CN108535239A - Micro-fluidic chip based on microlayer model and detecting system - Google Patents

Micro-fluidic chip based on microlayer model and detecting system Download PDF

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CN108535239A
CN108535239A CN201810265745.9A CN201810265745A CN108535239A CN 108535239 A CN108535239 A CN 108535239A CN 201810265745 A CN201810265745 A CN 201810265745A CN 108535239 A CN108535239 A CN 108535239A
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microlayer model
module
micro
drop
detection
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CN108535239B (en
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王陈成
罗贞
王茜
马云林
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SHANGHAI AIRUIDE BIOTECHNOLOGY Co Ltd
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SHANGHAI AIRUIDE BIOTECHNOLOGY Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

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Abstract

The present invention provides the microfluidic control systems based on microlayer model for biology sample detection.Specifically, the present invention provides a kind of micro-fluidic chips, the micro-fluidic chip includes micro-fluidic flow-through channel, reaction microlayer model generation module, cleaning microlayer model generation module, shearing microlayer model generation module, capture cleaning shear module, signal-obtaining detection module, drop control of queue module, optionally includes and is incubated control module and waste collection module.

Description

Micro-fluidic chip based on microlayer model and detecting system
Technical field
The invention belongs to biotechnology, clinical vitro detection technical field more particularly to a kind of miniflows based on microlayer model Control chip and detecting system.
Background technology
In-vitro diagnosis (In Vitro Diagnosis, IVD) refers to except human body, by human sample (blood, body Liquid, tissue etc.) it is detected and obtains clinical diagnosis information, and then judge disease or the products ＆ services of body function.It is existing Conventional method in technology for biological detection diagnosis has very much, such as biochemical detection methods, and Molecular Detection analysis method is immunized Blotting, chemiluminescence immunoassay detection method, enzyme-linked immunization (ELISA method) etc..But these detection methods need long point The time is analysed, Liquid-treatment processes are cumbersome, and flux is smaller, and accuracy is insufficient.
Recent microfluidic system as a kind of platform that can gather multiple functions module be widely used in biology and Chemical field is known as " influencing one of 15 most important inventions of human future ".It is diagnosed relative to traditional biological detection Method, the method based on microfluidic system have the advantages that apparent several, including can gather various functions component, expensive life Object reagent consumption is few, can avoid sample cross contamination, and analysis time is shorter etc..
Have some immune detection platforms for using microflow control technique at present.However, exempting from currently based on microfluidic platform There are still some apparent disadvantages for epidemic disease checkout and diagnosis method or system.For example, antibody to be embedded in the somewhere of solid phase carrier in advance In channel or chamber, react for heterogeneous reaction, and therefore can not effectively and whether accurately determine antigen-antibody fully anti- It answers；Although required sample size is slightly reduced compared to conventional method but aequum is still relatively more；Most of micro-fluidic chip For disposable products, it is very high to reuse or reuse cost, and disposable micro-fluidic chip manufacturing cost is also higher.
In addition, there are also the microfluidic high-throughput bioanalysis platforms based on drop to deliver.Wherein use drop miniflow The immune detection platform of control technology is representative to be had：It is immunized based on nanometer homogeneous phase time discrimination fluorescence and is examined with drop microflow control technique Survey large biological molecule method, based on novel sensor (sensor of such as encapsulated) high-throughput liquid drop microfluidic system or Method, the liquid drop microfluidic system of detection quantum dot and bio-molecular interaction and method etc..However, these systems are still difficult to Satisfactory, there is also different disadvantages, such as：Some micro-fluidic chips can carry out high-throughput detection, but complicated, Of high cost and sensitivity is relatively low；Some uses heterogeneous reaction, therefore sample requirement is big and repeatability is relatively low；And absolutely mostly The above-mentioned micro-fluidic chip of number is disposable, is unfavorable for promoting.
In conclusion there is an urgent need in the art to develop it is a kind of and meanwhile have high throughput, high accuracy, high sensitivity, it is low at This advantages that and reusable micro-fluidic chip and detecting system based on microlayer model.
Invention content
Having many advantages, such as high throughput, high accuracy, high sensitivity simultaneously it is an object of the invention to provide one kind and can weigh The micro-fluidic chip and detecting system based on microlayer model used again.
In the first aspect of the present invention, providing a kind of micro-fluidic chip includes：
(a) micro-fluidic flow-through channel, the micro-fluidic flow-through channel are used for for entrained in continuous phase and continuous phase Microlayer model flowing；
(b) microlayer model generation module is reacted, the reaction microlayer model generation module is for generating the micro- liquid reacted Drop, the reaction microlayer model contain：Sample to be detected, the reaction reagent reacted with the sample to be detected, wherein institute State the detection product for being formed after the substance to be detected in sample to be detected is reacted with reaction reagent and carrying detectable marker；
(c) microlayer model generation module is cleaned, the cleaning microlayer model generation module is for generating the cleaning cleaned Liquid microlayer model；
(d) microlayer model generation module is sheared, the shearing microlayer model generation module is for generating the shearing sheared Liquid microlayer model；
(e) capture-cleaning-shear module, also referred to as extracts module, and the capture-cleaning-shear module is used for from through incubating It carries out capturing the detection product for carrying detectable marker in reaction microlayer model after educating, and micro- with the cleaning solution Drop cleans the captured detection product for carrying detectable marker；And with the shearing microlayer model pair The detection product of once purged carrying detectable marker is sheared, to generate the microlayer model for detection；
(f) signal-obtaining detection module, the signal-obtaining detection module are used to carry out the microlayer model for detection Signal-obtaining；With
(g) drop control of queue module, the drop control of queue module and cleaning microlayer model generation module and shearing Microlayer model generation module is connected, and for after reaction microlayer model is moved to precalculated position, controlling the cleaning microlayer model The work of generation module and the shearing microlayer model generation module, thus in liquid flowing side in the micro-fluidic flow-through channel Microlayer model queue to formation " reaction microlayer model-cleaning microlayer model-shearing microlayer model ", wherein reaction microlayer model is located at flowing Forefront.
In another preferred example, the micro-fluidic flow-through channel is capillary channel.
In another preferred example, the micro-fluidic flow-through channel is that depth is 10 to 1000 μm and/or width is 10 μm To 1000 μm of microchannel；Preferably, the micro-fluidic flow-through channel is that depth is 50 μm and/or width is 100 μm micro- Channel.
In another preferred example, the micro-fluidic flow-through channel is selected from：Straight channel, circular passage are folding shape channel, back-shaped Channel, cavity or combinations thereof channel.
In another preferred example, the reaction microlayer model generation module further includes droplet counting labeling submodule, the liquid Drop counts labeling submodule and is used to that reaction microlayer model to be counted and be numbered.
In another preferred example, the reaction microlayer model generation module further includes microlayer model fusion submodule, described Sample microlayer model and respectively, simultaneously or sequentially generate reaction reagent microlayer model is first generated in reaction microlayer model generation module, then is led to The microlayer model fusion submodule is crossed, so that reaction reagent microlayer model is merged with sample microlayer model becomes the micro- liquid of reaction Drop.
In another preferred example, the reaction microlayer model generation module further includes valve member, and the valve member is used In controlling the reaction reagent microlayer model with the generation of the sample microlayer model or for the microlayer model that reacts described in controlling It generates.
In another preferred example, the dispersed phase sample introduction submodule include a dispersed phase injection port, a waste liquid outlet and Two valves；The dispersed phase injection port is used for dispersed phase into original mold for adding sample, the valve and waste liquid outlet The Rapid Cleaning of block, the waste liquid outlet are connected with waste collection module.
In another preferred example, the dispersed phase injection port can be bonded closed with sample needle, be made in the same of addition sample When ensure enough positive pressures.
In another preferred example, the reaction microlayer model generation module, cleaning microlayer model generation module and the micro- liquid of shearing Drip generation module, have T-type interface structure, flowing altogether focusing structure, cross drop formation structure, coaxial fluidal texture or its Combination, the structure is for generating microlayer model；Preferably, using T-type drop formation structure, cross drop formation structure or A combination thereof.
In another preferred example, the signal is selected from：The light letter that chemiluminescence signal, fluorophor are stimulated emitted Number, quantum dot microsphere light excite emitted visible light signal, absorbance change signal, magnetic field intensity signal or combinations thereof.
In another preferred example, the cleaning microlayer model is one or more.
In another preferred example, the reaction drop is one or more.
In another preferred example, the shearing drop is one.
In another preferred example, the micro-fluidic flow-through channel includes following paragraphs：Continuous phase sample introduction section, detection sample Sample introduction section, reaction reagent addition section, incubation reaction section, cleaning microlayer model addition section, shearing microlayer model addition section, capture-cleaning- Shear treatment section, signal detection section and optional waste fluid channel section.
In another preferred example, the capture-cleaning-shear module further includes field regions, and the field regions are can Control the field regions of open and close.
In another preferred example, the field regions are equipped with moveable permanent magnet or electromagnet, the magnetic field The magnetic field in area is generated by moveable permanent magnet or electromagnet.
In another preferred example, the cleaning microlayer model generation module and the shearing microlayer model generation module also wrap It includes：Control valve component, the control valve component are controlled by drop control of queue module.
In another preferred example, the control valve component is selected from：Electromagnetic valve, paraffin valve, paraffin hot melt valve, Magnet mobile valve, operated pneumatic valve, diaphragm valve, hydrophobic valve, machinery valve or combinations thereof；Preferably, the control valve Door component is electromagnetic valve.
In another preferred example, the driving force component is pressure-driven component.
In another preferred example, the micro-fluidic chip further includes：(i) control module, incubation control are incubated Module is used to control the incubation parameter for the micro-fluidic flow-through channel for being incubated section.
In another preferred example, the micro-fluidic chip further includes：(j) waste collection module, the waste collection mould Block is for collecting the waste liquid generated in detection process.
In another preferred example, the signal-obtaining detection module further includes post-processing submodule, in the post-processing Into the microlayer model for detection, addition is used to generate the substance of detection signal in submodule.
In another preferred example, the post-processing submodule is additionally provided with whether the microlayer model for detection for identification is effective The sensor of drop, and in the post-processing submodule to addition in effectively for the microlayer model of detection for generating detection The substance of signal.
In another preferred example, the material of the micro-fluidic chip be selected from glass substrate material, glass, silica-base material, Dimethyl silicone polymer, acrylic plastics, cycloolefin copolymer species material, polypropylene plastics, polystyrene plastics or its group It closes；Preferably, it is glass substrate material.
The second aspect of the present invention provides a kind of detection method based on micro-fluidic chip, and the method includes following Step：
A. mobile phase is added, and the mobile phase is made to be full of micro-fluidic flow-through channel；
B. reagent is pre-processed；
C. prepare the biological sample to be detected, the sample contains target substance to be detected；
D. sample and pretreated reagent is added, and sample microlayer model is generated by the reaction microlayer model generation module And reagent microlayer model, and it is fused into reaction microlayer model in the reaction microlayer model generation module；Or by sample and preprocessed Reagent be pre-mixed as reaction liquid, and the reaction liquid is added and is generated by the reaction microlayer model generation module React microlayer model；
E. under the control of drop control of queue module, microlayer model generation module is cleaned, generates cleaning microlayer model；
F. under the control of drop control of queue module, microlayer model generation module is sheared, generates shearing microlayer model；
G. the microlayer model and reaction microlayer model that step e, step f are generated form microlayer model queue；
H. the microlayer model queue enters capture-cleaning-shear module, and forms the microlayer model for detection；
I. for the microlayer model of detection by signal-obtaining detection module, the reading detection module detects and reads signal.
In another preferred example, the reagent is the reagent that can be reacted with target substance in sample.
In another preferred example, the reagent is the reagent that can combine magnetic bead or generation detectable signal.
In another preferred example, the target substance is selected from：Protein, nucleic acid, liposome, peptide fragment, nucleotide, amino Acid, virus, bacterium, parasite, cell；Preferably peptide fragment or protein substance.
In another preferred example, the sample is selected from：The blood of people or other animals, blood plasma, serum, tissue fluid, Lymph, urine or the culture solution for cultivating microorganism/cell.
In another preferred example, the biological sample is pre-processed.
In another preferred example, after step d, injection port is cleaned with micro-fluidic chip cleaning agent, cleaning agent gives up Liquid enters waste collection module, repeats step d, generates multiple reaction microlayer models with different samples.
In another preferred example, after step d, the micro-fluidic chip cleaning agent is selected from buffer system, surfactant, egg White matter or combinations thereof；Preferably, buffer system is selected from：Phosphate system, acetic acid salt system, borate salt system or Tris-HCl bodies System and/or surfactant are selected from：PEG, PVP, polysorbas20, Tween 80, triton X-100 or combinations thereof and/or protein choosing From seralbumin, casein or combinations thereof.
In another preferred example, it being generated in step d, the reaction microlayer model fully reacts in incubation reaction section/ Even mixing.
In another preferred example, the condition of the incubation reaction section is controlled by the incubation control module.
In another preferred example, the condition of the incubation reaction section is temperature condition.
In another preferred example, in single detection, 1-3 cleaning microlayer model is generated.
In another preferred example, in single detection, 1 shearing microlayer model is generated.
In another preferred example, when drop queue enters the capture-cleaning-shear module, magnetic field is opened, and reaction is micro- The sample containing magnetic bead is captured by magnetic field in drop, is then cleaned microlayer model and is cleaned captured sample, then shears droplet shearing Lower detectable marker, and form the microlayer model for detection.
In another preferred example, the cleaning microlayer model after use and the reaction microlayer model without sample, pass through waste liquid Channel enters waste liquid module.
In another preferred example, closing discarded magnetic bead in magnetic field enters waste liquid module with continuous phase.
In another preferred example, there is the microlayer model post-processing step for detection in step i, to micro- liquid for detection The substance for generating detection signal is added in drop.
In another preferred example, the substance of the excitation-detection signal is that chemiluminescence excites substrate.
In another preferred example, the microlayer model for having read signal enters through waste fluid channel and enters liquid waste processing module.
In another preferred example, in step b, the pretreatment to reagent is coating or coupled bead.
In another preferred example, in step b, the magnetic bead is the compound of iron, cobalt, nickel；Preferably di-iron trioxide Or ferroso-ferric oxide compound；
In another preferred example, in step b, the magnetic bead is Superparamagnetic particulates.
In another preferred example, in step b, wherein the size of magnetic bead is 0.01-10 μm, more preferably 1-2.8 μm.
In another preferred example, the volume ratio of the sample microlayer model and reagent microlayer model is 1:0.5 to 1:2.
In another preferred example, the method is the detection method based on double antibody sandwich method or competitive binding method.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.
Description of the drawings
Fig. 1 shows the structural schematic diagram of the micro-fluidic chip in one embodiment of the invention.Wherein, each mark is as follows：
1- continuous phase injection ports, 2- control valves a, 3- flow duct, 4- waste liquid ports, 5- control valves b, 6- dispersed phase into Sample mouth, 7- control valves c, 8- drop generate mouth, 9- sensors As, and 10- sensors B, 11- reagent merges mouth, the fusion of 12- reagents Injection port 1,13- reagents merge injection port 2, and 14- control valves d, 15- first is incubated pipeline, 16- the first drop of testing liquid liquid sample introductions Mouth (or pretreatment liquid injection port), the first column construction area 17- control valves e, 18-, the field regions 19-, 20- sensors C, 21- Sensor D, 22- drop of testing liquid liquid merge mouth, the second drop of testing liquid liquid injection port of 23- control valves f, 24- (or aftertreatment fluid into Sample mouth), 25- second is incubated pipeline, the second column constructions of 26- area, 27- sensors E, 28- outlet.
Fig. 2 shows the diagrammatic cross-section of the dispersed phase injection port in one embodiment.
Fig. 3 shows the work flow diagram of micro-fluidic chip of the present invention.
Fig. 4 shows that in the micro-fluidic chip of one embodiment of the invention, the activity in each stage of microlayer model is illustrated Figure.Wherein, the A- drop formations stage, B- reagent fusing stages, in the C- blending incubation stages, in the D- droplet extraction stages, E- drops are cut Cut the stage.In C, it is shown that three kinds of different representative reactions：Blending incubation rank based on antigen-antibody reaction in C1- drops Section, is based on loop-mediated isothermal amplification in C2- drops, and BCA methods measure the blending incubation rank of total protein concentration in C3- drops Section.
Fig. 5 shows the status diagram of extraction process microlayer model in an embodiment of the invention.
Fig. 6 shows the experimental result extracted in an embodiment of the invention.
Fig. 7 shows the block diagram of the gray value of the optical signal detected under different extraction times of the invention.
Fig. 8 shows the standard curve of different extraction times.
Specific implementation mode
The present inventor's in-depth study by long-term devises a kind of module integrated level height based on the micro-fluidic of microlayer model Chip and its control system and device and method.Accurately controlling and realizing in list to drop may be implemented by the design Various required processes are completed on one chip.On this basis, the present invention is completed.
Term
As used herein, term " micro-fluidic chip of the present invention ", " micro-fluidic chip of the invention based on microlayer model " or " chip of the present invention " is used interchangeably, and refers to the micro-fluidic chip of the present invention.
As used herein, term " drop micro-fluidic chip " refers to the chip (or system) based on drop microflow control technique, It is middle using immiscible two phase flow, under the action of hydrodynamic shear and interfacial tension, wherein a phase dispersion in the other phase, A series of monodispersed drops are formed, and each drop includes specific biochemical reaction.Compared to the micro-fluidic system of continuous fluid System, the microfluidic system based on drop can with the frequency of up to kHz generate containing nanoliter to picoliters volume drop, often One drop all can serve as a microreactor, to realize that high throughput provides specific scheme, and can be in the short time It is interior to realize to the independent operation of single drop, including the generation of drop, fusion, incubation, aggregation, division, sorting, extraction and divide Analysis.The generation of drop relies on the shearing force and surface tension collective effect of two-phase.
As used herein, term " fusion of drop " refers to being integrated two or more droplet coalescence, can To carry out the hybrid reaction between different component drop.
As used herein, it is two or more liquid that term " division of drop ", which refers to by a drop breakup, Drop realizes the concentration for further regulating and controlling content in droplet size and drop after drop formation.
As used herein, term " incubation of drop " refers within a certain period of time being uniformly mixed the content in drop.
As used herein, term " continuous phase " refers to carrier liquid, for example, be oil phase in the embodiments herein, but simultaneously It is not limited to oil phase.
As used herein, term " dispersed phase " refer to include detection sample, reaction reagent, cleaning solution or shearing liquid etc. liquid Body, the liquid can produce microlayer model in the continuous phase, for example, be water-soluble liquid in the embodiments herein, but simultaneously It is not limited to water-soluble liquid.Wherein, continuous phase is selected according to the property of dispersed phase, and continuous phase is immiscible with dispersed phase.
As used herein, term " microlayer model queue " is only represented by microlayer model by the sequence in precalculated position, " micro- liquid Microlayer model in drop queue " can exist in microchannel simultaneously, and followed by precalculated position；Alternatively, successively generating micro- liquid Drop, and followed by precalculated position.
Micro-fluidic chip
The present invention provides a kind of micro-fluidic chip, and the functional integration of micro-fluidic chip of the invention is high, can be automatically real Include now a series of functions such as generation, fusion, extraction, incubation, division, signal detection.
The quantity and sequence of the micro-fluidic chip modules of the present invention are not particularly limited, can be according to the specific of detection Situation is adjusted.
In another preferred example, micro-fluidic chip of the invention uses a kind of valve mechanism of optimization, in the valve Control under, accurately control drop.
In another preferred example, micro-fluidic chip of the invention is furnished with power source, and the power source is for making each micro- liquid Drip (dispersed phase) and continuous opposite required direction movement, wherein the power source can provide forward drive power or reversed Driving force；The power source can be the common various power resources in field, such as, but not limited to, by centrifuging power drive, electricity Wetting driving, pressure-driven obtain power；Preferably (pressure is selected from for pressure-driven acquisition：Electrolytic pressure, compressed gas pressure Power, direct draught head).
In another preferred example, the reading of content of the signal-obtaining detection module of micro-fluidic chip of the invention includes but not It is limited to：Chemiluminescence signal, fluorophor be stimulated emitted optical signal, quantum dot microsphere light emitted it is visible Optical signal etc..
In another preferred example, the signal-obtaining detection module of micro-fluidic chip of the invention can be according to specific detection side Method determines that representative example includes (but being not limited to)：Induced with laser multi-channel spectral camera, induced with laser multi-channel spectral Sensor, photomultiplier or combinations thereof.
In another preferred example, in chip of the present invention multiple positions be equipped with sensor (such as each microlayer model generate mould Block, capture-cleaning-shear module entrance and exit)；The sensor can count drop and mark, confirm Droplet size whether meet examine needed for, calculate drop by frequency (or speed) and feed back to control system.
In another preferred example, micro-fluidic chip of the invention can pass through general control system (e.g., self-programmed software or business Software etc.) realize modules cooperation；The general control system receives the signal that each sensor obtains in chip And generation control signal is handled, the general control system is by controlling the various pieces of entire chip, such as power source and each valve Deng, the drop control of queue module of cooperation chip of the present invention, detection of the realization to various biologies.
In another preferred example, micro-fluidic chip of the invention can be made of common materials in field, these material packets It includes (but being not limited to)：Glass, dimethyl silicone polymer, acrylic plastics, cycloolefin copolymer species material, gathers silica-base material Acrylic plastic, polystyrene plastics, rubber, or combinations thereof；These materials itself can be selected, can also select it is doped, Modification, modified material.
In another preferred example, the shape of micro-fluidic chip micro-fluidic flow-through channel of the invention can be that this field is various Common channel shape includes but not limited to channel, circular passage, folding shape channel, back-shaped channel or cavity；Wherein, cavity body structure Including but not limited to single layer height, the double-deck height or multilayer height.
In another preferred example, micro-fluidic chip micro-fluidic flow-through channel of the invention can etch shape by material internal It to be formed at that can also be connected by capillary.
In another preferred example, each microlayer model generation module, which has, to form drop knot by fluid shearing active force Structure, these structures include but not limited to T-type interface structure, cross interface structure, flowing focusing structure or coaxial flowing knot altogether Structure.
In another preferred example, the microlayer model generation module of micro-fluidic chip forms microlayer model structure through the invention, raw At the diameter of microlayer model be about 1 μm -1000 μm, preferably diameter is about 50- μm, it is necessary to when can also produce liquid column.
In another preferred example, micro-fluidic chip of the invention can be realized not according to different objects on chip With detection, including but not limited to based on nucleic acid detection method (for example, PCR, PT-PCR, loop-mediated isothermal amplification technique or Person's strand displacement) or based on protein-specific reaction detection side's (for example, double antibody sandwich method, competitive immunization combined techniques etc.).
In another preferred example, micro-fluidic chip of the invention is suitable for disposable, is also applied for reusing.
Detection method
The present invention also provides the detection methods based on micro-fluidic chip of the present invention.
There is provided a kind of extracting operations of high automation for a prominent features in the method for the present invention.For the ease of reason Solution, is described below in conjunction with attached drawing.It should be understood that these attached drawings do not limit protection scope of the present invention in any way.
Referring to Fig. 3, the workflow of micro-fluidic chip of the invention is sample introduction, drop generates, reagent merges, is incubated, liquid It drips pre-treatment, drop post-processing, signal observation and enters waste liquid pool.
Referring to Fig. 4, on the chip of the present invention, the generation of microlayer model is as shown in Figure 4 A, and sample and continuous phase are driven in external force It is flowed along micro-fluidic flow-through channel under dynamic, in drop formation interface, sample is generating the shearing work formed by dispersed phase at mouth With lower formation microlayer model.Fusion process between microlayer model is as shown in Figure 4 B, and microlayer model and reagent microlayer model containing sample are a pair of One fusion.Various incubation reactions are as shown in Figure 4 C inside microlayer model.As shown in Figure 4 D, the microlayer model containing the sample with magnetic bead, warp The substance with magnetic bead is retained in the region when crossing field regions.As shown in Figure 4 E, the liquid of the lower target object to be checked of shearing microlayer model shearing It drips (the shearing liquid after i.e. clipped reaction).
Referring to Fig. 5, wherein with 3 microlayer models for having carried out respective reaction, 1 cleaning solution drop and 1 shearing liquid drop For, extraction process is described, main includes capture, cleaning and shearing.
By taking paramagnetic particle method as an example, in chip channel, the reagent droplet of the sample droplets of generation and the reagent generation of addition is melted It closes and forms mixing drop i.e. reaction microlayer model (microlayer model reacted), mixing drop enters chip extraction after the completion of being incubated Module.
After mixed liquor drip crosses precalculated position, is transmitted in accurate signal and successively open required valve under operation and control Door generates cleaning solution drop and shearing liquid drop, to form microlayer model, cleaning solution drop and shearing liquid in microfluidic channel Team's sequence of drop (Fig. 5 a).
Under magnetic fields, when the drop containing the magnetic bead for being combined with antigen-antibody and fluorescent marker flows through field regions Domain, first magnetic bead mixed in drop are attracted on the side wall of chip channel (Fig. 5 b), and (are schemed with flowing drop separation 5c, 5d).Similarly, second and third mix the magnetic bead in drop and are also attracted on the side wall of chip channel and (do not show Go out), and with flowing drop separation.
With flowing of the continuous phase (oil phase) in chip channel, by drop breakup will mix the magnetic bead cluster in drop with Other drop contents detach, and then wash magnetic bead cluster successively with the drop containing cleaning solution, fully remove interference residue (figure 5e, 5f).
Finally, shearing liquid drop contacts fusion with magnetic bead cluster in the same way, then divides, shears in this process simultaneously Take away the substance (Fig. 5 g, 5h) for signal detection.Later, the shearing liquid drop continuation after shearing is flowed forward with the state of drop It is dynamic to carry out signal detection, or enter chip post-conditioning region and carry out operation (not shown) in next step.Magnetic bead cluster is closing magnetic field Afterwards, the state dripped with waste liquid flows forwardly into waste liquid pool.(not shown)
In another preferred example, primitive organism sample or biological sample after processing, are entered by sample injection port In drop micro-fluidic chip, the control flowed to sample is realized by valve.Sample introduction of the continuous phase (such as oil phase) in continuous phase Mouth enters in drop micro-fluidic chip, and the control flowed to continuous phase is realized by valve.
In another preferred example, the biological sample is biological liquid sample, can be people or the blood of other animals Liquid, blood plasma, serum, tissue fluid, lymph, urine etc., either cultivate microorganism either the culture solution of cell or other compare Precious or rare environmental samples.
In another preferred example, the biological liquid sample contains target object to be checked, and the method for the present invention can be to target Detectable substance carries out qualitative, sxemiquantitative or quantitative detection.
Wherein optional, target object to be checked can be, but not limited to be protein, nucleic acid, liposome, peptide fragment, nucleotide, ammonia Base acid, virus, bacterium, parasite, cell and some other unimolecule or complex etc..
In another preferred example, the biological sample is conventional in biological detection by pretreatment, the pretreatment Preprocess method (for example, anti-coagulants is added, lysate, adjustment concentration is added etc.).
In another preferred example, chip cleaning agent can be added after adding a sample in the sample injection port, cleaning Sample injection port, and controlled by valve control or driving force, it is discharged in waste liquid module from connected waste fluid channel section.
In another preferred example, the microlayer model for detection stops in the signal detection section and is read letter Number or the microlayer model for detection flowed in the signal detection section and be read signal.
In another preferred example, microlayer model can make microlayer model rest on incubation when in the stage of incubation by control Conversion zone is sufficiently mixed or reacts；Alternatively, microlayer model keeps flow regime in incubation reaction section, it is sufficiently mixed or reacts.
In another preferred example, the method for the present invention can make mobile phase take away capture-cleaning-shearing by closing magnetic field The remaining magnetic bead of processing section carries out the preparation for carrying out extraction processing next time.
In another preferred example, raising module can be controlled and keep constant temperature (temperature is according to specific selecting response).
In another preferred example, method of the invention can carry out monitoring and control by computer software in entire test process System.
In another preferred example, the sample size volume of the method for the present invention single detection is 0.5-10 μ L, preferably 0.5-5 μ L.
In another preferred example, the single detection time of the method for the present invention single sample is 3-15min.
In another preferred example, the method for the present invention can be simultaneously to multiple samples into detection.
Microfluidic control chip based on microlayer model or method
A kind of microfluidic control chip or method based on microlayer model, the microfluidic control chip includes flow injecting mould Block, microlayer model generation module, reagent Fusion Module, raising module, microlayer model processing module, signal-obtaining detection module, waste liquid Pond module.But it is not limited to this kind of module to combine successively, also being used in mixed way including this kind of module and multiplex system.It is special Sign is, including module design and step in detailed below：
A) flow injecting module：Biological initial liquid sample or biological liquid sample after processing, pass through dispersed phase Inlet port enters in drop micro-fluidic chip, and the control flowed to sample is realized by valve.Oil phase as continuous phase exists The injection port of continuous phase enters in drop micro-fluidic chip, and the control flowed to continuous phase is realized by valve.
It is wherein optional, waste fluid channel can be added at the dispersed phase inlet port, remnants can be discharged in waste fluid channel Sample.
And it is optional, cleaning solution is added from dispersed phase inlet port, dispersed phase inlet port is cleaned, cleaning solution passes through Waste fluid channel is discharged.And it is optional, opening or closing for valve control waste fluid channel can be used.
The power source of the power source and discharge waste liquid of the dispersed phase and continuous phase flowing, is for drop micro-fluidic chip Interior fluid provides forward thrust, makes the uniform flow forward of fluid, avoids because of test value difference caused by different in flow rate.Its In it is optional, the power source can be, but not limited to be centrifugation power drive, electrowetting driving, pressure-driven (electrolytic pressure, compression Gas pressure, chemical breakdown pressure, the driving of direct draught head).Wherein optional, the power source can be forward drive power, It can also be reverse actuating force.
The dispersed phase and continuous phase also have the valve being discharged in the channel of waste liquid, can be, but not limited to be paraffin valve, Paraffin heats valve, magnet mobile valve, operated pneumatic valve, diaphragm valve, hydrophobic valve, machinery valve, can be therein one Kind or multiple combinations use, or directly use dynamic Control without using valve.
B) microlayer model generation module：The dispersed phase and continuous phase liquid are linked together by channel, pass through flowing Fluid shearing active force forms drop, and the mode of droplet formation can be all structures for forming drop, it is preferred that including T Type interface, flowing copolymerization coke, coaxial fluidal texture.
C) reagent Fusion Module：It is added and can be combined with object to be detected in the drop that the biological sample is formed Labeled substance, pass through droplet coalescence, contact fusion method realize reagent and sample drop fusion.
Wherein optional, the reagent refers to the mark substance that can be combined with target object to be checked being added in drop, Can also be the medium that auxiliary object to be checked is combined with mark substance, or the detectable letter of the generation that can react with object to be checked Number reactant or other similar functions reagent.
Wherein optional, the amalgamation mode of the reagent and drop can be produced by the structure or drop of chip channel The fusion of raw velocity interpolation drop, or the method that is contacted with each other by drop and reagent carry out the fusion of reagent and drop, Active control fusion or passive exposure fusion can be selected herein.
D) raising module：The module provides a region, can so that solution carries out mixing in drop or reaction is filled Point, it can be completed in the flowing of drop, the flowing that can also stop drop fully being completed.
Wherein optional, the module provides or does not provide thermostat module, in auxiliary droplet reagent it is abundant react or Person reaches the reaction condition of the reaction.
And optional, the module can provide one or more thermostat module, to the progress of assisted reaction.
E) drop of testing liquid module, each module in micro-fluidic chip is (for example, capture-cleaning-shearing mould through the invention Block, drop control of queue module etc.) it realizes to the extraction for reacting microlayer model through incubation：The drop of testing liquid module includes drop The input of process chamber (for example, capture-cleaning-shear treatment section) and drop cleaning agent/inorganic agent.The module provides an area Domain can be handled the target detection thing fully reacted in drop, keep it more accurate in signal-obtaining, fast It is fast, efficient.
In another preferred example, pre-treatment and post-processing can be divided into, the pre-treatment is that extraction or capture-are cleaned-cut It cuts.
Design or additional driving force wherein optional, that the drop of testing liquid room that the module provides can be by channel It realizes control of the drop in the region, drop is further processed to convenient.
And optional, the design by channel can be that double-layer structure control drop rests on drop of testing liquid room, can be with It is the movement velocity for designing column structure and slowing down drop, can also realizes same or similar function using other designs.
And it is optional, it can realize the control of the movement to microlayer model by additional driving force, added driving force can be with It is magnetic force or other can travel the power of same or similar function.
Wherein optional, the input of drop cleaning agent/inorganic agent, can be handled drop in the module.Cleaning The input of agent may be implemented, on the cleaning of the unreacted substance that can influence signal-obtaining in drop, to go deimpurity influence.Liquid Other following functions of needing, such as the shearing etc. between magnetic bead and antibody may be implemented in drop inorganic agent.
It is wherein optional, the step of auxiliary signal is read, such as homogeneous immunization method can be provided in drop of testing liquid room In light excitation processing is either other same or similar step process.
F) signal-obtaining detection module：The module is that one piece of region is provided in chip, to having reacted in the region At drop pre-processed, later chip exterior signal detection apparatus or instrument such as sensor or sensing system read The index of reaction target detection thing content in coring piece in the region can be, but not limited to be optical signal, fluorophor letter Number, quantum dot signal, fluorescent microsphere signal etc..
Wherein optional, the sensor can be, but not limited to be microscope, optical fiber etc..
Pretreatment to the drop of internal-response completion refers to being removed in drop to have reaction target detection thing with cleaning solution Other substances of interference avoid causing false sun or false cloudy signal.
G) waste liquid pool module：The module is for collecting all waste liquids generated in sample continuous mode.
In another preferred example, all modules in the microfluidic control system or device or method based on microlayer model Operating status is monitored and is controlled by computer software.For example, the switch of the valve in control chip system；Signal is controlled to read Detection module is taken, carrying out processing to measurement data provides visual information.
Wherein optional, computer control software can be, but not limited to be business control software and self-programming software, can be with It is one such or two kinds are applied in combination.
In another preferred example, the fluid for generating drop described in foregoing teachings is biological initial liquid sample, Or other treated biological initial liquid samples.Including but not limited to the blood of people or other animals, blood plasma, blood Clearly, tissue fluid, lymph, urine etc..
Wherein optional, the sample can be the culture solution for cultivating microorganism or cell.
Wherein optional, the sample can also be other more precious or rare environmental samples.
It in another preferred example, may be to be checked containing target in the fluid for generating drop described in foregoing teachings Object carries out qualitative, sxemiquantitative or quantitative detection using the system to target detection thing.
Wherein optional, target object to be checked can be, but not limited to be protein, nucleic acid, liposome, peptide fragment, nucleotide, ammonia Base acid, virus, bacterium, parasite, cell and some other unimolecule or complex etc..
In another preferred example, reagent described in foregoing teachings fusion part reagent in, containing can and target detection The marker that object be combined with each other, for example it is combined with the antibody of magnetic bead or fluorescence signal object.
And optional, which is that can react with sample, generates other substances or the chela of detectable signal Close object.
Wherein optional, magnetic bead is Superparamagnetic particulates, includes the compound of iron, cobalt, nickel, includes but not limited to mainly three oxygen Change two iron and ferroso-ferric oxide compound.Wherein the size of magnetic bead is 0.1-10 μm, preferred 0.5-3 μm.
And it is optional, the movement or aggregation of magnetic bead can be manipulated with permanent magnet or electromagnet.
Wherein optional, fluorescence signal object includes but not limited to mainly fluorescent microsphere and fluorescence signal group.
In another preferred example, it is carried out with microchannel between the micro-fluid chip system modules described in foregoing teachings Connection, the drop that the sample generates can be along passage flow to each module.The microchannel still can not only limit Then：Material internal etches or the microchannel of other methods formation, and the microchannel formed between different components uses capillary Microchannel of making etc..
Wherein optional, the shape of microchannel can be, but not limited to be straight channel, and shape channel is rolled in circular passage, back-shaped logical Road etc..
It is wherein optional, it is 1 μm -10 μm, 5 μm -50 μm and 30 μm that the depth of microchannel, which can be, but not limited to, - 300 μm and 100 μm -1000 μm etc..
And it is optional, it is 1 μm -10 μm, 5 μm -50 μm and 30 μm that the width of microchannel, which can be, but not limited to, - 300 μm and 100 μm -1000 μm etc..
7：Microlayer model leading in invention content 5 is formed under the action of continuous phase according to fluid described in invention content 1 Flowed in road, the diameter for generating drop can be, but not limited to be 1 μm -50 μm, 50 μm --- 1000 μm etc..
In another preferred example, the modules described in foregoing summary can be combined sequentially into system, can also Selection a portion is combined into system, forms system after can also mixing, some module and other moulds can also be used for multiple times Block forms system, these block combiners can also be utilized at multiplex system etc..
It is wherein optional, in above system also include being used for multiple times in systems of part of module combination, for example try The combination of agent Fusion Module and raising module is incubated again after being separately added into different reagents.
In another preferred example, the continuous phase described in foregoing teachings can be the oil phase being previously mentioned in invention content 1, Can also be other and another immiscible liquid of dispersed phase, continuous phase can be selected according to the property of sample to be tested.
In another preferred example, the injection port cleaning solution of the proposition described in foregoing teachings, can be used for cleaning sample and exists Residual at injection port removes the analyte of non-specific adsorption and the substance of other influences testing result, avoids generating friendship Fork pollution.
Wherein optional, the cleaning solution includes buffer system, surfactant and protein, wherein optionally, buffering System can be, but not limited to be phosphate, acetate, borate, Tris-HCl etc.；Protein can be, but not limited to be cow's serum Albumin, casein etc.；Surfactant can be, but not limited to be PEG, PVP, polysorbas20, Tween 80, triton X-100 etc.；Institute State the pH ranging from 6-10 of cleaning solution.
In another preferred example, the waste fluid channel described in foregoing teachings, this channel can with but be not limited only to and dispersed phase Injection port be connected, by waste fluid channel can be discharged extra sample or clean injection port cleaning solution.
It is wherein optional, this waste fluid channel can with but be not limited only to be：What material internal etching or other methods were formed Microchannel, the microchannel formed between different components, the microchannel etc. for using capillary to make.
And optional, the shape of waste fluid channel can be, but not limited to be straight channel, and shape channel is rolled in circular passage, back-shaped Channel etc..
In another preferred example, the raising module described in foregoing teachings, the module can be used for carrying out mixing in drop, And it is adequately reacted.
Wherein optional, which can be, but not limited to be split as two modules, mixing module and raising module, but go Make and raising module same or similar function described in invention content 1.
Wherein optional, which can be, but not limited to be straight channel, circular passage, folding shape channel, back-shaped channel, Or storage cavity etc..
In another preferred example, the signal-obtaining detection module described in foregoing teachings is the sensing using chip exterior The drop of device or sensor system senses after incubation.Sample is by generating microlayer model, being added and can detect in drop The reagent of target detection thing, and after raising module reaction is completed, signal-obtaining detection module can utilize sensor or sensing Device system detects the signal of target detection thing.
It is wherein optional, the method for signal is detected according to different target detection things, including but not limited to based on nucleic acid Detection method, based on the detection method of the albumen such as antibody and enzyme, molecular beacon detects the sensor of fluorescence, and immunostaining measures, Interlayer structure measuring method, chemical measure based on small molecule etc. and their combine detection.
And optional, the detection method based on nucleic acid includes but not limited to PCR, PT-PCR, ring mediated isothermal expansion Increasing technology, strand displacement etc..
And optional, the detection method based on antibody includes but not limited to ELISA, the detection based on interlayer structure Method, immunostaining measure, and antibody capture measures etc..
Wherein optional, the signal of target detection thing includes but not limited to electric signal, quality, turbidity (absorbance), magnetic letter Number, fluorescence, quantum dot, chemiluminescence, fluorescent microsphere etc.
In another preferred example, the signal acquisition module described in foregoing teachings can carry out signal in droplet flow Acquisition and reading, or drop is preserved or carries out signal acquisition and reading after stopping.
It is wherein optional, signal is acquired in the droplet flow, refers to that drop is flowing through sensor or sensor system When system, the useful signal in drop is collected.
And the channel for optionally, flowing through sensor can be still not limited only to：Material internal etches or its other party The microchannel that method is formed, the microchannel formed between different components, the microchannel etc. for using capillary to make.
And optional, the shape for flowing through sensor passage can be, but not limited to be straight channel, and it is logical to roll over shape for circular passage Road, back-shaped channel etc..
It is wherein optional, the part that drop is preserved or stopped can be storage chamber either memory channel or its The component of his similar functions.Storage chamber can be single layer height, and retention may be used in the double-deck height and multilayer height, memory channel Design, can also directly be controlled using power source, or otherwise realize identical function.
In another preferred example, the material of the drop micro-fluidic chip module described in foregoing teachings can be, but not limited to be Glass, silicon chip, ceramics, plastics, paper and rubber can be one such or multiple combinations.
It is wherein optional, it can be described material itself, can also be the material through overdoping, modification, modification.
And optional, the drop micro-fluidic chip made of the material, is suitable for disposable, is also applied for weight It is multiple to use.
The testing process based on microlayer model microfluidic control system or device or method includes：
Flow injecting module will be added as the test sample of dispersed phase and oil phase in step 1), opens at dispersed phase injection port Valve, test sample and oil phase successively enter microlayer model generation module；
Step 2) generates the microlayer model of test sample in microlayer model generation module；
Step 3) microlayer model enters reagent Fusion Module, test sample and the detectable substance that can be specifically bound with determinand Fusion starts to react；
Step 4) microlayer model enters raising module, and determinand is fully reacted with detectable substance inside microlayer model；
The complete microlayer model entering signal of step 5) internal-response reads detection module, is pre-processed to microlayer model, profit The signal to be detected in microlayer model is read out with sensor or sensing system, synchronous driving to computer carries out at numerical value Reason provides visual information；
Step 6) can synchronize the addition at dispersed phase inlet port in the state that dispersed phase valve is closed in progress step 3) Cleaning solution, cleaning solution are flowed out from waste fluid channel, can start second of test, this step is repeatable；
Step 7) waste liquid pool module collects all waste liquids during the test；
Step 8) computer software is monitored and controlled in the implementation of entire test process.
In another preferred example, the step 1) of the aforementioned testing process test sample amount volume is 0.5-10 μ L, preferably 0.5-5μL。
In another preferred example, aforementioned testing process, single sample testing time are 3-10min, but the test process It can synchronize and carry out multiple sample tests, greatly reduce the population sample testing time.
Main advantages of the present invention include：
1) the highly sensitive detection to biological sample, and also reproducible, strong antijamming capability, detection time can be achieved The advantages that short, flux is big, and sample consumption is minimum；
2) drop microfluidic chip technology is used, sample is generated, is mixed, is reacted, is collected and detection is integrated on chip, All reagent components needed for reaction can be accurately controlled by valve to be added in chip, easy to operate, and chip interior is tested Sample or reagent are not in direct contact with chip and instrument, and will not both pollute instrument will not generate cross contamination and seriously do It disturbs.
3) micro-fluidic chip can both reuse, and can also be disposable.
4) the function integration degree higher of chip of the present invention, realize to include generation simultaneously on the same chip, merge, The functions such as extraction, incubation, division, signal detection.
5) chip of the present invention have many advantages, such as high throughput, high accuracy, high sensitivity and it is reusable the advantages that.
6) the online addition reagent that the chip can also be controllable, so that a variety of detectable substances can be realized on a single die It detects simultaneously.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) or Roitt et al., immunology (Immunology, 6thEdition the condition described in), Or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number are weight percent and parts by weight Number.
Embodiment 1
BNP in human blood sample is detected using chemistry method of giving out light
In the present embodiment, using micro-fluidic chip shown in FIG. 1, method is given out light to the progress of human blood sample using chemistry BNP is detected.
One, experimental method
1. carrying out the pre-treatment of blood sample and reagent, that is, human blood sample first, second is taken, EDTA- anticoagulant blood-collecting pipes are added Interior anti-hemostasis-coagulation.
The processing of 2.BNP reagents：BNP antibody one is coated with magnetic bead, and BNP antibody two marks a word used for translation to sting ester.
3. the preparation of chemiluminescent substrate：Isometric 0.1M HNO3, 0.1%H2O2Shine exciting liquid A and 0.25M The luminous exciting liquid B mixing of NaOH, 2%Tween-20, forms chemiluminescent excimer.
4. sample sample introduction：Sample first, continuous phase sample introduction shown in Fig. 1 are added in dispersed phase injection port 6 shown in Fig. 1 Continuous phase mineral oil is added in mouth 1, control valve 2 and 5 is closed at this moment, and control valve 7 is opened, outlet shown in Fig. 1 28 and waste liquid port 4 give negative pressure as power source, the design of injection port is as shown in Figure 2.
5. drop formation：Drop formation process is as shown in Figure 4 A, and mineral oil and sample design channel in outer power drive lower edge Flowing, at drop formation interface 8, sample forms microlayer model generating under the shear action that is formed by dispersed phase at mouth, and Along channel flow forward under the sweeping along of mobile phase.
6. injection port cleans：After microlayer model is formed, injection port cleaning solution is added in closing control valve 7.Open control Valve 5 cleans injection port and waste liquid is quickly discharged.After completing injection port cleaning, closing control valve 5 is added again Cleaning solution is opened control valve 7, is cleaned to sample runner, cleaning solution can also form drop at 8, this partial drop quilt Sensor and control system identification, will not add reagent or carry out other processing, directly form waste liquid drop queue by going out sample Mouth 28 is discharged.The sample introduction that sample second starts new samples can be added after the completion of sample introduction module cleaning.
7. droplet counting and mark：The drop of generation is measured it by sensor 9 mark and drop number, with this It determines and generates whether drop is effective sample drop, and instruct the addition of follow-up reagent according to the mark and number.Following steps It is to be carried out for effective drop (sample), is such as detected as invalid drop (waste liquid), then follows directly after drop queue in outlet 28 Discharge.
8. droplet coalescence：Sensor 10, reagent fusion mouth 11, reagent fusion injection port 12,13 and control valve 14 and its The pipeline of connection together constitutes reagent Fusion Module.It, can be as schemed institute when detecting the multiple markers in a kind of sample simultaneously Show that increasing reagent Fusion Module is added corresponding reagent.The antibody one of coating magnetic bead is added in the present embodiment in injection port 12, The antibody two that label a word used for translation stings ester is added in injection port 13, while under the response of sensor 10 feedback, being controlled by control valve 14 Two kinds of antibody of system generate drop, the one-to-one fusion of drop progress of the drop and package sample.Fusion process is as shown in Figure 4 B, it Raising module is flowed to before backward.
9. the incubation of sample：Raising module is made of incubation pipeline 15 and extraneous temperature controller, which passes through liquid The turbulent motion of drop makes the liquid in drop be sufficiently mixed and completes reaction at a suitable temperature.In reaction process such as Fig. 4 C Shown in C1, refer to BNP for the experiment fully reacts with antibody one and antibody two.Drop fully after reaction enters at drop Manage module.
10. the pre-treatment of drop：Drop of testing liquid module is divided into the input of drop of testing liquid room and drop cleaning/inorganic agent.Liquid Drop process chamber is made of the cavity 18 of a distribution column structure and the magnetic field 19 wrapped up, and entire cavity is in magnetic field 19 In.The input of drop cleaning agent/inorganic agent is made of injection port 16 and valve 17 and relevant pipeline, which can be according to need It accelerates.The input of the input and a luminous substrate liquid of a cleaning agent is added in the present embodiment.Liquid drop movement is to liquid Process chamber is dripped, column structure can be such that liquid drop speed slows down, and magnetic bead is acted on by magnetic field in drop, and the substance with magnetic bead exists Drop of testing liquid room retains, such as Fig. 4 D.Control valve 17 is opened at this time, cleaning solution is passed through under pressure-activated, and generated at 12 Cleaning solution microlayer model, cleaning microlayer model rinse out that the indoor a word used for translation for having neither part nor lot in reaction stings ester label BNP antibody two and other are unbonded Substance, or occur non-specific binding substance.It is passed through the shearing liquid containing inorganic agent at subsequent identical structure, inorganic agent can So that magnetic bead is detached with antibody one, the new drop (the shearing liquid after i.e. clipped reaction) containing target object to be checked is formed, such as Fig. 4 E.
11. the post-processing of drop：Drop is detected by sensor 20, the information of drop is captured, judges whether it is effective Drop (is formed by antigen-antibody sandwich complex) containing target object to be checked.If drop is effective drop, pass through adding mouth 24 Chemiluminescent substrate is added with control valve 23.If being waste liquid in drop, without following the steps below, follow drop queue from going out Sample mouth 28 is discharged.
12. the reading of drop signal：Signal-obtaining detection module includes column structure region 26 and for detecting signal Sensor 27.After the completion of the reading of module progress chemiluminescence signal, signal-obtaining, drop enters 28 row of outlet Go out.
13. waste liquid is discharged：Outlet 28 connects waste liquid pool module.It is useless with other for collecting the drop of testing result Liquid.
Two, result：
Through the above steps.The micro-fluidic chip realizes the chemiluminescence detection of BNP, detection speed, testing result It is as shown in table 1 with required amount of antibody and the comparison of current main-stream detection method：
Table 1
From table 1 it follows that in terms of detection result, detection method based on micro-fluidic chip of the present invention and current Detection method is suitable, but in detection repeatability and antibody dosage, method of the invention has obviously advantage.Especially It is for single test, the antibody dosage needed for the method for the present invention declines to a great extent, and only 0.45-2.8pg is conventional method About the 1/100 to 1/1000 of dosage.
And it can be done directly the operation of cleaning in the micro-fluidic chip of the present invention, reduce additional manual operation, The integrated level for improving entire detection process simplifies the operation of detection process, and extraction process by the method for the invention, It is greatly improved accuracy of detection.
Embodiment 2：
Utilize double antibody sandwich method detection b-type natriuretic peptide (BNP)
In the present embodiment, using micro-fluidic chip shown in FIG. 1, human blood sample is carried out using double antibody sandwich method BNP is detected.
One, experimental method
1. and embodiment 1 is identical, carries out the pre-treatment of blood sample and reagent first：Human blood is taken in EDTA anticoagulant tubes Sample first, to prevent hemostasis-coagulation.
2. two kinds of antibody for double antibody sandwich method are handled：Fluorescent labeled antibody one and magnetic bead coupled antibody two.
3. sample sample introduction：Sample first, continuous phase sample introduction shown in Fig. 1 are added in dispersed phase injection port 6 shown in Fig. 1 Continuous phase mineral oil is added in mouth 1, control valve 5 is closed at this moment, and control valve 7 is opened, 28 He of outlet shown in Fig. 1 Waste liquid port 4 gives negative pressure as power source, and the design of injection port is as shown in Figure 2.
4. drop formation：Drop formation process is as shown in Figure 4 A, and mineral oil and sample are given birth under driving force effect in drop At interface 8, microlayer model is formed.
5. injection port cleans：After microlayer model is formed, injection port cleaning solution is added in closing control valve 7.Open control Valve 5 cleans injection port and waste liquid is quickly discharged.After completing injection port cleaning, closing control valve 5 is added again Cleaning solution is opened control valve 7, is cleaned to sample runner, cleaning solution can also form drop at 8, this partial drop quilt Sensor and control system identification, will not add reagent or carry out other processing, directly form waste liquid drop queue by going out sample Mouth 28 is discharged.The sample introduction that sample second starts new samples can be added after the completion of sample introduction module cleaning.
6. droplet counting and mark：The drop of generation is measured it by sensor 9 mark and drop number, with this It determines and generates whether drop is effective sample drop, and instruct the addition of follow-up reagent according to the mark and number.Following steps It is to be carried out for effective drop (sample), is such as detected as invalid drop (waste liquid), then follows drop queue directly in outlet 28 Discharge.
7. droplet coalescence：Sensor 10, reagent fusion mouth 11, reagent fusion injection port 12,13 and control valve 14 and its The pipeline of connection together constitutes reagent Fusion Module.It, can be as schemed institute when detecting the multiple markers in a kind of sample simultaneously Show that increasing reagent Fusion Module is added corresponding reagent.The antibody one of coating magnetic bead is added in the present embodiment in injection port 12, The antibody two of mark fluorescent is added in injection port 13, while under the response of sensor 10 and feedback, being controlled by control valve 14 Two kinds of antibody of system generate drop, and the one-to-one fusion of drop progress of the drop and package sample, fusion process is as shown in Figure 4 B, so Raising module is flowed to before backward.
8. sample incubation：Raising module is made of incubation pipeline 15 and extraneous temperature controller, which passes through drop Turbulent motion make the liquid in drop be sufficiently mixed and at a suitable temperature complete reaction.Process is as shown in Figure 4 C, right In the experiment refer to BNP fully reacts with antibody one and antibody two.Drop fully after reaction enters drop of testing liquid module.
9. drop of testing liquid：Drop of testing liquid module is divided into the input of drop of testing liquid room and drop cleaning/inorganic agent.Drop of testing liquid Room is made of the cavity 18 of a distribution column structure and the magnetic field 19 wrapped up, and entire cavity is in magnetic field 19.Cleaning The input of liquid is made of injection port 16 and valve 17 and cleaning solution drop formation mouth 12, which can increase number as needed Amount.Liquid drop movement to drop of testing liquid room, column structure can be such that liquid drop speed slows down, and magnetic bead is acted on by magnetic field in drop, Substance with magnetic bead retains in drop of testing liquid room, and it is anti-that cleaning microlayer model rinses out the indoor fluorescent marker BNP for having neither part nor lot in reaction Body two and other unbonded substances, such as Fig. 4 D.Be passed through inorganic agent at subsequent identical structure, inorganic agent can cut off magnetic bead and Contact between antibody one forms the new drop containing target object to be checked, such as Fig. 4 E.
10. signal-obtaining:Signal-obtaining detection module includes column structure region 26 and the sensing for detecting signal Device 27.Emit fluorescence in the excitation fluorescent material of the module suitable wavelength, photoexitation fluorescence sensor completes signal-obtaining Afterwards, drop enters the discharge of outlet 28.
11. waste liquid is discharged：Outlet 28 connects waste liquid pool module.It is useless with other for collecting the drop of testing result Liquid.
Two, result：
At present about there are many fluorescence detection methods of BNP, such as use magnetic bead+fluorescence, lateral flow chromatography and these sides The testing result of method compares, and the comparison of the detection result of the chip is as shown in table 2：
Table 2
In this table, it can be seen that in terms of detection result, the detection method based on micro-fluidic chip of the present invention and mesh Preceding detection method compared to effect it is suitable or close, but single detection duration and detection flux in terms of on, side of the present invention Method has a clear superiority.In addition to this, since it is drop homogeneous reaction, showed in terms of the stability of detection it is excellent, And the huge amplitude of amount of antibody needed for single detection reduces.For being tested especially for single, needed for the method for the present invention Antibody dosage declines to a great extent, only 0.45-2.8pg, is about the 1/100 to 1/1000 of conventional method dosage.And the present invention's Microlayer model chip and based on the operation that can be done directly cleaning in its detection method, reduces additional manual operation, improves The integrated level of entire detection process, simplifies the operation of detection process, and extraction process by the method for the invention, can be big It is big to improve accuracy of detection.
Embodiment 3
Utilize the making of the standard curve of double antibody sandwich method detection b-type natriuretic peptide (BNP)
In the present embodiment, for the double antibody sandwich method detection b-type natriuretic peptide (BNP) based on immune response, in difference Comparative result in extraction process, and standard curve is made to it.In addition, being based on testing result, cleaning performance is compared.
Method is as follows：First prepare a series of BNP calibration objects of concentration；It is clear in (a) extraction process using same calibration product Wash one times that microlayer model quantity is sample droplets；(b) extraction process cleaning microlayer model quantity is two times of feelings of sample droplets It under condition, is measured, the result of different extraction process is compared, and make standard curve respectively.
Shown in wherein comparing result such as Fig. 6, Fig. 7 and table 3.
Diagram 6 is the picture in the chip sample introduction and after completing experiment to the solution of the various concentration of BNP, wherein left side After the identical reagent of BNP antigens addition that three figures are 400pg/mL is reacted, extraction cleaning, extraction cleaning are not carried out once (one times of cleaning microlayer model) and the extraction cleaning baseline results picture that (two times of cleaning microlayer models) obtains twice.Three figure of right side is After the identical reagent of BNP solution addition of 0pg/mL is reacted, is not cleaned, extracts to clean and once be cleaned twice with extraction Obtained result picture.
Table 3 is the corresponding data obtained after picture is handled using image processing software.
Table 3
Fig. 7 is the visual analyzing result obtained using above-mentioned data.
By Fig. 6, Fig. 7 and table 3, in the case where not extracting, the BNP testing results of a concentration of 400pg/mL and 0pg/mL It differs minimum, target detection thing (BNP) can not be detected.In the case where extraction is primary, the BNP detections of 400pg/mL and 0pg/mL As a result difference is apparent, the difference bigger of result relative extractive that can obviously distinguish, and be extracted twice, therefore can reach more Big sensitivity.It can therefore be seen that if without extraction, it is unable to get reliable testing result, and being extracted twice can To there is higher sensitivity.
In fig. 8, the comparison of degree of cleaning in extraction process different (cleaning microlayer model number is different) is carried out with calibration object Detection, the testing result in the case of two kinds of comparison find the letter of when cleaning microlayer model negligible amounts (one times of sample droplets) Number testing result be slightly above clean microlayer model quantity it is more when (twice of sample droplets) signal detecting result, but pass through song Line fitting after cleaning microlayer model negligible amounts when (one times of sample droplets) R2When more much smaller than cleaning microlayer model quantity The R of (twice of sample droplets)2, show that degree of cleaning directly affects accuracy of detection, the removal degree of the substance of interferer signal detection Higher, detection sensitivity is also higher.Although extraction times are not The more the better, its process is indispensable, and the chip can be with Extraction times, the signal strength being optimal and signal-to-noise ratio are flexibly controlled as needed.To improve the detection of detected sample Sensitivity, repeatability and accuracy in detection.
It discusses
In the prior art, the micro-fluidic chip based on microlayer model is often complicated for operation, and accuracy is insufficient.For example, normal In rule method, substance usually to be reacted generates drop (such as the libraries addition IDE, target molecules and fluorescent enzyme substrate), then, All drops are collected after being incubated, and are sorted and (for example, sub-electing the drop of needs using FACS, such as contain aptamer Fluorescence drop), then ruptured, diluted and subregion encapsulates again in chip exterior (such as EP pipe).Later other one It is generated again on chip comprising the drop for waiting for reactant (such as sorted go out aptamer, target molecules and fluorescent enzyme substrate), so It collects after being incubated afterwards and is further detected.Whole process is complicated, and the function of needing cannot be completed on one single chip and is integrated, is needed Additional manual operation is carried out in chip exterior, the degree of automation is relatively low.
And correspondingly, at present in the method for existing in-vitro diagnosis, the conventional method for biological detection diagnosis has very much, Such as Western blot, chemiluminescence immunoassay detection method, enzyme-linked immunization (ELISA method) etc..These behaviour based on detection method Long analysis time is needed as mode, flux is smaller, and accuracy is insufficient, and equipment is huge.And in the drop core of the present invention In piece, a kind of new extraction module is on the one hand provided, and integrate to other function modules, so as in same chip On extraction process as described above is carried out to the drop of generation, removal has the substance of non-specific binding or Interference Detection, liquid The substance for signal detection is left behind in drop, whole process is more integrated and automates.Also, it on the same chip can be real Existing sample drop generation, the addition of required reagent, incubation and above-mentioned cleaning and extraction process and last detection process, are not necessarily to The operation bidirectional of chip exterior is carried out, whole process is that liquid phase reactor is fully reacted conducive to reaction system, and required reagent dosage is minimum, because This realizes the microfluidic chip analysis detection of the advantages such as high throughput, high accuracy, high sensitivity, low cost.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Claims (10)

1. a kind of micro-fluidic chip, which is characterized in that the micro-fluidic chip includes：
(a) micro-fluidic flow-through channel, the micro-fluidic flow-through channel are used for for entrained micro- in continuous phase and continuous phase Droplet flow；
(b) microlayer model generation module is reacted, the reaction microlayer model generation module is for generating the microlayer model reacted, institute The reaction microlayer model stated contains：Sample to be detected, the reaction reagent reacted with the sample to be detected, wherein described to be checked Substance to be detected in test sample sheet forms the detection product for carrying detectable marker after being reacted with reaction reagent；
(c) microlayer model generation module is cleaned, the cleaning microlayer model generation module is used to generate the cleaning solution cleaned micro- Drop；
(d) microlayer model generation module is sheared, the shearing microlayer model generation module is used to generate the shearing liquid sheared micro- Drop；
(e) capture-cleaning-shear module, also referred to as extracts module, and the capture-cleaning-shear module is used for from after incubation Reaction microlayer model in capture the detection product for carrying detectable marker, and with the cleaning solution microlayer model The captured detection product for carrying detectable marker is cleaned；And with the shearing microlayer model to through clear The detection product of carrying detectable marker after washing is sheared, to generate the microlayer model for detection；
(f) signal-obtaining detection module, the signal-obtaining detection module are used to carry out signal to the microlayer model for detection It reads；With
(g) drop control of queue module, the drop control of queue module and cleaning microlayer model generation module and the micro- liquid of shearing Drop generation module is connected, and for after reaction microlayer model is moved to precalculated position, controlling the cleaning microlayer model and generating The work of module and the shearing microlayer model generation module, thus in liquid flow direction shape in the micro-fluidic flow-through channel At the microlayer model queue of " reaction microlayer model-cleaning microlayer model-shearing microlayer model ", wherein reaction microlayer model be located at flowing most before Side.
2. micro-fluidic chip as described in claim 1, which is characterized in that the micro-fluidic flow-through channel includes following Section：Continuous phase sample introduction section, reaction reagent addition section, incubation reaction section, cleaning microlayer model addition section, is cut at detection sample feeding section Cut microlayer model addition section, capture-cleaning-shear treatment section, signal detection section and optional waste fluid channel section.
3. micro-fluidic chip as described in claim 1, which is characterized in that the capture-cleaning-shear module further includes magnetic Place, the field regions are the field regions of controllable open and close.
4. micro-fluidic chip as described in claim 1, which is characterized in that the cleaning microlayer model generation module and described Shearing microlayer model generation module further includes：Control valve component, the control valve component is by drop control of queue module control System.
5. micro-fluidic chip as described in claim 1, which is characterized in that the micro-fluidic chip further includes：(i) it is incubated control Molding block, the incubation control module are used to control the incubation parameter for the micro-fluidic flow-through channel for being incubated section.
6. micro-fluidic chip as described in claim 1, which is characterized in that the micro-fluidic chip further includes：(j) waste liquid is received Collect module, the waste collection module is for collecting the waste liquid generated in detection process.
7. micro-fluidic chip as described in claim 1, which is characterized in that the signal-obtaining detection module further includes post-processing Submodule, into the microlayer model for detection, addition is used for the substance of excitation-detection signal in the post-processing submodule.
8. a kind of detection method based on micro-fluidic chip as described in claim 1, which is characterized in that include the following steps：
A. mobile phase is added, and the mobile phase is made to be full of micro-fluidic flow-through channel；
B. reagent is pre-processed；
C. prepare the biological sample to be detected, the sample contains target substance to be detected；
D. sample and pretreated reagent is added, and sample microlayer model and examination are generated by the reaction microlayer model generation module Agent microlayer model, and it is fused into reaction microlayer model in the reaction microlayer model generation module；Or by sample and pretreated examination Agent is pre-mixed as reaction liquid, and the reaction liquid is added and passes through the reaction microlayer model generation module reaction of formation Microlayer model；
E. under the control of drop control of queue module, microlayer model generation module is cleaned, generates cleaning microlayer model；
F. under the control of drop control of queue module, microlayer model generation module is sheared, generates shearing microlayer model；
G. the microlayer model and reaction microlayer model that step e, step f are generated form microlayer model queue；
H. the microlayer model queue enters capture-cleaning-shear module, and forms the microlayer model for detection；
I. for the microlayer model of detection by signal-obtaining detection module, the reading detection module detects and reads signal.
9. detection method as claimed in claim 8, which is characterized in that in step b, the pretreatment to reagent be coating or Coupled bead.
10. detection method as claimed in claim 8, which is characterized in that the method is based on double antibody sandwich method or competing The detection method of striving property combined techniques.
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