CN107044972A - A kind of micro-fluidic chip fluorescence immunoassay quick detection kit and its preparation and detection method - Google Patents
A kind of micro-fluidic chip fluorescence immunoassay quick detection kit and its preparation and detection method Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/571—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses for venereal disease, e.g. syphilis, gonorrhoea
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
- G01N33/5764—Hepatitis B surface antigen
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2446/00—Magnetic particle immunoreagent carriers
- G01N2446/80—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids
- G01N2446/90—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids characterised by small molecule linker used to couple immunoreagents to magnetic particles
Abstract
A kind of micro-fluidic chip fluorescence immunoassay quick detection kit and its detection method of preparation, the kit contains micro-fluidic chip, the micro-fluidic chip includes one or more detection unit, the detection unit is located in half journal axle of micro-fluidic chip, dissipated and arranged with micro-fluidic chip central point, the detection unit is arranged in sequence with loading slot, fluorescence probe groove, reaction detection groove and waste liquid tank, and the microchannel being connected with each other from chip center's point to edge.Use the kit, reagent dosage is few, integrated height, reaction system is homogeneous, all reagents solidify in chip in advance, and antigen-antibody reaction, no cross contamination can be carried out in relatively closed space, it is free from environmental pollution, there is high efficiency, high flux, high sensitivity, quick, sensitive, stabilization.
Description
Technical field
The invention belongs to field of immunodetection, and in particular to a kind of micro-fluidic chip fluorescence immunoassay quick detection kit and
It is prepared and detection method.
Background technology
Micro-fluidic chip (microfluidic chips) technology is quickly grown in recent years, in medical science, life science
Application Deng field is constantly extended.Micro-fluidic chip refers to use Micrometer-Nanometer Processing Technology, by microchannel network structure and its
His function element is integrated on several square centimeters of substrate, by being controlled to the fluid in microchannel, with realize sample introduction,
The micro-total analysis system of a variety of functions such as dilution, mixing, reaction, separation, detection, with miniaturization, integrated, analyze speed
It hurry up, reagent consumes the remarkable advantage such as few.
Immunolabelling technique is using antigen and antibody specific reaction as general principle, using fluorescein, isotope or enzyme etc.
Probe material labelled antibody (or antigen) carries out antigen-antibody reaction, by the measure to the label in immune complex, reaches
To the purpose detected to immune response.
Immunolabelling technique is divided into homogeneous immune response and heterogeneous immune response by the physical state of detection reaction system.
Heterogeneous immune response refers to antigen or antibody being fixed on solid phase carrier（Microwell plate or filter paper）Surface, passes through
Specific immune response, by the antibody of required detection or antigen binding in solid phase surface, after cleaned, that is, can be achieved antigen
Physical separation between antibody complex and free antigen, antibody.Usually used immunochromatographic method, ELISA, plate
Formula chemoluminescence method is heterogeneous immunoassay method.
" micro-fluidic chip immunoassay antibody fixing means compares "（《Chinese Medicine biotechnology》1st phase in 2015
）, " micro-fluidic chip immuno analytical method and its progress "（《Laboratory medicine》The 4th phases of volume 2007 the 22nd）Etc. document report profit
With micro-fluidic chip conduit wall it is solid phase carrier come coated antibody（Or antigen）Method, be exactly a kind of easy micro-fluidic chip
The heterogeneous immunoassay method being combined with microwell plate immunological technique.
Patent " double layer micro fluidic chip device and its purposes in immune detection "（CN 102749433）Use micro-
Fluidics and traditional immunolabelling technique（It is heterogeneous）It is combined.Two micro-fluidic chips above and below use, each lead into anti-
Former and antibody, by vacuumizing, makes antigen and antibody be enriched in respectively on polyester film and occurs immune response.The patent has following
It is not enough：（1）Antigen and antibody reagent used still needs to be stored in reagent bottle respectively（It is identical with conventional method）, in process of the test
Flowed, mixed, reacted by vacuumizing driving liquid, it is impossible to make reaction integrated；（2）Solid phase carrier used-polyester film is replaced
Microwell plate（Or test paper）, cost height；（3）The enrichment of polyester film pore size influences antigen or antibody；（3）Heterogeneous reaction is in solid phase
The surface of carrier is reacted, and reacts insufficient, sensitivity and repeatability are restricted.
Homogeneous immune response refers to antigen and antibody in same medium（Liquid system）The immune response of middle progress.At present should
It is exactly homogeneous immune response with wider magnetic microparticle chemiluminescence immunization method, i.e., by antibody（Or antigen）It is coupled at and has wrapped up magnetic
On the microballoon of particulate, antibody has been coupled by this during the course of the reaction（Or antigen）Magnetic microsphere be suspended in reaction solution so that
This magnetic microsphere antibody（Or antigen）Fully contacted with test substance in solution, make immune response more fully, completely.Additional
Under magnetic fields, the compound that immune response is formed is separated with uncombined other materials.This method is as a result of homogeneous
Reaction system, makes the precision and repeatability of reaction further improve.But the technology needs to match large-scale detecting instrument, check fee
With big, short time consumption is long, and reagent and amount of samples are big.
" the microflow controlled biochip systematic research based on magnetic bead technology "（《Shanghai Communications University》2007）Being will
Immunomagnetic beads is placed in microfluidic channel, due to there was only the shadow of transient contact by fluid-flow rate and antigen and antibody
Ring, antigen and antibody are it cannot be guaranteed that fully, thoroughly react.
There is presently no more for microflow control technique, magnetic particle immunological technique and the research of immunofluorence technic combined technology
Document report.
Therefore, a kind of convenient, fast, high flux, high sensitivity, integrated height, the miniflow for being adapted to industrialized production are developed
Control chip equal（Liquid）Phase fluorescence immunoassay quick detection kit is current urgent problem to be solved.
The content of the invention
It is an object of the invention to provide a kind of micro-fluidic chip fluorescence immunoassay quick detection kit, the kit is with glimmering
Stimulative substance is label, using magnetic microsphere as catches, and reagent dosage is few, and integrated height, all reagents solidify in core in advance
In piece；Mark fluorescent microballoon and capture magnetic bead solidification are easy, easily redissolve, easily suspend；It can be resisted in relatively closed space
Antigen-antibody reaction, no cross contamination is free from environmental pollution.With high efficiency, high flux, high sensitivity, quick, sensitive, stable etc.
Advantage.
The object of the present invention is achieved like this：A kind of micro-fluidic chip fluorescence immunoassay quick detection kit, its feature
It is：The kit contains micro-fluidic chip, and the micro-fluidic chip includes one or more detection unit, the detection unit
In half journal axle of micro-fluidic chip, dissipated and arranged with micro-fluidic chip central point, the detection unit is from chip center's point
Loading slot, fluorescence probe groove, reaction detection groove and waste liquid tank, and the microchannel being connected with each other are arranged in sequence with to edge；Institute
State fluorescence probe trench bottom and be solidified with fluorescence probe, the fluorescence probe is polystyrene fluorescence（Flu）Microballoon coupled antibody（Or it is anti-
Former or secondary antibody）Compound；The reaction detection in-tank-solidification has capture magnetic bead microballoon, and the capture magnetic bead microballoon is polystyrene
Magnetic bead microballoon coupled antibody（Or antigen or secondary antibody）Compound.
The micro-fluidic chip only loading slot is communicated by well with the external world, and remaining groove is closing.
The loading slot, fluorescence probe groove, reaction detection groove and waste liquid tank volume it is equal or unequal, two neighboring groove
Spacing it is equal or unequal.
The driving force of the micro-fluidic chip is centrifugal force.
A diameter of 10~300nm of described polystyrene fluorescent microsphere, preferably 50~200nm；Polystyrene fluorescence is micro-
Parcel fluorescent material in ball, the fluorescent material includes rhodamine, fluorescein isothiocynate, phycoerythrin, lanthanide series（Samarium
Sm, europium Eu, gadolinium Gd, terbium Tb etc.）, quantum dot etc.；The polystyrene magnetic beads microballoon is directly 1~10um, preferably 2um~
Parcel super-paramagnetic ferriferrous oxide particulate (Mag), particle average diameter 10nm in 5um, microballoon.
The micro-fluidic chip is by two boards up and down（A plates, B plates）Bonding is formed, and the material of the micro-fluidic chip is
The high polymer such as quartz, glass or PDMS, PMMA, PS, PC, COC, COP, the micro-fluidic chip is shaped as circular or circumscribed circle
Polygon.
The micro-fluidic chip centre bit is equipped with a fixing hole matched with centrifuge rotating shaft, the micro-fluidic chip
Thickness is 4mm~10mm, preferably a diameter of 50~200mm, 80~100mm, and the rooved face can be circular, oval, water
Shape or other irregular shapes are dripped, the volume of groove is 5~200ul.
The detection sample of the detection kit be whole blood, serum, blood plasma, saliva, sputum, tear, urine or other people
Body secretion, and aforesaid liquid extract solution or dilution or eluent；The detection kit can carry out 1 sample simultaneously
Multiple-factor detect respectively, the detection respectively of one or more factors of multiple samples can be also carried out simultaneously.
A kind of preparation method of micro-fluidic chip fluorescence immunoassay quick detection kit, this method comprises the following steps.
（1）Prepare A plates and B plates.
The detection unit that A plates and B plates contain on mutual corresponding one or more detection unit, A plates contains well, glimmering
Detection on part on light probe groove, part on part and waste liquid tank on reaction detection groove, and the passage being connected with each other, B plates
Unit contains under the loading slot corresponding with A plates, fluorescence probe groove part and waste liquid tank bottom under part, reaction detection groove
Point.
（2）The preparation of fluorescence probe polystyrene fluorescent microsphere.
Take parcel fluorescent material polystyrene fluorescent microsphere is scrubbed, ultrasonic, after stir process, with antibody 1（Or antigen
1 or secondary antibody）Coupling, adds BSA buffer solutions and makees Seal treatment to fluorescent microsphere, produce " antibody（Or antigen or secondary antibody）- fluorescence is micro-
Ball " [Ab1（Ag1）-Flu].
（3）Capture antibody 2（Or antigen 2 or secondary antibody）The preparation of magnetic microsphere.
Take that Magnetic Polystyrene Microsphere is scrubbed, ultrasonic, after stir process, with antibody 2（Or antigen 2 or secondary antibody）Mixing,
Add BSA buffer solutions and Seal treatment is made to microballoon, produce " capture antibody（Or antigen or secondary antibody）- magnetic microsphere " [Ab2 (Ag2)-
Mag]。
（4）By " antibody 1（Or antigen 1 or secondary antibody）- fluorescent microsphere " and " capture antibody 2（Or antigen 2 or secondary antibody）- magnetic
Each 1~20ul of microballoon " " is added separately in the fluorescence probe groove and reaction detection groove of B plates, 37 °C of dry solidifications.
（5）Chip A, B plate is bonded to by one piece of complete chip using ultrasonic bonding machine.
（6）Sample well is closed with special adhesion sealed membrane, in envelope aluminium foil bag, produced.
A kind of detection method of micro-fluidic chip fluorescence immunoassay quick detection kit, this method comprises the following steps.
(1) test substance solution is added in well, and solution to be measured is driven to reaching fluorescence probe groove simultaneously by centrifuging
Probe microballoon is dissolved, test substance is specifically bound with corresponding antibody 1 (or antigen 1 or secondary antibody), formation Ag-Ab-glimmering
Light microballoon (Flu-Ab- Ag) compound.
（2）Under centrifugation driving, the solution such as the compound in fluorescence probe groove are all delivered to reaction detection groove by continuation
In, and dissolve the (Flu-Ab- of Ag-Ab-fluorescent microsphere 1 in reaction detection groove in capture antibody magnetic bead microballoon, solution
Ag) compound and capture antibody 2（Or antigen 2 or secondary antibody）Specific immune response occurs for-magnetic microsphere, formed " fluorescent microsphere-
The double-antibody sandwich magnetic composite (Flu-Ab1- Ag-Ab2-Mag) or " glimmering of antibody 1- Ag-Ab 2- magnetic beads microballoon "
Double antigens sandwich magnetic composite (Flu-Ag1- the Ab-Ag2- of light microballoon-antigen 1-antibody-antigene 2- magnetic beads microballoon "
Mag), and suspend in the solution.
（3）In the presence of externally-applied magnetic field, magnetic composite is attracted to the bottom of reaction detection groove, in centrifugation driving
Under, the solution without magnetic microsphere is transported to waste liquid tank, magnetic fluorescence compound then stays in the bottom of detect tank.
（4）The magnetic fluorescence compound added by well in cleaning buffer solution, centrifugation, cleaning reaction detection groove, plus
Enter external magnetic field, centrifugation removes cleaning fluid, operated repeatedly.
（5）Detected by fluorescence detector, with the magnetic fluorescence compound in the photo-irradiation reaction detect tank of certain wavelength,
The luminous intensity of fluorescent microsphere is detected, fluorescence intensity is in correlation with testing concentration.
Compared with prior art, the present invention has advantages below.
（1）Using fluorescent material as label, using magnetic microsphere as catches, reagent dosage is few, integrated height.
（2）All reagents solidify in chip in advance, save the use of a large amount of test tubes of conventional reagents box, and save repeatedly
Sampling operation.
（3）The polystyrene fluorescent microsphere and polystyrene magnetic beads microballoon of solidification work as sample solution or cleaning buffer solution is flowed through
When easily redissolve and be suspended in liquid.
（4）Sample needs sample amount few, and single index only needs 10ul.
（5）Detection time is short, simple to operate.Add sample after, be put into detector, can complete within 5~10 minutes all from
Dynamic detection.
（6）Totally-enclosed system, can carry out the detection of minimum 1 sample, remaining detection unit not polluted.
（7）Multisample or multiple-factor can be carried out to detect simultaneously, the high flux of detection is realized.
（8）Homogeneous reaction system makes testing result sensitivity high（10-15mol/ml）, precision it is high（CV ＜ 5%）, repeatability
It is higher（CV ＜ 5%）.
Brief description of the drawings
Accompanying drawing 1 is the detection unit structural representation of micro-fluidic chip fluorescence immunoassay kit.
In figure, 1:Well, 2:Loading slot, 3:Fluorescence probe groove, 4:Reaction detection groove, 5：Waste liquid tank,
6：Microchannel.
Accompanying drawing 2 is the micro-fluidic chip internal structure schematic diagram of micro-fluidic chip fluorescence immunoassay kit.
Accompanying drawing 3 is 3 samples, the microfluidic chip structure schematic diagram of 4 factor micro-fluidic chip fluorescence immunoassay kits.
Accompanying drawing 4 is the fluorescence immunoassay Cleaning Principle schematic diagram of micro-fluidic chip fluorescence immunoassay kit.
Accompanying drawing 5 is the comparative result figure of the invention with Roche magnetic particle electrochemical luminescence kit uniformity.
Embodiment
Further illustrated the following is to the present invention, rather than limitation of the present invention.
Embodiment 1：N-terminal plasma pro-brain natriuretic peptide levels（NT-proBNP）Micro-fluidic chip fluorescence immunoassay kit reagent（A variety of samples, 1
Index detection kit）Preparation and detection.
1st, A plates and B plates are prepared.
A plates and B plates are prepared, the two detection unit contained on mutually 12 detection units of correspondence, A plates contains sample-adding
On part on hole, fluorescence probe groove, part on part and waste liquid tank on reaction detection groove, and the passage being connected with each other, B plates
Detection unit contains under the loading slot corresponding with A plates, fluorescence probe groove under part, reaction detection groove under part and waste liquid tank
Part.
2nd, the preparation of labelled antibody fluorescent microsphere.
A diameter of 100nm polystyrene is taken to wrap up quantum dot（Qd, fluorescence probe）Fluorescent microsphere it is scrubbed, ultrasonic,
After stir process, it is coupled with NT-proBNP monoclonal antibodies 1, " labelled antibody 1- fluorescent microspheres " is formed after coupling reaction, plus
Enter BSA buffer solutions and Seal treatment is made to fluorescent microsphere, obtain " microballoon of NT-proBNP monoclonal antibodies 1 of fluorescence labeling " (Qd-
Ab1)。
3rd, the preparation of antibody magnetic microsphere is captured.
Take that a diameter of 5um Magnetic Polystyrene Microsphere is scrubbed, ultrasonic, after stir process, with NT-proBNP Dan Ke
Grand antibody 2 is mixed, coupling reaction formation " capture antibody 2- magnetic microspheres ", is added BSA buffer solutions and is made Seal treatment to microballoon,
Obtain " the NT-proBNP monoclonal antibody 2- magnetic microspheres " for capture（Ab2-Mag）.
4th, by " the quantum dot-labeled microballoon of NT-proBNP monoclonal antibodies 1 " and " NT-proBNP monoclonal antibody 2- magnetic
Each 10ul of property microballoon " is added separately in the fluorescence probe groove and reaction detection groove of B plates, 37 °C of dry solidifications 1 hour.
5th, chip A, B plate is bonded to by one piece of complete chip, chip structure such as Fig. 2 using ultrasonic bonding machine；Chip is straight
There are 12 groups of identical detection units on footpath 80mm, thickness 6mm, each chip.
6th, seal, closed sample well with special adhesion sealed membrane, in envelope aluminium foil bag, storage.
7th, detect, take out the micro-fluidic chip kit of Packing Sound, tear the sealed membrane of sample well, be put into homemade complete
In the chip room of automatic fluid-phase micro-fluidic chip fluorescence analysis detector, machine automatic data collection whole blood sample adds micro-fluidic chip
Loading slot in, automatic centrifugation, cleaning, detection, print result, all detection 10 minutes complete.
8th, testing result and repetition, see the table below.
The comparative result of testing result and Roche magnetic particle electrochemical luminescence kit uniformity.
Testing result to two methods carries out linear regression analysis, sets up equation of linear regression：Y=a+bX.Coefficient correlation
R values ＞ 0.90.From result, the testing result of two methods has good correlation, sees Fig. 5.
Embodiment 2：Preoperative 4（Hepatitis B surface antigen/c-hepatitis antibody/human immune defect virus antibody/treponemal
Body antibody）Joint inspection kit（3 parts of samples, 4 index joint inspection kits）Preparation and detection.
1st, labelled antibody（Or antigen）The preparation of fluorescent microsphere:A diameter of 200nm polystyrene is taken to wrap up europium metal chelating
Compound（Eu, fluorescence probe）Fluorescent microsphere is scrubbed, ultrasonic, after stir process, respectively with 1. hepatitis B surface antibody 1 2. hepatitis
Recombinant antigen 1 3. human immunodeficiency virus recombinant antigen 1 4. microspironema pallidum recombinant antigen 1 is coupled, after coupling reaction
4 kinds of Fluorescent microsphere markers are formed respectively, it is standby.
2nd, antibody is captured（Or antigen）The preparation of microballoon:Take a diameter of 3um Magnetic Polystyrene Microsphere scrubbed, super
After sound, stir process, respectively with the 1. hepatitis B surface antibody 2 2. 3. human immunodeficiency virus recombinant antigen 2 of hepatitis recombinant antigen 2
4. microspironema pallidum recombinant antigen 2 is coupled, and 4 kinds of capture antibody are formed after coupling reaction（Or antigen）Magnetic microsphere, it is standby.
3rd, with polymethyl methacrylate（PMMA）For material, chip diameter 100mm, A, B plate is by 3 groups of structure identicals
Microchannel constitutes 3 groups of detection units, and every group of detection unit is made up of 4 groups of structure identical micro-structurals, can entered simultaneously, respectively again
Detected while each 4 kinds of indexs of the sample of row 3, chip structure is as shown in Figure 3.
4th, respectively by 4 kinds of labelled antibodies（Or antigen）Fluorescent microsphere solidifies every group of corresponding fluorescence probe groove in B plates
Bottom, 37 °C of dry solidifications 40 minutes.
5th, antibody is captured by 4 kinds respectively（Or antigen）Magnetic bead microballoon solidifies every group of corresponding reaction detection groove in B plates
Bottom, 37 °C of dry solidifications 40 minutes.
6th, chip A, B plate is bonded to by one piece of complete chip, chip thickness 5mm using ultrasonic bonding machine.
7th, seal, closed various kinds sample wells with special adhesion sealed membrane, in envelope aluminium foil bag, storage.
8th, detect, take out the micro-fluidic chip kit of Packing Sound, tear the sealed membrane of sample well, be put into homemade complete
In the chip room of automatic fluid-phase micro-fluidic chip fluorescence analysis detector, machine automatic data collection whole blood sample adds micro-fluidic chip
Well in, automatic centrifugation, cleaning, detection, print result.All detection is completed for 10 minutes.
Results contrast:This kit and Abbott Laboratories ARCHITECT-i2000SYSTEM particulate chemical luminescence reagent kits（Second
Liver surface antigen, c-hepatitis antibody, human immune defect virus antibody, 4 kinds of syphilis helicoid antibody）Compare, be shown in Table 2.
The fluorescent immune method of table 2 and chemoluminescence method test result compare.
From result, HBsAg, Anti-TP, Anti-HIV, Anti-HCV comparison result are that positive coincidence rate is
100%, negative match-rate is 100%, K=1.Two methods testing result has the uniformity of height.
Claims (10)
1. a kind of micro-fluidic chip fluorescence immunoassay quick detection kit, it is characterised in that：The kit contains micro-fluidic core
Piece, the micro-fluidic chip includes one or more detection unit, and the detection unit is located in half journal axle of micro-fluidic chip,
Dissipated and arranged with micro-fluidic chip central point, the detection unit is arranged in sequence with loading slot from chip center's point to edge, glimmering
Light probe groove, reaction detection groove and waste liquid tank, and the microchannel being connected with each other, the fluorescence probe trench bottom are solidified with fluorescence
Probe, the fluorescence probe is polystyrene fluorescence（Flu）Microballoon coupled antibody（Or antigen or secondary antibody）Compound；The reaction
Capture magnetic bead microballoon is solidified with detect tank, the capture magnetic bead microballoon is polystyrene magnetic beads microballoon coupled antibody（Or antigen or
Secondary antibody）Compound.
2. micro-fluidic chip fluorescence immunoassay quick detection kit according to claim 1, it is characterised in that the miniflow
Control chip only loading slot is communicated by well with the external world, and remaining groove is closing.
3. micro-fluidic chip fluorescence immunoassay quick detection kit according to claim 1, it is characterised in that the sample-adding
Groove, fluorescence probe groove, reaction detection groove and waste liquid tank volume it is equal or unequal, the spacing of two neighboring groove is equal or not phase
Deng.
4. micro-fluidic chip fluorescence immunoassay quick detection kit according to claim 1, it is characterised in that the miniflow
The driving force for controlling chip is centrifugal force.
5. micro-fluidic chip fluorescence immunoassay quick detection kit according to claim 1, it is characterised in that described is poly-
A diameter of 10~300nm of styrene fluorescent microballoon, preferably 50~200nm；Parcel fluorescent material in polystyrene fluorescent microsphere,
The fluorescent material includes rhodamine, fluorescein isothiocynate, phycoerythrin, lanthanide series（Samarium Sm, europium Eu, gadolinium Gd, terbium Tb
Deng）, quantum dot etc.；The polystyrene magnetic beads microballoon is directly 1~10um, preferably 2um~5um, the interior parcel superparamagnetic of microballoon
Ferroferric oxide particle (Mag), particle average diameter 10nm.
6. micro-fluidic chip fluorescence immunoassay quick detection kit according to claim 1, it is characterised in that the miniflow
Control chip is by two boards up and down（A plates, B plates）Bonding is formed, the micro-fluidic is quartz, glass or PDMS,
The high polymers such as PMMA, PS, PC, COC, COP, the polygon for being shaped as circular or circumscribed circle of the micro-fluidic chip.
7. micro-fluidic chip fluorescence immunoassay quick detection kit according to claim 1, it is characterised in that the miniflow
There is a fixing hole matched with centrifuge rotating shaft control chip center position, and the thickness of the micro-fluidic chip is 4mm~10mm,
A diameter of 50~200mm, preferably 80~100mm, the rooved face can be circular, ellipse, water-drop-shaped or other are irregular
Shape, the volume of groove is 5~200ul.
8. micro-fluidic chip fluorescence immunoassay quick detection kit according to claim 1, it is characterised in that the detection
The detection sample of kit is whole blood, serum, blood plasma, saliva, sputum, tear, urine or other human secretions, and above-mentioned
The extract solution or dilution or eluent of liquid；The multiple-factor that the detection kit can carry out 1 sample simultaneously detects respectively,
Also the detection respectively of one or more factors of multiple samples can be carried out simultaneously.
9. a kind of preparation method of the micro-fluidic chip fluorescence immunoassay quick detection kit described in claim 1, its feature exists
Comprise the following steps in this method：
（1）Prepare A plates and B plates:
The detection unit that A plates and B plates contain on mutual corresponding one or more detection unit, A plates, which contains well, fluorescence, to be visited
Detection unit on part on needle tray, part on part and waste liquid tank on reaction detection groove, and the passage being connected with each other, B plates
Contain part under part under part, reaction detection groove under the loading slot corresponding with A plates, fluorescence probe groove and waste liquid tank；
（2）The preparation of fluorescence probe polystyrene fluorescent microsphere:
Take parcel fluorescent material polystyrene fluorescent microsphere is scrubbed, ultrasonic, after stir process, with antibody 1（Or antigen 1 or
Secondary antibody）Coupling, adds BSA buffer solutions and makees Seal treatment to fluorescent microsphere, produce " antibody（Or antigen or secondary antibody）- fluorescent microsphere "
[Ab1（Ag1）-Flu]；
（3）Capture antibody 2（Or antigen 2 or secondary antibody）The preparation of magnetic microsphere:
Take that Magnetic Polystyrene Microsphere is scrubbed, ultrasonic, after stir process, with antibody 2（Or antigen 2 or secondary antibody）Mixing, is added
BSA buffer solutions make Seal treatment to microballoon, produce " capture antibody（Or antigen or secondary antibody）- magnetic microsphere " [Ab2 (Ag2)-
Mag]；
（4）By " antibody 1（Or antigen 1 or secondary antibody）- fluorescent microsphere " and " capture antibody 2（Or antigen 2 or secondary antibody）- magnetic is micro-
Each 1~20ul of ball " " is added separately in the fluorescence probe groove and reaction detection groove of B plates, 37 °C of dry solidifications；
（5）Chip A, B plate is bonded to by one piece of complete chip using ultrasonic bonding machine；
（6）Sample well is closed with special adhesion sealed membrane, in envelope aluminium foil bag, produced.
10. a kind of detection method of the micro-fluidic chip fluorescence immunoassay quick detection kit described in claim 1, its feature exists
Comprise the following steps in this method：
(1) test substance solution is added in well, and solution to be measured is driven to reaching fluorescence probe groove and dissolve by centrifuging
Probe microballoon, test substance is specifically bound with corresponding antibody 1 (or antigen 1 or secondary antibody), forms Ag-Ab-fluorescence micro-
Ball (Flu-Ab- Ag) compound；
（2）Under centrifugation driving, the solution such as the compound in fluorescence probe groove are all delivered in reaction detection groove by continuation, and
Dissolve the Ag-Ab-fluorescent microsphere 1 (Flu-Ab- Ag) captured in reaction detection groove in antibody magnetic bead microballoon, solution multiple
Compound and capture antibody 2（Or antigen 2 or secondary antibody）Specific immune response occurs for-magnetic microsphere, forms " fluorescent microsphere-antibody
The double-antibody sandwich magnetic composite (Flu-Ab1- Ag-Ab2-Mag) of 1- Ag-Ab 2- magnetic beads microballoon " or " fluorescence is micro-
The double antigens sandwich magnetic composite (Flu-Ag1- Ab-Ag2-Mag) of ball-antigen 1-antibody-antigene 2- magnetic beads microballoon "；
（4）In the presence of externally-applied magnetic field, magnetic composite is attracted to the bottom of reaction detection groove, under centrifugation driving, will
Solution without magnetic microsphere is transported to waste liquid pool, and magnetic fluorescence compound then stays in the bottom of detection cell；
（4）The magnetic fluorescence compound added by well in cleaning buffer solution, centrifugation, cleaning reaction detection groove, is added outer
Magnetic field, centrifugation removes cleaning fluid, operates repeatedly；
（5）Detected by fluorescence detector, with the magnetic fluorescent microspheres compound in the photo-irradiation reaction detect tank of certain wavelength,
The luminous intensity of fluorescent microsphere is detected, fluorescence intensity is in correlation with testing concentration.
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