CN205650213U - Myoglobin quantitative determination's magnetic particle chemiluminescence micro -fluidic chip - Google Patents
Myoglobin quantitative determination's magnetic particle chemiluminescence micro -fluidic chip Download PDFInfo
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Abstract
The utility model provides a myoglobin quantitative determination's magnetic particle chemiluminescence micro -fluidic chip, its structure mainly includes cover plate (1) and film (11), wherein air pump (3) on cover plate (1), air current microchannel (5), sample port (2), and reservoir (4) are deposited to liquid sample circulation way (6), a biological marker, and little blender (7) and transition district (10) connect gradually, filter (12) on film (11), pond (14) is washd in reaction tank (13), detects pond (15), and solution release channel (18) connect gradually, detection pond (15) on film (11) are deposited reservoir (16) and luminous liquid through solution release channel (18) and washing liquid and are deposited reservoir (17) and be connected, cover plate (1) and film (11) is sealed with sticky tape (20 and 22), the utility model provides a sensitivity of micro -fluidic chip testing result is high, and is accurate reliable, and the good reproducibility is with low costs.
Description
Technical field
The present invention relates to clinical medical inspection field; it is specifically related to the magnetic microparticle chemiluminescence micro-fluidic chip of a kind of Myoglobin detection by quantitative, it is possible to realizing the detection by quantitative of Myoglobin in biological sample in several minutes, this detection method has simple to operate; highly sensitive, the feature such as low cost.
Background technology
The associated proteins that Myoglobin (myoglobin, MYO, Mb) is made up of a peptide chain and a prosthetic heme group, is the protein of intramuscular storage oxygen, and its oxygen saturation curve is hyperbolic-type.Delivering the protein of oxygen in muscle, be made up of 153 amino acid residues, molecular weight is 16.7KD, containing haemachrome, and hemoglobin homology, and the binding ability of oxygen is between hemoglobin and cytochrome oxidase, and myocyte can be helped to mitochondrion by oxygen transport.Myoglobin is composition skeletal muscle and the main protein of cardiac muscle, when muscle injury, can drain in blood circulation from muscular tissue, make serum myoglobin concentration increase.Therefore, this index is used for judging whether muscle injury.In healthy human body, normal value, male 20～80 μ g/L；Women 10～70 μ g/L；Acute myocardial infarction early stage, acute muscle injury, muscular dystrophy, amyotrophy, polymyositis, acute or chronic renal failure, severe congestive heart failure and long-term shock etc. all may cause myoglobin content to raise.Measure early stage that serum myoglobin can diagnose as acute myocardial infarction (AMI) the sensitiveest index.But poor specificity, the disease such as Skeletal muscle injury, wound, renal failure, all may result in it and raise.Though the Myo positive can not make a definite diagnosis AMI, but can be used for getting rid of in early days the important indicator of AMI diagnosis.
The main method being currently used for detecting Myoglobin is euzymelinked immunosorbent assay (ELISA) (enzyme-linked immunosorbent assay, ELISA) and chemiluminescence immunoassay (chemiluminescence immunoassay, CLIA).ELISA, then owing to differing greatly between method, causes result heterogeneity, and the range of linearity is narrower, operation complexity, is unsuitable for doing single part or the detection of a small amount of specimen.It is continue Enzyme-multiplied immune technique and to put the emerging technology grown up after immune technology the eighties that chemiluminescence rises in eighties of last century, due to its high sensitivity, high specific, method is easy, quickly simultaneously, labelling conjugate is stable, the features such as the damage of "dead" isotope and pollution, were developed rapidly in recent years.Chinese patent (CN 103033627 A), disclose a kind of human muscle hemoglobin enzymatic chemical luminous immune detection method, specific practice is: on the polystyrene micropore plate being fixed with anti-human Myoglobin monoclonal antibody, add the anti-human Myoglobin monoclonal antibody of sample to be detected and horseradish peroxidase-labeled, hatch certain time, through washing, add luminescence reagent, luminous value is measured at Chemiluminescence Apparatus, according to the relation between antigen concentration and luminous intensity, draw the content of Myoglobin in measuring samples.This method is highly sensitive for the detection by quantitative of Myoglobin, good stability, but complex operation step during detection, needing professional to operate, be not suitable for middle and small hospital and different medical unit, the detection time is long simultaneously, it is impossible to for timely quick diagnosis.
In recent years, bioassay technique field has obtained quick development, occurs in that the most important research direction.Microfluidic chip analysis technology is the most most active one, all obtains in scientific research and practical application area and payes attention to widely.Micro-fluidic chip, as a kind of novel analysis test platform, has the advantages such as high flux, operation integrated, portable, easy, low cost, has been widely used in various fields, has especially shown up prominently in immunoassay field.
Surface-functionalized magnetic microsphere, as solid phase carrier, can be used to effectively capture nucleic acid, protein molecular, virion even cell, the field such as clinical diagnosis being widely used in various biochemical indicator.And micro-fluidic chip system has the features such as quick, efficient, integrated, both combine, a kind of novel high-performance detection method will be become, low to solve sensitivity present in current detection method, detection process is complicated, the problem being difficult to trace sample detection, is expected to promote Clinical detection instrument to portability and miniaturization further.
The biological micro-fluidic chip of immunomagnetic beads is by magnetic granule technology, a kind of analyzing detecting method that immunoassay is integrated on micro-fluidic chip, the Major Difficulties of current this comprehensive detection method shows themselves in that 1) liquid is in the Based Intelligent Control of chip internal microfluidic, at present frequently with method be that multiple Micropump and micro-valve are set at chip internal so that micro-fluidic system becomes more to complicate；2) undercompounding of reaction system, causes reacting insufficient；3) integration degree is the highest, causes non-specific background high.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, propose the magnetic microparticle chemiluminescence micro-fluidic chip of a kind of Myoglobin detection by quantitative, Clinical Laboratory personnel only can need to quickly realize the detection by quantitative of myoglobin concentration in sample through simple operations in several minutes.Testing result is highly sensitive, accurately and reliably, reproducible, low cost.
The technical scheme that the technical problem that the present invention relates to is used the following is:
A kind of magnetic microparticle chemiluminescence micro-fluidic chip of Myoglobin detection by quantitative; it is characterized in that; described microfluidic chip structure mainly includes cover plate (1) and egative film (11); wherein cover plate (1) overhead air pump (3); air-flow microchannel (5); adding mouth (2); sample fluid course (6); first biomarker storage pool (4), micro-mixer (7) and transition region (10) are sequentially connected with；The upper filter (12) of egative film (11), reaction tank (13), service sink (14), detection cell (15), solution release channel (18) is sequentially connected with；The upper detection cell (15) of egative film (11) is connected with cleanout fluid storage pool (16) and luminescent solution storage pool (17) by solution release channel (18)；
Described first biomarker storage pool (4) stores pre-packaged enzyme or luminous agent labelling anti-Myoglobin antibody-solutions；Described reaction tank (13) stores pre-packaged magnetic particle marker anti-Myoglobin antibody；Described cleanout fluid storage pool (14) and and luminescent solution storage pool (17) store pre-packaged cleanout fluid and luminescent solution respectively；Described cover plate (1) and egative film (11) adhesive tape (20 and 22) seal；In described micro-fluidic chip testing process, move with Magnet manipulation magnetic granule or assemble.
Specifically, the first biomarker storage pool (4) of the present invention, cleanout fluid storage pool (14) and luminescent solution storage pool (17) volume are 10～500 μ l, and preferably 10～300 μ l, for liquid capsule or cavity；The micro-mixer of described micro-fluidic chip be width be 20～300 μm, the degree of depth is the snakelike of 10～100 μm, fold-line-shaped or square structure；The filter of described micro-fluidic chip is made up of cavity and hemofiltration film.
Preferably, the micro-mixer width of described micro-fluidic chip is 150 μm, and the degree of depth is the square structure of 50 μm, under external pressure effect, sample and reagent can be made to be sufficiently mixed, and improves reaction efficiency.
Preferably, the cavity volume of the filter of described micro-fluidic chip be 3～10 times of sample volume, preferably cavity volume be 4～6 times of sample volume.
Specifically, in described micro-fluidic chip egative film, the hemofiltration membrane material in filter can be glass fibre membrane, polyester fiber film or CytoSep film etc., it is preferable that using glass fibre membrane as hemofiltration film.
Specifically, the filter of micro-fluidic chip of the present invention has outside the function filtering sample impurity, it is also possible to liquid leads into next stage micro structure and microchannel.
Specifically, the reaction tank of micro-fluidic chip of the present invention is a capillary microchannels, and this capillary microchannels is tubular conduit or rectangular channel, it is allowed to micro-liquid flows to or passes through.
Preferably, described reaction tank is tubular conduit, a diameter of 0.5～10mm；More preferably a diameter of 5mm of microchannel, further preferred 2mm or 1mm.
Preferably, described reaction tank is rectangular channel, a width of 0.1～5mm, and the degree of depth is 0.01～2mm, a length of 5～40mm.
Specifically, the volume of capillary channel of the present invention is less than 150 μ l, as preferably, the volume of capillary channel is less than 100 μ l, and further preferably, capillary channel volume is less than 50 μ l, even better less than 30 μ l, even better less than 20 μ l, such as less than 15 μ l, it is even less than 5 μ l less than 10 μ l.
Specifically, the magnetic granule that magnetic particle marker of the present invention anti-Myoglobin antibody uses is supperparamagnetic particles, can be paramagnetic Fe3O4Or γ-Fe2O3Compound, preferably Fe3O4Compound, magnetic grain diameter is 0.1～10 μm.Preferably magnetic grain diameter be 1～3 μm, more preferably particle diameter be the magnetic granule of 2.0 μm.
Specifically, in addition to above-mentioned main microchannel and micro structure, also have many air-vents and for assembling fixing resigning hole and column in micro-fluidic chip cover plate of the present invention and egative film.
Specifically, the air pump (3) on described micro-fluidic chip cover plate mainly transmits pressure by gas channel (5), and Main Function is for sample and the mixing of the first biomarker, improves one-level and hatches effect.
Specifically, the gas channel of micro-fluidic chip of the present invention a size of 0.1～100 μm, further preferred 2～50 μm.
Specifically, the transition region of micro-fluidic chip cover plate of the present invention is cover plate and the hinge of egative film connection, and first order reaction mixture is through transition region inflow filter, it is achieved that liquid is in the flowing of the fluctuating plate interlayer of micro-fluidic chip.
Need inside the reaction tank of micro-fluidic chip of the present invention to carry out certain surface modification treatment, described surface modifying treatment can be chemical reaction, face coat, plasma treatment etc., thus obtain good hydrophilic, liquid sample is promoted to flow under capillary force, Fast Filling microchannel.
Heretofore described enzyme, comprises but is not limited to catalase (HRP) and alkali phosphatase (ALP).Luminous substrate liquid is the luminous substrate (such as luminol or diamantane (obsolete)) and luminescence enhancement liquid (such as reinforcing agents such as benzene derivatives) that enzyme is corresponding, wherein luminous substrate and luminescence enhancement liquid can merge, and inject luminous substrate liquid storage pool (17) as shown in Figure 2 after mix homogeneously；But should separate when the mixed liquor shelf-life was less than 1 year, be injected separately into luminous substrate liquid storage pool (17) and luminous substrate liquid storage pool (24) as shown in Figure 4, by pre-mixing passages (23) mix homogeneously.
The assembling of micro-fluidic chip of the present invention, cover plate and egative film are binded by sealant tape, and sealant tape can be common double faced adhesive tape, and heat-sensitive glue and pressure sensitive adhesive etc. are heat-sensitive glue or pressure sensitive adhesive as preferred sealant tape.
Specifically, cleanout fluid of the present invention, it is used for cleaning magnetic granule, removes the impurity substances having neither part nor lot in reaction.Cleanout fluid mainly comprises buffer system, protein, surfactant and preservative, and wherein buffer system is including but not limited to borate, phosphate, Tris-HCl and acetate etc..Wherein protein is including but not limited to bovine serum albumin, casein etc.；Wherein surfactant is including but not limited to polysorbas20, Tween 80, triton x-100, Polyethylene Glycol and polyvinyl pyrrolidone etc..
The present invention provides a kind of method using above-mentioned micro-fluidic chip to detect sample to be tested, and concrete operation step includes:
Step 1) add sample from adding mouth, micro-fluidic chip is put in necessary instrument, after the first biomarker storage pool release enzyme or luminous agent labelling anti-Myoglobin antibody-solutions, pressing air pump, air-flow advances sample to flow through sample fluid course by air-flow microchannel, mix homogeneously reaction with enzyme or luminous agent labelling anti-Myoglobin antibody-solutions at micro-mixer, form a kind of immune complex, then flow into transition region and enter in base plate filter；
Step 2) sample is after filter, arrive reaction tank, redissolution is coated on the magnetic particle marker anti-Myoglobin antibody in this region, and react therewith, then Magnet collects magnetic granule and mobile to service sink, cleanout fluid is discharged from cleanout fluid storage pool, magnetic granule is after fully washing, it is displaced downwardly to detection cell in Magnet effect, luminous substrate liquid is discharged from luminescent solution storage pool, according to the proportionate relationship between relative luminous intensity (RLU) and Myoglobin antigen concentration, detecting system can convert automatically, can fast report test result, thus realize the detection by quantitative of Myoglobin.
Specifically, the detection sample used by the present invention includes that the whole blood from human body, serum and blood plasma, sample volume used are 5～200 μ l, preferably 150 μ l, further preferred 50 μ l.
Specifically, described necessary instrument is small portable device, and described equipment mainly includes controlling device and detection device.
Specifically, the control device of described supporting small portable device primarily serves the purpose of can be implemented the micro-fluidic chip placing its inside to comprise control liquid flowing, sample mixes, reagent in release storage pool, Magnet moves, primarily serving the purpose of collection luminous signal and being analyzed it of detection device, finally shows testing result.
Specifically, described magnet movement is that the speed of uniform motion is 1mm/s～50mm/s, and preferably magnet movement speed is 2mm/s～30mm/s relative to the linear uniform motion of reaction tank or accelerated motion or retarded motion.
Specifically, the device that controls of portable equipment of the present invention primarily serves the purpose of the mixing that micro-fluidic chip enforcement extruding air pump realizes sample and biomarker, control that the motion of Magnet realizes magnetic microsphere traget antibody and first order reaction thing is sufficiently mixed the collection with magnetic bead, the motion controlling Magnet realizes reactant mixture successively at reaction tank, service sink, movement between detection cell, effectively reduces nonspecific interference.
The preparation method of micro-fluidic chip of the present invention is as follows:
Step 1) enzyme or luminous agent labelling anti-Myoglobin antibody, magnetic particle marker anti-Myoglobin antibody, both traget antibodies can be same antibody or different antibodies；
Step 2) enzyme or luminous agent traget antibody solution are put in the traget antibody storage pool of cover plate, seal, magnetic particle marker antibody-solutions is put in the reaction tank of egative film, it is dried, cleanout fluid and luminous substrate liquid are injected separately in cleanout fluid storage pool and luminous substrate liquid storage pool, seal, with adhesive tape (20 and 22) sealed cover slip and egative film, and assemble one complete micro-fluidic chip of formation.
Compared with prior art, obtained has the beneficial effect that the present invention
1) without sample being carried out the pretreatment work of complexity before test, and sample type is suitable for the most in the method without limitation, whole blood, serum, blood plasma；
2) magnetic immunological technique is integrated on micro-fluidic chip, combines the advantage of two kinds of technology, make whole detection process have simple to operate, result accurately and reliably, highly sensitive feature.
3) small-sized detection equipment and the simple process of chip internal are coordinated, it is not necessary to complicated pump valve and electric field just can realize the Based Intelligent Control of liquid stream effectively inside micro-fluidic chip.
4) there is multistep mixed function, immunoreation efficiency can be improved to a certain extent.
5) the various reactions on micro-fluidic chip, cleaning and the process subregion of detection is carried out, integration degree is high, can effectively reduce nonspecific interference, improves the sensitivity of detection.
6) use micro-fluidic chip involved in the present invention to detect, it is possible to fast report testing result, the other detection of bed and various portable equipments can be further used for.
The core technology of the present invention is magnetic particle technology, chemiluminescence immunoassay detection technique and microfluidic chip technology three to be combined, and wherein the kind of magnetic granule, size, channel shape, the isoparametric trickle change of size all can the accuracys of extreme influence testing result.The present invention passes through Fine design and the processing of micro-fluidic chip, and coordinate small portable device, it is successfully realized microfluid in the Based Intelligent Control within micro-fluidic chip, make magnetic particle can realize controlled motion inside micro-fluidic chip by the effect of Magnet, mixing, collect and clean, improve reaction efficiency, reduce nonspecific interference, thus enhance detection signal, improve detection sensitivity.The simple superposition of the technology of the present invention not three, but it is integrated with the advantage of three, a kind of new method for Myoglobin fast quantification improved on the basis of prior art.
Accompanying drawing explanation
Fig. 1 is the cover plate structural representation of micro-fluidic chip, and wherein 2 is adding mouth, and 3 is air pump, and 4 is the first biomarker storage pool, 5 is gas channel, and 6 is sample fluid course, and 7 is micro-mixer, 8 is liquid storage tank resigning hole, and 9 is magnet movement chute, and 10 is transition region.
Fig. 2 is the chassis construction schematic diagram of micro-fluidic chip, and wherein 12 is filter, and 13 is reaction tank, and 14 is service sink, and 15 is detection cell, and 16 is cleanout fluid storage pool, and 17 is luminescent solution storage pool, and 18 is cleanout fluid and luminescence reagent release channel, and 19 is devil liquor recovery pond.
Fig. 3 is the complete structure schematic diagram of micro-fluidic chip, and wherein 1 is cover plate, and 11 is egative film, and 20 is top adhesive tape, and 22 is bottom tape.
Fig. 4 is the chassis construction schematic diagram of three liquid storage pools, and wherein 16 is cleanout fluid storage pool, and 17 and 24 is luminescent solution storage pool, and 18 is cleanout fluid, and 23 is luminescent solution release channel.
Detailed description of the invention:
The invention discloses the magnetic microparticle chemiluminescence micro-fluidic chip of a kind of Myoglobin detection by quantitative, those skilled in the art can use for reference present disclosure, is suitably modified technological parameter and realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are considered as being included in the present invention.Method and the application of the present invention are described by preferred embodiment, method described herein and application substantially can be modified in without departing from present invention, spirit and scope or suitably change and combine by related personnel, realizes and applies the technology of the present invention.
In order to make those skilled in the art be more fully understood that technical scheme, below against accompanying drawing and combine preferred embodiment the present invention is explained in detail.
Embodiment 1: horseradish peroxidase-luminol (HRP-luminol) system is for the detection of Myoglobin
1. the making of micro-fluidic chip
1) antibody labeling: i) enzyme labelled antibody: weigh 5mg HRP and be dissolved in 1ml distilled water；Adding 0.1M NaIO 4 solution that 0.2ml newly joins in upper liquid, under room temperature, lucifuge stirs 20 minutes；Being loaded by above-mentioned solution in bag filter, dialyse the sodium-acetate buffer of 1mM pH4.4,4 DEG C overnight；Adding 20 μ l 0.2M pH9.5 carbonate buffer solutions, make the pH of above hydroformylation HRP be increased to 9.0～9.5, add 10mg Myoglobin antibody immediately after, in 1ml 0.01M carbonate buffer solution, room temperature lucifuge is gently mixed 2 hours；Add the 4mg/ml NaBH4 liquid that 0.1ml newly joins, mixing, then put 4 DEG C 2 hours；Being loaded by above-mentioned liquid in bag filter, dialyse 0.15M pH7.4PBS, 4 DEG C overnight；Under agitation it is added dropwise over equal-volume saturated ammonium sulfate, puts 4 DEG C 1 hour；3000rpm is centrifuged half an hour, abandons supernatant；Precipitate semi-saturation ammonium sulfate washes secondary, and last precipitate is dissolved in the PBS of a small amount of 0.15M pH7.4；Being loaded by above-mentioned solution in bag filter, dialyse the PBS of 0.15M pH7.4, after removing ammonium ion, 10,000rpm are centrifuged 30 minutes removal precipitations, and supernatant is enzyme conjugates, after subpackage, and 2-4 DEG C of preservation.Ii) magnetic labeling antibody: accurately pipetting the marked by streptavidin magnetic bead that 30 μ l concentration are 1mg/ml, wherein this functionalization magnetic granule is with Fe3O4For core, polystyrene is shell, and particle diameter is 2.0 μm, in 2ml centrifuge tube, and vortex concussion 30min.Add the biotin labeled human muscle hemoglobin two that 30 μ l concentration are 10 μ g/ml to resist, incubated at room temperature 2 hours.Magnetic separator removes the liquid in magnetic bead, cleans 3 times with 0.01M PBS (containing 0.01%BSA/0.2%NaN3pH 7.4), magnetic bead is suspended in 0.01M PBS, overnight incubation, repeats above-mentioned separation, cleaning step, be finally diluted to prescribed concentration.
2) being coated of magnetic labeling antibody: use point sample instrument or specking instrument by 10 μ l magnetic microsphere point samples in the reaction tank of micro-fluidic chip, drying at room temperature more than 30 minutes.
3) assembling of micro-fluidic chip: by the HRP that 10 μ l concentration are 3 μ g/ml, 300 μ l cleanout fluid, 200 μ l luminescence reagents are individually enclosed in each storage pool of reagent card, are assembled by chip card miscellaneous part simultaneously, form a complete micro-fluidic chip.
4) storage: 4 DEG C, humidity is preservation under the conditions of 50%.
2. the detection by quantitative of Myoglobin
1) making of standard curve: Myoglobin standard substance calf serum is diluted, being made into concentration is 0ng/ml, 10ng/ml, 25ng/ml, 50ng/ml, 150ng/ml, the each 150 μ l of Myoglobin standard substance of 400ng/ml, take 30 μ l and add the adding mouth of the micro-fluidic chip of preparation in the embodiment of the present invention, cover blood lid, test kit is placed in supporting equipment, places 15min.Replication 3 times respectively of each sample.Reader shows myoglobin content automatically, according to concentration and the corresponding luminous intensity of Myoglobin, takes three times and measures meansigma methods, draw standard curve.
2) taking sample 200 μ l to be checked, be placed in the embodiment of the present invention in made micro-fluidic chip and detect, every sub-sampling 30 μ l, replication 3 times, according to 1) in the standard curve drawn obtain the concentration of Myoglobin in measuring samples.
3) result shows: using the method for the invention, the detection drawn is limited to 1ng/ml, and detection range is 0.5ng/ml-1000ng/ml, and between batch, CV is less than 10%, and in batch, CV is less than 5%.
Embodiment 2: alkali phosphatase-diamantane (obsolete) (ALP-AMPPD) system is for the detection of Myoglobin
1. the making of micro-fluidic chip
1) antibody labeling: i) enzyme labelled antibody: 2.5mg ALP (50IU/mg), adds the PB (pH6.8) of the 200 μ l 100mM containing 1.25% glutaraldehyde, and mixing, room temperature reaction is overnight；Under the conditions of 4 DEG C, electromagnetic stirr, dialysis, to 50mM PBS (pH7.2), 12 hours, changes liquid 4 times；1.5mg Myoglobin antibody is dissolved in the carbonate solution (pH9.0) of 100 μ l 1M；The AP of activation is added in the protein fluid prepared, mixing, react 24 hours under the conditions of 4 DEG C, add the lysine solution of 10 μ l 200mM, mixing, react 2 hours under the conditions of 22 DEG C；Dialyse under the conditions of 4 DEG C to 50mM PBS (pH7.2), 12 hours, change liquid 4 times；Centrifugal, take supernatant, use 50mM TB7.4+0.6%BSA+0.05%NaN3Dilution desired concn ,-20 DEG C of preservations.Ii) magnetic labeling antibody: accurately pipetting the marked by streptavidin magnetic bead that 30 μ l concentration are 1mg/ml, wherein this functionalization magnetic granule is with Fe3O4For core, polystyrene is shell, and particle diameter is 2.0 μm, in 2ml centrifuge tube, and vortex concussion 30min.Add the biotin labeled human muscle hemoglobin two that 30 μ l concentration are 10 μ g/ml to resist, incubated at room temperature 2 hours.Magnetic separator removes the liquid in magnetic bead, with 0.01M PBS (containing 0.01%BSA/0.2%NaN3PH 7.4) clean 3 times, magnetic bead is suspended in 0.01M PBS, overnight incubation, repeats above-mentioned separation, cleaning step, be finally diluted to prescribed concentration.
2) being coated of magnetic labeling antibody: use point sample instrument or specking instrument by 10 μ l magnetic microsphere point samples in the reaction tank of micro-fluidic chip, more than drying at room temperature 30min.
3) assembling of micro-fluidic chip: by the HRP that 10 μ l concentration are 3 μ g/ml, 300 μ l cleanout fluid, 200 μ l luminescence reagents are individually enclosed in each storage pool of reagent card, are assembled by chip card miscellaneous part simultaneously, form a complete micro-fluidic chip.
4) storage: 4 DEG C, humidity is preservation under the conditions of 50%.
2. the detection by quantitative of Myoglobin
1) making of standard curve: Myoglobin standard substance calf serum is diluted, being made into concentration is 0ng/ml, 10ng/ml, 25ng/ml, 50ng/ml, 150ng/ml, the each 150 μ l of Myoglobin standard substance of 400ng/ml, take 30 μ l and add the sample application zone of the test kit of preparation in the embodiment of the present invention, cover blood lid, test kit is placed in supporting equipment, places 15 minutes.Replication 3 times respectively of each sample.Reader shows myoglobin content automatically, according to concentration and the corresponding luminous intensity of Myoglobin, takes three times and measures meansigma methods, draw standard curve.
2) taking sample 200 μ l to be checked, be placed in the embodiment of the present invention in made micro-fluidic chip and detect, every sub-sampling 30 μ l, replication 3 times, according to 1) in the standard curve drawn obtain the concentration of Myoglobin in measuring samples.
3) result shows: using the method for the invention, the detection drawn is limited to 0.05ng/ml, and detection range is 0.01ng/ml-1500ng/ml, and between batch, CV is less than 8.5%, and in batch, CV is less than 2.4%.
Embodiment 3: magnetic particle particle size is screened
The particle diameter of magnetic microsphere is little, and specific surface area is big, and active group is contained on surface, therefore coupling capacity is big, but magnetic particle size is too small is unfavorable for that Magnet is collected, therefore carries out magnetic particle size selection.
Other experiment condition sees embodiment 2, and the size of magnetic particle granule is carried out according to below scheme.
Particle size is selected to be respectively 0.1 μm, 0.5 μm, 1.8 μm, 2 μm, 3 μm, 10 μm magnetic particle marker anti-c reactive protein antibody.Use magnetic size through preferred permanent magnet, fixed magnet height during detection.
Experimental result is as follows:
Magnetic particle grain size strengthens successively from 0.1 μm, 0.5 μm, 1.8 μm, 2 μm, 3 μm, and 3 μm interference strengthen, and 10 μm start to reduce, and signal value is minimum, and comprehensive each factor 1.8 μm signal is the strongest, interference minimum.
Interpretation of result: when magnetic particle size is less, specific surface area is relatively big, and the biotinylated molecular weight that surface is loaded is big, can be well dispersed in solution simultaneously, but magnetic microsphere to be ensured fully to be collected required magnetic field intensity big.Magnetic field force suffered by magnetic bead is it cannot be guaranteed that it is fully collected in the present embodiment, causes part effectively magnetic bead to run off in cleaning process, thus causes final detected signal value the highest.When magnetic grain diameter is bigger, specific surface area is little, the mark rate of surface biomolecules is relatively low, under the same terms, magnetic particle institute is magnetic field force induced, and scattered magnetic bead can sufficiently be collected greatly, but it is owing to easily settling, cause between biomolecule, reacting insufficient, thus weaken luminous signal.Considering, particle diameter is that 1～3 μm magnetic microsphere effects are preferable, and therefore in embodiments of the invention, the magnetic microsphere effect of 1.8 μm is best.
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 regarded as protection scope of the present invention.
Claims (7)
1. the magnetic microparticle chemiluminescence micro-fluidic chip of a Myoglobin detection by quantitative; it is characterized in that; described microfluidic chip structure mainly includes cover plate (1) and egative film (11); the wherein air pump (3) on cover plate (1); air-flow microchannel (5); adding mouth (2); sample fluid course (6); first biomarker storage pool (4), micro-mixer (7) and transition region (10) are sequentially connected with；Filter (12) on egative film (11), reaction tank (13), service sink (14), detection cell (15), solution release channel (18) is sequentially connected with；Detection cell (15) on egative film (11) is connected with cleanout fluid storage pool (16) and luminescent solution storage pool (17) by solution release channel (18)；
Described first biomarker storage pool (4) stores pre-packaged enzyme or luminous agent labelling anti-Myoglobin antibody-solutions；Described reaction tank (13) stores pre-packaged magnetic particle marker anti-Myoglobin antibody；Described cleanout fluid storage pool (16) and luminescent solution storage pool (17) store pre-packaged cleanout fluid and luminescent solution respectively；Described cover plate (1) and egative film (11) adhesive tape (20 and 22) seal.
2. micro-fluidic chip as claimed in claim 1, it is characterised in that described first biomarker storage pool (4), cleanout fluid storage pool (16) and luminescent solution storage pool (17) volume are 10～500 μ l, for liquid capsule or cavity；The micro-mixer of described micro-fluidic chip be width be 20～300 μm, the degree of depth is fold-line-shaped or the square structure of 10～100 μm；The filter of described micro-fluidic chip is made up of cavity and hemofiltration film；The reaction tank of described micro-fluidic chip is capillary microchannels, and this capillary microchannels is tubular conduit or rectangular channel, it is allowed to micro-liquid flows to or passes through.
3. micro-fluidic chip as claimed in claim 2, it is characterised in that described reaction tank is tubular conduit, a diameter of 0.5～10mm.
4. micro-fluidic chip as claimed in claim 2, it is characterised in that described reaction tank is rectangular channel, a width of 0.1～5mm, and the degree of depth is 0.01～2mm, a length of 5～40mm.
5. micro-fluidic chip as claimed in claim 1, it is characterised in that described magnetic granule is supperparamagnetic particles, for Fe3O4Or γ-Fe2O3Compound, magnetic grain diameter is 0.1～10 μm.
The magnetic microparticle chemiluminescence micro-fluidic chip of a kind of Myoglobin detection by quantitative the most as claimed in claim 1, it is characterised in that described sample volume is 5～200 μ l.
The magnetic microparticle chemiluminescence micro-fluidic chip of a kind of Myoglobin detection by quantitative the most as claimed in claim 1; it is characterized in that; described chip also comprises necessary instrument, and described necessary instrument is small portable device, and described equipment mainly includes controlling device and detection device.
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| CN201520828637.XU CN205650213U (en) | 2015-10-26 | 2015-10-26 | Myoglobin quantitative determination's magnetic particle chemiluminescence micro -fluidic chip |
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| CN201520828637.XU CN205650213U (en) | 2015-10-26 | 2015-10-26 | Myoglobin quantitative determination's magnetic particle chemiluminescence micro -fluidic chip |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105195243A (en) * | 2015-10-26 | 2015-12-30 | 深圳华迈兴微医疗科技有限公司 | Magnetic particulate chemiluminescent micro-fluidic chip for quantitatively detecting myohemoglobin |
| CN109999931A (en) * | 2019-04-18 | 2019-07-12 | 天津诺迈科技有限公司 | Chemiluminescence detection micro-fluidic chip and application method, reagent cleaning method |
| CN110208528A (en) * | 2019-06-27 | 2019-09-06 | 深圳华迈兴微医疗科技有限公司 | A kind of micro-fluidic chip |
| WO2021092801A1 (en) * | 2019-11-13 | 2021-05-20 | 京东方科技集团股份有限公司 | Assay chip |
| CN113101985A (en) * | 2019-06-26 | 2021-07-13 | 京东方科技集团股份有限公司 | Detection chip and detection system |
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2015
- 2015-10-26 CN CN201520828637.XU patent/CN205650213U/en not_active Withdrawn - After Issue
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105195243A (en) * | 2015-10-26 | 2015-12-30 | 深圳华迈兴微医疗科技有限公司 | Magnetic particulate chemiluminescent micro-fluidic chip for quantitatively detecting myohemoglobin |
| CN105195243B (en) * | 2015-10-26 | 2017-10-31 | 深圳华迈兴微医疗科技有限公司 | The magnetic microparticle chemiluminescence micro-fluidic chip that a kind of myoglobins is quantitatively detected |
| CN109999931A (en) * | 2019-04-18 | 2019-07-12 | 天津诺迈科技有限公司 | Chemiluminescence detection micro-fluidic chip and application method, reagent cleaning method |
| CN109999931B (en) * | 2019-04-18 | 2023-08-11 | 天津诺迈科技有限公司 | Microfluidic chip for chemiluminescence detection, use method and reagent cleaning method |
| CN113101985A (en) * | 2019-06-26 | 2021-07-13 | 京东方科技集团股份有限公司 | Detection chip and detection system |
| CN110208528A (en) * | 2019-06-27 | 2019-09-06 | 深圳华迈兴微医疗科技有限公司 | A kind of micro-fluidic chip |
| WO2021092801A1 (en) * | 2019-11-13 | 2021-05-20 | 京东方科技集团股份有限公司 | Assay chip |
| CN113115587A (en) * | 2019-11-13 | 2021-07-13 | 京东方科技集团股份有限公司 | Detection chip |
| US11986821B2 (en) | 2019-11-13 | 2024-05-21 | Beijing Boe Health Technology Co., Ltd. | Detection chip |
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