CN205449995U - Magnetic particle chemiluminescence micro -fluidic chip - Google Patents
Magnetic particle chemiluminescence micro -fluidic chip Download PDFInfo
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- CN205449995U CN205449995U CN201520828120.0U CN201520828120U CN205449995U CN 205449995 U CN205449995 U CN 205449995U CN 201520828120 U CN201520828120 U CN 201520828120U CN 205449995 U CN205449995 U CN 205449995U
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Abstract
The utility model discloses a magnetic particle chemiluminescence micro -fluidic chip, micro -fluidic chip includes roof (1) and bottom plate (2), wherein roof (1) contains air pump (3), sample port (4), sample fill area (12), tagged ligand deposits reservoir (5) and sample mixed zone (13), bottom plate (2) contain filtering area (6), magnetic particle peridium district (7), wash district (14), detection zone (8), reservoir (9) are deposited to the washing liquid, reservoir (10) and liquid release channel (16) are deposited to luminous basement liquid, roof and bottom plate all contain liquid sensing device, are used for confirming the flow state of the interior liquid of micro -fluidic chip and whether sneaking into bubble etc..
Description
Technical field
The invention belongs to fluidic chip chemiluminescence technical field of immunoassay, particularly to a kind of chip utilizing magnetic microparticle chemiluminescence technology and microfluidic chip technology to realize the highly sensitive detection by quantitative of analyte.
Background technology
At present, in-vitro diagnosis (IVD) mainly has two kinds of development trends: one is automatization, integrated integration, i.e. utilizes the large-scale instrument and equipment full-automatic, high-sensitive of the supporting central laboratory of large hospital, it is achieved high-precision diseases analysis diagnoses；Another kind of miniaturization, bed sideization, i.e. by the compact simplified equipment of palm, it is achieved on-the-spot quickly analyzing and diagnosing.Small hospital's insufficient funds, sample size are few, are not appropriate for the main equipment that purchasing price is expensive.Present stage, small-sized quick detection equipment was mainly test strips and corollary equipment thereof, but test strips can only realize qualitative or half-quantitative detection, and detection sensitivity is low, poor specificity, poor repeatability, be disturbed substantially.Owing to Chinese population is numerous, aging aggravates, and sickness rate increases severely, and simple dependence large hospital can't bear the heavy load.Therefore develop easy and simple to handle, highly sensitive, reproducible and quantitative method for quick accurately and equipment becomes the most urgent.
Micro-fluidic chip is also called chip lab (Lab-on-a-chip), refer to the involved sample in the fields such as biological, chemistry and medical science to prepare, react, separate, the basic operation unit such as detection is integrated on one piece of chip with micro-meter scale microchannel, is automatically performed reaction and the overall process analyzed.The advantage of analysis and detection device based on micro-fluidic chip realization is: sample consumption is few, analyzes speed fast, it is simple to make portable instrument, is highly suitable for instant, field assay.
Chemiluminescence refers to that chemical energy is changed into the phenomenon of luminous energy by the reaction intermediate in chemical reaction process, product or additional luminescence reagent.Compared with fluorescence and absorbing light, chemiluminescence does not has external excitation source background signal to disturb, and cross interference is little, has the advantages such as highly sensitive, range of linearity width.The chemiluminescence analysis thus set up oneself be widely used in the fields such as clinical diagnosis.Chemiluminescence Apparatus is big type analysis detection equipment main for IVD.
But, data micro-fluidic chip and chemiluminescence combined and document are the most few, practical and can industrialization less, as Chinese patent 200910114403.8 describes a kind of method that fluidic chip chemiluminescence measures materials inside human single blood erythrocyte by mocro, it need to rely on microscope stage or lens and the complex optical path of optical filter composition；Chinese patent 200910154432.7 discloses the micro-fluidic chip of capillary electrophoresis separation and chemiluminescence detection, and its flow passage structure is single, and sample introduction is not adequately mixed thus causes reaction efficiency relatively low, it is impossible to reach maximum emission intensity.
Additionally, the reaction realized based on micro-fluidic chip and analysis, its basic process is pre-packaged in chip or is introduced from outside into one or more reaction reagents, then liquid sample is added chip, sample contacts with reagent according to microchannel set in advance stream and reacts, and is read out result by instrument or naked eyes.The detection means of micro-fluidic chip main flow includes laser-induced fluorescence (LIF), chemiluminescence and uv absorption etc. at present.Precision of analysis to be realized, in most important microchannel, plurality of liquid wants arriving within the time specified according to passage set in advance of accurate quantitative analysis to specify position.But practical situation might not be preferable, due to chip or the reason of sample, the process that reaction in micro-fluidic chip may not have according to setting is carried out, stop as produced liquid flowing in bubble causes chip, or chip generation seepage causes liquid cannot fill each pre-routing etc., these all can make reaction between predetermined reagent or the substrate reacted incomplete so that analysis result produces mistake.But owing to the reaction in chip is to be automatically performed, the simply final result that experimenter or tester see, lack the monitoring to pilot process, so inevitably result in the error analysis to result, thus have impact on the accuracy of analysis.
Summary of the invention
It is an object of the invention to provide a kind of magnetic microparticle chemiluminescence micro-fluidic chip; it is characterized in that this chip, by integrated chip (all components in addition to test sample is all integrated in chip) supporting small portable device, thus realizes the detection by quantitative quick, accurate, highly sensitive of analyte in field samples.
Another object of the present invention is to provide a kind of micro-fluidic chip possessing liquid sensing system.
A kind of magnetic microparticle chemiluminescence micro-fluidic chip; it is characterized in that; described micro-fluidic chip includes top board (1) and base plate (2), and wherein said top board comprises air pump (3), adding mouth (4), tagged ligand storage pool (5), sample fill area (12) and sample mixed zone (13)；Described base plate comprises filtering area (6), magnetic particle is coated district (7), chemiluminescence detection district (8), cleanout fluid storage pool (9), luminous substrate liquid storage pool (10), cleaning area (14), waste liquid pool (15) and liquid release channel (16)；Described top board (1) and base plate (2) two-sided tape (19) and one-faced tapes (20) seal, and are assembled into micro-fluidic chip；Described top board (1) and base plate (2) all comprise liquid sensing system；Described liquid sensing system is made up of one or more liquid sensing devices；Described liquid sensing device includes: conductive pin (31), contact probe (30), metal basal board (28) and support circuit plate (29)；Described conductive pin is embedded in by micro-fluidic chip liquid release channel, communicates with microchannel, and is exposed on the surface of chip；Described contact probe one end is arranged on support circuit plate, and the other end passes metal basal board, contacts one to one with the conductive pin on micro-fluidic chip；Described metal basal board is used for loading micro-fluidic chip and fixing circuit board；Described support circuit plate is used for realizing detecting the signal of telecommunication on probe；
Described air pump (3) and adding mouth (4) UNICOM, adding mouth (4) and tagged ligand storage pool (5) are connected by sample fill area (12), and tagged ligand storage pool (5) is connected with sample mixed zone (13)；The detection zone (8) of described base plate is connected by liquid release channel (16) with cleanout fluid storage pool (9) and luminous substrate liquid storage pool (10)；Described filtering area (6), magnetic particle are coated district (7), cleaning area (14), detection zone (8) are sequentially connected with；Described sample mixed zone (13) end connects with filtering area (6)；
It is covered with one layer of elastic rubber belt (23) on described air pump (3), micro-fluidic chip is formed an air chamber sealed, by pressing and unclamping elastic rubber belt, it is provided that positive and negative pressure air-flow, thus drive sample to flow forward or backward；
Described tagged ligand storage pool (5), cleanout fluid storage pool (9), luminous substrate liquid storage pool (10) and magnetic particle are coated pre-packaged in district (7) has reagent；Liquid in storage pool is to seal, and can be extruded by external force and make partial fracture and releasing liquid；
Described filtering area (6) comprises hemofiltration film, is retained the erythrocyte in whole blood sample by physical separation mode；
Described micro-fluidic chip, when test, need to move with Magnet manipulation magnetic particle or assemble.
As in figure 2 it is shown, the moulding material of described top board (1) and described base plate (2) is polymer, including but not limited to polystyrene, polrvinyl chloride, polypropylene, epoxy resin etc.；Described adhesive tape (19 and 20) can be double faced adhesive tape or one side glue, and wherein double faced adhesive tape can substitute with two panels one side glue.Micro-fluidic chip is in test process, need to control up movement or the gathering of magnetic particle with Magnet, therefore, in luminous substrate liquid and cleanout fluid storage pool region, and Magnet slide rail region, top board needs to reserve the resigning hole (respectively 17 and 18) of storage pool and Magnet slide rail, double adhesive tape should reserve the resigning hole (respectively 21 and 22) when storage pool and sample mixed liquor inflow filtering area.
The material of the conductive pin (31) of described liquid sensing device is simple metal or the alloy conductive materials such as gold, copper, aluminum；Described contact probe (30) can use electrical testing probe, probe interior band spring, and probe can be round end or tack；By between detection conductive pin and reference point, or resistance value between conductive pin two-by-two, and the comparison between canonical parameter, determine the flow regime of liquid in micro-fluidic chip and whether be mixed into bubble etc..
The testing process of described micro-fluidic chip includes:
(1) top board flow process: whole blood sample is instilled adding mouth (4), sample flows into sample fill area (12), micro-fluidic chip is put in necessary instrument, enzyme or luminous agent standard configuration body are after tagged ligand storage pool (5) discharges, air pump (3) makes sample and enzyme or luminous agent standard configuration body in sample mixed zone (13) mix homogeneously, then injects base plate filtering area (6) by resigning hole (22)；
(2) base plate flow process: sample, after filtering area (6) filter, arrives magnetic particle and is coated district (7), dissolve magnetic standard configuration body, controls Magnet and moves, accelerates analyte and magnetic standard configuration precursor reactant in sample, and then Magnet collects magnetic particle；Cleanout fluid storage pool (9) release cleanout fluid, after magnetic particle cleans, magnetic particle is moved to detection zone (8) by Magnet；Luminous substrate liquid storage pool (9) release luminous substrate liquid, instrument detecting system detection luminous signal intensity, thus realize the detection by quantitative of analyte.
In described base plate flow process, analyte and the reaction of part, comprise analyte and form sandwich structure with enzyme labelled antibody and magnetic particle traget antibody, and analyte is competed with enzyme labelled antibody, reduces the combination of magnetic particle and enzyme labelled antibody.
The analyte part of the present invention comprises antigen, hapten, monoclonal antibody, polyclonal antibody and hormone receptor.This part can be combined (such as double antibody sandwich method) or compete (such as competition law) with analyte with analyte.Wherein the antibody of luminous agent labelling can be identical with the antibody of magnetic particle marker, it is also possible to different.
As shown in Figure 3, described luminous substrate liquid is the most removable is divided into substrate solution and luminescence enhancement liquid, it is injected separately into luminous substrate liquid storage pool A (24), luminous substrate liquid storage pool B (25), by luminous substrate liquid pre-mixing passages (26) mix homogeneously after release.
The sample volume of the present invention is at 10～500 μ L, preferably 20～100 μ L.As preferably, injection volume is 50 μ L in an embodiment.
Magnetic granule used by the present invention comprises the compound of ferrum, cobalt, nickel, mainly including but not limited to iron sesquioxide and ferroso-ferric oxide compound.Preferably magnetic granule be polystyrene be shell, iron sesquioxide is the granule of core.Owing to magnetic granule easily precipitates, traditional chemical light-emitting appearance use manual mixing, and with persistent oscillation maintain magnetic granule suspended state, but in micro-fluidic chip magnetic granule be mixed operation be difficult in miniature portable instrument realize.Magnetic granule is coated, is dried in micro-fluidic chip raceway groove by the present invention, and devise Magnet active drive magnetic granule (and traditional microfluidic chip typically uses fluid drives or electric drive), so that magnetic granule redissolves, and realize immunoreation, cleaning, luminescence in micro-fluidic chip zones of different.This design not only solve magnetic granule be applied to during micro-fluidic chip easily to precipitate, the problem such as poor repeatability, also achieve more controlled immunoreation and physical cleaning, improve sensitivity and repeatability.Wherein Detection results is had a significant effect by Magnet magnetic and magnetic particle size, and select magnet magnetic induction of the present invention is 500-30000 Gauss, preferably 1000-8000 Gauss；Magnetic particle size is 0.1-10 μm, preferably 0.5-3 μm.
Magnetic microparticle chemiluminescence micro-fluidic chip of the present invention needs a supporting small portable device, is used for realizing air pump extruding on micro-fluidic chip, the release of multiple storage pool liquid, Magnet move the functions such as control and chemiluminescence signal detection.The micro-fluidic chip of the present invention is quickly detection, and the detection time should be less than 30 minutes, as preferably, uses 15 minutes in embodiment.
A kind of magnetic microparticle chemiluminescence micro-fluidic chip that the present invention provides is a kind of based on chemiluminescence, it is achieved analyte is quick, accurate, highly sensitive detection micro-fluidic chip.Can be used for the analyte in quantitative sample.Analyte includes little molecule (medicine and drugs), antigen, antibody, hormone, antibiotic, antibacterial and virus and other biochemical markers.Wherein, other biochemical markers include cardiovascular mark, tumor markers and autoimmune disease mark.
The core of the present invention is to use magnetic microparticle chemiluminescence immunoassay technology to realize quick, the detection of high sensitivity, accurate quantitative analysis of object at micro-fluidic chip.
Utilize liquid sensing device provided by the present invention, liquid flowing state in chip can be monitored, by reading the detection data of liquid sensing device, may determine that whether liquid is reacted according to the process set, and whether fluid path exists bubble etc., thus add the accuracy of microfluidic chip analysis result.
The micro-fluidic chip of the present invention is by all reagent component (the enzyme standard configuration body needed for detection process, magnetic particle marker part, cleanout fluid, luminous substrate liquid etc.) the most integrated, it is built in micro-fluidic chip, and designed by ingenious raceway groove, under the operation of necessary instrument, the operating in a key realizing micro-fluidic chip (only need to can be achieved with detection by initiating key, without complex operations), realize detection sample to separate, immunoreation, clean and separate, chemiluminescence detection, thus it is simple to avoid structure design in existing micro-fluidic chip, not enough and the defects such as complicated are operated during detection.The micro-fluidic chip of the present invention may be used for whole blood test, overcomes traditional chemical light-emitting appearance and can only carry out serum or blood plasma detection, and the shortcoming that can not detect whole blood sample.
In the present invention, micro-fluidic chip necessary instrument contacts with micro-fluidic chip no liquid, it is not necessary to parts to be cleaned, it is to avoid cross interference that traditional giant chemical light-emitting appearance needs stirring or sample-adding, cleaning etc. to operate and produces and pollution.
The present invention is not simple superposition magnetic microparticle chemiluminescence technology and microfluidic chip technology; but designed by liquid seal design, raceway groove; required for detection all chemical constituents are integrated, it is built in micro-fluidic chip; and with Magnet active drive; realize one-touch magnetic microparticle chemiluminescence immune detection, thus in portable necessary instrument, realize quick, the detection of high sensitivity, accurate quantitative analysis of analyte in whole blood.
Accompanying drawing explanation
Fig. 1 is the structural representation of magnetic microparticle chemiluminescence micro-fluidic chip; wherein 1 is top board; 2 is base plate, and 3 is air pump, and 4 is adding mouth; 5 is tagged ligand storage pool; 6 is filtering area, and 7 are coated district for magnetic granule, and 8 is detection zone; 9 is cleanout fluid storage pool; 10 is luminous substrate liquid storage pool, and 11 is lid, and 12 is sample fill area; 13 is sample mixed zone; 14 is cleaning area, and 15 is waste liquid pool, and 16 is liquid release channel; 17 is luminous substrate liquid and cleanout fluid storage pool resigning hole (in top board), and 18 is Magnet slide rail resigning hole.
Fig. 2 is the package assembly schematic diagram of magnetic microparticle chemiluminescence micro-fluidic chip top board and base plate; wherein 1 is top board; 2 is base plate; 19 is two-sided tape; 20 is one-faced tapes; 21 is luminous substrate liquid and cleanout fluid storage pool resigning hole, and 22 flow into resigning hole during filtering area for mixed liquor, and 23 is the elastic packing adhesive tape on air pump.
Fig. 3 is the micro-fluidic chip base arrangement schematic diagram of a kind of pair of luminous substrate liquid of present pre-ferred embodiments, and wherein 24 is luminous substrate liquid storage pool A, and 25 is luminous substrate liquid storage pool B, and 26 is luminescent solution pre-mixing passages.
Fig. 4 is the liquid sensing device structural section schematic diagram that the present invention provides.27 is micro-fluidic chip (top board or base plate), and 28 is metal basal board, and 29 is support circuit plate, and 30 is contact probe, and 31 is conductive pin.
Fig. 5 is the liquid sensor apparatus structure schematic diagram of the micro-fluidic chip top board of present pre-ferred embodiments.A1, b1, c1, d1 are four conductive pins being embedded on microchannel.
Fig. 6 is the liquid sensor apparatus structure schematic diagram of the micro-fluidic chip base plate of present pre-ferred embodiments.A2, b2, c2, d2, e2 are five conductive pins being embedded on microchannel.
Detailed description of the invention
The invention discloses a kind of magnetic microparticle chemiluminescence micro-fluidic chip, 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 in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment 1: with the micro-fluidic chip of liquid sensor device
As shown in Figure 4, for a kind of liquid sensing device structural section schematic diagram for micro-fluidic chip.A conductive pin (31) it is inlaid with on micro-fluidic chip (27), contact probe (30) one end is arranged on supporting circuit board (29), the other end passes metal basal board (28), and the conductive pin exposed with on micro-fluidic chip contacts.Metal basal board top is used for carrying micro-fluidic chip and fixing circuit board, and support circuit plate is used for detecting the signal of telecommunication on probe.
As shown in Figure 5 and Figure 6, the diverse location of micro-fluidic chip is inlaid with multiple conductive pin, is respectively positioned on micro-fluidic chip the side of the microchannel having liquid process, communicates with microchannel.Conductive pin uses aluminum to be processed into the acicular, columnar body of diameter 1mm, and conductive pin height is equal to chip thickness.Conductive pin is mounted in the mould of chip as inserts during chip injection molding, makes conductive pin and chip be integrally forming, it is ensured that the microchannel with conductive pin will not occur leakage.The function realized according to chip, four conductive pins it are designed with on top board, position is as follows: conductive pin a1 is near sample application mouth (4), it is positioned at the initiating terminal of sample fill area (12), conductive pin b1 is positioned at sample fill area end, and near tagged ligand storage pool (5), conductive pin c1 and d1 then lays respectively at initiating terminal and the end of sample mixed zone (13)；The Position Design of five conductive pins of base plate is as follows: conductive pin a2 is positioned at sample filtering area (6) outlet, magnetic particle is coated the initiating terminal of district (7), conductive pin b2 is positioned at magnetic particle and is coated the end of district (7), conductive pin c2 and d2 lays respectively in cleanout fluid storage pool (9) and luminous substrate liquid storage pool (10) outlet, and conductive pin e2 is positioned at the end of liquid release channel (16), the front position of detection zone (8).According to the structure of the liquid sensing device shown in Fig. 4, needing there being a contact probe bottom each conductive pin, what contact probe shown here was selected is the round end battery probe of electrical testing, probe interior band spring.Measure resistance value between conductive pin two-by-two by support circuit, thus judge the liquid flowing state between corresponding microchannel.
For top board (1): it is enough that the resistance value between conductive pin a1 and b1 is used for judgment sample amount after sample-adding, and whether air pump drives sample completely into sample mixed zone (13), resistance value between conductive pin c1 and d1 is used for the occupied state of judgment sample mixed zone (13), and has mixed whether rear air pump drives sample downwardly into base plate.
For base plate (2): whether the resistance value judgment sample between conductive pin a2 and b2 is full of magnetic particle after filtering is coated district (7), resistance value between conductive pin c2 and e2 and d2 and e2 is then respectively intended to judge cleanout fluid and the mobility status of luminous substrate liquid, if discharges from storage pool and fills up whole liquid release channel (16).
All of resistance value has a standard value when liquid is full of passage, and actual measured value is compared with standard value, if measured value and standard value has obvious deviation, then error occurs in the liquid flowing state in explanation microchannel.Such as, if fluid flow is not enough to fill up microchannel, then the resistance value recorded is generally higher than standard value；Equally, if being mixed into bubble in passage, the resistance value recorded also can be higher than standard value.If the result that liquid sensing device records and standard value have obvious deviation, then the last testing result of this micro-fluidic chip then can not react the actual value of sample, and needs again to detect.
Embodiment 2: double-antibody sandwich measures super quick Troponin I (cTnI)
(1) antibody labeling
Take 5 μ gHRP to be dissolved in 1mL distilled water, add 0.2mL0.1M and newly join NaIO4 solution, after room temperature lucifuge reaction 20min, with 1mMpH4.4 sodium-acetate buffer dialysis purification solution.Regulate pH to 9.0 with 0.2MpH9.5 carbonate buffer solution again, add 10 μ g anti-cTnI monoclonal antibody, room temperature lucifuge reaction 2h.Add the 4mg/mLNaBH4 liquid that 0.1mL newly joins, mixing, react 2h in 4 DEG C.Above-mentioned solution being loaded bag filter, dialyses with 0.15MpH7.4PBS, 4 DEG C overnight, obtains HRP labelling cTnI antibody.
1mg magnetic granule (a size of 2 μm), 10 μ gEDC and 15 μ gNHS solution and 15 μ g anti-cTnI monoclonal antibody (different from the antibody of HRP labelling) solution is added in 1ml10mMpH7.4 phosphate buffer, mix homogeneously also reacts 4h at room temperature, adds 1mg glycine and closes.It is enriched with Magnet, removes unreacted anti-cTnI monoclonal antibody, obtain magnetic particle marker cTnI antibody.
(2) micro-fluidic chip assembles
Containing 0.1% bovine serum albumin, 0.1% polysorbas20 and the pH7.4 phosphate buffer of 0.01%Proclin300 in HRP labelling cTnI antibody-solutions；Magnetic particle marker cTnI antibody-solutions is the pH7.4 phosphate buffer comprising 0.5% bovine serum albumin, 0.1% casein, 0.2% polysorbas20 and 0.01%Proclin300.
Cleanout fluid is the pH7.4 phosphate buffer comprising 0.2%BSA, 0.5% polysorbas20 and 0.01%Proclin300.Luminous substrate liquid divides A liquid and B liquid, and A liquid is the acid solution containing luminol, and B liquid is the alkaline solution containing benzene derivative.
HRP traget antibody solution is put in top board enzyme standard configuration body storage pool (5), seal.Magnetic particle marker antibody-solutions is put into base plate be coated in district (7), drying at room temperature.Cleanout fluid is injected in cleanout fluid storage pool (9), A liquid and the B liquid of luminous substrate liquid are injected separately into luminous substrate liquid storage pool A (24) and luminous substrate liquid storage pool B (25), seal.As shown in Figure 1, hemofiltration film is glued in base plate filtering area, storage pool is built into base plate.The most as shown in Figure 2, with one-faced tapes and two-sided tape, top board and base plate are assembled into micro-fluidic chip.Load in aluminium foil bag, seal 4 DEG C of preservations.
(3) pattern detection
Make diluent with human normal plasma, cTnI standard substance are diluted to following concentration: 0pg/ml, 5pg/ml, 50pg/ml, 500pg/ml, 5ng/ml, 50ng/ml and 200ng/ml.
After 50 μ l samples are instilled adding mouth, close the lid.Micro-fluidic chip is put in necessary instrument (Magnet magnetic induction is 6000 Gausses), instrument extrusion HRP labelling monoclonal antibody, and make injection base plate filtering area after sample and HRP labelling monoclonal antibody mix homogeneously.After sample filters, arriving microchannel, and dissolve magnetic particle marker monoclonal antibody, Magnet accelerates sample reaction, forms the sandwich structure of HRP labelling monoclonal antibody-cTnI antigen-magnetic particle marker monoclonal antibody, and then Magnet collects magnetic granule.Storage pool release cleanout fluid, after magnetic particle cleaning, luminous substrate liquid discharges, instrument detecting system detection luminous signal intensity.Total detection time 15min.Each sample measures 3 times with 3 micro-fluidic chips respectively, averages, and draws standard curve.
50 μ l whole blood samples are instilled adding mouth, and instrument detecting system detection luminous signal intensity in 15 minutes, establishing criteria curve obtains cTnI concentration in sample.
Cleaning Principle is: after whole blood adds micro-fluidic chip, and whole blood first mixes with HRP traget antibody, and after being then passed through filtering district, the blood plasma being mixed with HRP traget antibody arrives microchannel, and blood plasma dissolves magnetic marker antibody.When blood sample contains cTnI, then form the sandwich structure (double antibody sandwich method) of HRP traget antibody-cTnI-magnetic particle marker antibody.Once purged more luminous under luminous substrate liquid effect, instrument detecting system test luminous signal.The standard curve obtained according to necessary instrument, and then analyze cTnI concentration in blood sample.In sample, cTnI content is the highest, then luminous signal is the strongest.
Result shows, its lowest detection is limited to 10pg/ml, minimum is quantitatively limited to 50pg/ml, in the range of detection by quantitative, coefficient R2, there is not HOOK effect in ＞ 0.99, and batch in and batch between repeatability all preferable, can be that heart infarction heart failure medical diagnosis on disease provides reference.
Embodiment 3: magnetic particle particle size is screened
Other experiment condition sees embodiment 2, magnetic particle size and Magnet magnetic induction to be carried out according to below scheme.
Particle size is 0.1 μm, 0.5 μm, 1.8 μm, 2.4 μm, 1 μm, 3 μm, 10 μm.Magnet magnetic induction is 500 Gausses, 1000 Gausses, 4000 Gausses, 8000 Gausses, 12000 Gausses.The magnetic granule of seven kinds of sizes is driven respectively with these five kinds of Magnet.
Experimental result shows: when 0.1 μm magnetic granule and the combination of 500 Gauss Magnet, its lowest detection is limited to 500pg/ml, and detection by quantitative scope is 0.5～50ng/ml, linearly dependent coefficient R2＞ 0.95；Batch in and batch between repeatability be respectively less than 20%.That is: chemiluminescence signal is more weak, and sensitivity is the highest, and repeatability is poor.
When 10 μm magnetic granules and the combination of 12000 Gauss Magnet, its lowest detection is limited to 150pg/ml, and detection by quantitative scope is 0.1～10ng/ml, linearly dependent coefficient R2＞ 0.95；Batch in and batch between repeatability be respectively less than 20%.That is: negative sample signal higher (cleaning insufficient), the range of linearity is the widest.
When the magnetic granule of 0.5～3 μm and the Magnet of 1000～8000 Gausses are applied in combination, its lowest detectable limit is respectively less than 100pg/ml, and detection by quantitative scope can reach 0.1～50ng/ml, linearly dependent coefficient R2＞ 0.98；Batch in and batch between repeatability be respectively less than 12%.Meet the needs that reference is provided for clinical heart infarction heart failure medical diagnosis on disease.
According to result above, magnetic particle size preferably 0.5～3 μm, Magnet magnetic induction preferably 1000～8000 Gauss.
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 (5)
1. a magnetic microparticle chemiluminescence micro-fluidic chip; it is characterized in that; described micro-fluidic chip includes top board (1) and base plate (2), and wherein said top board (1) comprises air pump (3), adding mouth (4), tagged ligand storage pool (5), sample fill area (12) and sample mixed zone (13)；Described base plate (2) comprises filtering area (6), magnetic particle is coated district (7), chemiluminescence detection district (8), cleanout fluid storage pool (9), luminous substrate liquid storage pool (10), cleaning area (14), waste liquid pool (15) and liquid release channel (16)；Described top board (1) and base plate (2) two-sided tape (19) and one-faced tapes (20) seal, and are assembled into micro-fluidic chip；Described top board (1) and base plate (2) all comprise liquid sensing system；Described liquid sensing system is made up of one or more liquid sensing devices；Described liquid sensing device includes: conductive pin (31), contact probe (30), metal basal board (28) and support circuit plate (29)；Described conductive pin is embedded in by micro-fluidic chip liquid release channel, communicates with microchannel, and is exposed on the surface of chip；Described contact probe one end is arranged on support circuit plate, and the other end passes metal basal board, contacts one to one with the conductive pin on micro-fluidic chip；Described metal basal board is used for loading micro-fluidic chip and fixing circuit board；Described support circuit plate is used for realizing detecting the signal of telecommunication on probe；
Described air pump (3) and adding mouth (4) UNICOM, adding mouth (4) and tagged ligand storage pool (5) are connected by sample fill area (12), and tagged ligand storage pool (5) is connected with sample mixed zone (13)；The detection zone (8) of described base plate is connected by liquid release channel (16) with cleanout fluid storage pool (9) and luminous substrate liquid storage pool (10)；Described filtering area (6), magnetic particle are coated district (7), cleaning area (14), detection zone (8) are sequentially connected with；Described sample mixed zone (13) end connects with filtering area (6)；
It is covered with one layer of elastic rubber belt (23) on described air pump (3), micro-fluidic chip is formed an air chamber sealed, by pressing and unclamping elastic rubber belt, it is provided that positive and negative pressure air-flow, thus drive sample to flow forward or backward；
Described tagged ligand storage pool (5), cleanout fluid storage pool (9), luminous substrate liquid storage pool (10) and magnetic particle are coated pre-packaged in district (7) has reagent；Liquid in storage pool is to seal, and can be extruded by external force and make partial fracture and releasing liquid；
Described filtering area (6) comprises hemofiltration film, is retained the erythrocyte in whole blood sample by physical separation mode.
2. magnetic microparticle chemiluminescence micro-fluidic chip as claimed in claim 1, it is characterised in that the material of the conductive pin (31) of described liquid sensing device is gold, copper, aluminum or alloy conductive material；Described contact probe (30) is electrical testing probe, probe interior band spring, and probe can be round end or tack；By between detection conductive pin and reference point, or resistance value between conductive pin two-by-two, and the comparison between canonical parameter, determine the flow regime of liquid in micro-fluidic chip and whether be mixed into bubble.
3. magnetic microparticle chemiluminescence micro-fluidic chip as claimed in claim 1 or 2, it is characterised in that sample volume described in top board flow process is 10-500 μ L.
4. magnetic microparticle chemiluminescence micro-fluidic chip as claimed in claim 3, it is characterised in that the μm of magnetic particle a size of 0.1～10 described in base plate flow process.
5. magnetic microparticle chemiluminescence micro-fluidic chip as claimed in claim 4, it is characterised in that the μm of magnetic particle a size of 0.5～3 described in base plate flow process.
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| CN201520828120.0U CN205449995U (en) | 2015-10-26 | 2015-10-26 | Magnetic particle chemiluminescence micro -fluidic chip |
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| CN105214744A (en) * | 2015-10-26 | 2016-01-06 | 深圳华迈兴微医疗科技有限公司 | A kind of magnetic microparticle chemiluminescence micro-fluidic chip |
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