CN105435868A - Magnetic particle chemiluminescent microfluidic chip for quantitatively detecting troponin I in whole blood - Google Patents
Magnetic particle chemiluminescent microfluidic chip for quantitatively detecting troponin I in whole blood Download PDFInfo
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Abstract
The present invention discloses a magnetic particle chemiluminescent microfluidic chip for quantitatively detecting troponin I in whole blood. The microfluidic chip comprises a head plate (1) structure and a bottom plate (2) structure, and a gas pump (3), a sampling port (4), a sample filling area (12), a marked antibody storage pool (5) and a sample mixing region (13) on the head plate (1) are connected successively; a filtration zone (6), a magnetic particle coating region (7), a washing zone (14), a detection zone (8) and a liquid release channel (16) on the bottom plate are connected successively; the detection area (8) of the bottom plate is connected with a washing fluid storage pool (9) and a lighting substrate fluid storage pool (10) through the fluid release channel (16), respectively.
Description
Technical field
The present invention relates to a kind of method utilizing magnetic microparticle chemiluminescence technology and microfluidic chip technology to realize the highly sensitive quantitative detection of cTnI in whole blood sample; particularly disclose the magnetic microparticle chemiluminescence micro-fluidic chip of Troponin I in a kind of quantitative detection whole blood; accurate, the highly sensitive quantitative detection of cTnI in whole blood sample can be realized, belong to fluidic chip chemiluminescence technical field of immunoassay.
Background technology
The prevention and control situation of current China angiocardiopathy is still severe, and cardiovascular disease incidence rate is in the situation that constantly rises.According to statistics, cardiovascular death rate accounts for human mortality's 40%, therefore carries out early discovery, early prevention, early treatment, and raising preventing and treating cardiovascular disease level is crucial.Angiocardiopathy traditional detection project is mostly myocardial enzymes.But there is enzymatic activity to raise and occur the deficiencies such as more late, specificity is poor and the duration is short.And cardiac troponin is the contractile protein being uniquely present in cardiac muscle, there are high susceptibility and specificity to myocardial necrosis.
Cardiac troponin is by cTnI, cTnC and cTnT tri-kinds, in muscle diastole and contraction process, play important regulative.But cTnC is without Cardiac-specific, is generally not used in myocardial damage and detects.Under normal condition, cTnI and cTnT all permeates cell membranes can not enter blood, so cTnI and cTnT is extremely low in healthy human blood; As myocardial cell damage, cTnI and cTnT enters people's cytoplasm and blood.In the diseases such as kidney failure, pneumonia and septicemia, in blood, cTnT content also can raise, so its specificity is not as cTnI.CTnI raises for 3 ~ 5 hours after morbidity, and within 15 ~ 24 hours, reach peak, the duration is of a specified duration, can be down to normal after 5 ~ 10 days.CTnI is one of best at present myocardial injury markers.
Multiplex ELISA, chemoluminescence method and immunochromatographic method (test strips) etc. detect cTnI traditionally.But ELISA complicated operation, detection time is long; Chemoluminescence method needs supporting Large expensive instrument, and the testing time is long, not easily realizes instant fast detection.Though colloidal gold immunity chromatography is fast easy, poor repeatability, sensitivity are low, easily occur erroneous judgement.
Chinese patent 200780015772.0 discloses a kind of troponin High Sensitive Analysis system, adopts microtiter plate (microwell plate) to detect, highly sensitive, but complicated operation, the testing time is long, detection range is narrow.Chinese patent 200610028913.X describes a kind of kit realizing detecting cTnI with colloidal gold immunochromatographimethod technology, but can only carry out qualitative detection, and cannot be quantitative.Chinese patent 201010619731.6 discloses the immuno-chromatographic test paper strip that a kind of whole process quantitatively detects cTnI, substitutes collaurum with fluorescent latex particles, realizes the quantitative detection to cTnI, but still cannot solve the defects such as test strips poor repeatability.
Therefore develop quick, accurate, highly sensitive detection method, there is great potential and application prospect.With fluorescence with absorb light and compare, chemiluminescence does not have external excitation source background signal to disturb, and cross jamming is little, highly sensitive, the range of linearity is wide.Microfluidic chip technology is integrated into basic operation units such as sample preparation, reaction, separation, detections on the chip of one piece of micro-meter scale, can complete whole process analysis.
For deficiency and the defect of existing cTnI detection method, micro-fluidic magnetic microparticle chemiluminescence method utilizes magnetic microparticle chemiluminescence and microflow control technique, can realize accurate to cTnI, highly sensitive quantitative detection.
Summary of the invention
The technical problem to be solved in the present invention is, poor repeatability low for existing fast diagnosis method sensitivity, is disturbed obviously; and the problem that existing chemiluminescence necessary instrument is expensive, detection time is long; the magnetic microparticle chemiluminescence micro-fluidic chip of Troponin I in a kind of quantitative detection whole blood is provided; by integrated chip (all components except test sample book is all integrated in chip) and supporting small portable device, thus realize quick, accurate, the highly sensitive quantitative detection of cTnI in on-the-spot whole blood sample.
For solving the problems of the technologies described above, technical scheme provided by the invention is:
The magnetic microparticle chemiluminescence micro-fluidic chip of Troponin I in a kind of quantitative detection whole blood, it is characterized in that, described micro-fluidic chip comprises top board (1) structure and base plate (2) structure, and wherein the upper air pump (3) of top board (1), adding mouth (4), sample fill area (12), labelled antibody storage pool (5) are connected successively with sample mixed zone (13); On base plate, filtering area (6), magnetic particle Bao Bei district (7), cleaning area (14), detection zone (8), liquid release channel (16) connect successively; The detection zone (8) of base plate is connected by liquid release channel (16) with cleaning fluid storage pool (9) and luminous substrate liquid storage pool (10) respectively;
Described labelled antibody storage pool (5) stores pre-packaged enzyme or luminous agent marks anti-cTnI antibody, magnetic particle Bao Bei district (7) bag is by the anti-cTnI antibody of pre-packaged magnetic particle marker, and cleaning fluid storage pool (9) and luminous substrate liquid storage pool (10) store pre-packaged cleaning fluid and luminous substrate liquid; In described micro-fluidic chip testing process, with magnet manipulation magnetic particle moving or gathering; Described labelled antibody storage pool, cleaning fluid storage pool and luminous substrate liquid storage pool are hydraulic seal pond, the partial fracture by external force extruding, releasing liquid; Described filtering area comprises top board (1) described in hemofiltration film and seals with base plate (2) adhesive tape (19 and 20).
Particularly, micro-fluidic chip of the present invention, the luminous substrate liquid shelf-life should separate when being less than 1 year, substitute luminous substrate liquid storage pool (10) with luminous substrate liquid storage pool A (23) and luminous substrate liquid storage pool B (24), described luminous substrate liquid storage pool A (23) is connected by pre-mixing passages (25) with luminous substrate liquid storage pool B (24).
Particularly, the present invention's magnetic particle used comprises the compound of iron, cobalt, nickel, mainly including but not limited to di-iron trioxide and tri-iron tetroxide compound.Preferred magnetic particle is polystyrene is shell, and di-iron trioxide is the particle of core, and the magnetic induction intensity of magnetic particle size and magnet has obvious impact to testing result.
Particularly, the magnetic particle size that the anti-cTnI antibody of described magnetic particle marker uses is 0.1 ~ 10 μm; The magnet magnetic induction intensity mated with magnetic bead is 500 ~ 30000 Gausses.
Preferably, the magnetic particle size that the anti-cTnI antibody of described magnetic particle marker uses is 0.5 ~ 3 μm, and the magnet magnetic induction intensity mated with magnetic bead is 1000 ~ 8000 Gausses.
Particularly, described enzyme or luminous agent mark cTnI antibody-solutions, magnetic particle marker cTnI antibody-solutions and cleaning fluid all comprise buffer solution, protein, surfactant and anticorrisive agent, and luminous agent mark cTnI antibody-solutions also comprises glycerine, magnetic particle marker cTnI antibody-solutions also comprises carbohydrate.
Particularly, described enzyme labeling cTnI antibody-solutions comprises the borate buffer of bovine serum albumin(BSA) (BSA), Tween-20 and Proclin300; Magnetic particle marker cTnI antibody-solutions comprises the borate buffer of BSA, glucose, Tween-20 and Proclin300; Described cleaning fluid comprises the borate buffer of BSA, Tween-20 and Proclin300.
Particularly, described luminous agent mark cTnI antibody-solutions comprises the phosphate buffer of BSA, glycerine, Tween-20, triton x-100 and Sodium azide; Described magnetic particle marker cTnI antibody-solutions comprises the phosphate buffer of BSA, casein, sucrose, Tween-20, triton x-100 and Sodium azide; Described cleaning fluid comprises the phosphate buffer of BSA, polysorbas20, triton x-100, polyethylene glycol and Sodium azide.
Particularly, described luminous substrate liquid comprises the substrate corresponding with enzyme and luminescence enhancement liquid, rear injection luminous substrate liquid storage pool (10) can be merged, or inject luminous substrate liquid storage pool A (23) and luminous substrate liquid storage pool B (24) respectively.
Particularly, described luminous substrate liquid comprises hydrogen peroxide solution corresponding to luminous agent and alkaline solution, rear injection luminous substrate liquid storage pool (10) can be merged, or inject luminous substrate liquid storage pool A (23) and luminous substrate liquid storage pool B (24) respectively.
Micro-flow control chip preparation method of the present invention is as follows:
Step 1) enzyme or luminous agent mark anti-cTnI antibody, the anti-cTnI antibody of magnetic particle marker, and these two kinds of antibody may be the same or different;
Step 2) enzyme or luminous agent labelled antibody solution are put into the labelled antibody storage pool of top board, sealing, magnetic particle marker antibody-solutions is put into the Bao Bei district of base plate, dry, cleaning fluid and luminous substrate liquid are injected respectively cleaning fluid storage pool and luminous substrate liquid storage pool, sealing, is assembled into micro-fluidic chip by top board and base plate.
In a kind of quantitative detection whole blood provided by the invention, the magnetic microparticle chemiluminescence micro-fluidic chip of Troponin I is a kind of micro-fluidic chip realizing quick, accurate, the highly sensitive detection of cTnI based on chemiluminescence, on micro-fluidic chip.
This chip is that anti-cTnI antibody modification, on magnetic particle, utilizes antigen-antibody effect by anti-cTnI antibody modification enzyme, as whether double antibody sandwich method principle contains cTnI in conjunction with in magnetic particle rich, chemiluminescence detection whole blood sample, and its content of accurate analysis.
Enzyme described in the present invention, including but not limited to catalase (HRP) and alkaline phosphatase (ALP).Luminous substrate liquid is the luminous substrate (as luminol or adamantane) and luminescence enhancement liquid (as reinforcing agents such as benzene derivatives) that enzyme is corresponding, wherein luminous substrate and luminescence enhancement liquid can merge, and mix rear injection luminous substrate liquid storage pool (10) as shown in Figure 1; But should separate when the mixed liquor shelf-life is less than 1 year, inject luminous substrate liquid storage pool A (23) and luminous substrate liquid storage pool B (24) respectively as shown in Figure 3, as shown in Figure 3, mixed by pre-mixing passages (25) during test, then flow into detection zone participation reaction.One embodiment of the invention adopts horseradish peroxidase (HRP).
Luminous agent of the present invention, including but not limited to acridinium ester and acridine sulfonamide.After luminous agent and the effect of luminous substrate liquid, do not need the catalytic action of enzyme, participate in luminescence-producing reaction directly.One embodiment of the present of invention adopt acridinium ester.Luminous substrate liquid comprises H
2o
2solution and alkaline solution, can be merged into alkaline H
2o
2solution, injects luminous substrate liquid storage pool (10); But time unstable after mixing, hydrogen peroxide solution and alkaline solution should inject luminous substrate liquid storage pool A (23) and luminous substrate liquid storage pool B (24) respectively, as shown in Figure 3, first mixed by pre-mixing passages (25) during test, then flow into detection zone participation reaction.
CTnI antibody of the present invention comprises monoclonal antibody and polyclonal antibody.This antibody can be combined with cTnI (as double antibody sandwich method).Wherein the antibody of enzyme or luminous agent mark can be identical with the antibody of magnetic particle marker, also can be different.
Enzyme of the present invention or luminous agent labelled antibody solution and magnetic particle marker antibody-solutions all comprise buffer solution, protein, surfactant and anticorrisive agent, and magnetic particle marker antibody-solutions also comprises carbohydrate.Wherein HRP labelled antibody, can not contain NaN in buffer system
3; ALP labelled antibody, buffer system can not be Phosphoric Acid.
The top board of micro-fluidic chip of the present invention and the moulding material of base plate are polymer, including but not limited to polystyrene, polyvinyl chloride, polypropylene, epoxy resin etc.Two-sided tape can be replaced by two one-faced tapes.As shown in Figure 1, top board structure is made up of air pump (3), adding mouth (4), labelled antibody storage pool (5), lid (11), sample fill area (12) and sample mixed zone (13).Base arrangement is by filtering area (6), magnetic particle Bao Bei district (7), detection zone (8), cleaning fluid storage pool (9), luminous substrate liquid storage pool (10), cleaning area (14), waste liquid pool (15) and liquid release channel (16).As shown in Figure 2, in luminous substrate liquid and cleaning fluid storage pool region, and magnet slide rail region, top board needs the resigning hole (being respectively 17 and 18) reserving storage pool and magnet slide rail, double adhesive tape should reserve the resigning hole (being respectively 21 and 22) when storage pool and sample mixed liquor inflow filtering area, certain region is got out of the way in the effect of resigning hole, does not disturb liquid flow path, or necessary instrument parts act on the path of micro-fluidic chip.
Storage pool of the present invention is hydraulic seal pond, and encapsulant used comprises glass, plastics, rubber, aluminium foil and high-isolation film, and wherein encapsulant can be same material composition, also can be multiple material and combines.Under physical impact, storage pool can partial fracture, thus the liquid of sealing is discharged.Wherein enzyme mark cTnI antibody storage pool, cleaning fluid storage pool, luminous substrate liquid storage pool can adopt identical or different materials and methods to make.In one embodiment of the invention, enzyme mark cTnI antibody storage pool, cleaning fluid storage pool, luminous substrate liquid storage pool all adopt plastics and elastic caoutchouc to be sealed to form.In an alternative embodiment of the invention, enzyme mark cTnI antibody storage pool adopts plastics and elastic caoutchouc to be sealed to form, and cleaning fluid storage pool, luminous substrate liquid storage pool adopt high-isolation film to be sealed to form.
Filtering area of the present invention comprises hemofiltration film, wherein hemofiltration film makes liquid and cell separation by physical pore size or biology/chemical reagent, realizes blood plasma and is separated with red blood cell, and blood plasma flows to magnetic particle Bao Bei district, and red blood cell rests on hemofiltration film, thus reduce red blood cell to the interference of result of the test.Wherein said biology/chemical reagent comprises coagulant etc., can make to connect between red blood cell, and form grumeleuse, increased in size, is more easily stopped by the network structure of hemofiltration film.
Micro-fluidic chip of the present invention, when there is luminous substrate liquid storage pool A (23) and luminous substrate liquid storage pool B (24), luminous substrate liquid pre-mixing passages (25) should be increased on base plate, this pre-mixing passages can be serpentine channel or up-down structure hybrid channel, as shown in Figure 3.
In one embodiment, labelled antibody storage pool (5) is enclosed HRP and is marked anti-cTnI antibody, and Bao Bei district bag, by magnetic particle marker anti-cTnI antibody (different from enzyme labelled antibody), detects cTnI with magnetic particle enzyme-catalyzed chemical luminescence method.In another embodiment, labelled antibody storage pool (5) encloses the anti-cTnI antibody of acridinium ester label, Bao Bei district bag, by magnetic particle marker anti-cTnI antibody (different from acridinium ester label antibody), detects cTnI content with magnetic particle enzyme-catalyzed chemical luminescence method.
Cleaning fluid of the present invention, for cleaning magnetic particle, removes the material of the cTnI of non-specific adsorption, enzyme marker and other influences testing result.Cleaning fluid mainly comprises buffer system, protein and surfactant, and wherein buffer system is including but not limited to borate, phosphate, Tris-HCl and acetate etc.Cleaning fluid pH6.0 ~ 10.0, when detect sample be strong acid or strong basicity time, pH scope can be relaxed.Wherein protein is including but not limited to bovine serum albumin(BSA), casein etc.Wherein surface-active is including but not limited to comprising polysorbas20, Tween 80, triton x-100, polyethylene glycol and PVP etc.
Sample volume of the present invention at 10 ~ 500 μ l, preferably 20 ~ 100 μ l.As preferably, injection volume is 50 μ l in an embodiment.
Micro-fluidic chip of the present invention is detect fast, and detection time should be less than 30 minutes, as preferably, adopts 15 minutes in embodiment.
Antibody instrument of the present invention comprises extruding air pump and storage pool, and the functions such as magnet moves, luminescent detection system, should comprise pressurizing unit, magnet and mobile device, detection system, control analysis module and software systems.
The magnetic microparticle chemiluminescence micro-fluidic chip that cardiac muscle troponin I quantitatively detects, is characterized in that, the testing process of described micro-fluidic chip comprises:
Step 1) sample is instilled adding mouth after, cover lid, micro-fluidic chip puts into necessary instrument, after enzyme or luminous agent labelled antibody discharge, air pump makes sample and labelled antibody mix, and then injects base plate filtering area, described necessary instrument is small portable device, comprise extruding air pump and storage pool, the functions such as magnet moves, luminescent detection system;
Step 2) sample is after filtration behind district, arrive Bao Bei district, dissolve magnetic labeling antibody, after abundant reaction, magnet collects magnetic particle, and storage pool release cleaning fluid, after magnetic particle cleaning, move to detection zone, release luminous substrate liquid, instrument detection system detects luminous signal intensity, and then realizes the quantitative detection of cTnI.
Core of the present invention adopts magnetic microparticle chemiluminescence immunoassay technology to detect at quick, high sensitivity, the accurate quantitative analysis of micro-fluidic chip realize target thing.
Microfluidic chip technology is that biological, chemistry, medical analysis process the basic operation unit such as sample preparation, reaction, separation, detection is integrated on the chip of one piece of micro-meter scale, automatically completes analysis overall process.
Micro-fluidic chip of the present invention is by all reagent component (the enzyme mark cTnI antibody needed for testing process, magnetic particle marker cTnI antibody, cleaning fluid, luminous substrate liquid etc.) all integrated, be built in micro-fluidic chip, and by ingenious channel design, under the operation of necessary instrument, the one-touch detection realizing micro-fluidic chip (only need press beginning key just can realize detecting, without the need to complex operations), realize separation of whole blood, immune response, cleaning is separated, chemiluminescence detection, thus it is simple to avoid structural design in existing micro-fluidic chip, the deficiency such as complicated operation and defect during detection.Also overcome traditional chemical light-emitting appearance and can only carry out serum or blood plasma detection, and the shortcoming that can not detect whole blood sample.
Because magnetic particle easily precipitates, traditional chemical light-emitting appearance adopts manual mixing, and maintains the suspended state of magnetic particle with persistent oscillation, but in micro-fluidic chip, the operation that is mixed of magnetic particle is difficult to realize in miniature portable instrument.
The present invention by magnetic particle bag by, dry in micro-fluidic chip raceway groove, and devise magnet active drive magnetic particle (and traditional microfluidic chip generally adopts fluid to drive or electric drive), thus magnetic particle is redissolved, and realize immune response, cleaning, luminescence in micro-fluidic chip zones of different.This design not only solve when magnetic particle is applied to micro-fluidic chip easily precipitate, the problem such as poor repeatability, also achieve more controlled immune response and physical cleaning, improve sensitivity and repeatability.
In the present invention, micro-fluidic chip necessary instrument contacts with micro-fluidic chip no liquid, the parts that need not clean, and avoiding traditional giant chemical light-emitting appearance needs stirring or application of sample, cleaning etc. to operate and the cross jamming of generation and pollution.
So the present invention is not simple superposition magnetic microparticle chemiluminescence technology and microfluidic chip technology; but by hydraulic seal design, channel design; integrated for required for detection all chemical constituents, be built in micro-fluidic chip; and with magnet active drive; realize one-touch magnetic microparticle chemiluminescence immune detection, thus in portable necessary instrument, realize quick, the detection of high sensitivity, accurate quantitative analysis of cTnI in whole blood.
The present invention can be applicable to the angiocardiopathy especially quantitative detection of cTnI in heart failure.
Major advantage of the present invention is as follows:
1) the present invention adopts chemiluminescence method, has the advantage that background is low, highly sensitive, the range of linearity is wide.
2) the present invention adopts magnetic granule technology, has magnetic enrichment function, strengthens and amplifying signal; And magnet can be utilized magnetic transfer of granules region (as by Bao Bei district-cleaning area-detection zone), reduce the impact of sample matrix.
3) the present invention adopts microfluidic chip technology, sample is mixed, reacts, separation and detection is integrated on chip, and all reagent component needed for reaction are integrated on chip.
4) the present invention is easy and simple to handle, during detection, only need add sample, cover lid, chip is put into miniature portable necessary instrument.
5) necessary instrument of the present invention is miniature portable instrument, instrument only with chip generation physical contact, in chip liquid not with instrument contacts, can not instrument be polluted and produce cross jamming.
Accompanying drawing explanation
Fig. 1 is that brain natriuretic peptide quantitatively detects micro-fluidic chip body structural representation, wherein 1 is top board, 2 is base plate, and 3 is air pump, and 4 is adding mouth, 5 is labelled antibody storage pool, 6 is filtering area, and 7 is magnetic particle Bao Bei district, and 8 is detection zone, 9 is cleaning fluid storage pool, 10 is luminous substrate liquid storage pool, and 11 is lid, and 12 is sample fill area, 13 is sample mixed zone, 14 is cleaning area, and 15 is waste liquid pool, and 16 is liquid release channel, 17 is luminous substrate liquid and cleaning fluid storage pool resigning hole (in top board), and 18 is magnet slide rail resigning hole.
Fig. 2 is the complete microfluidic chip structure schematic diagram that brain natriuretic peptide quantitatively detects, wherein 1 is top board, 2 is base plate, 19 is two-sided tape, 20 is one-faced tapes, 21 is luminous substrate liquid and cleaning fluid storage pool resigning hole (in two-sided tape), and 22 is resigning hole during mixed liquor inflow filtering area.
Fig. 3 is the micro-fluidic chip base arrangement schematic diagram of two luminous substrate liquid, and wherein 23 is luminous substrate liquid storage pool A, and 24 is luminous substrate liquid storage pool B, and 25 is pre-mixing passages.
Detailed description of the invention
The invention discloses micro-fluidic magnetic microparticle chemiluminescence method and micro-fluidic chip special thereof that a kind of cTnI quantitatively detects, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
In order to make those skilled in the art understand technical scheme of the present invention better, below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment 1: enzyme-catalyzed chemical luminescence measures cTnI
(1) antibody labeling
Get 50 μ gHRP to be dissolved in 1mL distilled water, then add 10 μm of ol and newly join NaIO
4solution, after room temperature lucifuge reaction 20min, with 1mMpH4.4 sodium-acetate buffer dialysis purification solution.With pH9.5 carbonate buffer solution, pH is adjusted to 9.0 again, adds the anti-cTnI monoclonal antibody of 100 μ g, room temperature lucifuge reaction 2h.Add 0.1mL4mg/mL and newly join NaBH
4, in 4 DEG C of reaction 2h after mixing.Above-mentioned solution is loaded bag filter, and with 0.15MpH7.4PBS dialysis, 4 DEG C are spent the night, and obtain HRP and mark cTnI antibody.
1mg magnetic particle (diameter is 2 μm), 10 μ gEDC and 15 μ gNHS solution and 10 ~ 30 μ g anti-cTnI monoclonal antibody (different from the antibody that HRP marks) solution is added in pH7.4 phosphate buffer, mix and react 4h under room temperature, adding 1mg glycine and close.With magnet enriching and purifying, remove unreacted cTnI monoclonal antibody, obtain magnetic particle marker cTnI antibody.
(2) micro-fluidic chip assembling
HRP marks the pH7.4 borate buffer containing 1%BSA, 0.2% polysorbas20 and 0.05%Proclin300 in cTnI antibody-solutions; Magnetic particle marker cTnI antibody-solutions is the pH7.4 borate buffer comprising 0.5%BSA, 2% glucose, 1% Tween-20 and 0.05%Proclin300.
HRP labeling antibody solution is put into top board labelled antibody storage pool, sealing.Magnetic labeling antibody solution is put into base plate magnetic particle Bao Bei district, drying at room temperature.
Cleaning fluid is the pH7.2 borate buffer of 0.3%BSA, 0.2% Tween-20 and 0.03%Proclin300.Cleaning fluid is injected cleaning fluid storage pool.Luminous substrate liquid is divided into HRP substrate (hydrogen peroxide solution of luminol) and alkalescence to strengthen liquid (alkaline solution of benzene derivative), inject luminous substrate liquid storage pool A (23) and luminous substrate liquid storage pool B (24) respectively, sealing.Shown in Fig. 1, filtering area is glued in base plate.Then press shown in Fig. 2, with one-faced tapes and two-sided tape, top board and base plate are assembled into micro-fluidic chip.Load in aluminium foil bag, seal 4 ° of preservations.
(3) pattern detection
Make dilution with human normal plasma, cTnI standard items are diluted to following concentration: 0pg/ml, 50pg/ml, 100pg/ml, 500pg/ml, 1ng/ml, 5ng/ml, 10ng/ml and 50ng/ml.
After 50 μ l sample instillation adding mouths, cover lid.Micro-fluidic chip is put into necessary instrument (magnet magnetic induction intensity is 6000 Gausses), instrument is extruded HRP and is marked monoclonal antibody, and makes sample and HRP mark monoclonal antibody mix rear injection base plate filtering area.After sample filters, arrive microchannel, and dissolve magnetic particle marker monoclonal antibody, magnet accelerates sample reaction, and form the sandwich structure that HRP marks monoclonal antibody-cTnI antigen-magnetic particle marker monoclonal antibody, then magnet collects magnetic particle.Storage pool release cleaning fluid, after magnetic particle cleaning, luminous substrate liquid discharges, and instrument detection system detects luminous signal intensity.Total detection time 15min.Each sample measures 3 times with 3 micro-fluidic chips respectively, averages, drawing standard curve.
By 50 μ l whole blood sample instillation adding mouths, in 15 minutes, instrument detection system detects luminous signal intensity, and establishing criteria curve obtains cTnI concentration in sample.
Cleaning Principle is: after whole blood adds micro-fluidic chip, and whole blood first mix with HRP labelled antibody, then after filtration behind district, is mixed with the blood plasma arrival microchannel of HRP labelled antibody, blood plasma dissolving magnetic labelled 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.After cleaning, more luminous under the effect of luminous substrate liquid, instrument detection system test luminous signal.According to the calibration curve that necessary instrument obtains, and then analyze cTnI concentration in blood sample.In sample, cTnI content is higher, then luminous signal is stronger.
Result shows, its lowest detection is limited to 50pg/ml, is minimumly quantitatively limited to 200pg/ml, and quantitative detection range is 0.05 ~ 50ng/ml, linearly dependent coefficient R
2> 0.99; In detection range, there is not HOOK effect; And batch in and batch between repeatability be all less than 10%.Can be the medical diagnosis on disease of heart stalk heart failure and reference is provided.
Embodiment 2: directly chemical luminescent detecting cTnI
(1) antibody labeling
In phosphate buffer, add acridinium ester and the 100 μ g anti-cTnI monoclonal antibody solution of appropriate activation, after mixing, under room temperature, react 3h, add 1mg glycine and close.Dialysis separation and purification, obtains acridinium ester label cTnI antibody.
In 1ml10mMpH7.4 phosphate buffer, add 1mg magnetic particle (diameter is 1 μm), 20 μ gEDC and 20 μ gNHS solution and 30 μ g Streptavidins, mix and react 4h under room temperature, add 1mg glycine and close.With magnet adsorption enrichment, remove unreacted Streptavidin, obtain magnetic particle marker Streptavidin.
The anti-cTnI monoclonal antibody of 20 μ g is added in 10 μ L0.25mg/mLSulfo-NHS-LC-biotin solution, reaction 1h.Ultrafiltration centrifugal purification, removes unreacted biotin, obtains biotin-cTnI antibody.
By the interaction between Avidin-Biotin, anti-cTnI antibody is connected to magnetic particle surface, obtains magnetic particle marker cTnI antibody.Wherein Avidin mark magnetic particle and biotinylated antibody ratios 1: 10
4~ 2: 10
5.
(2) micro-fluidic chip assembling
Containing the pH7.4 phosphate buffer of 0.5%BSA, 1% glycerine, 0.2% polysorbas20,1% triton x-100 and 0.1% Sodium azide in acridinium ester label cTnI antibody-solutions; Magnetic particle marker cTnI antibody-solutions is the pH7.4 phosphate buffer comprising 0.2%BSA, 0.2% casein, 1% sucrose, 0.5% polysorbas20,0.5% triton x-100 and 0.1% Sodium azide.Acridinium ester label antibody-solutions is put into top board labelled antibody storage pool, sealing.Magnetic labeling antibody solution is put into base plate magnetic particle Bao Bei district, drying at room temperature.
Cleaning fluid is the pH7.2 phosphate buffer of 0.3%BSA, 0.5% polysorbas20,1% triton x-100 and 0.02% Sodium azide.Cleaning fluid is injected cleaning fluid storage pool.Luminous substrate liquid is divided into and comprises hydrogen peroxide solution and alkaline solution, injects luminous substrate liquid storage pool A (23) and luminous substrate liquid storage pool B (24) respectively, sealing.Shown in Fig. 1, hemofiltration film is glued in base plate filtering area, inserts base plate by storage pool.Then press shown in Fig. 2, with one-faced tapes and two-sided tape, top board and base plate are assembled into micro-fluidic chip.Load in aluminium foil bag, seal 4 ° of preservations.
(3) pattern detection
Make dilution with human normal plasma, cTnI standard items are diluted to following concentration: 0pg/ml, 100pg/ml, 500pg/ml, 1ng/ml, 5ng/ml, 10ng/ml and 50ng/ml.
After 50 μ l sample instillation adding mouths, cover lid.Micro-fluidic chip is put into necessary instrument (magnet magnetic induction intensity is 4000 Gausses), acridinium ester label monoclonal antibody extruded by instrument, and makes sample and acridinium ester label monoclonal antibody mix rear injection base plate filtering area.After sample filters, arrive microchannel, and dissolve magnetic particle marker monoclonal antibody, magnet accelerates sample reaction, and form the sandwich structure of acridinium ester label antibody-cTnI antigen-magnetic particle marker antibody, then magnet collects magnetic particle.Storage pool release cleaning fluid, after magnetic particle cleaning, luminous exciting liquid release, instrument detection system detects luminous signal intensity.Total detection time 15min.Each sample measures 3 times with 3 micro-fluidic chips respectively, averages, drawing standard curve.
By 50 μ l plasma sample instillation adding mouths, in 15 minutes, instrument detection system detects luminous signal intensity, and establishing criteria curve obtains cTnI concentration in sample.
Cleaning Principle is: after whole blood adds micro-fluidic chip, and whole blood first mix with acridinium ester label antibody, then after filtration behind district, is mixed with the blood plasma arrival microchannel of acridinium ester label antibody, blood plasma dissolving magnetic labelled antibody.When containing cTnI in blood sample, then form the sandwich structure (double antibody sandwich method) of acridinium ester label antibody-cTnI-magnetic particle marker antibody.After cleaning, luminous exciting liquid release, produces direct chemiluminescence with acridinium ester effect after mixing, instrument detection system test luminous signal.According to the calibration curve that necessary instrument obtains, and then cTnI concentration in analysed for plasma.In blood plasma, cTnI content is higher, then luminous signal is stronger.
Result shows, its lowest detection is limited to 80pg/ml, is minimumly quantitatively limited to 300pg/ml, and quantitative detection range is 0.08 ~ 50ng/ml, linearly dependent coefficient R
2> 0.99; In detection range, there is not HOOK effect; And batch in and batch between repeatability be all less than 10%.Can be the medical diagnosis on disease of heart stalk heart failure and reference is provided.
Embodiment 3: magnetic particle particle size is screened
Other experiment condition is see embodiment 2, and magnetic particle size and magnet magnetic induction intensity carry out according to following scheme.
Particle size is 0.1 μm, 0.5 μm, 1.0 μm, 2.0 μm, 2.4 μm, 3 μm, 10 μm.Magnet magnetic induction intensity is 500 Gausses, 1000 Gausses, 4000 Gausses, 8000 Gausses, 12000 Gausses, 30000 Gausses.The magnetic particle of seven kinds of sizes is driven respectively respectively with these six kinds of magnet.
Experimental result shows: when 0.1 μm of magnetic particle and the combination of 500 Gauss's magnet, its lowest detection is limited to 500pg/ml, and quantitative detection range is 0.5 ~ 50ng/ml, linearly dependent coefficient R
2> 0.95; Batch in and batch between repeatability be all less than 20%.That is: chemiluminescence signal is more weak, and sensitivity is not high, and repeatability is poor.
When 10 μm of magnetic particles and the combination of 30000 Gauss's magnet, its lowest detection is limited to 400pg/ml, and quantitative detection range is 0.4 ~ 5ng/ml, linearly dependent coefficient R
2> 0.95; Batch in and batch between repeatability be all less than 20%.That is: negative sample signal higher (cleaning insufficient), the range of linearity is not wide.
The magnetic particle of 0.5 ~ 3 μm is when combinationally using with the magnet of 1000 ~ 8000 Gausses, and its LDL is all less than 150pg/ml, and quantitative detection range can reach 0.15 ~ 50ng/ml, linearly dependent coefficient R
2> 0.97; Batch in and batch between repeatability be all less than 12%.Meet for the clinical heart obstructs the needs that heart failure medical diagnosis on disease provides reference.
According to above result, magnetic particle size preferably 0.5 ~ 3 μm, magnet magnetic induction intensity is 1000 ~ 8000 Gausses preferably; Magnetic particle size more preferably 1.0 ~ 3 μm, magnet magnetic induction intensity 4000 ~ 8000 Gauss.According to magnetic particle size used, magnet magnetic induction intensity can be determined further.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. one kind is quantitatively detected the magnetic microparticle chemiluminescence micro-fluidic chip of Troponin I in whole blood, it is characterized in that, described micro-fluidic chip comprises top board (1) structure and base plate (2) structure, and the air pump (3) wherein on top board (1), adding mouth (4), sample fill area (12), labelled antibody storage pool (5) are connected successively with sample mixed zone (13); Filtering area (6) on base plate, magnetic particle Bao Bei district (7), cleaning area (14), detection zone (8), liquid release channel (16) connect successively; The detection zone (8) of base plate is connected by liquid release channel (16) with cleaning fluid storage pool (9) and luminous substrate liquid storage pool (10) respectively;
Described labelled antibody storage pool (5) stores pre-packaged enzyme or luminous agent marks anti-cTnI antibody, magnetic particle Bao Bei district (7) bag is by the anti-cTnI antibody of pre-packaged magnetic particle marker, and cleaning fluid storage pool (9) and luminous substrate liquid storage pool (10) store pre-packaged cleaning fluid and luminous substrate liquid; In described micro-fluidic chip testing process, with magnet manipulation magnetic particle moving or gathering; Described labelled antibody storage pool, cleaning fluid storage pool and luminous substrate liquid storage pool are hydraulic seal pond, the partial fracture by external force extruding, releasing liquid; Described filtering area comprises hemofiltration film, and described top board (1) seals with base plate (2) adhesive tape (19 and 20).
2. micro-fluidic chip as claimed in claim 1, should separate when it is characterized in that the luminous substrate liquid shelf-life is less than 1 year, substitute luminous substrate liquid storage pool (10) with luminous substrate liquid storage pool A (23) and luminous substrate liquid storage pool B (24), described luminous substrate liquid storage pool A (23) is connected by pre-mixing passages (25) with luminous substrate liquid storage pool B (24).
3. chip as claimed in claim 1, is characterized in that, the magnetic particle size that the anti-cTnI antibody of described magnetic particle marker uses is 0.1 ~ 10 μm; The magnet magnetic induction intensity mated with magnetic bead is 500 ~ 30000 Gausses.
4. chip as claimed in claim 3, is characterized in that, the magnetic particle size that the anti-cTnI antibody of described magnetic particle marker uses is 0.5 ~ 3 μm, and the magnet magnetic induction intensity mated with magnetic bead is 1000 ~ 8000 Gausses.
5. micro-fluidic chip as claimed in claim 1, it is characterized in that, described enzyme or luminous agent mark cTnI antibody-solutions, magnetic particle marker cTnI antibody-solutions and cleaning fluid all comprise buffer solution, protein, surfactant and anticorrisive agent, and luminous agent mark cTnI antibody-solutions also comprises glycerine, magnetic particle marker cTnI antibody-solutions also comprises carbohydrate.
6. the micro-fluidic chip as described in claim 1 or 5, is characterized in that, described enzyme labeling cTnI antibody-solutions comprises the borate buffer of bovine serum albumin(BSA) (BSA), Tween-20 and Proclin300; Magnetic particle marker cTnI antibody-solutions comprises the borate buffer of BSA, glucose, Tween-20 and Proclin300; Described cleaning fluid comprises the borate buffer of BSA, Tween-20 and Proclin300.
7. micro-fluidic chip as claimed in claim 1, is characterized in that, described luminous agent mark cTnI antibody-solutions comprises the phosphate buffer of BSA, glycerine, Tween-20, triton x-100 and Sodium azide; Described magnetic particle marker cTnI antibody-solutions comprises the phosphate buffer of BSA, casein, sucrose, Tween-20, triton x-100 and Sodium azide; Described cleaning fluid comprises the phosphate buffer of BSA, polysorbas20, triton x-100, polyethylene glycol and Sodium azide.
8. micro-fluidic chip as claimed in claim 1, it is characterized in that, described luminous substrate liquid comprises the substrate corresponding with enzyme and luminescence enhancement liquid, rear injection luminous substrate liquid storage pool (10) can be merged, or inject luminous substrate liquid storage pool A (23) and luminous substrate liquid storage pool B (24) respectively.
9. micro-fluidic chip as claimed in claim 1, it is characterized in that, described luminous substrate liquid comprises hydrogen peroxide solution corresponding to luminous agent and alkaline solution, rear injection luminous substrate liquid storage pool (10) can be merged, or inject luminous substrate liquid storage pool A (23) and luminous substrate liquid storage pool B (24) respectively.
10. the magnetic microparticle chemiluminescence micro-fluidic chip that quantitatively detects of cardiac muscle troponin I, it is characterized in that, the testing process of described micro-fluidic chip comprises:
Step 1) sample is instilled adding mouth after, cover lid, micro-fluidic chip puts into necessary instrument, after enzyme or luminous agent labelled antibody discharge, air pump makes sample and labelled antibody mix, and then injects base plate filtering area, described necessary instrument is small portable device, comprise extruding air pump and storage pool, the functions such as magnet moves, luminescent detection system;
Step 2) sample is after filtration behind district, arrive Bao Bei district, dissolve magnetic labeling antibody, after abundant reaction, magnet collects magnetic particle, and storage pool release cleaning fluid, after magnetic particle cleaning, move to detection zone, release luminous substrate liquid, instrument detection system detects luminous signal intensity, and then realizes the quantitative detection of cTnI.
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