CN105259163B - The direct chemiluminescence micro-fluidic chip of magnetic particle for whole blood sample detection - Google Patents
The direct chemiluminescence micro-fluidic chip of magnetic particle for whole blood sample detection Download PDFInfo
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- CN105259163B CN105259163B CN201510696709.4A CN201510696709A CN105259163B CN 105259163 B CN105259163 B CN 105259163B CN 201510696709 A CN201510696709 A CN 201510696709A CN 105259163 B CN105259163 B CN 105259163B
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Abstract
The present invention relates to a kind of direct chemiluminescence micro-fluidic chip of magnetic particle for whole blood sample detection, the chip is made of top adhesive tape (10), chip substrate (1) and bottom tape (13)；The chip substrate includes filtering area (2), coating area (3), cleaning area (4), detection zone (5), cleaning solution storage pool (7) and luminous substrate liquid storage pool (8) and waste liquid pool (11) composition, and wherein detection zone is connected with cleaning solution storage pool and luminous substrate liquid storage pool by liquid release channel (9) respectively；Top adhesive tape includes adding mouth (11) and cleaning solution storage pool relief hole (12).
Description
Technical field
The present invention relates to one kind analyte height is realized using the direct chemiluminescence of magnetic particle and microfluidic chip technology
The sensitive chip quantitatively detected, particularly discloses a kind of micro-fluidic core of the direct chemiluminescence of magnetic particle for whole blood sample detection
Piece, can be applied to biomedical research, clinical diagnosis, biochemistry detection, judicial expertise, belong to fluidic chip chemiluminescence 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, realizes 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 large scale equipment of purchasing price costliness.Quick detection device small-sized at this stage mainly tries
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.Since Chinese population is numerous, aging aggravation, incidence 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 examines
The field such as disconnected.Chemiluminescence Apparatus is the main large-scale analysis and testing equipments 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.Since it is in biology, chemistry, doctor
The great potential in etc. field, has been developed as the multi-crossed disciplines such as biology, chemistry, medicine, 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, 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 the 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, complicated, detection time length during detection, 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..
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, a kind of the problem of and existing chemiluminescence micro-fluidic chip necessary instrument is expensive, detection time is long, there is provided 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 fulfill quick, accurate, the highly sensitive quantitative detection of analyte in field samples.
In order to solve the above technical problems, technical solution provided by the invention is：
A kind of direct chemiluminescence micro-fluidic chip of magnetic particle for whole blood sample detection, it is characterised in that the core
Piece is made of top adhesive tape 10, chip substrate 1 and bottom tape 13；Filtering area 2, coating on the chip substrate 1
Area 3, cleaning area 4, detection zone 5, liquid release channel 6 are sequentially connected, and detection zone 5 is deposited by liquid release channel 6 with cleaning solution
Reservoir 7 and luminous substrate liquid storage pool 8 connect；Top adhesive tape includes adding mouth 11 and cleaning solution storage pool relief hole 12；It is described
Coating area 3 is coated with pre-packaged magnetic particle tagged ligand and enzyme or luminous agent tagged ligand；Cleaning solution storage pool 7 and luminous base
Bottom liquid storage pool 8 stores pre-packaged cleaning solution and luminous substrate liquid.Top adhesive tape includes adding mouth 11 and cleaning solution storage pool allows
Position hole 12.
Specifically, the luminous substrate liquid storage pool 8 is by luminous substrate liquid storage pool A16 and luminous substrate liquid storage pool
B17 is substituted, and luminous substrate liquid storage pool A16 and luminous substrate liquid storage pool B17 are connected by luminescent solution pre-mixing passages 18.
Specifically, the luminous substrate liquid includes acid solution and alkaline solution, can merge the liquid storage of injection luminous substrate
Pond (8), can also be injected separately into luminous substrate liquid storage pool A16, luminous substrate liquid storage pool B17, pass through luminous substrate after release
Liquid pre-mixing passages 18 are uniformly mixed.
Specifically, cleaning solution storage pool 7 and luminous substrate the liquid storage pool 8 is hydraulic seal pond, can be squeezed by external force
Press and partial fracture, discharge liquid；The filtering area includes hemofiltration film.
Specifically, the magnetic particle tagged ligand and luminous agent tagged ligand can all be put into coating area 3, also can be by magnetic particle
Tagged ligand and luminous agent tagged ligand are respectively put into magnetic particle coating area 14 and luminous agent tagged ligand coating area 15.
Specifically, the luminous agent tagged ligand solution includes buffer solution, protein, glycerine, surfactant and anti-corrosion
Agent, magnetic particle marker ligand solution include buffer solution, protein, carbohydrate, surfactant and preservative, and cleaning solution includes buffering
Liquid, protein, surfactant and preservative.
Specifically, the luminous agent that luminous agent tagged ligand of the present invention uses includes acridinium ester and acridine sulfonamide；Institute
It is supperparamagnetic particles to state the magnetic particle that magnetic particle tagged ligand uses, and is made of iron, cobalt or nickel compound, and magnetic particle size is
0.1~10 μm, magnet magnetic induction intensity is 500~10000 Gausses.
Preferably, the magnetic particle is supperparamagnetic particles, is made of iron, cobalt or nickel compound, and magnetic particle size is 0.5
~3 μm, magnet magnetic induction intensity is 1000~8000 Gausses.
Specifically, sample volume of the invention is in 10~500 μ l, preferably 20~100 μ l.
The preparation method of micro-fluidic chip of the present invention is as follows：
A kind of ligand that step 1) luminous agent mark can be combined or competed with analyte, obtains luminous agent tagged ligand, magnetic
Another ligand that particle marker can be combined or competed with analyte, obtains magnetic particle tagged ligand, both ligands can be identical
It is or different；
Magnetic particle labelled antibody solution is put into coating area by step 2), dry, then drips luminous agent label solution
Enter to be coated with area, it is dry, cleaning solution and luminous exciting liquid are injected separately into cleaning solution storage pool and luminous substrate liquid storage pool,
Sealing, assembles micro-fluidic chip.
Micro-fluidic chip testing process includes：
Micro-fluidic chip is put into necessary instrument by step 1), after sample is instilled adding mouth 11, starts to test, and sample passes through
After filtering area, coating area is reached, dissolves magnetic particle and luminous agent label, fully magnet collects magnetic particle after reaction；
Step 2) cleaning solution storage pool discharges cleaning solution, after magnetic particle cleaning, moves to detection zone, discharges the exciting liquid that shines,
Instrument detecting system detects luminous signal intensity, and then realizes the quantitative detection of analyte.
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 ligand (such as antigen, antibody) of analyte is modified luminous agent, and another ligand of analyte is repaiied
Decorations are acted on magnetic particle using ligand, such as the enrichment of double antibody sandwich method principle combination magnetic particle, chemiluminescence detection whole blood
Whether contain analyte in this.
Micro-fluidic chip of the present invention is used for the analyte 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 marker.
The direct chemiluminescence micro-fluidic chip of magnetic particle that the present invention is used for whole blood sample detection can solve existing chemistry
Luminescence technology Instrumental is expensive, the deficiency of detection time length and defect are, it can be achieved that detection to trace sample.Due to chemiluminescence
High sensitivity, its sensitivity are more than 100 times of fluorescence detection method.
Luminous agent of the present invention, including but not limited to acridinium ester and acridine sulfonamide, the embodiment of the present invention uses a word used for translation
Pyridine 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 labeling method that the present invention uses will be analyzed comprising specific effect between physical absorption, chemical crosslinking or biomolecule
The ligand 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 ligand, and non-specific adsorption arrives
Particle surface, forms the compound of ligand and magnetic particle.
Heretofore described chemical crosslinking is：, can be with ligand 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) on 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, using buffer solution as anti-matter, training
4h is educated, adds the closing of L- glycine, is purified in a manner of chromatography, chromatographic column or ultrafiltration centrifugation etc., so as to obtain protein modification
Magnetic particle.
Specific effect includes biotin-avidin system and Ag-Ab system between biomolecule described in bright.As
It is preferred that in another embodiment of the present invention, magnetic is carried out to analyte ligand using biotin-avidin system combination
Particulate is modified, which has the function that amplified signal, is specially：Streptavidin is connected to by magnetic particle table with EDC
Face, biotin are connected to protein molecule surface, and by the interaction between Avidin-Biotin, protein molecule is connected
To magnetic particle surface.
The analyte ligand of the present invention includes antigen, haptens, monoclonal antibody, polyclonal antibody and hormone receptor.Should
Ligand can be combined (such as double antibody sandwich method) with analyte or compete (such as competition law) with analyte.Wherein luminous agent marks
The antibody that antibody is marked with magnetic particle may be the same or different.
Preferably, in one embodiment of the invention, selecting two kinds of different antibodies, mark luminous agent and magnetic micro- 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 labelled antibody solution be comprising bovine serum albumin(BSA), casein, polysorbas20 and
The pH7.4 phosphate buffers of Proclin300.In another embodiment, acridinium ester label antibody-solutions are pure comprising ox blood
Albumen, glycerine, the pH7.4Tris-HCl buffer solutions of triton x-100 and Proclin300；Magnetic particle marker antigenic solution is bag
PH7.4Tris-HCl buffer solutions containing 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 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 solution storage pool 7 and luminous substrate liquid storage pool 8 and gives up
Liquid pool 11 forms, and wherein detection zone is connected with cleaning solution storage pool and luminous substrate liquid storage pool by liquid release channel 9 respectively
Connect.
The storage pool of the present invention is hydraulic seal pond, and sealing material used includes glass, plastics, rubber, aluminium foil and high resistant
Every film, wherein sealing material can be that same material forms, or multiple material is composed.Under physical impact, storage
Pond can partial fracture, so that the liquid of sealing is discharged.Wherein enzyme standard configuration body storage pool, cleaning solution 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 solution storage pool, luminous substrate liquid storage pool are sealed to form using plastics and elastic rubber.Another reality of the present invention
Apply in example, enzyme standard configuration body storage pool is sealed to form using plastics and elastic rubber, and cleaning solution 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 is separated with cell, realizes that blood plasma is separated 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 net structure of hemofiltration film.
The micro-fluidic chip of the present invention, can mix magnetic particle tagged ligand solution and luminous agent tagged ligand solution or first
The coating area 3 on chip substrate is added afterwards, it is dry, as shown in Figure 1；When two kinds of solution, there are certain interference, or detection result
When 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 cannot 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 A16 and luminous substrate liquid storage pool B17, and in core
On piece increases luminous substrate liquid and luminescence enhancement liquid pre-mixing passages 18, the pre-mixing passages can be by serpentine channel or up-down structures
Hybrid channel, as shown in Figure 4.
The cleaning solution of the present invention, for cleaning magnetic particle, removes analyte, the luminous agent label of non-specific adsorption
And the material of _ other influences testing result.Cleaning solution mainly includes buffer system, protein and surfactant, wherein buffering
System is including but not limited to borate, phosphate, Tris-HCl and acetate etc., cleaning solution pH 6.0~10.0.Wherein albumen
Matter is including but not limited to bovine serum albumin(BSA), casein etc..Wherein surface-active is including but not limited to may include polysorbas20, tween
80th, triton x-100, polyethylene glycol and polyvinylpyrrolidone etc..
Preferably, in the embodiment of the present invention, using cleaning solution be comprising bovine serum albumin(BSA), polysorbas20 and
The pH7.0 phosphate buffers of Proclin300.
In one embodiment, magnetic particle coating area has been coated with magnetic particle labelled 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 labelled 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 ligand 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 mark
Body, cleaning solution, 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,
Realize whole blood separation, immune response, cleaning separation, chemiluminescence detection, set so as to avoid structure in existing micro-fluidic chip
Meter is simple, detection when it is complicated 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 cannot being detected.
Since magnetic particle easily precipitates, traditional chemical light-emitting appearance uses manual mixing, and maintains magnetic particle with persistent oscillation
Suspended state, but the magnetic particle operation that is mixed is difficult to realize in miniature portable instrument in micro-fluidic chip.
Magnetic particle is coated with, is dry in micro-fluidic chip raceway groove by the present invention, and devises magnet active drive magnetic particle
(and traditional microfluidic chip is generally 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 shone.This design not only solve magnetic particle be applied to easily to precipitate during micro-fluidic chip,
The problems such as poor repeatability, also achieve more controllable immune response and physical cleaning, improve sensitivity and repeatability.Wherein magnetic
Ferromagnetism and magnetic particle size significantly affect detection result, and select magnet magnetic induction intensity of the present invention is high for 500~30000
This, preferably 1000~8000 Gausses；Magnetic particle size is 0.1~10 μm, preferably 0.5~3 μm.
Micro-fluidic chip necessary instrument is contacted with micro-fluidic chip no liquid in the present invention, woth no need to the component of cleaning, is kept away
Having exempted from traditional giant chemical light-emitting appearance needs stirring or sample-adding, cleaning etc. to operate and the cross jamming produced 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 that low background, high sensitivity, the range of linearity are wide.
2) present invention uses magnetic particle technology, has the function of magnetic enrichment, strengthens simultaneously amplified signal；And magnet can be utilized magnetic
Particulate 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, is 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 micro-fluidic chip substrate schematic diagram, wherein 1 is chip substrate, 2 be filtering area, and 3 be coating area, and 4 be cleaning
Area, 5 be detection zone, and 6 be liquid release channel, and 7 be cleaning solution storage pool, and 8 be luminous substrate liquid storage pool, and 9 be waste liquid pool.
Fig. 2 is micro-fluidic chip overall structure diagram, wherein 1 is chip substrate, 10 be top adhesive tape, and 11 be sample-adding
Mouthful, 12 be luminous substrate liquid and cleaning solution storage pool relief hole, and 13 be bottom tape.
Fig. 3 is micro-fluidic chip board structure schematic diagram, wherein 1 is chip substrate, 2 be filtering area, and 4 be cleaning area, and 5 are
Detection zone, 6 be liquid release channel, and 7 be cleaning solution storage pool, and 8 be luminous substrate liquid storage pool, and 9 be waste liquid pool, and 14 is micro- for magnetic
Grain coating area, 15 are coated with area for luminous agent tagged ligand.
Fig. 4 is the micro-fluidic chip substrate schematic diagram of double luminous substrate liquid storage pools, wherein 16 store for luminous substrate liquid
Pond A, 17 be luminous substrate liquid storage pool B, and 18 be luminous substrate liquid pre-mixing passages.
Embodiment
The invention discloses a kind of direct chemiluminescence micro-fluidic chip of magnetic particle for whole blood sample detection, this area
Technical staff can use for reference present disclosure, be suitably modified technological parameter realization.In particular, all similar replacements
Apparent to those skilled in the art with changing, they are considered as being included in the present invention.The method of the present invention
And application is described by preferred embodiment, related personnel can substantially not depart from present invention, spirit and model
Enclose it is interior method described herein and application are modified or suitably change with combining, to realize and using the technology of the present invention.
In order to make those skilled in the art more fully understand technical scheme, with reference to specific embodiment pair
The present invention is described in further detail.
Embodiment 1：Double-antibody sandwich measure Procalcitonin (PCT)
(1) antibody marks
Appropriate acridinium ester, 10 μ g EDC and 15 μ g NHS solution are added into phosphate buffer and 10~30 anti-PCT of μ g are mono-
Anti- solution, is uniformly mixed and reacts 4h at room temperature, adds the closing of 1mg glycine.With chromatographic column or chromatography column separating purification, obtain
To acridinium ester label PCT antibody.
1mg magnetic particles (size is 2 μm), 10 μ g EDC and 15 μ g NHS solution and 10~30 are added into phosphate buffer
The anti-PCT monoclonal antibodies of μ g (different from the antibody of acridinium ester label) solution, is uniformly mixed and reacts 2h at room temperature, add the sweet ammonia of 2mg
Acid seal is closed.Magnet adsorption purifies, and obtains magnetic particle mark PCT antibody.
(2) micro-fluidic chip assembles
In acridinium ester label PCT antibody-solutions containing 0.1% bovine serum albumin(BSA), 5% glycerine, 1% triton x-100,
The pH7.4 phosphate buffers of 0.1% polysorbas20 and 0.01%Proclin300；Magnetic particle mark PCT antibody-solutions be comprising
0.5% bovine serum albumin(BSA), 0.1% casein, the pH7.4 phosphate buffers of 0.2% polysorbas20 and 0.01%Proclin300.
Cleaning solution is the pH7.4 phosphate buffers comprising 0.4%BSA, 0.2% polysorbas20 and 0.01% Proclin300.
Luminous substrate liquid.Luminous substrate liquid divides A liquid and B liquid, and A liquid is acid solution, and B liquid is alkaline solution.
Magnetic particle labelled antibody solution is put into magnetic particle coating area, drying at room temperature.By acridinium ester label antibody-solutions
It is put into luminous agent label coating area, drying at room temperature.Cleaning solution is injected into cleaning solution storage pool, by the A liquid of luminous substrate liquid
Luminous substrate liquid storage pool A16 and luminous substrate liquid storage pool B17 sealings are injected separately into B liquid.As shown in Figure 1, by hemofiltration film
Glued respectively into chip with storage pool, and be assembled into micro-fluidic chip.It is fitted into aluminium foil bag, seals 4 DEG C of preservations.
(3) pattern detection
Make dilution with human normal plasma, PCT standard items are diluted to following concentration：Opg/ml、20pg/ml、50pg/
Ml, 500pg/ml, 5ng/ml, 50ng/ml and 200ng/m1.
Micro-fluidic chip is put into necessary instrument (magnet magnetic induction intensity is 6000 Gausses), 50 μ l are instilled toward adding mouth
Sample.After sample filtering, microchannel is reached, magnetic particle and luminous agent label is dissolved and reacts, then magnet collects magnetic particle.
Cleaning solution storage pool discharges cleaning solution, and after magnetic particle is cleaned, shine exciting liquid release, instrument detecting system detection luminous signal
Intensity.Total detection time 15min.Each sample is measured 3 times with 3 micro-fluidic chips respectively, is averaged, and it is bent to draw standard
Line.
50 μ l whole blood samples are added drop-wise to adding mouth, instrument detecting system detects luminous signal intensity, foundation in 15 minutes
Standard curve obtains PCT concentration in whole blood sample.
Testing principle is：After whole blood adds micro-fluidic chip, behind filtered area, microchannel, blood plasma dissolving magnetic mark are reached
Remember antibody and luminous agent label, magnet accelerates sample reaction, then collects magnetic particle.When containing PCT in blood sample, then formed
The sandwich structure (double antibody sandwich method) of HRP labelled antibody PCT magnetic particle labelled antibodies.It is once purged, in luminous exciting liquid
Effect is lower to shine, instrument detecting system test luminous signal.The standard curve obtained according to necessary instrument, and then analyze in blood sample
PCT concentration.PCT contents are higher in sample, then luminous signal is stronger.
It is minimum to be quantitatively limited to 70pg/ml the result shows that its lowest detection is limited to 20pg/ml, detection range for 0.02~
200ng/ml, linearly dependent coefficient R2＞ 0.99；In detection range, do not occur HOOK effects；And batch in batch between it is repeated
Respectively less than 10%.Reference can be provided for heart infarction heart failure medical diagnosis on disease.
Embodiment 2：Competition law measures tacrolimus blood concentration
(1) antibody/antigen marks
Appropriate magnetic particle (size is 0.5 μm), glutaraldehyde and tacrolimus monoclonal antibody solution are added into phosphate buffer, is mixed
Close uniformly and react 4h at room temperature, add glycine closing.With chromatographic column or chromatography column separating purification, magnetic particle mark is obtained
Antibody.
With 1: 0.5~1: 2 ratio mixing Avidin mark acridinium ester and biotinylation tacrolimus, acridinium ester mark is obtained
Remember tacrolimus.
(2) micro-fluidic chip assembles
In acridinium ester label antigenic solution containing 0.1% bovine serum albumin(BSA), 5% glycerine, 1% triton x-100 and
The pH7.4 phosphate buffers of 0.01%Proclin300；Magnetic particle labelled antibody solution 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 solution is to include 0.1%BSA, 0.1% polysorbas20,0.3% Tween 80 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 acid solution, and B liquid is molten for alkalescence
Liquid.
Magnetic particle labelled antibody is put into coating area, drying at room temperature.Acridinium ester label tacrolimus solution is put into hair
In photo etching label coating area, drying at room temperature.Cleaning solution is injected into cleaning solution storage pool, by the A liquid of luminous substrate liquid and B liquid point
Not Zhu Ru luminous substrate liquid storage pool A16 and luminous substrate liquid storage pool B17, sealing.As shown in Figure 1, by hemofiltration film and storage
Pond is respectively implanted in chip substrate, and is assembled into micro-fluidic chip.It is fitted into aluminium foil bag, seals 4 DEG C of preservations.
(3) pattern detection
Make dilution with phosphate buffer, it is as follows that tacrolimus standard items are prepared series concentration standard solution：0ng/
mL、0.1ng/mL、1ng/mL、5ng/mL、10ng/mL、50ng/mL、100ng/mL。
Micro-fluidic chip is put into necessary instrument (magnet magnetic induction intensity is 2000 Gausses), 50 μ l are instilled toward adding mouth
Sample, after sample filtering, reaches microchannel, dissolves magnetic particle and luminous agent label and reacts, then magnet collects magnetic particle.
The magnetic particle labelled antibody that luminous agent mark tacrolimus competition is combined with tacrolimus.Cleaning solution storage pool discharges cleaning solution,
After magnetic particle is cleaned, shine exciting liquid release, instrument detecting system detection luminous signal intensity.Total detection time 15min.Often
A sample is measured 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 tacrolimus concentration in sample.
Testing principle is：Microchannel, sample dissolving magnetic particle mark are reached after sample addition micro-fluidic chip, filtered area
Note antibody and luminous agent label simultaneously react.When containing tacrolimus in sample, luminous agent mark tacrolimus competition with he gram
The magnetic particle labelled antibody (competition law) of combination is not taken charge of.Magnet collects magnetic particle, once purged, is issued in the effect of luminous exciting liquid
Light, instrument detecting system test luminous signal.The standard curve obtained according to necessary instrument, and then analyze tacrolimus in sample
Concentration.Tacrolimus content is higher in sample, then luminous signal is weaker.
The result shows that tacrolimus lowest detection is limited to 0.2ng/mL, minimum to be quantitatively limited to 0.4ng/mL, detection range is
0.2~100ng/ml, linearly dependent coefficient R2＞ 0.99；In quantitative detection range, do not occur HOOK effects；And with criticizing in criticizing
Between repeatability be respectively less than 15%, 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, 2.0 μm, 2.8 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 100pg/ml, fixed
Amount detection range is 0.1~100ng/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, less reproducible.
When 10 μm of magnetic particles and 30000 Gauss magnet combine, its lowest detection is limited to 150pg/ml, and quantitative detection range is
0.15~100ng/ml, linearly dependent coefficient R2＞ 0.92；Batch in batch between repeatability respectively less than 20%.I.e.：Negative sample is believed
Number higher (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
50pg/ml, quantitative detection range can reach 0.05~200ng/ml, linearly dependent coefficient R2＞ 0.96；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.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered within the scope of protection of the present invention.
Claims (10)
- A kind of 1. direct chemiluminescence micro-fluidic chip of magnetic particle for whole blood sample detection, it is characterised in that the chip It is made of top adhesive tape (10), chip substrate (1) and bottom tape (13)；Filtering area (2), coating on the chip substrate (1) Area (3), cleaning area (4), detection zone (5), liquid release channel (6) are sequentially connected, and detection zone (5) passes through liquid release channel (6) it is connected with cleaning solution storage pool (7) and luminous substrate liquid storage pool (8)；Top adhesive tape includes adding mouth (11) and cleaning solution Storage pool relief hole (12)；Coating area (3) is coated with pre-packaged magnetic particle tagged ligand and enzyme or luminous agent mark is matched somebody with somebody Body；Cleaning solution storage pool (7) and luminous substrate liquid storage pool (8) store pre-packaged cleaning solution and luminous substrate liquid；Top adhesive tape (10) adding mouth (11) and cleaning solution storage pool relief hole (12) are included.
- 2. micro-fluidic chip as claimed in claim 1, it is characterised in that the luminous substrate liquid storage pool (8) is by the base that shines Bottom liquid storage pool A (16) and luminous substrate liquid storage pool B (17) is substituted, luminous substrate liquid storage pool A (16) and luminous substrate liquid Storage pool B (17) is connected by luminescent solution pre-mixing passages (18).
- 3. micro-fluidic chip as claimed in claim 1 or 2, it is characterised in that the luminous substrate liquid include acid solution and Alkaline solution, can merge injection luminous substrate liquid storage pool (8), can also be injected separately into luminous substrate liquid storage pool A (16), shine Substrate liquid storage pool B (17), is uniformly mixed after release by luminous substrate liquid pre-mixing passages (18).
- 4. micro-fluidic chip as claimed in claim 1 or 2, it is characterised in that the cleaning solution storage pool (7) and luminous substrate Liquid storage pool (8) is hydraulic seal pond, can be extruded by external force and partial fracture, discharge liquid；The filtering area includes hemofiltration Film.
- 5. micro-fluidic chip as claimed in claim 1 or 2, it is characterised in that the magnetic particle tagged ligand and luminous agent mark Note ligand can all be put into coating area (3), also magnetic particle tagged ligand and luminous agent tagged ligand can be respectively put into magnetic particle bag By area (14) and luminous agent tagged ligand coating area (15).
- 6. micro-fluidic chip as claimed in claim 1 or 2, it is characterised in that the luminous agent tagged ligand solution includes slow Fliud flushing, protein, glycerine, surfactant and preservative, magnetic particle tagged ligand solution include buffer solution, protein, carbohydrate, Surfactant and preservative, cleaning solution include buffer solution, protein, surfactant and preservative.
- 7. micro-fluidic chip as claimed in claim 1 or 2, it is characterised in that what the luminous agent tagged ligand used shines Agent includes acridinium ester and acridine sulfonamide；The magnetic particle that the magnetic particle tagged ligand uses is supperparamagnetic particles, by iron, cobalt Or nickel compound composition, magnetic particle size are 0.1~10 μm, magnet magnetic induction intensity is 500~10000 Gausses.
- 8. micro-fluidic chip as claimed in claim 7, it is characterised in that the magnetic particle is supperparamagnetic particles, by iron, cobalt Or nickel compound composition, magnetic particle size are 0.5~3 μm, magnet magnetic induction intensity is 1000~8000 Gausses.
- 9. a kind of direct chemiluminescence micro-fluidic chip of magnetic particle for whole blood sample detection as claimed in claim 1 or 2, It is characterized in that, the sample volume of the micro-fluidic chip is 10~500 μ l.
- 10. a kind of direct chemiluminescence micro-fluidic chip of magnetic particle for whole blood sample detection as claimed in claim 9, its It is characterized in that, the sample volume of the micro-fluidic chip is 20~100 μ l.
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