with the experimental result of history, it is equal to amplification is improved to study extra small drop
The influence of even property.By bioinformatic analysis, verifies the extra small drop that the embodiment of the present invention generates and the uniformity is expanded to raising
Effect.By carrying out the combination experiment of different genes group segment, droplet size, Optimal Parameters are found out, to improve sequencing result
Accuracy.
It should be noted that the system of the embodiment of the present invention further includes by mature unicellular capture and separation module, cracking
Chamber and micro-valve door etc. are integrated into micro-fluidic chip, to realize the capture of single piece type high-flux cell sequence, crack and pass through
Electron spray generates extra small drop and carries out whole genome amplification operation.
To sum up, system of the invention is electrically generated drop using exchange, and the comprehensive adjustment controlled with electricity is controlled in conjunction with liquid,
Microlayer model generation module is integrated into mems chip, the stable control advantage of fluid method and electricity modulator approach are utilized
Speed and flux advantage, the final generation for realizing the submicron droplets (such as less than 5 microns) of a large amount of uniform particle diameters in the short time.This hair
Bright system has the characteristics that flux is high, easy to operate, size droplet diameter homogeneity is high.
The embodiment of the present invention also provides a kind of single cell whole genome amplification method based on micro-fluidic electron spray, referring to figure
Shown in 3, method includes the following steps:
Step S310 generates the extra small drop of expectation target partial size using micro-fluidic chip by exchange electric drive.
For example, by the setting temperature of sample solution, the shape of electrode, the distance to electrode, the conductivity of sample solution,
The sticking property of sample solution, the shape of sample flow channel, the size of sample flow channel, the flow velocity of continuous phase, discrete phase flow velocity, continuous
The amount of the surfactant of phase internal mix, the voltage of the alternating current of application and frequency etc. come control generate have desired partial size and
Internal active extra small drop.
Step S320 determines extra small size droplet diameter, the incidence relation between nucleic acid fragment size and amplification efficiency.
For example, realizing the condition of constant temperature nucleic acid amplification (MDA) in extra small drop, and carry out single cell whole genome amplification
Reaction, determines droplet size, the incidence relation between nucleic acid fragment size and amplification efficiency, and then determines amplification ratio, extra small
Size droplet diameter, fragmentation DNA fragmentation between equilibrium relation, find an efficiency optimum interval.
Step S330 carries out gene sequencing to single cell whole genome amplification result, is further adjusted according to sequencing result
Control the parameter of drop formation.
After completing amplification, sequencing is delivered, and determine extra small drop to raising amplification uniformity based on sequencing result
It influences, and then advanced optimizes the parameter for influencing extra small drop formation, for example, inside the voltage of AC field, frequency, continuous phase
The amount etc. of mixed surfactant.
In conclusion the present invention utilizes micro-fluidic chip, under AC field effect, realize that the drop in water-oil phase is split
Solution, realizes electron spray emulsion for the first time in oily phase, overcomes the problems, such as that traditional electrospray microlayer model is easy evaporation in air,
And make size droplet diameter uniform, adjustable；According to biomolecule such as nucleic acid, enzymes in the character variation after AC field, explain
The affecting laws of electric fields on biological molecule are proposed micro- liquid based on the fact generated drop can carry out nucleic acid constant-temperature amplification
Drop is applied to single cell whole genome amplification, to reduce amplification preference.The drop cleavage method of exchange electron spray of the invention, it is right
In electron spray field or even a strong supplement of entire microfluid drop formation theoretical system, solves unicellular sequencing
In amplification prejudice problem, the more accurately quantitative sequencing analysis of unicellular full-length genome is helped to realize, thus early for tumour
The Personalized medicines such as sieve, pre-natal diagnosis provide help.
It should be understood that in embodiments of the present invention, the size design of sample flow channel is matched with the size of drop, for example,
The size of drop is generally less than 100 microns, and sample flow channel is also in 100 microns.In practical applications, two-phase liquid is from sample introduction
Mouth arrives intersection, it may be necessary to flow through one section of main pipeline.The width of main pipeline is traditionally arranged to be that (such as 200 is micro- greater than 100 microns
Rice~500 microns), to reduce the flow resistance of fluid motion.In addition, extra small drop described herein, ultra micro droplet or ultra micro liquid
Drop has the same meaning, and the extra small drop after cracking by AC field is indicated, unless based on context referring else.
It should be noted that, although each step is described according to particular order above, it is not intended that must press
Each step is executed according to above-mentioned particular order, in fact, some in these steps can concurrently execute, or even is changed suitable
Sequence, as long as can be realized required function.
Various embodiments of the present invention are described above, above description is exemplary, and non-exclusive, and
It is not limited to disclosed each embodiment.Without departing from the scope and spirit of illustrated each embodiment, for this skill
Many modifications and changes are obvious for the those of ordinary skill in art field.The selection of term used herein, purport
In principle, the practical application or to the technological improvement in market for best explaining each embodiment, or make the art its
Its those of ordinary skill can understand each embodiment disclosed herein.
Claims (10)
1. a kind of single cell whole genome amplification system based on micro-fluidic electron spray, which is characterized in that including micro-fluidic chip
And AC power supply module, wherein the micro-fluidic chip is equipped with the sample flow channel for injecting discrete phase and continuous phase, the friendship
Galvanic electricity source module is used to apply alternating current to the sample solution in sample flow channel, the sample solution in sample flow channel in electric field force and
Drop is generated under the action of hydraulic power, stretching of the drop in a distance Jing Guo AC field is cracked into extra small drop.
2. system according to claim 1, which is characterized in that be fixed with first electrode and second on the micro-fluidic chip
Electrode, one end of the first electrode are electrically connected the sample flow channel of continuous phase, and the other end is for accessing alternating voltage, and described the
One end of two electrodes is electrically connected the sample flow channel for generating drop, and the other end is for accessing alternating voltage.
3. system according to claim 1, which is characterized in that the sample flow channel is shaped to drop formation hair
It is born in the tip of Taylor cone, the discrete phase of injection with ion and has the conductivity set, mixed inside the continuous phase of injection
Conjunction has surfactant.
4. system according to claim 3, which is characterized in that according to the electric field force and sample applied on the micro-fluidic chip
The subtended angle of hydraulic power control Taylor cone in product runner, and then control the partial size and frequency of generated drop.
5. system according to claim 2, which is characterized in that one or more super to control in lower list by controlling
The generation of droplet: the distance between temperature, the shape of electrode, first electrode and second electrode of sample solution, sample solution
Conductivity, the sticking property of sample solution, the shape of sample flow channel, the size of sample flow channel, the flow velocity of continuous phase, discrete phase
Flow velocity, application alternating current voltage and frequency.
6. system according to claim 1, which is characterized in that the system further includes the extra small drop for generation, is used
Ultraviolet light triggering curing mode is solidified into solid globules, and carries out table with scanning electron microscope or atomic force microscope characteristic manner
Face morphology analysis obtains the grain diameter characteristic of extra small drop, and carries out constant temperature nucleic acid amplification reaction, is judged by fluorescence signal
The bioactivity of extra small drop internal.
7. system according to claim 6, which is characterized in that grain diameter characteristic based on extra small drop obtained and extra small
Bioactivity inside liquid controls the generation for optimizing extra small drop to the influence of amplified reaction.
8. system according to any one of claims 1 to 7, which is characterized in that the partial size of extra small drop generated is less than 5
Micron.
9. a kind of single cell whole genome amplification method based on micro-fluidic electron spray, comprising:
Extra small drop is generated using the described in any item systems of claim 1 to 8；
Determine extra small size droplet diameter, the incidence relation between nucleic acid fragment size and amplification efficiency；
Gene sequencing is carried out to single cell whole genome amplification result, the extra small drop of generation is controlled to adjust according to sequencing result
Characteristic.
10. according to the method described in claim 9, it is characterized in that, the friendship being applied on the micro-fluidic chip by control
The voltage of galvanic electricity and frequency control the partial size and frequency of extra small drop formation.
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| CN112557379A (en) * | 2020-12-08 | 2021-03-26 | 深圳先进技术研究院 | Biochemical luminescence detection system based on liquid drop injection |
| CN115178198A (en) * | 2022-05-30 | 2022-10-14 | 中国科学院深圳先进技术研究院 | Polymer microsphere preparation device and preparation method |
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