CN105233892B - Magnetic microparticle chemiluminescence double layer micro fluidic chip for whole blood sample detection - Google Patents
Magnetic microparticle chemiluminescence double layer micro fluidic chip for whole blood sample detection Download PDFInfo
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- CN105233892B CN105233892B CN201510696707.5A CN201510696707A CN105233892B CN 105233892 B CN105233892 B CN 105233892B CN 201510696707 A CN201510696707 A CN 201510696707A CN 105233892 B CN105233892 B CN 105233892B
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Abstract
The present invention relates to a kind of magnetic microparticle chemiluminescence double layer micro fluidic chip for whole blood sample detection; the micro-fluidic chip includes top plate (1) and bottom plate (2), and wherein top plate includes adding mouth (4), sample fill area (12), tagged ligand storage pool (5) and sample mixed zone (13)；Bottom plate includes filtering area (6), magnetic particle coating area (7), cleaning area (14), detection zone (8), cleaning fluid storage pool (9), luminous substrate liquid storage pool (10), liquid release channel (16).
Description
Technical field
Realize that analyte is highly sensitive using magnetic microparticle chemiluminescence technology and micro-fluidic lamination technology the present invention relates to one kind
The chip quantitatively detected, a kind of magnetic microparticle chemiluminescence double layer micro fluidic chip for whole blood sample detection is particularly disclosed,
Biomedical research, clinical diagnosis, biochemistry detection, judicial expertise are can be applied to, belongs to the immune inspection of fluidic chip chemiluminescence
Survey technology field.
Background technology
At present, in-vitro diagnosis (IVD) mainly has two kinds of development trends：One kind is automation, integrated integration, i.e., using big
Full-automatic, the highly sensitive large-scale instrument and equipment of supporting central laboratory of type hospital, realize that high-precision diseases analysis is examined
It is disconnected；Another kind miniaturization, bed sideization, i.e., by the compact simplified equipment of palm, realize the quick analyzing and diagnosing in scene.Small hospital provides
Golden deficiency, sample size are few, are not appropriate for the expensive large scale equipment of purchasing price.Small-sized quick detection equipment is mainly tried at this stage
Paper slip and its corollary equipment, but test strips can only realize qualitative or half-quantitative detection, detection sensitivity is low, poor specificity, repetition
Property it is poor, be disturbed it is obvious.Because Chinese population is numerous, aging aggravation, the incidence of disease increases severely, can't bear by large hospital merely
Heavy burden.Therefore developing easy to operate, high sensitivity, reproducible and quantitative accurate quick determination method and equipment becomes extremely
Urgently.
Chemiluminescence refers to reaction intermediate, reaction product or additional luminescence reagent in chemical reaction process by chemical energy
It is changed into the phenomenon of luminous energy.Compared with fluorescence and absorbing light, chemiluminescence does not have external excitation source background signal to disturb, and intersects
Disturb small, have the advantages that high sensitivity, the range of linearity are wide.Thus the chemiluminescence analysis established is widely used to clinic and examined
The field such as disconnected.Chemiluminescence Apparatus is the main big type analysis detection devices of IVD.
Microfluidic chip technology is bases such as biology, chemistry, the sample preparation of medical analysis process, reaction, separation, detections
This operating unit is integrated on the chip of one piece of micro-meter scale, is automatically performed analysis overall process.Because it is in biology, chemistry, doctor
The great potential in etc. field, has been developed as the multi-crossed disciplines such as biology, chemistry, medical science, fluid, material, a machinery
Research field, be applied to the fields such as biomedical research, biochemistry detection, judicial expertise.Such as Chinese patent
201110006837.3 describe a kind of orientable micro-fluidic chip, and the micro-fluidic chip is by fence array layer, egative film and lid
Layer composition, available in vitro fertilization, detection Deiter's cells to neuron operation, structure neutral net and detection cell growth
State etc..
But the data and document that combine chemiluminescence and micro-fluidic chip are simultaneously few, it is practical and can industrialization more
It is few, as Chinese patent 200910114403.8 describes thing in a kind of fluidic chip chemiluminescence measure human single blood erythrocyte by mocro
The method of matter, its need to rely on microscope stage or lens and filter set into complex optical path；Chinese patent
200910154432.7 disclose the micro-fluidic chip of capillary electrophoresis separation and chemiluminescence detection, and its flow passage structure is single,
Sample introduction is not adequately mixed so as to cause reaction efficiency relatively low, is unable to reach maximum emission intensity.
Therefore prior art is primarily present following shortcoming：
1) existing fast diagnosis method main qualitative or the test strips of sxemiquantitative, its sensitivity is low, poor repeatability, is disturbed
Substantially.
2) design of existing chemiluminescence microfluidic chip structure is simple, complex operation during detection, detection time length, required examination
Agent is more to inject chip by external pressure.
3) existing its detecting system of chemiluminescence micro-fluidic chip relies on large scale equipment, such as microscope stage, biochip
Scanner etc..
In addition, based on micro-fluidic chip realize reaction and analysis, its basic process be it is pre-packaged in chip or from
Outside introduces one or more reaction reagents, liquid sample then is added into chip, sample flows according to microchannel set in advance
Road is contacted and reacted with reagent, and result is read out by instrument or naked eyes.The inspection of micro-fluidic chip main flow at present
Survey means include LIF, chemiluminescence and UV absorption etc..Precision of analysis is realized, it is most important micro-
What plurality of liquid wanted accurate quantitative analysis in passage reaches specified location according to passage set in advance within the specified time.It is but real
Border situation might not be preferable, and due to chip or sample, the reaction in micro-fluidic chip may not have according to setting
Fixed process is carried out, and such as producing bubble causes liquid flowing in chip to stop, or chip occurs seepage and causes liquid not fill out
Each pre- routing etc. is filled, these can all make to react incomplete between the reagent or substrate of predetermined reaction so that analysis result produces
Mistake.But because the reaction in chip is to be automatically performed, simply final result that experimenter or tester see lacks centering
Between process monitoring, the error analysis to result is so inevitably resulted in, so as to have impact on the accuracy of analysis.
The content of the invention
The technical problem to be solved in the present invention is for existing fast diagnosis method sensitivity is low, poor repeatability, is disturbed
Substantially, and existing chemiluminescence micro-fluidic chip necessary instrument is expensive, the problem of detection time is long, there is provided a kind of micro-fluidic magnetic
The detection of particulate chemistry electrochemiluminescent immunoassay integrated chip (in addition to test sample all components be integrated into chip) it is and supporting
Small portable device, so as to realize quick, accurate, the highly sensitive quantitative detection of analyte in field samples.
In order to solve the above technical problems, technical scheme provided by the invention is：
A kind of magnetic microparticle chemiluminescence double layer micro fluidic chip for whole blood sample detection, it is characterised in that described micro-
Fluidic chip includes top plate (1) and bottom plate (2), and wherein top plate includes adding mouth (4), sample fill area (12), tagged ligand and deposited
Reservoir (5) and sample mixed zone (13)；Bottom plate includes filtering area (6), magnetic particle coating area (7), cleaning area (14), detection zone
(8), cleaning fluid storage pool (9), luminous substrate liquid storage pool (10), liquid release channel (16)；
The adding mouth (4) and tagged ligand storage pool (5) are connected by sample fill area (12), tagged ligand storage pool
(5) it is connected with sample mixed zone (13)；The detection zone (8) of the bottom plate stores with cleaning fluid storage pool (9) and luminous substrate liquid
Pond (10) is connected by liquid release channel (16)；The filtering area (6), magnetic particle coating area (7), cleaning area (14), detection
Area (8) is sequentially connected；Sample mixed zone (13) end connects with filtering area (6)；
The tagged ligand storage pool (5), cleaning fluid storage pool (9), luminous substrate liquid storage pool (10) are hydraulic seal
Pond, it can be extruded by external force and partial fracture, discharge liquid；
The tagged ligand storage pool (5), cleaning fluid storage pool (9), luminous substrate liquid storage pool (10) and magnetic particle bag
By the pre-packaged reagent of storage in area (7)；The filtering area (6) includes hemofiltration film, and wherein hemofiltration film can pass through physical pore size or life
Thing/chemical reagent makes liquid be separated with cell, and the biology/chemical reagent includes coagulant etc.；
In the micro-fluidic chip testing process, the movement of magnetic particle or aggregation are manipulated with magnet.
It is mobile or poly- that the movement of micro-fluidic chip magnet manipulation magnetic particle or aggregation are divided into top magnet manipulation magnetic particle
Collection and two kinds of bottom magnet manipulation magnetic particle movement or aggregation.
The testing process of the micro-fluidic chip includes：
Whole blood sample is instilled adding mouth (4) by step 1), and sample flows into sample fill area (12), and close the lid (11), micro-
Fluidic chip is put into necessary instrument, and enzyme or luminous agent standard configuration body are after tagged ligand storage pool (5) release, sample and enzyme or hair
Photo etching standard configuration body is well mixed in sample mixed zone (13), is then injected into bottom plate filtering area (6)；
Behind the filtered area of step 2) sample (6), magnetic particle coating area (7) is reached, dissolves magnetic standard configuration body, magnet accelerates sample
Middle analyte and magnetic standard configuration precursor reactant, then magnet collection magnetic particle, cleaning fluid storage pool (9) release cleaning fluid, magnetic particle are clear
After washing, magnetic particle is moved to detection zone (8), luminous substrate liquid storage pool (9) release luminous substrate liquid, instrument detecting system by magnet
Luminous signal intensity is detected, and then realizes the quantitative detection of analyte.
The testing process step 1) sample volume of the micro-fluidic chip is 10~500 μ l, preferably 20~100 μ l.
Preferably, injection volume is 50 μ l in embodiment.
Testing process step 2) the magnetic particle size of the micro-fluidic chip is 0.1~10 μm, preferably 0.5~3 μm.
Wherein the magnetic induction intensity of magnetic particle size and magnet has obvious influence to testing result, to match with magnetic particle, magnet
Magnetic induction intensity is 500~30000 Gausses, preferably 1000~8000 Gausses.Preferably, 2 μ are used in one of embodiment
M magnetic particle, magnetic induction intensity are 6000 Gausses.0.5 μm of magnetic particle is used in another embodiment, magnetic induction intensity is
2000 Gausses.
Magnetic particle supperparamagnetic particles used in the present invention, comprising iron, cobalt, nickel compound, mainly including but not limited to three
Aoxidize two iron and ferroso-ferric oxide compound.Magnetic particle used is that polystyrene is shell in the embodiment of the present invention, di-iron trioxide
For the particle of core.
The testing process step 1) necessary instrument of the micro-fluidic chip is small portable device, includes extruding air pump
And storage pool, magnet move, the function such as luminescent detection system, after micro-fluidic chip is put into instrument, click starts to test, and instrument can
It is automatically performed all operations；Step 1) the air pump carries elastic rubber belt (23) sealing comprising micro-fluidic chip and forms air sky
Chamber, and necessary instrument provide positive and negative two kinds of pressure gas stream；The reaction of the step 2) analyte and part, includes analyte and enzyme
Labeling antibody and magnetic particle marker antibody form sandwich structure, and analyte competes with enzyme labelled antibody, reduces magnetic particle and enzyme
The combination of labeling antibody.
The preparation method of micro-fluidic chip of the present invention is as follows：
A kind of part that step 1) luminous agent mark can be combined or competed with analyte, obtains luminous agent tagged ligand, magnetic
Another part that particle marker can be combined or competed with analyte, magnetic particle marker part is obtained, both parts can be identical
It is or different；
Magnetic particle marker antibody-solutions are put into coating area by step 2), are dried, and then drip luminous agent label solution
Enter to be coated with area, dry, cleaning fluid and luminous exciting liquid are injected separately into cleaning fluid storage pool and luminous substrate liquid storage pool,
Sealing, assemble micro-fluidic chip.
Micro-flow control chip preparation method Zhang Suoshu of the present invention step 2) the tagged ligand solution, magnetic particle marker part
Solution and cleaning fluid include buffer solution, protein, surfactant and preservative, and magnetic particle marker antibody-solutions also include
Carbohydrate；Step 2) the luminous substrate liquid includes substrate corresponding with enzyme and luminescence enhancement liquid；Step 2) the luminous substrate liquid
It is removable to be divided into substrate solution and luminescence enhancement liquid, it is injected separately into luminous substrate liquid storage pool A (24), luminous substrate liquid storage pool B
(25) it is, well mixed by luminous substrate liquid pre-mixing passages (26) after release.
The micro-flow control chip preparation method Zhang Suoshu of the present invention step 1) enzyme includes catalase (HRP) and alkalescence
Phosphatase (ALP)；The luminous agent uses acridine including but not limited to acridinium ester and acridine sulfonamide, embodiments of the invention
Ester.Acridinium ester is with after the effect of luminous exciting liquid, being not required to the catalytic action of enzyme, directly participating in luminescence-producing reaction.The magnetic particle includes
Di-iron trioxide and ferroso-ferric oxide compound.
The direct chemiluminescence micro-fluidic chip of magnetic particle provided by the present invention for whole blood sample detection is one kind with straight
The novel chip of quick, accurate, the highly sensitive detection of analyte is realized based on connecing chemiluminescence, on micro-fluidic chip.
This chip is that the part (such as antigen, antibody) of analyte is modified into luminous agent, and another part of analyte is repaiied
Decorations are acted on, such as double antibody sandwich method principle combination magnetic particle rich, chemiluminescence detection whole blood on magnetic particle using part
Whether contain analyte in this.
The analyte that micro-fluidic chip of the present invention is used in quantitative whole blood sample.Analyte include medicine, drugs, antigen,
Antibody, hormone, antibiotic, bacterium and virus and other biochemical markers.Wherein other biochemical markers include cardiovascular indicate
Thing, tumor markers and autoimmune disease mark.
The direct chemiluminescence micro-fluidic chip of magnetic particle that the present invention is used for whole blood sample detection can solve the problem that existing chemistry
Luminescence technology Instrumental is expensive, the deficiency and defect of detection time length, and the detection to trace sample can be achieved.Due to chemiluminescence
High sensitivity, its sensitivity are more than 100 times of fluorescence detection method.
The labeling method that the present invention uses will be analyzed comprising specific effect between physical absorption, chemical crosslinking or biomolecule
The part of thing is connected to luminous agent or magnetic particle surface, obtains the luminous agent of ligand-labeled or the magnetic particle of ligand-labeled, wherein
Specifically bind or compete with physical efficiency and analyte.
Heretofore described physical absorption is mainly the surface charge difference by particle and part, and non-specific adsorption arrives
Particle surface, form the compound of part and magnetic particle.
Heretofore described chemical crosslinking is：, can be with part or Quality Control when the active group of magnetic particle or luminous agent surface
When molecule directly reacts, it is not required to use chemical cross-linking agent, otherwise with chemical cross-linking agent ligand modified to magnetic particle surface or luminous
In agent.
Use in an embodiment of the present invention chemical crosslink technique to magnetic particle carry out protein molecule modification method for：Profit
It is crosslinked with 1- ethyls -3- (3- DimethylAminopropyls) carbodiimides (EDC)/n-hydroxysuccinimide (NHS), glutaraldehyde etc.
Agent by the functional group on functional group's (such as carboxyl, amino) of magnetic particle surface and protein molecule (such as antigen, antibody) surface (such as
Amino, carboxyl, aldehyde radical etc.) connection.
Use in an embodiment of the present invention chemical crosslink technique to luminous agent carry out protein molecule modification method for：Profit
It is crosslinked with 1- ethyls -3- (3- DimethylAminopropyls) carbodiimides (EDC)/n-hydroxysuccinimide (NHS), glutaraldehyde etc.
Luminous agent is tagged on protein molecule (such as antigen, antibody) by agent.
Preferably, in one embodiment of the invention, magnetic particle is modified using EDC/NHS cross-linking methods, one
As step be：Magnetic particle solution is mixed with EDC and NHS, then adds a certain amount of protein, is situated between by reaction of buffer solution
Matter, 4h is cultivated, add the closing of L- glycine, purified in a manner of chromatogram, chromatographic column or ultrafiltration centrifugation etc., repaiied so as to obtain protein
The magnetic particle of decorations.
Specific effect includes biotin-avidin system and Ag-Ab system between heretofore described biomolecule.
Preferably, in another embodiment of the present invention, analyte ligand is entered using biotin-avidin system combination
Row magnetic particle modification, the combination have the function that amplified signal, are specially：Streptavidin is connected to by magnetic with EDC
Grain surface, biotin is connected to protein molecule surface, by the interaction between Avidin-Biotin, protein molecule
It is connected to magnetic particle surface.
The analyte ligand of the present invention includes antigen, haptens, monoclonal antibody, polyclonal antibody and hormone receptor.Should
Part can be combined (such as double antibody sandwich method) with analyte or compete (such as competition law) with analyte.Wherein luminous agent marks
Antibody can be with identical with the antibody of magnetic particle marker, can also be different.
Preferably, in one embodiment of the invention, selecting two kinds of different antibodies, luminous agent and magnetic are marked respectively
Grain, thing is tested and analyzed with double antibody sandwich method.In an alternative embodiment of the invention, a kind of antigen and a kind of antibody are selected, point
Not Biao Ji luminous agent and magnetic particle, with competition law test and analyze thing.
The tagged ligand solution of the present invention includes buffer solution, protein, glycerine, surfactant and preservative；Magnetic particle
Tagged ligand solution includes buffer solution, protein, carbohydrate, surfactant and preservative.Preferably, in the implementation of the present invention
In example, acridinium ester label antibody-solutions are to include bovine serum albumin(BSA), glycerine, triton x-100, polysorbas20 and Proclin300
PH7.4 phosphate buffers；Magnetic particle marker antibody-solutions be comprising bovine serum albumin(BSA), casein, polysorbas20 and
Proclin300 pH7.4 phosphate buffers.In another embodiment, acridinium ester label antibody-solutions are pure comprising ox blood
Albumen, glycerine, triton x-100 and Proclin300 pH7.4 Tris-HCl buffer solutions；Magnetic particle marker antigenic solution is
PH7.4 Tris-HCl buffer solutions comprising bovine serum albumin(BSA), casein, polysorbas20 and Proclin300.
The micro-fluidic chip of the present invention is as shown in Fig. 2 chip is made up of top adhesive tape, chip substrate and bottom tape.Into
Section bar material is polymer, including but not limited to polystyrene, polyvinyl chloride, polypropylene, epoxy resin etc..As shown in figure 1, chip
Substrate includes filtering area (2), coating area (3), cleaning area (4), detection zone (5), cleaning fluid storage pool (7) and luminous substrate liquid and deposited
Reservoir (8) and waste liquid pool (11) composition, wherein detection zone passes through liquid with cleaning fluid storage pool and luminous substrate liquid storage pool respectively
Body release channel (9) connects.
The storage pool of the present invention is hydraulic seal pond, and encapsulant used includes glass, plastics, rubber, aluminium foil and high resistant
Every film, wherein encapsulant can be that same material forms, or multiple material combines.Under physical impact, storage
Pond can partial fracture, so that the liquid of sealing is discharged.Wherein enzyme standard configuration body storage pool, cleaning fluid storage pool, luminous base
Bottom liquid storage pool can use identical or different material and method to make.In one embodiment of the invention, enzyme standard configuration body stores
Pond, cleaning fluid storage pool, luminous substrate liquid storage pool are sealed to form using plastics and elastic caoutchouc.Another reality of the present invention
Apply in example, enzyme standard configuration body storage pool is sealed to form using plastics and elastic caoutchouc, and cleaning fluid storage pool, luminous substrate liquid store
Pond is sealed to form using high-isolation film.
The filtering area of the present invention includes hemofiltration film, and wherein hemofiltration film can make liquid by physical pore size or biology/chemical reagent
Body separates with cell, realizes that blood plasma separates with red blood cell, and blood plasma flows to magnetic particle coating area, and red blood cell rests on hemofiltration film
On, so as to reduce interference of the red blood cell to result of the test.Wherein described biology/chemical reagent includes coagulant etc., can make red thin
Intercellular connects, and forms grumeleuse, increased in size, it is easier to is stopped by the network structure of hemofiltration film.
The micro-fluidic chip of the present invention, magnetic particle marker ligand solution and luminous agent tagged ligand solution can be mixed or first
The coating area (3) added afterwards on chip substrate, dry, as shown in Figure 1；When two kinds of solution have certain interference, or detection effect
When fruit is bad, magnetic particle coating area (14) and luminous agent tagged ligand coating area (15) can be separately added into.
The micro-fluidic chip of the present invention, when luminous substrate liquid due to can not mix and and the reason such as preserve for a long time, and need
When being split as two kinds of solution, it can be injected separately into luminous substrate liquid storage pool A (16) and luminous substrate liquid storage pool B (17), and
Increase luminous substrate liquid and luminescence enhancement liquid pre-mixing passages (18) on chip, the pre-mixing passages can by serpentine channel or on
Lower structure hybrid channel, as shown in Figure 4.
The cleaning fluid of the present invention, for cleaning magnetic particle, remove the analyte of non-specific adsorption, luminous agent label and
The material of other influences testing result.Cleaning fluid mainly includes buffer system, protein and surfactant, wherein buffer system
Including but not limited to borate, phosphate, Tris-HCl and acetate etc., cleaning fluid pH 6.0~10.0.Wherein protein bag
Contain but be not limited to bovine serum albumin(BSA), casein etc..Wherein surface-active including but not limited to may include polysorbas20, Tween 80,
Triton x-100, polyethylene glycol and PVP etc..
Preferably, in the embodiment of the present invention, using cleaning fluid be comprising bovine serum albumin(BSA), polysorbas20 and
Proclin300 pH7.0 phosphate buffers.
In one embodiment, magnetic particle coating area has been coated with magnetic particle marker antibody, luminous agent label coating area bag
By acridinium ester label antibody, Procalcitonin (PCT) is detected with double antibody sandwich method.In another embodiment, magnetic particle coating
Area has been coated with magnetic particle marker antibody, and luminous agent label coating area has been coated with acridinium ester label antigen, him is detected with competition law
Ke Mosi.
The micro-fluidic chip of the present invention is quick detection, and detection time should be less than 30 minutes, preferably, being adopted in embodiment
With 15 minutes.
The part instrument of the present invention includes the functions such as extruding storage pool, magnet movement, luminescent detection system, should can include and squeeze
Pressure device, magnet and mobile device, detecting system, control analysis module and software systems.
It the composite can be widely applied to pathogen, major disease (such as tumour, angiocardiopathy), illegal drug, drugs
The quantitative detection of the multiple analytes such as detection, food security.
The core of the present invention realizes object using magnetic microparticle chemiluminescence immunoassay technology in micro-fluidic chip
Quickly, high sensitivity, accurate quantitative analysis detection.
Microfluidic chip technology is bases such as biology, chemistry, the sample preparation of medical analysis process, reaction, separation, detections
This operating unit is integrated on the chip of one piece of micro-meter scale, is automatically performed analysis overall process.
The micro-fluidic chip of the present invention by all reagent components needed for detection process, (match somebody with somebody by enzyme standard configuration body, magnetic particle marker
Body, cleaning fluid, luminous substrate liquid etc.) integrate, be built into micro-fluidic chip, and designed by ingenious raceway groove, in supporting instrument
Under the operation of device, the operating in a key (need to only can be achieved with detecting by start button, without complex operations) of micro-fluidic chip is realized,
Whole blood separation, immune response, cleaning separation, chemiluminescence detection are realized, is set so as to avoid structure in existing micro-fluidic chip
Meter is simple, detection when complex operation the deficiencies of and defect.Serum or blood plasma inspection can only be carried out by also overcoming traditional chemical light-emitting appearance
The shortcomings that surveying, and whole blood sample can not being detected.
Because magnetic particle easily precipitates, traditional chemical light-emitting appearance uses manual mixing, and maintains magnetic particle with persistent oscillation
Suspended state, but the operation that is mixed of magnetic particle is difficult to realize in miniature portable instrument in micro-fluidic chip.
Magnetic particle is coated with, dried in micro-fluidic chip raceway groove by the present invention, and devises magnet active drive magnetic particle
(and traditional microfluidic chip is typically using fluid driving or electric drive), so that magnetic particle redissolves, and in micro-fluidic chip not
Immune response, cleaning are realized with region, are lighted.This design not only solve magnetic particle be applied to easily to precipitate during micro-fluidic chip,
The problems such as poor repeatability, more controllable immune response and physical cleaning are also achieved, improves sensitivity and repeatability.Wherein magnetic
Ferromagnetism and magnetic particle size significantly affect to Detection results, and select magnet magnetic induction intensity of the present invention is that 500-30000 is high
This, preferred 1000-8000 Gausses；Magnetic particle size is 0.1-10 μm, preferably 0.5-3 μm.
Micro-fluidic chip necessary instrument contacts with micro-fluidic chip no liquid in the present invention, no part for needing to clean, keeps away
Having exempted from traditional giant chemical light-emitting appearance needs stirring or sample-adding, cleaning etc. to operate and caused cross jamming and pollution.
So the present invention is not simple superposition magnetic microparticle chemiluminescence technology and microfluidic chip technology, but pass through liquid
Seal Design, raceway groove design, integrate all chemical constituents needed for detection, are built into micro-fluidic chip, and with magnet actively
Driving, realizes one-touch magnetic microparticle chemiluminescence immune detection, so as to realize analyte in whole blood in portable necessary instrument
Quick, high sensitivity, accurate quantitative analysis detection.
Main advantages of the present invention are as follows：
1) present invention uses direct chemiluminescence method, has the advantages of low background, high sensitivity, wide range of linearity.
2) present invention uses magnetic granule technology, has magnetic enrichment function, strengthens simultaneously amplified signal；And magnet can be utilized magnetic
Particle transport zone (such as by being coated with area-cleaning area-detection zone), reduces the influence of sample matrix.
3) present invention uses microfluidic chip technology, and sample is mixed, reacted, is separated and detection is integrated on chip, and
All reagent components needed for reaction are integrated on chip.
4) present invention is easy to operate, during detection, only need to add sample, close the lid, it is supporting that chip is put into miniature portable
In instrument.
5) necessary instrument of the present invention is miniature portable instrument, and instrument is only physically contacted with chip, and liquid is not in chip
With instrument contacts, instrument will not be polluted and produce cross jamming.
Brief description of the drawings
Fig. 1 is the micro-fluidic chip double-decker schematic diagram of top magnet manipulation, wherein 1 is top plate, 2 be bottom plate, and 3 be gas
Pump, 4 be adding mouth, and 5 be tagged ligand storage pool, and 6 be filtering area, and 7 be that magnetic particle is coated with area, and 8 be detection zone, and 9 be cleaning fluid
Storage pool, 10 be luminous substrate liquid storage pool, and 11 be lid, and 12 be sample fill area, and 13 be sample mixed zone, and 14 be cleaning
Area, 15 be waste liquid pool, and 16 be liquid release channel, and 17 be luminous substrate liquid and cleaning fluid storage pool resigning hole (in top plate), 18
For magnet slide rail resigning hole.
Fig. 2 is the structural representation of the complete micro-fluidic chip of the micro-fluidic chip of top magnet manipulation, wherein 1 is top
Plate, 2 be bottom plate, and 19 be two-sided tape, and 20 be one-faced tapes, and 21 be luminous substrate liquid and cleaning fluid storage pool resigning hole (in double
Face adhesive tape), 22 be resigning hole when mixed liquor flows into filtering area.
Fig. 3 is the micro-fluidic chip top board structure schematic diagram for carrying air pump, wherein 23 be diaphragm seal.
Fig. 4 is the micro-fluidic chip base arrangement schematic diagram of double luminous substrate liquid, wherein 24 be luminous substrate liquid storage pool
A, 25 be luminous substrate liquid storage pool B, and 26 be luminescent solution pre-mixing passages.
Fig. 5 is the micro-fluidic chip double-decker schematic diagram of bottom magnet manipulation, wherein 1 is top plate, 2 be bottom plate, and 3 be gas
Pump, 4 be adding mouth, and 5 be tagged ligand storage pool, and 6 be filtering area, and 7 be that magnetic particle is coated with area, and 8 be detection zone, and 9 be cleaning fluid
Storage pool, 10 be luminous substrate liquid storage pool, and 11 be lid, and 12 be sample fill area, and 13 be sample mixed zone, and 14 be cleaning
Area, 15 be waste liquid pool, and 16 be liquid release channel, and 27 be magnet sliding-rail groove.
Fig. 6 is the structural representation of the complete micro-fluidic chip of the micro-fluidic chip of bottom magnet manipulation, wherein 1 is top
Plate, 2 be bottom plate, and 19 be two-sided tape, and 20 be one-faced tapes, and 22 be resigning hole when mixed liquor flows into filtering area, and 28 be magnet
Slide rail resigning hole, 29 be luminous substrate liquid and cleaning fluid storage pool resigning hole (in one-faced tapes).
Embodiment
The invention discloses a kind of magnetic microparticle chemiluminescence double layer micro fluidic chip, those skilled in the art can use for reference this
Literary content, it is suitably modified technological parameter realization.In particular, all similar replacements and change are to art technology
It is it will be apparent that they are considered as being included in the present invention for personnel.The method of the present invention and application are by preferable
Embodiment is described, related personnel substantially can not depart from present invention, in spirit and scope to side as described herein
Method and application are modified or suitably changed with combining, to realize and using the technology of the present invention.
In order that those skilled in the art more fully understands technical scheme, with reference to specific embodiment pair
The present invention is described in further detail.
Embodiment 1：Double-antibody sandwich determines super quick Troponin I (cTnI)
(1) antibody labeling
Take 5 μ g HRP to be dissolved in 1mL distilled water, add 0.2mL0.1M and newly match somebody with somebody NaIO4Solution, the reaction of room temperature lucifuge
After 20min, with 1mM pH4.4 sodium-acetate buffer dialysis purification solution.PH is adjusted with 0.2M pH9.5 carbonate buffer solutions again
To 9.0, the anti-cTnI monoclonal antibodies of 10 μ g, room temperature lucifuge reaction 2h are added.Add the 4mg/mL NaBH that 0.1mL newly matches somebody with somebody4Liquid, mix, in 4
DEG C reaction 2h.Above-mentioned solution is loaded into bag filter, with 0.15M pH7.4 PBSs, 4 DEG C overnight, obtains HRP marks cTnI and resists
Body.
1mg magnetic particle (size is 2 μm), 10 μ g EDC and 15 μ g are added into 1ml 10mM pH7.4 phosphate buffers
NHS solution and the anti-cTnI monoclonal antibodies of 15 μ g (different from the antibody of HRP marks) solution, are well mixed and react 4h at room temperature, add
Enter the closing of 1mg glycine.It is enriched with magnet, removes unreacted anti-cTnI monoclonal antibodies, obtain magnetic particle marker cTnI antibody.
(2) micro-fluidic chip assembles
Containing 0.1% bovine serum albumin(BSA), 0.1% polysorbas20 and 0.01% in HRP mark cTnI antibody-solutions
Proclin300 pH7.4 phosphate buffers；Magnetic particle marker cTnI antibody-solutions be comprising 0.5% bovine serum albumin(BSA),
The pH7.4 phosphate buffers of 0.1% casein, 0.2% polysorbas20 and 0.01%Proclin300.
Cleaning fluid is the pH7.4 phosphate buffers comprising 0.2%BSA, 0.5% polysorbas20 and 0.01%Proclin300.
Luminous substrate liquid divides A liquid and B liquid, and A liquid is the acid solution containing luminol, and B liquid is the alkaline solution containing benzene derivative.
HRP labelled antibody solution is put into top plate enzyme standard configuration body storage pool (5), sealed.Magnetic particle marker antibody is molten
Liquid is put into bottom plate coating area (7), drying at room temperature.Cleaning fluid is injected in cleaning fluid storage pool (9), by the A of luminous substrate liquid
Liquid and B liquid are injected separately into luminous substrate liquid storage pool A (24) and luminous substrate liquid storage pool B (25), sealing.As shown in Figure 1, will
Hemofiltration film is glued in bottom plate filtering area, and storage pool is built into bottom plate.Then as shown in Figure 2, with one-faced tapes and two-sided tape,
Top plate and bottom plate are assembled into micro-fluidic chip.It is fitted into aluminium foil bag, seals 4 ° of preservations.
(3) pattern detection
Make dilution with human normal plasma, cTnI standard items are diluted to following concentration：0pg/ml、5pg/ml、50pg/
Ml, 500pg/ml, 1ng/ml, 10ng/ml and 50ng/ml.
After 50 μ l samples are instilled into adding mouth, close the lid.Micro-fluidic chip is put into necessary instrument, and (magnet magnetic induction is strong
Spend for 6000 Gausses) in, instrument extrusion HRP mark monoclonal antibodies, and make sample and HRP mark monoclonal antibody to inject bottom plate mistake after being well mixed
Filter area.After sample filtering, microchannel is reached, and dissolves magnetic particle marker monoclonal antibody, magnet accelerates sample reaction, forms HRP marks
The sandwich structure of monoclonal antibody-cTnI antigens-magnetic particle marker monoclonal antibody, then magnet collect magnetic particle.Storage pool discharges cleaning fluid,
After magnetic particle cleaning, the release of luminous substrate liquid, instrument detecting system detection luminous signal intensity.Total detection time 15min.Often
Individual sample is determined 3 times with 3 micro-fluidic chips respectively, is averaged, and draws standard curve.
50 μ l whole blood samples are instilled into adding mouth, instrument detecting system detects luminous signal intensity in 15 minutes, according to mark
Directrix curve obtains cTnI concentration in sample.
Cleaning Principle is：After whole blood adds micro-fluidic chip, whole blood first mixes with HRP labelled antibodies, then filtered
Qu Hou, the blood plasma for being mixed with HRP labelled antibodies reach microchannel, blood plasma dissolving magnetic marker antibody.When containing cTnI in blood sample, then
Form the sandwich structure (double antibody sandwich method) of HRP labelled antibody-cTnI- magnetic particle marker antibody.It is once purged, then light
The effect of substrate liquid is lower luminous, instrument detecting system test luminous signal.The standard curve obtained according to necessary instrument, and then analyze
CTnI concentration in blood sample.CTnI contents are higher in sample, then luminous signal is stronger.
As a result showing, its lowest detection is limited to 10pg/ml, minimum to be quantitatively limited to 50pg/ml, in quantitative detection range,
Coefficient R2＞ 0.99, does not occur HOOK effects, and batch in batch between it is repeated preferable, can be heart infarction heart failure medical diagnosis on disease
Reference is provided.
Embodiment 2：Competition law determines tacrolimus blood concentration
(1) antibody/antigen marks
Weigh ALP 0.1mg to be dissolved in the pH6.8 0.1M PBS solutions of the glutaraldehydes of 0.1ml 12.5%, ambient temperature overnight.Instead
Enzyme solutions after answering are eluted with physiological saline through SephadexG-25 chromatographic columns, collect brown liquid.22.5 μ g are resisted him under stirring
Ke Mosi antibody is added dropwise in enzyme solutions, reacts 3h.Add 0.2M lysines to close, after mixing, put room temperature 2h.Under agitation by
Isometric saturated ammonium sulfate is added dropwise to, puts 4 DEG C of 1h.3000rpm/min centrifuges 30min, abandons supernatant.Sediment semi-saturation sulfuric acid
Ammonium is washed secondary, and last sediment is dissolved in 0.15M pH7.4 PBS.Above-mentioned solution is fitted into bag filter, to 0.15M pH7.4
PBS is dialysed, and after removing ammonium ion, except precipitation, supernatant is that ALP marks anti-tacrolimus for 10000rpm/min centrifugations
Antibody.
1mg magnetic particle (size is 0.5 μm), 10 μ g EDC and 15 μ g are added into 1ml 10mM pH7.4 phosphate buffers
NHS solution and 20 μ g Streptavidins, it is well mixed and reacts 4h at room temperature, adds the closing of 1mg glycine.With magnet adsorption
Enrichment, removes unreacted Streptavidin, obtains magnetic particle marker Streptavidin.
10 μ g tacrolimus antigens are added in 5 μ L 0.25mg/mL Sulfo-NHS-LC-biotin solution, react 1h.
Purified with ultra-filtration centrifuge tube, remove unreacted biotin.Obtain biotinylated antigen.
By the interaction between Avidin-Biotin, antigen is connected to magnetic particle surface.Wherein Avidin marks
Magnetic particle and biotinylated antibody ratios are 1: 104。
(2) micro-fluidic chip assembles
ALP is marked in anti-tacrolimus antibody-solutions containing 0.1% bovine serum albumin(BSA), polysorbas20 and Proclin300
PH7.4 Tris-HCl buffer solutions；Magnetic particle marker ligand solution include 0.5% bovine serum albumin(BSA), 0.2% casein,
The pH7.4 Tris-HCl buffer solutions of 0.1% polysorbas20 and 0.02%Proclin300.
Cleaning fluid is to include 0.2%BSA, 0.5% polysorbas20,0.5% triton x-100 and 0.01%Proclin300
PH7.4Tris-HCl buffer solutions.Luminous substrate liquid.Luminous substrate liquid divides A liquid and B liquid, and A liquid is the acid solution containing adamantane, B
Liquid is alkaline solution.
ALP labelled antibody solution is put into top plate enzyme standard configuration body storage pool (5), sealed.The magnetic that tacrolimus is marked
Particle solution is put into bottom plate coating area (7), drying at room temperature.Cleaning fluid is injected in cleaning fluid storage pool (9), by luminous substrate
The A liquid and B liquid of liquid are injected separately into luminous substrate liquid storage pool A (24) and luminous substrate liquid storage pool B (25), sealing.By Fig. 1 institutes
Show, hemofiltration film is glued in bottom plate filtering area, storage pool is built into bottom plate.Then as shown in Figure 2, with one-faced tapes and two-sided
Adhesive tape, top plate and bottom plate are assembled into micro-fluidic chip.It is fitted into aluminium foil bag, seals 4 ° of preservations.
(3) pattern detection
Make dilution with phosphate buffer, it is as follows that tacrolimus standard items are prepared into series concentration standard solution：0ng/
mL、0.1ng/mL、1ng/mL、5ng/mL、10ng/mL、50ng/mL、100ng/mL。
After 50 μ l samples are instilled into adding mouth, close the lid.Micro-fluidic chip is put into necessary instrument, and (magnet magnetic induction is strong
Spend for 2000 Gausses) in, instrument extrusion ALP labelled antibodies, and make to inject bottom plate mistake after sample and ALP labelled antibodies are well mixed
Filter in area.Sample filtering after, reach microchannel, and dissolve tacrolimus mark magnetic particle, magnet accelerate sample reaction, he gram
The magnetic particle competition binding ALP labelled antibodies with tacrolimus mark are not taken charge of, then magnet collects magnetic particle.Storage pool release is clear
Washing lotion, after magnetic particle cleaning, the release of luminous substrate liquid, instrument detecting system detection luminous signal intensity.Total detection time
15min.Each sample is determined 3 times with 3 micro-fluidic chips respectively, is averaged, and draws standard curve.
50 μ l samples are instilled into adding mouth, instrument detecting system detects luminous signal intensity in 15 minutes, and establishing criteria is bent
Line obtains tacrolimus concentration in sample.
Cleaning Principle is：After sample adds micro-fluidic chip, sample is well mixed with ALP labelled antibodies, filtered area
Afterwards, mixture reaches microchannel, the magnetic particle of sample dissolving tacrolimus mark.When containing tacrolimus in sample, then he gram
Not department and the magnetic particle competition binding ALP labelled antibodies (competition law) of tacrolimus mark.It is once purged, then luminous substrate liquid work
With lower luminous, instrument detecting system test luminous signal.According to necessary instrument obtain standard curve, and then analyze sample in he
Ke Mosi concentration.Tacrolimus content is higher in sample, then luminous signal is weaker.
As a result show, tacrolimus lowest detection is limited to 0.05ng/mL, minimum to be quantitatively limited to 0.1ng/mL, detection range
Interior linearly dependent coefficient R2＞ 0.98；In quantitative detection range, do not occur HOOK effects；And batch in batch between repeatability it is equal
Less than 10%, detected available for tacrolimus blood concentration.
Embodiment 3：Magnetic particle particle size is screened
Other experiment conditions are carried out referring to embodiment 1, magnetic particle size and magnet magnetic induction intensity according to following scheme.
Particle size is 0.1 μm, 0.5 μm, 1.0 μm, 3.0 μm, 3.4 μm, 5 μm, 10 μm.Magnet magnetic induction intensity is 500
Gauss, 1000 Gausses, 4000 Gausses, 8000 Gausses, 12000 Gausses, 30000 Gausses.Driven respectively with this six kinds of magnet respectively
The magnetic particle of seven kinds of sizes.
Experimental result is shown：When 0.1 μm of magnetic particle and 500 Gauss magnet combine, its lowest detection is limited to 500pg/ml, fixed
Amount detection range is 0.5~50ng/ml, linearly dependent coefficient R2＞ 0.95；Batch in batch between repeatability respectively less than 20%.I.e.：
Chemiluminescence signal is weaker, and sensitivity is not high, and repeatability is poor.
When 10 μm of magnetic particles and 30000 Gauss magnet combine, its lowest detection is limited to 300pg/ml, and quantitative detection range is
0.1~5ng/ml, linearly dependent coefficient R2＞ 0.95；Batch in batch between repeatability respectively less than 20%.I.e.：Negative sample signal compared with
High (cleaning is insufficient), the range of linearity is not wide.
0.5~3 μm of magnetic particle is and the magnet of 1000~8000 Gausses is combined in use, its minimum detection limit is respectively less than
150pg/ml, quantitative detection range can reach 0.15~50ng/ml, linearly dependent coefficient R2＞ 0.97；Repeated in batch and between criticizing
Property is respectively less than 12%.Meet the needs that reference is provided for clinical heart infarction heart failure medical diagnosis on disease.
According to result above, preferably 0.5~3 μm of magnetic particle size, magnet magnetic induction intensity preferably 1000~8000 Gausses.
Can according to used in magnetic particle size, further determine that magnet magnetic induction intensity.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
- A kind of 1. magnetic microparticle chemiluminescence double layer micro fluidic chip for whole blood sample detection, it is characterised in that the miniflow Control chip includes top plate (1) and bottom plate (2), and wherein top plate includes adding mouth (4), sample fill area (12), tagged ligand storage Pond (5) and sample mixed zone (13)；Bottom plate include filtering area (6), magnetic particle coating area (7), cleaning area (14), detection zone (8), Cleaning fluid storage pool (9), luminous substrate liquid storage pool (10), liquid release channel (16)；The adding mouth (4) and tagged ligand storage pool (5) are connected by sample fill area (12), tagged ligand storage pool (5) with Sample mixed zone (13) connects；The detection zone (8) of the bottom plate and cleaning fluid storage pool (9) and luminous substrate liquid storage pool (10) Connected by liquid release channel (16)；The filtering area (6), magnetic particle coating area (7), cleaning area (14), detection zone (8) according to Secondary connection；Sample mixed zone (13) end connects with filtering area (6)；The tagged ligand storage pool (5), cleaning fluid storage pool (9), luminous substrate liquid storage pool (10) are hydraulic seal pond, can Extruded by external force and partial fracture, discharge liquid；The tagged ligand storage pool (5), cleaning fluid storage pool (9), luminous substrate liquid storage pool (10) and magnetic particle coating area (7) the pre-packaged reagent of storage in；The filtering area (6) includes hemofiltration film, and wherein hemofiltration film can pass through physical pore size or biology/change Learning reagent makes liquid be separated with cell, and the biology/chemical reagent includes coagulant；In the micro-fluidic chip testing process, the movement of magnetic particle or aggregation are manipulated with magnet.
- 2. micro-fluidic chip as claimed in claim 1, it is characterised in that the micro-fluidic chip magnet manipulation magnetic particle movement Or aggregation is divided into top magnet and manipulates mobile magnetic particle or aggregation and two kinds of bottom magnet manipulation magnetic particle movement or aggregation.
- 3. micro-fluidic chip as claimed in claim 1, it is characterised in that the testing process of the micro-fluidic chip includes：Whole blood sample is instilled adding mouth (4) by step 1), and sample flows into sample fill area (12), and close the lid (11), micro-fluidic Chip is put into necessary instrument, and enzyme or luminous agent standard configuration body are after tagged ligand storage pool (5) release, sample and enzyme or luminous agent Standard configuration body is well mixed in sample mixed zone (13), is then injected into bottom plate filtering area (6)；Behind the filtered area of step 2) sample (6), magnetic particle coating area (7) is reached, dissolves magnetic standard configuration body, magnet accelerates to divide in sample Thing and magnetic standard configuration precursor reactant are analysed, then magnet collects magnetic particle, cleaning fluid storage pool (9) release cleaning fluid, after magnetic particle cleaning, Magnetic particle is moved to detection zone (8), luminous substrate liquid storage pool (10) release luminous substrate liquid, the detection of instrument detecting system by magnet Luminous signal intensity, and then realize the quantitative detection of analyte.
- 4. micro-fluidic chip as claimed in claim 3, it is characterised in that the step 1) sample volume is 10~500 μ l.
- 5. micro-fluidic chip as claimed in claim 3, it is characterised in that step 2) the magnetic particle size is 0.1~10 μm； The magnetic induction intensity of the step 2) magnet is 500~30000 Gausses.
- 6. micro-fluidic chip as claimed in claim 3, it is characterised in that step 2) the magnetic particle size is 0.5~3 μm； The magnetic induction intensity of the step 2) magnet is 1000~8000 Gausses.
- 7. the micro-fluidic chip as described in claim any one of 3-6, it is characterised in that the step 1) necessary instrument is small-sized Portable equipment, comprising extruding air pump and storage pool, magnet moves, luminescent detection system function, after micro-fluidic chip is put into instrument, Click starts to test, and instrument can be automatically performed all operations；Step 1) the air pump carries elastic rubber belt comprising micro-fluidic chip (23) sealing forms air cavity, and necessary instrument provides positive and negative two kinds of pressure gas stream；Step 2) the analyte and magnetic standard configuration Precursor reactant, sandwich structure is formed comprising analyte and enzyme labelled antibody and magnetic particle marker antibody, and analyte resists with enzyme mark Body competes, and reduces the combination of magnetic particle and enzyme labelled antibody.
- 8. micro-fluidic chip as claimed in claim 1, it is characterised in that the micro-flow control chip preparation method is as follows：Prepared by step 1) tagged ligand, the tagged ligand includes two kinds of tagged ligands, and a kind of part is that enzyme or luminous agent mark Part, another part are magnetic particle marker part；Described two parts are identical or different；Enzyme or luminous agent standard configuration liquid solution are put into the tagged ligand storage pool of top plate by step 2), sealing, by magnetic particle marker Ligand solution is put into the magnetic particle coating area of bottom plate, is dried, and cleaning fluid and luminous substrate liquid are injected separately into cleaning fluid storage In pond and luminous substrate liquid storage pool, sealing, top plate and bottom plate are sealed with adhesive tape (19 and 20), and be assembled into micro-fluidic chip.
- 9. micro-fluidic chip as claimed in claim 8, it is characterised in that step 2) the tagged ligand solution, magnetic particle mark Note ligand solution and cleaning fluid include buffer solution, protein, surfactant and preservative, and magnetic particle marker antibody-solutions Also include carbohydrate；Step 2) the luminous substrate liquid includes substrate corresponding with enzyme and luminescence enhancement liquid；Step 2) is described luminous Substrate liquid is removable to be divided into substrate solution and luminescence enhancement liquid, is injected separately into luminous substrate liquid storage pool A (24), the storage of luminous substrate liquid Pond B (25), it is well mixed by luminous substrate liquid pre-mixing passages (26) after release.
- 10. micro-fluidic chip as claimed in claim 8, it is characterised in that the step 1) enzyme includes catalase (HRP) With alkaline phosphatase (ALP)；The luminous agent includes acridinium ester and acridine sulfonamide；The magnetic particle include di-iron trioxide and Ferroso-ferric oxide compound.
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