CN205650215U - C reaction albumen quantitative determination's magnetic particle chemiluminescence micro -fluidic chip - Google Patents
C reaction albumen quantitative determination's magnetic particle chemiluminescence micro -fluidic chip Download PDFInfo
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Abstract
The utility model provides a C reaction albumen quantitative determination's magnetic particle chemiluminescence micro -fluidic chip, its structure mainly includes cover plate (1) and film (11), reservoir (4) are deposited to wherein sample port (2) on cover plate (1), liquid sample circulation way (6), a biological marker, and little blender (7), transition district (10) connects gradually, filter (12) on film (11), pond (14) is washd in reaction tank (13), detects pond (15), and solution release channel (18) connect gradually, detect pond (15) on film (11) and deposit reservoir (16) and luminous liquid through solution release channel (18) and washing liquid and deposit reservoir (17) and be connected, cover plate (1) and film (11) is with the sealed equipment of sticky tape (20 and 22), micro -fluidic chip detectivity is high, and the result is accurate reliable, the good reproducibility.
Description
Technical field
The present invention relates to clinical medicine in-vitro diagnosis field; it is specifically related to the magnetic microparticle chemiluminescence micro-fluidic chip of a kind of c reactive protein detection by quantitative, it is possible to realize, to the detection by quantitative of c reactive protein in biological sample, having simple to operate at short notice; highly sensitive, the feature such as low cost.
Background technology
C reactive protein (C-reaction protein, CRP) often occurs in acute inflammation patients serum, stem cell synthesize, be the non-specificity index of systemic inflammatory reaction.CRP content when infecting is significantly raised, and during acute inflammatory reaction, CRP is one of most sensitive protein of body, and is that body inflammatory reacts accurate objective indicator.CRP is one of independent risk factor of coronary heart disease, has important value in the prognosis of coronary heart disease；In acute myocardial ischemia and myocardial infarction, CRP content quickly raises.The CRP value of normal healthy people is the lowest, the normal Human C-reactiveprotein ＜ 1.0mg/l of 90%, after inflammation or acute tissue injury, the synthesis of CRP increased sharply in 4-6 hour, within 36-50 hour, peak, peak value is 100-1000 times of normal value, through positive rational therapy, within 3-7 days, can be rapidly reduced to normal value.
The detection of tradition CRP is based on Immunity transmission turbidity and Immune scatter turbidimetry, and detection range is 3-5mg/L, and these method sensitivity are the highest, it is difficult to for prediction and the diagnosis of the aspects such as infection of newborn disease and cardiovascular disease.In recent years, occur in that the new method of multiple detection CRP, such as immunofluorescence and Time-resolved Fluoimmunoassay, lowest detectable limit 0.0005-0.1mg/l, greatly improving the sensitivity of detection, CRP that these methods are measured is we term it super quick CRP (hs-CRP).Chinese patent (CN101769932) provides full-range C RP detection kit by using the styrene latex of different-grain diameter, not only can detect the CRP of low concentration but also the CRP of high concentration can be detected, the method will not be because of the accuracy of HOOK effects detection, but operation complexity, the detection time is long.Therefore, it is short to set up a kind of detection time, and detects except can be in addition to laboratory is carried out, and also requirement can be carried out by bed, is simultaneously suitable for the detection method of low value CRP, has great importance.
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, has been widely used in the clinical diagnosis field of 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
For the problems referred to above; the present invention proposes the magnetic microparticle chemiluminescence micro-fluidic chip of a kind of c reactive protein detection by quantitative; use simple method can realize the Based Intelligent Control of the internal microfluid of micro-fluidic chip; reaction liquid can be made to be sufficiently mixed simultaneously; ensure that reaction system is efficiently carried out, can quickly realize the detection by quantitative of CRP concentration in sample.Detection sensitivity is high, and result is accurately and reliably, reproducible.
The technical scheme that the technical problem of the present invention is used is as follows:
The magnetic microparticle chemiluminescence micro-fluidic chip of a kind of c reactive protein detection by quantitative, it is characterised in that described microfluidic chip structure mainly includes cover plate (1) and egative film (11)；The wherein adding mouth (2) on cover plate (1), sample fluid course (6), the first biomarker storage pool (4), micro-mixer (7), transition region (10) is 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, and 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 reaction tank (13) is coated the anti-c reactive protein antibody of magnetic particle marker；Described first biomarker storage pool (4), cleanout fluid storage pool (16) and luminescent solution storage pool (17) store pre-packaged reagent；Described cover plate (1) and egative film (11) seal with adhesive tape (20 and 22) and assemble.
Specifically, the volume of the first biomarker storage pool (4), cleanout fluid storage pool (16) and the luminescent solution storage pool (17) of described micro-fluidic chip is 10～500 μ l, for liquid capsule or cavity.
Specifically, the micro-mixer (7) 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 wave-shaped configuration.The most snakelike or square wave-shaped configuration, further preferred square wave-shaped configuration.
Preferably, micro-mixer is wide 150 μm, and the degree of depth is the square wave-shaped configuration of 50 μm, under outside pressures cycle effect, sample and reagent can be made to be sufficiently mixed, improve reaction efficiency.
Specifically, the filter (12) of described micro-fluidic chip is had figurate cavity by one and hemofiltration film forms；Described filter hemofiltration membrane material is glass fibre membrane, polyester fiber film or CytoSep film etc..Preferably glass fibre membrane is as hemofiltration film.Described cavity volume be 3～10 times of sample volume, preferably cavity volume be 4～6 times of sample volume.
The filter of micro-fluidic chip of the present invention is in addition to having the function filtering sample impurity, it is also possible to reaction liquid leads into next stage micro structure and microchannel.
Specifically, the capillary microchannels that reaction tank (13) is tubular conduit or rectangle of described micro-fluidic chip.
Specifically, the reaction tank (13) of described micro-fluidic chip is tubular conduit, a diameter of 0.5～10mm.
Preferably, a diameter of 5mm of tubulose microchannel, further preferred 2mm or 1mm.
Specifically, the reaction tank (13) of described micro-fluidic chip is rectangular channel, a width of 0.1～5mm, and the degree of depth is 0.01～2mm, a length of 5～40mm.
Preferably, rectangular channel a width of 0.3～2mm, the degree of depth is 0.2～1mm, a length of 5～20mm.
Specifically, magnetic granule of the present invention is hud typed supperparamagnetic particles, and magnetic core is Fe3O4Or γ-Fe2O3Compound, shell is polystyrene, and magnetic grain diameter is 0.1～10 μm.
Preferably, described magnetic grain diameter be 1～3 μm, more preferably particle diameter be the magnetic granule of 2.0 μm.
Specifically, described magnetic granule is nucleocapsid structure type, and magnetic microsphere surface is modified with various active functional group, such as-COOH ,-COH ,-NH2Deng, or other biological molecule, such as biotin, Streptavidin etc., preferred surface functionalization group is-COOH or biotin or Streptavidin.
The preparation method of described micro-fluidic chip is as follows:
Step 1) antibody labeling: enzyme or luminous agent labelling anti-c reactive protein antibody, magnetic particle marker anti-c reactive protein antibody, both antibody can same antibody or variety classes antibody；
Step 2) assembling of micro-fluidic chip: by enzyme or luminous agent traget antibody solution subpackage to the storage pool of cover plate, seal, magnetic particle marker antibody-solutions is coated the reaction tank to 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 miscellaneous part is assembled one complete micro-fluidic chip of formation simultaneously.
Microchannel and the processing technique of micro structure in micro-fluidic chip cover plate of the present invention and egative film include method of molding, pressure sintering, laser ablation method and soft lithography etc., in embodiments of the invention, preferred method of molding makes micro-fluidic chip, can effectively reduce cost of manufacture.
Need inside the reaction tank of micro-fluidic chip of the present invention to carry out certain surface modification treatment, described surface modifying treatment includes chemical reaction, face coat, plasma treatment etc., thus obtain good hydrophilic, liquid sample is promoted to flow under capillary force, Fast Filling microchannel, the magnetic labeling antibody of redissolution solidification.
Micro-fluidic chip testing process of the present invention is as follows:
Step 1) add sample from adding mouth (2), micro-fluidic chip is put in necessary instrument, after the first biomarker storage pool (4) release mark part, sample and tagged ligand enter mix homogeneously reaction in micromixer (7), form a kind of immune complex and pass through transition region (10), then flow in egative film filter (12)；
Step 2) sample is after filter, redissolution is coated on the anti-c reactive protein antibody of the magnetic particle marker of reaction tank (13), and react therewith, external magnet collects magnetic granule, after magnetic powder collection completes, service sink (14) it is transferred under external magnet effect, cleanout fluid storage pool (16) releasing liquid enters service sink (14) by solution release channel (18), magnetic granule is after fully washing, detection cell (15) it is transferred under external magnet effect, luminescent solution storage pool (17) releasing liquid enters detection cell (15) by solution release channel (18)；Measure relative luminous intensity (RLU), the quantitative test result of report c reactive protein.
The cover plate (1) of micro-fluidic chip of the present invention is upper also has air pump (3) and air-flow microchannel (5), and pressing air pump (3) causes air flow through air-flow microchannel (5) and drives sample by sample fluid course (6).
In addition to above-mentioned main microchannel and micro structure, also have many air-vents in micro-fluidic chip cover plate of the present invention and egative film, for getting rid of the bubble produced in liquid flow process, also have for assembling fixing resigning hole and column simultaneously.
Air pump (3) on micro-fluidic chip cover plate of the present invention 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 described micro-fluidic chip a size of 0.1～100 μm, further preferred 2～50 μm.
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 micro-fluidic chip fluctuating plate interlayer.
The number of the luminescent solution storage pool of micro-fluidic chip of the present invention, kind according to selected luminescence reagent can be one, two or three, as egative film during main luminescence reagent there are two luminescent solution storage pools using luminol as preferred one embodiment of the present of invention, be that main luminescence reagent egative film only needs a luminescent solution storage pool with diamantane (obsolete) in another embodiment.
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 (as to iodophenol or to reinforcing agents such as phenyl phenols) 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 luminescent solution storage pool (17) and luminescent solution 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.
Detection sample used by the present invention is predominantly from the whole blood of human body, and volume used is 150 μ l, preferably 100 μ l, further preferred 50 μ l, the most preferably 10 μ l.
Necessary instrument of the present invention is a small portable device, mainly comprises control device and detection device.
The device that controls of portable equipment of the present invention primarily serves the purpose of and micro-fluidic chip is implemented extruding air pump realizes sample and the first biomarker is sufficiently mixed, 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.
Cleanout fluid of the present invention, 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..
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；
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; not simple superposition magnetic microparticle chemiluminescence technology and microfluidic chip technology; the setting of magnetic granular size, the isoparametric trickle change of each channel shape, size all can cause analysis result accuracy to reduce.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 technology in technology involved in the present invention the most above-mentioned three, but it is integrated with the advantage of three, a kind of new method for c reactive protein Quantitative detection 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 release channel, 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 c reactive protein 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 c reactive protein
1. the making of micro-fluidic chip
1) antibody labeling: i) enzyme labelled antibody: weigh HRP 25mg and be dissolved in 1.25% glutaraldehyde solution, stand overnight in room temperature；Reacted enzymatic solution, through Sephadex G-25 chromatographic column, uses normal saline eluting, and flow speed control, at 1ml/min, collects brown effluent；To treat that anti-c reactive protein antibody 12.5mg normal saline dilution is added dropwise in HRP solution to 5ml, stirring；With 1M pH9.5 carbonic acid buffer 0.25ml, continue stirring 3 hours；Adding 0.2M lysine 0.25ml, after mixing, room temperature stands 2 hours；Under agitation it is added dropwise over equal-volume saturated ammonium sulfate, places 1 hour in 4 DEG C；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, (detect with Nai Shi reagent) after removing ammonium ion, 10000rpm is centrifuged 30 minutes and removes precipitation, 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 granule that 30 μ l concentration are 1mg/l, wherein this functionalization magnetic granule is with Fe3O4For core, polystyrene is shell, and particle diameter is 2.0 μm, and in 2ml centrifuge tube, vortex shakes 30 minutes.Add the biotin labeled CRP monoclonal antibody that 30 μ l concentration are 5mg/l, 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) pretreatment of micro-fluidic chip: using injection moulding to prepare cover plate and the egative film of micro-fluidic chip, respectively with ethanol, each ultrasonic cleaning of pure water 10 minutes, nitrogen dries up, and dustfree environment preserves, standby to make.
3) being coated of magnetic-particle: use point sample instrument or specking instrument by 10 μ l magnetic microsphere point samples in the reaction tank of chip, drying at room temperature more than 30 minutes.
4) assembling of micro-fluidic chip: by the HRP traget antibody that 10 μ l concentration are 3mg/l, 300 μ l cleanout fluid, 200 μ l luminol luminous agent A liquid and B liquid are individually enclosed in three storage pools of reagent card, are assembled by reagent card miscellaneous part simultaneously, form complete micro-fluidic chip.
The detection by quantitative of 2.C reactive protein
1) making of standard curve: by CRP standard substance employment serum-dilution, being made into concentration is 0mg/l, 1mg/l, 10mg/l, 20mg/l, 100mg/l, the each 150 μ l of CRP standard substance (setting 3 repetitions) of 200mg/l, take 30 μ l and add the micro-fluidic chip adding mouth of preparation in the embodiment of the present invention, cover blood lid, micro-fluidic chip is placed in supporting instrument, places 15 minutes.Replication 3 times respectively of each sample.Instrument is according to the concentration of CRP and corresponding luminous intensity, and the CRP content in report sample, takes three times and measure meansigma methodss, draw standard curve automatically.
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 CRP in measuring samples.
3. result shows: use detection method of the present invention, and the detection drawn is limited to 0.05mg/l, and detection range is 0.01mg/l-400mg/l, and between batch, CV is less than 12%, and in batch, CV is less than 10%.
Embodiment 2: alkali phosphatase-diamantane (obsolete) (ALP-AMPPD) system is for the detection of c reactive protein
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 c reactive protein 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, with 50mM TB pH7.4+0.6%BSA+0.05%NaN3Dilute 10 times ,-20 DEG C of preservations.Ii) magnetic labeling antibody: accurately pipetting the marked by streptavidin magnetic granule that 30 μ l concentration are 1mg/l, wherein this functionalization magnetic granule is with Fe3O4For core, polystyrene is shell, and particle diameter is 2.0 μm, and in 2ml centrifuge tube, vortex shakes 30 minutes.Add the biotin labeled CRP monoclonal antibody that 30 μ l concentration are 5mg/l, 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) pretreatment of micro-fluidic chip: using injection moulding to prepare cover plate and the egative film of micro-fluidic chip, respectively with ethanol, pure water each ultrasonic cleaning 10min, nitrogen dries up, and dustfree environment preserves, standby to make.
3) being coated of magnetic-particle: use point sample instrument or specking instrument by 10 μ l magnetic microsphere point samples in the reaction tank of chip, drying at room temperature 30min.
4) assembling of micro-fluidic chip: the HRP that 10 μ l concentration are 3mg/l is sealed in the first biomarker storage pool of cover plate；By 300 μ l cleanout fluid, 200 μ l alkaline phosphatase substrate liquid are individually enclosed in two storage pools of reagent card egative film, are assembled by miscellaneous part simultaneously, form complete micro-fluidic chip.
The detection by quantitative of 2.C reactive protein
1) making of standard curve: by CRP standard substance employment serum-dilution, being made into concentration is 0mg/l, 1mg/l, 10mg/l, 20mg/l, 100mg/l, the each 150 μ l of CRP standard substance (setting 3 repetitions) of 200mg/l, take 30 μ l and add the micro-fluidic chip adding mouth of preparation in the embodiment of the present invention, cover blood lid, micro-fluidic chip is placed in supporting instrument, places 15 minutes.Replication 3 times respectively of each sample.Instrument is according to the concentration of CRP and corresponding luminous intensity, and the CRP content in report sample, takes three times and measure meansigma methodss, draw standard curve automatically.
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 CRP in measuring samples.
3) result shows: use detection method of the present invention, and detection sensitivity is 0.01mg/l, and specificity and stability are high, and without HOOK effect.
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, 1.6 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, 1.6 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 1.6 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 1.6 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 (9)
1. the magnetic microparticle chemiluminescence micro-fluidic chip of a c reactive protein detection by quantitative, it is characterised in that described microfluidic chip structure mainly includes cover plate (1) and egative film (11)；The wherein adding mouth (2) on cover plate (1), sample fluid course (6), the first biomarker storage pool (4), micro-mixer (7), transition region (10) is 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, and 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 reaction tank (13) is coated the anti-c reactive protein antibody of magnetic particle marker；Described first biomarker storage pool (4), cleanout fluid storage pool (16) and luminescent solution storage pool (17) store pre-packaged reagent；Described cover plate (1) and egative film (11) seal with adhesive tape (20 and 22) and assemble.
2. micro-fluidic chip as claimed in claim 1, it is characterized in that, the volume of the first biomarker storage pool (4), cleanout fluid storage pool (16) and the luminescent solution storage pool (17) of described micro-fluidic chip is 10～500 μ l, for liquid capsule or cavity.
3. as claimed in claim 1 micro-fluidic chip, it is characterised in that the micro-mixer (7) of described micro-fluidic chip be width be 20～300 μm, the degree of depth is the fold-line-shaped structure of 10～100 μm.
4. micro-fluidic chip as claimed in claim 1, it is characterised in that the filter (12) of described micro-fluidic chip is made up of cavity and hemofiltration film；Described filter hemofiltration membrane material is glass fibre membrane, the one in polyester fiber film or CytoSep film.
5. micro-fluidic chip as claimed in claim 1, it is characterised in that the capillary microchannels that reaction tank (13) is tubular conduit or rectangle of described micro-fluidic chip.
6. micro-fluidic chip as claimed in claim 5, it is characterised in that the reaction tank (13) of described micro-fluidic chip is tubular conduit, a diameter of 0.5～10mm.
7. micro-fluidic chip as claimed in claim 5, it is characterised in that the reaction tank (13) of described micro-fluidic chip is the capillary microchannels of rectangle, a width of 0.1～5mm, and the degree of depth is 0.01～2mm, a length of 5～40mm.
8. micro-fluidic chip as claimed in claim 1, it is characterised in that described magnetic granule is hud typed supperparamagnetic particles, and magnetic core is Fe3O4Or γ-Fe2O3Compound, shell is polystyrene, and magnetic grain diameter is 0.1～10 μm.
9. micro-fluidic chip as claimed in claim 1, it is characterized in that, described cover plate (1) is upper also has air pump (3) and air-flow microchannel (5), and pressing air pump (3) causes air flow through air-flow microchannel (5) and drives sample by sample fluid course (6).
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105195242A (en) * | 2015-10-26 | 2015-12-30 | 深圳华迈兴微医疗科技有限公司 | Magnetic micro-particle chemiluminescence micro-fluidic chip for quantitatively detecting C-reaction protein |
| CN106902901A (en) * | 2017-01-23 | 2017-06-30 | 上海埃文生物科技有限公司 | Micro flow control chip device and its application |
| 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 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105195242A (en) * | 2015-10-26 | 2015-12-30 | 深圳华迈兴微医疗科技有限公司 | Magnetic micro-particle chemiluminescence micro-fluidic chip for quantitatively detecting C-reaction protein |
| CN105195242B (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 c reactive protein is quantitatively detected |
| CN106902901A (en) * | 2017-01-23 | 2017-06-30 | 上海埃文生物科技有限公司 | Micro flow control chip device and its application |
| CN106902901B (en) * | 2017-01-23 | 2024-03-08 | 上海埃文生物科技有限公司 | Microfluidic chip device and application thereof |
| 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 |
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