CN205317785U - Former quantitative determination's of calcitonin magnetic particle chemiluminescence micro -fluidic chip - Google Patents
Former quantitative determination's of calcitonin magnetic particle chemiluminescence micro -fluidic chip Download PDFInfo
- Publication number
- CN205317785U CN205317785U CN201520828640.1U CN201520828640U CN205317785U CN 205317785 U CN205317785 U CN 205317785U CN 201520828640 U CN201520828640 U CN 201520828640U CN 205317785 U CN205317785 U CN 205317785U
- Authority
- CN
- China
- Prior art keywords
- micro
- fluidic chip
- storage pool
- magnetic particle
- magnetic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Automatic Analysis And Handling Materials Therefor (AREA)
Abstract
The utility model discloses a former quantitative determination's of calcitonin magnetic particle chemiluminescence micro -fluidic chip, its structure mainly includes cover plate (1) and film (11), wherein air pump (3) on cover plate (1), and air current microchannel (5), the circulation of sample port (2), liquid sample says that (6), a biological marker deposit reservoir (4), little blender (7) and transition district (10) and 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, it deposits reservoir (16) and luminous liquid through solution release channel (18) and washing liquid and deposits reservoir (17) and be connected to detect pond (15).
Description
Technical field
The present invention relates to medical immunology in-vitro diagnosis field; it is specifically related to the magnetic microparticle chemiluminescence micro-fluidic chip of a kind of Procalcitonin detection by quantitative, it is possible within very short time, realize the detection by quantitative to Procalcitonin in biological sample, has simple to operate; highly sensitive, the features such as low cost.
Technical background
Procalcitonin (procalcitonin, PCT) is a small molecular protein, and containing 116 amino-acid residues, molecular weight is about 13kDa. The aminoacid sequence of PCT was found by people such as Moullec in 1984 first. It belongs to the family (CAPA peptide family) of a class associated protein, comprises calcitonin-gene-related peptide I and II, starch not lysin, adrenomedullin and thyrocalcitonin. Other peptides being similar to CAPA family, PCT derives from a precursor molecule-Preprocalcitonin. Preprocalcitonin contains 141 amino acid, and its N end obtains Procalcitonin after removing 25 amino-acid residues.
1993, PCT level raised to some extent in bacterial system infected patient to have report to find. Nowadays PCT has become the primary marking thing being attended by systemic inflammatory and septicemia disease. The value of PCT in clinical diagnosis, mainly being closely related of Based PC T concentration and severity of inflammation. PCT in normal human blood concentration lower than 0.05ng/ml, when PCT concentration in serum is higher than occurring during 0.5ng/ml that the risk of severe septicemia and/or septic shock is lower; Blood-serum P CT concentration >=2ng/mL occurs that the risk of severe septicemia and/or septic shock is higher, significantly increases in pyemia, its concentration of septic patient, can reach 1000ng/ml, is 2000 times of normal people. PCT can detect after 2 hours after infection, and clinical early diagnosis is significant, and within 12-24 hour, peaks after infection, the inside and outside good stability of body.
PCT is as the experimental index of the diseases such as a kind of sexy dye of the serious bacterial with innovative significance, it is to increase the accuracy of clinical diagnosis. Therefore, the diagnostic value of PCT is very high, it may also be useful in monoclonal antibody detection by quantitative blood samples of patients, the importance of PCT is apparent especially.
At present, the method detected for PCT mainly contains euzymelinked immunosorbent assay (ELISA), immunoturbidimetry, colloidal gold immunochromatographimethod, immunofluorescence technique etc., these methods can realize the detection to PCT to a certain extent, but also there is complicated operation, sensitivity is low, the high deficiency of testing cost. Chemiluminescence immune assay (chemiluminescenceimmunoassay, CLIA), it is that the chemical luminescent detecting technology with highly sensitive is combined with the immune response of high specific, technology is analyzed in detection for various antigen, haptens, antibody, hormone, enzyme, lipid acid, VITAMIN and medicine etc., is the up-to-date immunoassay grown up after exempting from analysis, fluoroimmunoassay and time-resolved fluoroimmunoassay analysis continue radioimmunology analysis, enzyme. Chinese patent (CN102359958A) discloses a kind of test kit detecting Procalcitonin, enzyme-catalyzed chemical luminescence method is adopted to realize the detection by quantitative to Procalcitonin, present method compares sensitivity and the accuracy that other traditional methods improve detection, but need detection sample is carried out complicated pre-treatment, need by large-scale chemiluminescence detector, required reagent consumption is big simultaneously, and detection time is long.
In recent years, bioassay technique field obtains to be developed fast, a lot of important research direction has occurred. Microfluidic chip analysis technology is wherein the 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-throughput, 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 is as solid phase carrier, it is possible to be used for 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 in current detection method the sensitivity existed, testing process is complicated, the problem being difficult to realize trace pattern 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, immunoassay is integrated into a kind of analysing and detecting method on micro-fluidic chip, the main difficult point of this kind of comprehensive detection method shows as at present: 1) liquid is in the intelligent control of chip internal microfluidic, the normal method adopted arranges multiple Micropump and micro-valve at present so that miniflow control system becomes more complicated; 2) mixing of reaction system is insufficient, causes reaction insufficient; 3) integration degree is not high, causes non-specific background height.
Summary of the invention
It is an object of the invention to as solving the deficiencies in the prior art; the magnetic microparticle chemiluminescence micro-fluidic chip of a kind of Procalcitonin detection by quantitative is proposed; Clinical Laboratory personnel only through simple operations, can need to realize the detection by quantitative of Procalcitonin concentration in sample fast in 15 minutes. Detected result is highly sensitive, and accurately and reliably, repeatability is good, and crossed contamination rate is low.
The technical scheme that the technical problem of the present invention adopts the following is:
The magnetic microparticle chemiluminescence micro-fluidic chip of a kind of Procalcitonin detection by quantitative, described microfluidic chip structure mainly comprises lid sheet (1) and egative film (11), pneumatic pump (3) on its middle cover sheet (1), air-flow microchannel (5), adds sample mouth (2), sample flow passage (6), the first biomarker storage pool (4), micro mixer (7) and zone of transition (10) and connects successively;Strainer (12) on egative film (11), reaction tank (13), service sink (14), detection cell (15), solution release channel (18) connects successively; Detection cell (15) is connected with scavenging solution storage pool (16) and luminescent solution storage pool (17) by solution release channel (18); First biomarker storage pool (4) stores pre-packaged enzyme or the anti-Procalcitonin antibody-solutions of luminous agent mark; Reaction tank (13) stores the anti-Procalcitonin antibody of pre-packaged magnetic particle marker; Described scavenging solution storage pool (16) and luminescent solution storage pool (17) store pre-packaged scavenging solution and luminous substrate liquid; Lid sheet (1) zone of transition (10) and egative film (11) strainer (12) connect; Described tagged ligand storage pool (5), scavenging solution storage pool (9), luminous substrate liquid storage pool (10) are hydraulic seal pond, the partial fracture by external force extruding, releasing liquid; Strainer (12) is made up of cavity and filter blood film; In described micro-fluidic chip testing process, manipulate magnetic particle moving or gathering with magnet.
Specifically, described first biomarker storage pool (4), scavenging solution storage pool (16) and luminescent solution storage pool (17) are liquid capsule or cavity, and volume is 10～500 μ l, further preferred 10～300 μ l.
Specifically, the micro mixer of described micro-fluidic chip is width is 20～300 μm, and the degree of depth is 10～100 μm snakelike, broken line shape or square structure.
Preferably, micro mixer is wide 150 μm, and the degree of depth is the square structure of 50 μm, under external pressure effect, sample and reagent can be made fully to mix, it is to increase reaction efficiency.
Specifically, the reaction tank of described micro-fluidic chip is capillary microchannels, and this capillary microchannels is tubular channel or rectangular channel, allows micro-liquid flow to or pass through.
Specifically, the reaction tank of described micro-fluidic chip is tubular channel, and diameter is 0.5～10mm, and the diameter as preferred microchannel is 5mm, further preferred 2mm or 1mm.
Specifically, the size range when capillary microchannels of described micro-fluidic chip is rectangular channel: wide is 0.1～5mm, and the degree of depth is 0.01～2mm, and length is 5～40mm.
Preferably, the size range when capillary microchannels of described micro-fluidic chip is rectangular channel: wide is 0.3～2mm, and the degree of depth is 0.2～1mm, and length is 5～20mm.
Specifically, the strainer of described micro-fluidic chip mainly comprises the cavity with a shaped and filter blood film, and described cavity volume is 3～10 times of sample volume, it is preferable that cavity volume is 4～6 times of sample volume.
Specifically, the filter blood membrane material in described micro-fluidic chip egative film middle filtrator can be glass fibre membrane, trevira film or CytoSep film etc.
Preferably, using glass fibre membrane as filter blood film.
Specifically, the strainer of micro-fluidic chip of the present invention has outside the function of filtering sample impurity, it is also possible to is guided by liquid and enters next stage microstructure and microchannel.
The volume of capillary channel of the present invention is less than 150 μ l, and as preferably, the volume of capillary channel is less than 100 μ l, further preferably, capillary channel volume is less than 50 μ l.
Specifically, described luminous substrate liquid comprises the substrate corresponding with enzyme and luminescence enhancer, can inject same luminescent solution storage pool after mixing, or injects two different luminescent solution storage pools respectively;
Specifically, the magnetic particle that the anti-Procalcitonin antibody of described magnetic particle marker uses is supperparamagnetic particles, is the Fe of paramagnetism3O4Or γ-Fe2O3Compound, magnetic grain diameter is 0.1～10 μm.Preferred Fe3O4Compound, and magnetic grain diameter is 1～3 μm, it is more preferable to particle diameter is the magnetic particle of 2.0 μm.
Specifically, micro-fluidic chip of the present invention, is connected by microchannel and microstructure between each functional zone, an inner formation complete liquid stream and air flow system.
Specifically, microchannel and the complete processing of microstructure in micro-fluidic chip lid sheet of the present invention and egative film comprise moulding method, pressure sintering, laser ablation method and soft lithography etc., and in embodiments of the invention, preferred moulding method makes micro-fluidic chip.
The lid sheet of micro-fluidic chip of the present invention and egative film material can be polymeric amide (PI), polymethylmethacrylate (PMMA), polycarbonate (PC), polydimethylsiloxane (PDMS), epoxy resin, polypropylene (PP) and ABS resin etc., preferred epoxy and ABS resin, further preferred epoxy.
Specifically, in micro-fluidic chip lid sheet of the present invention and egative film, except above-mentioned main microchannel and microstructure, also have many breather holes for eliminating the bubble of generation in liquid-flow process, and for assembling fixing resigning hole and column.
Specifically, the pneumatic pump (3) on described micro-fluidic chip lid sheet mainly transmits pressure by air-flow passage (5), and main effect is for the mixing of sample and the first biomarker, it is to increase one-level hatches effect.
Specifically, the air-flow channel size of described micro-fluidic chip is 0.1～100 μm, preferably 2～50 μm further.
Specifically, the zone of transition of described micro-fluidic chip lid sheet is the hinge that lid sheet and egative film connect, and first order reaction mixture is through zone of transition inflow filter, it is achieved that liquid is in the flowing between lamella up and down of micro-fluidic chip.
The reaction tank inside of micro-fluidic chip of the present invention need to carry out certain surface modification treatment, described surface modifying treatment can be chemical reaction, top coat, plasma treatment etc., thus obtain good wetting ability, impel liquid sample in capillary action current downflow, Fast Filling microchannel.
Enzyme described in the present invention, comprises but is not limited to catalase (HRP) and alkaline phosphatase (ALP). Luminous substrate liquid is the luminous substrate (such as luminol,3-aminophthalic acid cyclic hydrazide or diamantane) and luminescence enhancement liquid (such as tougheners 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) after mixing as shown in Figure 2; But when the mixed solution quality guaranteed period should separate less than when 1 year, inject luminous substrate liquid storage pool (17) and luminous substrate liquid storage pool (24) as shown in Figure 4 respectively, mixed by pre-mixing passages (23).
The assembling of micro-fluidic chip of the present invention, lid sheet and egative film are binded by sealant tape, and sealant tape can be common two-sided glue, heat-sensitive glue and pressure sensitive adhesive etc., are heat-sensitive glue or pressure sensitive adhesive as preferred sealant tape.
Micro-fluidic chip of the present invention carries out sample determination and mainly comprises following operation:
Step 1) add sample from adding sample mouth, cover blood lid, micro-fluidic chip is put into necessary instrument, after marking anti-Procalcitonin antibody from the first biomarker storage pool release enzyme or luminous agent, pressing pneumatic pump makes sample and enzyme labelled antibody mix and chemical reaction occurs, form a kind of immunocomplex, then flow in egative film strainer;
Step 2) sample is after filter, arrive reaction tank, redissolution is coated on the anti-Procalcitonin antibody of magnetic particle marker in this region, and react with it, external magnet sends out relative movement in some way on the motion chute of micro-fluidic chip, collect magnetic particle and by magnetic transfer of granules to service sink, system release scavenging solution, magnetic particle is after fully washing, it is transferred to detection cell under magnet effect, system release luminous substrate liquid, according to the proportionlity between relative luminous intensity (RLU) and Procalcitonin antigen concentration, detection system can convert automatically, test result can be reported fast, thus realize the detection by quantitative of Procalcitonin.
Specifically, step 1) described necessary instrument is small portable device, described equipment mainly comprises control device and detection device.
Specifically, the control device of described supporting small portable device mainly act as the micro-fluidic chip to placing its inside can be implemented to comprise control liquid-flow, sample mixes, reagent in release storage pool, magnet moves, detection device mainly act as gather luminous signal and it is analyzed, finally show detected result.
The present invention's detection sample used comprises the whole blood from human body, serum and plasma, and volume used is 150 μ L, it is preferable that 100 μ L, further preferred 50 μ L, more preferred 10 μ L further.
The control device of portable equipment of the present invention mainly act as implements the mixing that extruding pneumatic pump realizes sample and biomarker to micro-fluidic chip, the motion of controlling magnet realizes the abundant mixing of magnetic microsphere traget antibody and first order reaction thing and the collection of magnetic bead, the motion of controlling magnet realizes reaction mixture reaction tank successively, service sink, movement between detection cell, effectively reduces nonspecific interference.
The motion that magnet in control device of the present invention occurs is for move relative to the chute of micro-fluidic chip, and movement velocity is 1mm/s～50mm/s, is 4mm/s～30mm/s as preferred magnet movement speed.
Scavenging solution of the present invention, for cleaning magnetic particle, removes the impurity substances not participating in reaction. Scavenging solution mainly comprises buffer system, protein, tensio-active agent and sanitas, and wherein buffer system is including but not limited to borate, phosphoric acid salt, Tris-HCl and acetate etc. Wherein protein is including but not limited to bovine serum albumin, casein etc.; Wherein tensio-active agent is including but not limited to polysorbas20, tween 80, triton x-100, polyoxyethylene glycol and Polyvinylpyrolidone (PVP) etc.
The preparation method of micro-fluidic chip of the present invention is as follows:
Step 1) enzyme or luminous agent mark anti-Procalcitonin antibody, the anti-Procalcitonin antibody of magnetic particle marker, and these two kinds of antibody can same antibody or different sorts antibody;
Step 2) enzyme or luminous agent traget antibody solution are put into the storage pool of lid sheet, sealing, magnetic particle marker antibody-solutions is put into the reaction tank of egative film, dry, scavenging solution and luminous substrate liquid are injected respectively scavenging solution storage pool and luminous substrate liquid storage pool, sealing, with adhesive tape (20 and 22) sealed cover slip and egative film, and is assembled into micro-fluidic chip.
Compared with prior art, the useful effect obtained is in the present invention:
1) magnetic immunological technique is integrated on micro-fluidic chip, combines the advantage of two kinds of technology, whole testing process is had simple to operate, result accurately and reliably, highly sensitive feature.
2) micro-fluidic chip internal microstructure and microchannel are through reasonably design, can eliminate bubble completely to the impact of detected result in liquid-flow process.
3) simple process of small-sized test set and chip internal is coordinated, it is not necessary to complicated pump valve and electric field just can realize the intelligent control of liquid stream effectively in micro-fluidic chip inside.
4) there is multistep mixing functions, immune response efficiency can be improved to a certain extent.
5) the various reactions on micro-fluidic chip, cleaning and testing process subregion carry out, integration degree height, can effectively reduce nonspecific interference, it is to increase the sensitivity of detection.
6) adopt micro-fluidic chip involved in the present invention to detect, it is possible to report detected result fast, the other detection of bed and various portable equipment can be further used for.
The core technology of the present invention is combined at magnetic particle technology, chemiluminescence immunoassay detection technique and microfluidic chip technology three, by the Fine design of micro-fluidic chip and processing, and coordinate small portable device, successfully achieve the intelligent control of microfluid in micro-fluidic chip inside, make magnetic particle can realize controlled motion in micro-fluidic chip inside by the effect of magnet, mixing, collect and clean, improve reaction efficiency, reduce nonspecific interference, thus enhance detection signal, it is to increase detection sensitivity. The technology of the present invention is not the simple superposition of above-mentioned three kinds of technology, but it is integrated with the advantage of three, the isoparametric trickle change of the kind of magnetic particle, size, the shape of passage, size can greatly affect the accuracy of detected result, a kind of method for Procalcitonin fast quantification newly that the present invention has improved on prior art basis.
Accompanying drawing explanation
Fig. 1 is the lid chip architecture schematic diagram of micro-fluidic chip, and wherein 2 for adding sample mouth, and 3 is pneumatic pump, and 4 is the first biomarker storage pool, 5 is air-flow passage, and 6 is sample flow passage, and 7 is micro mixer, 8 is liquid storage tank resigning hole, and 9 is magnet movement chute, and 10 is zone of transition.
Fig. 2 is the chassis construction schematic diagram of micro-fluidic chip, and wherein 12 is strainer, and 13 is reaction tank, and 14 is service sink, and 15 is detection cell, and 16 is scavenging solution storage pool, and 17 is luminescent solution storage pool, and 18 is scavenging solution 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 lid sheet, and 11 is egative film, and 20 is top adhesive tape, and 22 is bottom adhesive tape.
Fig. 4 is the chassis construction schematic diagram in three liquid storage ponds, and wherein 16 is scavenging solution storage pool, and 17 and 24 is luminescent solution storage pool, and 18 is scavenging solution, and 23 is luminescent solution release channel.
Embodiment:
The present invention discloses the magnetic microparticle chemiluminescence micro-fluidic chip of a kind of Procalcitonin detection by quantitative, and those skilled in the art can use for reference content herein, 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 and the application of the present invention are described by better 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 the technician of this area understand the technical scheme of the present invention better, compare accompanying drawing below and in conjunction with preferred embodiment, the present invention is explained in detail.
Embodiment 1: horseradish peroxidase-luminol,3-aminophthalic acid cyclic hydrazide (HRP-luminol) system is used for the detection of Procalcitonin
1. the making of micro-fluidic chip
1) antibody labeling:
Ii) enzyme labelled antibody: take HRP25mg and be dissolved in 1.25% glutaraldehyde solution, in room temperature hold over night; Reacted enzyme solution, through SephadexG-25 chromatography column, uses physiological saline wash-out, and flow rate control, at 1ml/min, collects brown effluent liquid; By PCT monoclonal antibody 12.5mg normal saline dilution to 5ml, dropwise add under stirring in HRP solution; With 1MpH9.5 carbonate buffer solution 0.25ml, continue to stir 3 hours; Add 0.2M Methionin 0.25ml, after mixed even, put room temperature 2 hours; Under agitation dropwise add equal-volume saturated ammonium sulphate, place 1 hour in 4 DEG C;3000rpm centrifugal half an hour, abandon supernatant; Throw out cleans twice with half saturated ammonium sulphate, and last throw out is dissolved in the PBS of a small amount of 0.15MpH7.4; Being loaded in dialysis tubing by above-mentioned solution, dialyse with the PBS of 0.15MpH7.4, after removing ammonium ion, 10000rpm removes precipitation in centrifugal 30 minutes, and supernatant liquor is enzyme conjugates, after packing, in 2-4 DEG C of preservation.
Ii) magnetic labeling antibody: accurately pipette the marked by streptavidin magnetic bead that 50 μ L concentration are 1mg/mL, in 2mL centrifuge tube, vortex concussion 30min; Add the vitamin H PCT monoclonal antibody that 200 μ L concentration are 3mg/mL again, mix, react 2 hours in shaking table in 37 DEG C. Reaction mixture is separated at magnetic separator, removes supernatant liquor, by 0.01MPBS (containing 0.01%BSA/0.2% tween 20, pH7.4) repeated washing 3 times. Finally magnetic bead is suspended in 0.01MPBS (containing 0.1%BSA) to final prescribed concentration.
2) the bag quilt of magnetic labeling antibody: adopt point sample instrument or specking instrument to drip in the reaction tank being added in micro-fluidic chip by 10 μ L1mg/mL magnetic labeling antibody solution, drying at room temperature more than 30 minutes.
3) assembling of micro-fluidic chip: be the HRP of 5mg/mL by 10 μ L concentration, 300 μ L scavenging solutions, 200 μ L luminescence reagents are encapsulated in the corresponding stored pond of chip respectively, are assembled by other parts simultaneously, form a complete micro-fluidic chip.
2. the detection by quantitative of Procalcitonin
1) making of typical curve: PCT standard substance calf serum is diluted, being made into some row concentration of concentration is 0ng/mL, 5ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, the each 150 μ L of the Procalcitonin standard substance of 300ng/mL, get the sample application zone that 30 μ L add the micro-fluidic chip test card of preparation in the embodiment of the present invention, cover blood lid, test kit is placed in supporting bantam, places 15min. Each sample is replication 3 times respectively. Necessary instrument goes out PCT concentration according to the proportionlity between RLU and PCT concentration, automatic the Fitting Calculation, gets three times and measures mean value, drawing standard curve.
2) getting sample 200 μ L to be checked, the micro-fluidic chip being placed in the embodiment of the present invention made detects, every sub-sampling 30 μ L, and replication 3 times can obtain the concentration value of Procalcitonin in sample to be checked according to the typical curve drawn in 1.
3) result shows: adopting the method for the invention by further test, the minimum detection drawn is limited to 0.03ng/mL, and sensing range is 0.01ng/mL-1000ng/mL, and between batch, CV is less than 10%, and in batch, CV is less than 4%.
Embodiment 2: alkaline phosphatase-diamantane (ALP-AMPPD) system is used for the detection of Procalcitonin
1. the making of micro-fluidic chip
1) antibody labeling: i) enzyme labelled antibody: 2.5mgALP (50IU/mg), adds the PB (pH6.8) of 200uL containing the 100mM of 1.25% glutaraldehyde, and mixed even, room temperature reaction spends the night; Under 4 DEG C of conditions, electromagnetism stirs, and dialysis, to 50mMPBS (pH7.2), 12 hours, changes liquid 4 times; 1.5mg Procalcitonin antibody is dissolved in the carbonate solution (pH9.0) of 100uL1M; The AP of activation is added in the protein liquid prepared, mixed even, react 24 hours under 4 DEG C of conditions, add the lysine solution of 10 μ L200mM, mixed even, react 2 hours under 22 DEG C of conditions; Dialyse under 4 DEG C of conditions to 50mMPBS (pH7.2), 12 hours, change liquid 4 times; Centrifugal, get supernatant, use 50mMTB7.4+0.6%BSA+0.05%NaN3It is diluted to test desired concn ,-20 DEG C of preservations.Ii) magnetic labeling antibody: accurately pipette the marked by streptavidin magnetic bead that 50 μ L concentration are 1mg/mL, in 2mL centrifuge tube, vortex concussion 30min; Add the vitamin H PCT monoclonal antibody that 200 μ L concentration are 3mg/mL again, mix, react 2 hours in shaking table in 37 DEG C. Reaction mixture is separated at magnetic separator, removes supernatant liquor, by 0.01MPBS (containing 0.01%BSA/0.2% tween 20, pH7.4) repeated washing 3 times. Finally magnetic bead is suspended in 0.01MPBS (containing 0.1%BSA) to final prescribed concentration.
2) the bag quilt of magnetic labeling antibody: adopt point sample instrument or specking instrument to drip in the reaction tank being added in micro-fluidic chip by 10 μ L1mg/mL magnetic labeling antibody solution, more than drying at room temperature 30min.
3) assembling of micro-fluidic chip: be the HRP of 5mg/mL by 10 μ L concentration, 300 μ L scavenging solutions, 200 μ L luminescence reagents are encapsulated in the corresponding stored pond of chip respectively, are assembled by other parts simultaneously, form a complete micro-fluidic chip.
2. the detection by quantitative of Procalcitonin
1) making of typical curve: PCT standard substance calf serum is diluted, being made into some row concentration of concentration is 0ng/mL, 5ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, the each 150 μ L of the Procalcitonin standard substance of 300ng/mL, get the sample application zone that 30 μ L add the micro-fluidic chip test card of preparation in the embodiment of the present invention, cover blood lid, test kit is placed in supporting bantam, places 15min. Each sample is replication 3 times respectively. Necessary instrument goes out PCT concentration according to the proportionlity between RLU and PCT concentration, automatic the Fitting Calculation, gets three times and measures mean value, drawing standard curve.
2) getting sample 200 μ L to be checked, the micro-fluidic chip being placed in the embodiment of the present invention made detects, every sub-sampling 30 μ L, and replication 3 times can obtain the concentration value of Procalcitonin in sample to be checked according to the typical curve drawn in 1.
3) result shows: adopt the method for the invention by further test, detection sensitivity is 0.006ng/mL, sensing range is 0.001ng/mL-2000ng/mL, between batch, CV is less than 5%, in batch, CV is less than 1.5%, and HOOK effect does not occur, it is not necessary to detection sample is carried out dilution process.
Embodiment 3: magnetic particle particle size is screened
The particle diameter of magnetic microsphere is little, and specific surface area is big, and surface is containing active group, therefore coupling capacity is big, but magnetic particle size is excessively little is unfavorable for that magnet is collected, therefore carries out magnetic particle size selection.
Other experiment condition carries out according to following scheme see embodiment 2, the size of magnetic particle particle.
Particle size is selected to be respectively 0.1 μm, 0.5 μm, 2 μm, 2.2 μm, 3 μm, the 10 μm anti-c reactive protein antibody of magnetic particle marker. Adopt magnetic size through preferred permanent magnet during detection, fixed magnet height.
Experimental result is as follows:
Magnetic particle grain size strengthens successively from 0.1 μm, 0.5 μm, 2 μm, 2.2 μm, 3 μm, and 3 μm of interference strengthen, and 10 μm start to reduce, and signal value is minimum, and comprehensive each factor 2.2 μm of signals are the strongest, disturb minimum.
Result is analyzed: when magnetic particle size is less, and specific surface area is relatively big, and the biotinylated molecular weight of surface institute load is big, can disperse in the solution well, but it is big to be ensured that magnetic microsphere is fully collected required magneticstrength simultaneously. Magnetic field power suffered by magnetic bead can not ensure that it is fully collected in the present embodiment, cause part effectively magnetic bead run off in cleaning process, thus cause final detected signal value not high.When magnetic grain diameter is bigger, specific surface area is little, the mark rate of surface biological molecule is relatively low, under the same terms, magnetic particle institute is greatly magnetic field force induced, and the magnetic bead of dispersion can be collected fully, but it is owing to sedimentation very easily occurring, cause between biomolecules, reacting insufficient, thus weaken luminous signal. Considering, particle diameter is that 1～3 μm of magnetic microsphere effect is better, and therefore in embodiments of the invention, the magnetic microsphere effect of 2.2 μm is best.
The above is only the preferred embodiment of the present invention; it is noted that for those skilled in the art, under the premise without departing from the principles of the invention; can also making some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (7)
1. the magnetic microparticle chemiluminescence micro-fluidic chip of a Procalcitonin detection by quantitative, it is characterized in that, described microfluidic chip structure mainly comprises lid sheet (1) and egative film (11), pneumatic pump (3) on its middle cover sheet (1), air-flow microchannel (5), adds sample mouth (2), sample flow passage (6), the first biomarker storage pool (4), micro mixer (7) and zone of transition (10) and connects successively; Strainer (12) on egative film (11), reaction tank (13), service sink (14), detection cell (15), solution release channel (18) connects successively; Detection cell (15) is connected with scavenging solution storage pool (16) and luminescent solution storage pool (17) by solution release channel (18); First biomarker storage pool (4) stores pre-packaged enzyme or the anti-Procalcitonin antibody-solutions of luminous agent mark; Reaction tank (13) stores the anti-Procalcitonin antibody of pre-packaged magnetic particle marker; Described scavenging solution storage pool (16) and luminescent solution storage pool (17) store pre-packaged scavenging solution and luminous substrate liquid; Lid sheet (1) zone of transition (10) and egative film (11) filtrating area (10) connect; Described tagged ligand storage pool (5), scavenging solution storage pool (9), luminous substrate liquid storage pool (10) are hydraulic seal pond, the partial fracture by external force extruding, releasing liquid; Filtrating area (10) is made up of cavity and filter blood film; In described micro-fluidic chip testing process, manipulate magnetic particle moving or gathering with magnet.
2. micro-fluidic chip as claimed in claim 1, it is characterised in that, described first biomarker storage pool (4), scavenging solution storage pool (16) and luminescent solution storage pool (17) they are liquid capsule or cavity, and volume is 10～500 μ.
3. micro-fluidic chip as claimed in claim 1, it is characterised in that, the micro mixer of described micro-fluidic chip is width is 20～300 μm, and the degree of depth is broken line shape or the square structure of 10～100 μm.
4. micro-fluidic chip as claimed in claim 1, it is characterised in that, the reaction tank of described micro-fluidic chip is capillary microchannels, and this capillary microchannels is tubular channel or rectangular channel, allows micro-liquid flow to or pass through.
5. micro-fluidic chip as claimed in claim 4, it is characterised in that, described reaction tank is tubular channel, and diameter is 0.5～10mm.
6. micro-fluidic chip as claimed in claim 4, it is characterised in that, described reaction tank is rectangular channel, and wide is 0.1～5mm, and the degree of depth is 0.01～2mm, and length is 5～40mm.
7. micro-fluidic chip as claimed in claim 1, it is characterised in that, the magnetic particle that the anti-Procalcitonin antibody of described magnetic particle marker uses is supperparamagnetic particles, is Fe3O4 or the γ-Fe2O3 compound of paramagnetism, and magnetic grain diameter is 0.1～10 μm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201520828640.1U CN205317785U (en) | 2015-10-26 | 2015-10-26 | Former quantitative determination's of calcitonin magnetic particle chemiluminescence micro -fluidic chip |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201520828640.1U CN205317785U (en) | 2015-10-26 | 2015-10-26 | Former quantitative determination's of calcitonin magnetic particle chemiluminescence micro -fluidic chip |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN205317785U true CN205317785U (en) | 2016-06-15 |
Family
ID=56188174
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201520828640.1U Active CN205317785U (en) | 2015-10-26 | 2015-10-26 | Former quantitative determination's of calcitonin magnetic particle chemiluminescence micro -fluidic chip |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN205317785U (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105203775A (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 procalcitonin |
-
2015
- 2015-10-26 CN CN201520828640.1U patent/CN205317785U/en active Active
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105203775A (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 procalcitonin |
| CN105203775B (en) * | 2015-10-26 | 2017-10-17 | 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 Procalcitonin is quantitatively detected |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105203775B (en) | The magnetic microparticle chemiluminescence micro-fluidic chip that a kind of Procalcitonin is quantitatively detected | |
| CN105259163B (en) | The direct chemiluminescence micro-fluidic chip of magnetic particle for whole blood sample detection | |
| CN107942050B (en) | A kind of detection method of microfluidic chip based on magnetic bead technology | |
| CN105195242B (en) | The magnetic microparticle chemiluminescence micro-fluidic chip that a kind of c reactive protein is quantitatively detected | |
| CN105195243B (en) | The magnetic microparticle chemiluminescence micro-fluidic chip that a kind of myoglobins is quantitatively detected | |
| CN105241871B (en) | A kind of magnetic microparticle chemiluminescence micro-fluidic chip for whole blood sample detection | |
| CN109870582A (en) | A kind of more target magnetic immunochemiluminescence micro-fluidic chip detection platforms and method | |
| AU553376B2 (en) | Integrated single tube plunger immunoassay system | |
| CN105233892A (en) | Magnetic particle chemiluminescence double-layer micro-fluidic chip used for whole-blood sample detection | |
| CN105259164B (en) | The micro-fluidic chip that multiple target objects based on magnetic microparticle chemiluminescence quantitatively detect | |
| CN205650214U (en) | D - dimer quantitative determination's magnetic particle chemiluminescence micro -fluidic chip | |
| CN205317674U (en) | A direct chemiluminescence micro -fluidic chip of magnetic particle for whole blood sample test | |
| CN105597846B (en) | The magnetic microparticle chemiluminescence micro-fluidic chip that a kind of D dimers quantitatively detect | |
| CN205650213U (en) | Myoglobin quantitative determination's magnetic particle chemiluminescence micro -fluidic chip | |
| CN105785054A (en) | Chemical-luminescent microfluidic disk for quantitative detection of procalcitonin and using method thereof | |
| CN105259162A (en) | Magnetic particulate chemiluminiscence micro-fluidic chip capable of quantitatively detecting brain natriuretic peptide in whole blood | |
| EP4012410A1 (en) | Magnetic particle luminescence micro-fluidic chip for multi-marker detection, and detection device | |
| CN205650212U (en) | A double -deck micro -fluidic chip of magnetic particle chemiluminescence for whole blood sample test | |
| CN108051588A (en) | For the antibody fixing means on the micro-fluidic chip of whole blood sample separation detection | |
| CN205317673U (en) | Multiple target thing quantitative determination's micro -fluidic chip based on magnetic particle chemiluminescence | |
| WO2021068912A1 (en) | Magnetic particle luminescence micro-fluidic chip for multi-marker detection, and detection device | |
| CN112237948B (en) | Fluorescent magnetic bead micro-fluidic chip and analytical instrument thereof | |
| CN109211869A (en) | A kind of micro-fluidic fluorescence immunoassay chip of rapid quantitative detection d-dimer | |
| CN107655879B (en) | For detecting the micro-fluidic chemiluminescence detection system of the magnetic particle of sexual gland series | |
| WO2021068914A1 (en) | Magnetic particle light-emitting double-layer micro-fluidic chip and detection system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |