CN105424922A - Micro-fluidic chip based on magnetic bead coated antibody and method for capturing cardiac markers - Google Patents
Micro-fluidic chip based on magnetic bead coated antibody and method for capturing cardiac markers Download PDFInfo
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Abstract
The embodiment of the invention discloses a micro-fluidic chip based on a magnetic bead coated antibody and a method for capturing cardiac markers. The micro-fluidic chip based on the magnetic bead coated antibody and the method for capturing cardiac markers are applied to the field of immunomagnetic bead coated binding antibodies, and prevent coated antibodies from being redissolved in a fluid system or being captured by antigens in fluid and disengaged from a base material. The micro-fluidic chip comprises a sample injection chamber, a Y-shaped structure channel and recycling chambers. The Y-shaped structure channel comprises a funnel-shaped channel at the upper portion and a columnar channel at the lower portion. An immunolabelling module is arranged at the bottom of the funnel-shaped channel. A blood filtering module is arranged at the upper portion of the immunolabelling module. The bottom end of the columnar channel at the lower portion is forked to form a pair of parallel immunity channels. The tail end of each immunity channel is connected with one recycling chamber. The columnar channel is provided with an upper cavity and a lower cavity. The upper cavity is a strong magnetism cavity, and the lower cavity is a quality control cavity.
Description
Technical field
The present invention relates to the combined antibody art of immunomagnetic beads bag, particularly relate to a kind of micro-fluidic chip based on magnetic bead coated antibody and catch the method for Applications of Cardiac Markers.
Background technology
At present, microflow control technique has impressive progress in fields such as biochemical analysis, immunodiagnosis, environment measurings, as under the prerequisite of precise controlling flow velocity, controls antigen-antibody reaction, thus reaches and control immunoreactive high sensitivity.Coated in reaction microchamber by means heart antibodies such as Van der Waals force, Electrostatic Absorption, macromolecule embeddings, carry out antigen capture.For reaching the instant object detected, reaching immune substrate by high mass transfer in microchannel and mixing fully, so exist coated antibody redissolve in fluid system and by fluid in antigen capture depart from bag by problems such as base materials.
Summary of the invention
This patent object is to provide a kind of micro-fluidic chip based on magnetic bead coated antibody and catches the method for Applications of Cardiac Markers, catches the existence of center of a sample's flesh mark (antigen) in high sensitivity.By carrying out pre-service and balance to sample, as erythrocytic removal, pH balance, Background are eliminated, finally obtain the similar reaction system of system to enter sensing chamber and carry out target acquistion, prevent coated antibody redissolve in fluid system and by fluid in antigen capture depart from bag by base material.
For reaching above object, the application adopts immunomagnetic ca pture method, realizes micro-fluidic chip and the method for catching Applications of Cardiac Markers based on this chip.Immunomagnetic ca pture method a kind ofly to be learned a skill by the Novel immune that magnetic bead magnetic responsiveness ability combines with immunologic opsonin.Immunomagnetic beads has the advantage such as immobilization reagent speciality and immunological response high sensitivity, high selectivity, in field widespread uses such as immunology detection, microorganism detection, cell separation.Utilize composite structure iron content, immunomagnetic beads can be drawn by magnetic field, and its top layer has specific function group, can with the covalent bond having biological activity protein and carry out chemical group, as antibody supporting body.In immune response magnetic bead coated antibody with there is the antigen agreeing with determinant be combined, be separated efficiently from the complex systems such as blood plasma, and the antibody institute recognition quantitative being labeled marker detects.
First aspect present invention provides a kind of micro-fluidic chip, comprising:
Sample addition zone, y-type structure passage and recovery room;
Y-type structure passage is divided into the funnel channel on top and the cylindrical passageway of bottom; The bottom of described funnel channel is immune labeled module; Immune labeled module top is hemofiltration module;
The cylindrical passageway bottom fork of bottom is the immune passage walked abreast for a pair; Each immune channel end connects one and reclaims room;
Cylindrical passageway arranges upper and lower two chambers, and upper chamber is strong magnetic room, and lower chamber is Quality Control room.
Preferably, described hemofiltration module is glass or nonwoven cloth material.
Preferably, described hemofiltration module is with rabbit anti-human erythrocyte albumen and agglutinin 2:1 process in mass ratio, and freeze-drying obtains.
Preferably, described hemofiltration module middle part also comprises the immunomagnetic beads of binding antibody, and described immunomagnetic beads activates through activator.
Preferably, the bottom at described micro-fluidic chip strong magnetic room place is embedded with strong magnetic piece.
Second aspect present invention provides a kind of method of catching Applications of Cardiac Markers, and described method is applied to the micro-fluidic chip described in above-mentioned any embodiment, and described method comprises:
Make be coated with can with the immunomagnetic beads of the antibody of Blood Center flesh mark specific binding;
To the contraposition antibody labeling immune marking thing of described Applications of Cardiac Markers;
The immunomagnetic beads of coated antibody is inputted the hemofiltration module of described chip, the immune marking thing of mark contraposition antibody is inputted immune labeled module;
The mixed liquor of a certain amount of cardiac marker sample and equilibrium liquid is added to sample addition zone;
Start detection equipment, reaction Preset Time, detects predetermined detection project result.
Preferably, different Applications of Cardiac Markers has different magnetic bead bags by system.
Preferably, different Applications of Cardiac Markers has different markers;
Described marker comprises: quantum dot, fluorescent microsphere and collaurum.
Preferably, described Applications of Cardiac Markers comprises: cardiac troponin cTnI, myoglobins MYO, c reactive protein CRP, H-FABP FABP, N akrencephalon pro-BNP NT-proBNP, myeloperoxidase MPO.
Preferably, when described Applications of Cardiac Markers is cardiac troponin cTnI, the preparation of the immunomagnetic beads of coated antibody comprises:
Immunomagnetic beads balance MES damping fluid is 4 ~ 5 to the pH of described MES damping fluid, and ionic strength is 0.1M;
In solution after balance PH, add EDC and NHS respectively, be 1mg/mL to EDC and NHS concentration in the solution, react centrifugal after 20 minutes and abandon supernatant;
The sediment after supernatant is abandoned with the MES damping fluid redissolution before described balance;
The concentration adding antibody 8E10 to described antibody 8E10 in the solution that redissolution obtains is 0.5mg/mL;
Mixing bag was by 1 hour, and the concentration adding BSA to described BSA is 1% cessation reaction.
Preferably, when described Applications of Cardiac Markers is cardiac troponin cTnI, described Applications of Cardiac Markers labelled immune marker comprises:
1mg quantum dot balance pH is 7.5 ~ 8.5, the PBS damping fluid of concentration 0.01M is 0.1mg/mL to the concentration of described quantum dot;
In solution after balance, add EDC and NHS respectively, be 0.5mg/mL to the concentration of described EDC and NHS, activate and centrifugally after 30 minutes abandon supernatant;
The sediment after supernatant is abandoned with the PBS damping fluid redissolution before described balance;
In solution after redissolution, add antibody 7G2, mark 20 minutes, the concentration adding BSA to described BSA is 1% cessation reaction.
Preferably, when described Applications of Cardiac Markers is myoglobins MYO, the preparation of the immunomagnetic beads of coated antibody comprises:
Immunomagnetic beads balance PB damping fluid is 7 ~ 8 to the pH of damping fluid, and ionic strength is 0.1M;
Add EDC and NHS respectively in solution after balance, the concentration to described EDC and NHS is 1mg/mL, reacts centrifugal after 20 minutes and abandons supernatant;
Redissolve with the PB damping fluid before described balance and abandon supernatant postprecipitation thing;
In the solution that redissolution obtains, the concentration adding antibody 4E2 to described antibody 4E2 is 0.5mg/mL, and mixing bag was by 1 hour, and the concentration adding BSA to described BSA is 1% cessation reaction.
Preferably, when described Applications of Cardiac Markers is myoglobins MYO, described Applications of Cardiac Markers labelled immune marker comprises:
1mg fluorescent microsphere balance pH is 7 ~ 9, the PBS damping fluid of concentration 0.01M is 0.1mg/mL to the concentration of fluorescent microsphere;
Add EDC and NHS respectively in solution after balance, be 0.5mg/mL to the concentration of described EDC and NHS, activate and centrifugally after 30 minutes abandon supernatant;
The sediment after supernatant is abandoned with the PBS damping fluid redissolution before described balance;
In solution after redissolution, add antibody MYO, mark 20 minutes, the concentration adding BSA to described BSA is 1% cessation reaction.
Preferably, when described Applications of Cardiac Markers is c reactive protein CRP, the preparation of the immunomagnetic beads of coated antibody comprises:
Immunomagnetic beads balance MES damping fluid is 4.5 ~ 5.5 to pH, and ionic strength is 0.1M;
Add EDC and NHS respectively in solution after balance, the concentration to described EDC and NHS is 1mg/mL, reacts centrifugal after 20 minutes and abandons supernatant;
Abandon the sediment after supernatant with the MES damping fluid redissolution before described balance, the concentration added in antibody CRP135 to the solution of described antibody CRP135 after redissolution is 0.5mg/mL;
Mixing bag was by 1 hour, and the concentration added in BSA to the solution of described BSA after redissolution is 1% cessation reaction.
Preferably, when described Applications of Cardiac Markers is c reactive protein CRP, described Applications of Cardiac Markers labelled immune marker comprises:
40nm colloidal gold solution is with K
2cO
3solution equilibria is 8.5-9.5 to colloidal gold solution pH;
Add antibody CRP36 in solution after balance, mark 1 hour;
Mark after 1 hour, the concentration adding BSA to described BSA is in the solution 1% cessation reaction;
Centrifugal after cessation reaction, to take ionic strength as 0.1M, pH be 7 ~ 9 PBS damping fluid 0.1 times of redissolving to liquor capacity during cessation reaction.
Preferably, when described mark is H-FABP FABP, the preparation of the immunomagnetic beads of coated antibody comprises:
Immunomagnetic beads balance MES damping fluid is 4 ~ 5, ionic strength 0.1M to pH of buffer;
Add EDC and NHS respectively in solution after balance, the concentration to described EDC and NHS is 1mg/mL, reacts centrifugal after 20 minutes and abandons supernatant;
Abandon the sediment after supernatant with the MES damping fluid redissolution before described balance, adding antibody FABP5B5 in the solution after redissolution to concentration is 1.0mg/mL, and mixing bag was by 1 hour, and adding BSA to BSA concentration is 1% cessation reaction.
Preferably, when described mark is H-FABP FABP, described Applications of Cardiac Markers labelled immune marker comprises:
PBS solution to the quantum dot concentration that 1mg quantum dot balance pH is 7 ~ 8, concentration is 0.01M is 0.1mg/mL;
Add EDC and NHS respectively in solution after balance, be 0.5mg/mL to EDC and NHS concentration in the solution, activate after 30 minutes, centrifugally abandon supernatant;
Sediment after abandoning supernatant is redissolved with the PBS solution before described balance, adds antibody FABP28 and mark 20 minutes;
Adding BSA to described BSA concentration is 1% cessation reaction.
Preferably, when described Applications of Cardiac Markers is N akrencephalon pro-BNP NT-proBNP, the preparation of the immunomagnetic beads of coated antibody comprises:
Immunomagnetic beads balance PBS damping fluid is 6 ~ 7 to the pH of damping fluid, and ionic strength is 0.01M;
Add EDC and NHS respectively in solution after balance, be 1mg/mL to described EDC and NHS concentration in the solution, react centrifugal after 20 minutes and abandon supernatant;
Abandon the sediment after supernatant with the PBS damping fluid redissolution before described balance, the concentration added in antibody 18H5 to the solution of described antibody 18H5 after redissolution is 1mg/mL;
Mixing bag was by 1 hour, and the concentration added in the solution after BSA to described BSA redissolution is 1% cessation reaction.
Preferably, when described Applications of Cardiac Markers is N akrencephalon pro-BNP NT-proBNP, described Applications of Cardiac Markers labelled immune marker comprises:
The concentration 0.1mg/mL of PBS damping fluid to quantum dot that 1mg quantum dot balance pH is 7.5-8.5, concentration is 0.01M;
Add EDC and NHS respectively in solution after balance, be respectively 0.5mg/mL to the concentration of described EDC and NHS, activate and centrifugally after 30 minutes abandon supernatant;
Sediment after abandoning supernatant with the PBS damping fluid redissolution before described balance, add antibody 15F11, mark 30 minutes, the concentration added in BSA to the solution of described BSA after redissolution is 1% cessation reaction.
Preferably, when described Applications of Cardiac Markers is myeloperoxidase MPO, the preparation of the immunomagnetic beads of coated antibody comprises:
Immunomagnetic beads balance PB damping fluid is 7.5 ~ 8.5, ionic strength 0.01M to pH value of solution;
Add EDC and NHS respectively in solution after balance, be 1mg/mL to EDC and NHS concentration in the solution, react centrifugal after 20 minutes and abandon supernatant;
Abandon the sediment after supernatant with the PB damping fluid redissolution before described balance, the concentration adding antibody 19H2 to described antibody 19H2 in the solution after redissolution is 0.5mg/mL;
Mixing bag, by 1 hour, adds concentration 1% cessation reaction of BSA to described BSA.
Preferably, when described Applications of Cardiac Markers is myeloperoxidase MPO, described Applications of Cardiac Markers labelled immune marker comprises:
The PBS solution that 1mg fluorescent microsphere balance pH is 6.5 ~ 7.5, concentration is 0.1M is 0.1mg/mL to the concentration of fluorescent microsphere;
Add EDC and NHS respectively in solution after balance, be 0.5mg/mL to EDC and NHS concentration in the solution, activate after 30 minutes, centrifugally abandon supernatant;
Abandon the sediment after supernatant with the PBS solution redissolution before described balance, add antibody 18B7 in the solution after redissolution, mark 20 minutes, the concentration adding BSA to described BSA is 1% cessation reaction.
Third aspect present invention provides a kind of method of catching Applications of Cardiac Markers, and described method is applied to the micro-fluidic chip described in above-mentioned any embodiment, and described method comprises:
Make be coated with can with the immunomagnetic beads of the antibody of Blood Center flesh mark specific binding;
To the contraposition antibody labeling immune marking thing of described Applications of Cardiac Markers;
The immune marking thing of mark contraposition antibody is inputted immune labeled module;
The mixed liquor of a certain amount of cardiac marker sample and equilibrium liquid is added to sample addition zone, in described equilibrium liquid, comprises the immunomagnetic beads of described coated antibody;
Start detection equipment, reaction Preset Time, detects result.
Compared with prior art, technical scheme provided by the invention has the following advantages:
The invention provides in technical scheme, catch antigen in sample by immunomagnetic beads coated antibody, the covalent bond of magnetic bead and coated antibody, has the features such as high sensitivity, high specific, low background noise.Based on micro-fluidic detection chip, control immunoreaction process by fine-stabilization, reach the coefficient of variation effectively controlling micro-reaction.Powerful magnetic field, to the high capture ability of complex, can control immunomagnetic beads and be uniformly distributed, ensure that marking signal is detected recognition of devices in given area.By carrying out pre-service and balance to sample, as erythrocytic removal, pH balance, Background are eliminated, finally obtain the similar reaction system of system to enter sensing chamber and carry out target acquistion, prevent coated antibody redissolve in fluid system and by fluid in antigen capture depart from bag by base material.
Further, according to the different qualities of multiclass cardiac muscle marker, preferred differential responses system, such as immunomagnetic beads deposit position, marker kind and Mk system, thus reach good manipulation effect and transplantation effect.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below, apparently, accompanying drawing in the following describes is some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 is preferably a kind of microfluidic chip structure schematic diagram provided by the invention;
Fig. 2 is the micro-fluidic immune response schematic diagram of one of this patent preferably a kind of magnetic bead coated antibody.
Embodiment
The present invention program is understood better in order to make those skilled in the art person, below in conjunction with the accompanying drawing in the embodiment of the present invention, technical scheme in the embodiment of the present invention is clearly and completely described, obviously, described embodiment is only the embodiment of a part of the present invention, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, should belong to the scope of protection of the invention.
Below in conjunction with drawings and Examples, disclosed in detail further description is done to the specific embodiment of the present invention.
Shown in figure 1, be a kind of micro-fluidic chip based on magnetic bead coated antibody provided by the invention, the structure of this chip is as follows:
Sample addition zone, y-type structure passage and recovery room;
Y-type structure passage is divided into the funnel channel on top and the cylindrical passageway of bottom; The bottom of described funnel channel is immune labeled module; Immune labeled module top is hemofiltration module;
The cylindrical passageway bottom fork of bottom is the immune passage walked abreast for a pair; Each immune channel end connects one and reclaims room;
Arrange upper and lower two chambers between funnel channel bottom and cylindrical passageway bottom, upper chamber is strong magnetic room, and lower chamber is Quality Control room.
Wherein, preferably, described hemofiltration module can adopt glass or nonwoven cloth material.With rabbit anti-human erythrocyte albumen and agglutinin 2:1 process in mass ratio, freeze-drying obtains;
Immunomagnetic beads can be combined with Applications of Cardiac Markers before this chip at sample pipetting volume, preferably, according to the feature of different Applications of Cardiac Markers, can also magnetic bead after described hemofiltration module middle part arranges binding antibody, and described magnetic bead activates through activator.Be embedded with strong magnetic piece in the bottom at described micro-fluidic chip strong magnetic room place, be captured when immunomagnetic beads flows through strong magnetic room.
The method detecting Applications of Cardiac Markers based on above-mentioned micro-fluidic chip mainly contains following two kinds of modes, to those skilled in the art, under the prerequisite not paying creative work, can also obtain other method according to these methods.
The first: make be coated with can with the immunomagnetic beads of the antibody of Blood Center flesh mark specific binding;
To the contraposition antibody labeling immune marking thing of described Applications of Cardiac Markers;
The immunomagnetic beads of coated antibody is inputted the hemofiltration module of described chip, the immune marking thing of mark contraposition antibody is inputted immune labeled module;
The mixed liquor of a certain amount of cardiac marker sample and equilibrium liquid is added to sample addition zone;
Start detection equipment, reaction Preset Time, detects predetermined detection project result.
Practical operation based on said method is illustrated:
Step 1: sample (blood plasma 75 μ L or whole blood 100 μ L) mixes with equilibrium liquid, for subsequent use;
Step 2: sample this with equilibrium liquid mixed liquor 100 μ L application of sample in sample addition zone, mixed liquor enters hemofiltration module through passage, filtering haemocyte, and contacts with the immunomagnetic beads of coated antibody, and forming reactions liquid leaves;
Step 3: reactant liquor leaches strong magnetic room through passage, the magnetic bead of 95% ~ 100% is captured, and waste liquid leaves strong magnetic room through passage;
Step 4: waste liquid enters Quality Control room by passage, in system, immune marking thing at large is captured, and Indicator Reaction liquid is smoothly by strong magnetic room;
Step 5: waste liquid is entered by immune passage and reclaims room, is quantitatively detected after 15 minutes by set detector to result.
The second: make be coated with can with the immunomagnetic beads of the antibody of Blood Center flesh mark specific binding;
To the contraposition antibody labeling immune marking thing of described Applications of Cardiac Markers;
The immune marking thing of mark contraposition antibody is inputted immune labeled module;
The mixed liquor of a certain amount of cardiac marker sample and equilibrium liquid is added to sample addition zone, in described equilibrium liquid, comprises the immunomagnetic beads of described coated antibody;
Start detection equipment, reaction Preset Time, detects result.
The practical operation citing of said method:
Step I: sample (blood plasma 75 μ L or whole blood 100 μ L) mixes 1 minute with equilibrium liquid (comprising the immunomagnetic beads of coated antibody), for subsequent use;
Step II: sample this with equilibrium liquid mixed liquor 100 μ L application of sample in sample addition zone, mixed liquor enters hemofiltration module through passage, and filtering haemocyte, leaves native system;
Step III: reactant liquor leaches strong magnetic room through passage, the magnetic bead of 95% ~ 100% is captured, and waste liquid leaves strong magnetic room through passage;
Step IV: waste liquid enters Quality Control room by passage, in system, immune marking thing at large is captured, and Indicator Reaction liquid is smoothly by strong magnetic room;
Step V: waste liquid is entered by immune passage and reclaims room, is quantitatively detected after 15 minutes by set detector to result.
Different Applications of Cardiac Markers has different magnetic bead bags by system.Different Applications of Cardiac Markers can adopt different mark systems.Different markers can be adopted for different Applications of Cardiac Markers, mainly comprise quantum dot, fluorescent microsphere and collaurum.Catch antigen in sample by immunomagnetic beads coated antibody, the covalent bond of magnetic bead and coated antibody, has the features such as high sensitivity, high specific, low background noise.Based on micro-fluidic detection chip, control immunoreaction process by fine-stabilization, the coefficient of variation effectively controlling micro-reaction can be reached.The powerful magnetic field of profit, to the high capture ability of complex, can control immunomagnetic beads and be uniformly distributed, ensure that marking signal is detected recognition of devices in given area.According to the different qualities of multiclass cardiac muscle marker, preferred differential responses system, such as immunomagnetic beads deposit position, marker kind and Mk system, thus reach good manipulation effect and transplantation effect.
Below for concrete Applications of Cardiac Markers, be specifically described, the antibody be labeled of the immune labeled module of the input described in following examples is contraposition antibody.
embodiment 1
Be described for cardiac muscle troponin I (cTnI) in the present embodiment.
1) make the immunomagnetic beads of coated antibody, be coated with and the making of the immunomagnetic beads of the antibody that cTnI protein-specific is combined can comprise in blood:
Immunomagnetic beads (Cat.:LSKMAGA10, MerckMillipore), through MES damping fluid (2-(N-morpholine) ethyl sulfonic acid) balance, is 4-5 (preferably 4.5), ionic strength 0.1M to pH value of solution.Add EDC and NHS respectively in solution after balance, be 1mg/mL to EDC and NHS concentration in the solution, react centrifugal after 20 minutes and abandon supernatant;
Abandon the sediment after supernatant with described MES damping fluid (before balance) redissolution, the concentration adding antibody 8E10 (Cat.:4T13, Hytest) to 8E10 is 0.5mg/mL;
Mixing bag was by 1 hour, and the concentration then adding BSA (Cat.:10735108001, Roche) to BSA is 1%, cessation reaction.
2) for cTnI antigenic mark marker quantum dot (Cat.:F122270, aladdin) mark, comprising:
The PBS solution that 1mg quantum dot balance pH is 8.0, concentration is 0.01M is 0.1mg/mL to the final concentration of quantum dot; Then add EDC and NHS respectively, be 0.5mg/mL to EDC and NHS final concentration in the solution, activate after 30 minutes, centrifugally abandon supernatant.
Redissolved with described PBS solution by sediment after abandoning supernatant, add antibody 7G2 (Cat.:4T20, Hytest), mark 20 minutes, adding BSA to its concentration is 1% cessation reaction.
3) immune microfluidic chip structure is adopted as shown in Figure 1, prepared by micro-fluidic chip: PMMA through injection moulding, impression, modification, function input, bonding is shaping, wherein function input comprises, and wrap by 8E10 immunomagnetic beads input hemofiltration module, mark 7G2 quantum dot inputs immune labeled module.
Chip size 81mm × 32mm × 3mm, the full liquid measure in micro-fluidic indoor is 500 μ L.
4) sample detection: cTnI antigen (Cat.:8RTI7, hytest) is diluted as 0.1ng/mL ~ 50ng/mL, the cTnI antigen after dilution mixes with equilibrium liquid respectively, for subsequent use.
Get 100 μ L loadings to sample addition zone, and start detection equipment, timing 15 minutes, detects linear dependence and each concentration coefficient of variation respectively.
5) test 10 times at identical conditions, the statistics obtaining linear dependence and the coefficient of variation is as follows:
The cTnI antigen linear dependence of table 1 variable concentrations and the statistics of the coefficient of variation
embodiment 2
The present embodiment is described for myoglobins MYO.
1) be coated with and the immunomagnetic beads making of the antibody that MYO protein-specific is combined can comprise in blood:
Immunomagnetic beads, through PB (phosphate buffer, phosphatebuffersolution) damping fluid balance, is 7.4 (7.0-8.0), ionic strength 0.1M to pH value of solution.
Add EDC and NHS respectively in solution after balance, be 1mg/mL to EDC and NHS concentration in the solution, react centrifugal after 20 minutes and abandon supernatant.
The sediment after supernatant is abandoned with described PB damping fluid (before balance) redissolution, antibody (Cat.:4M23 is added in solution after redissolution, Hytest) to 4E2 concentration be 0.5mg/mL, mixing bag is by 1h, adding BSA (bovine serum albumin) to BSA ultimate density is 1%, cessation reaction.
2) for MYO antigenic mark marker fluorescent microsphere (Cat.:BM551, BangsLaboratories) mark, comprising:
1mg fluorescent microsphere balance to pH be 8.0, the PBS solution of concentration 0.01M to fluorescent microsphere concentration is 0.1mg/mL.
Then EDC (1 ethyl 3 (3 dimethyl aminopropyl) carbodiimides) and NHS (N N-Hydroxysuccinimide) is added respectively, 0.5mg/mL is to EDC and NHS concentration in the solution, activate after 30 minutes, centrifugally abandon supernatant.
Sediment after abandoning supernatant is redissolved with PBS (phosphate buffered saline(PBS), phosphatebuffersaline) solution, adds antibody MYO (Cat.:8M50, Hytest), mark 20 minutes, the concentration adding BSA to BSA is 1%, cessation reaction.
3) immune micro-fluidic chip is with implementing 1.Prepared by micro-fluidic chip: PMMA through injection moulding, impression, modification, function input, bonding is shaping, wherein function input comprises, and wrap by 4E2 immunomagnetic beads input hemofiltration module, mark MYO fluorescent microsphere inputs immune labeled module.
4) sample detection: MYO antigen (Cat.:8M50, hytest) is diluted as 50ng/mL ~ 1000ng/mL, mixes respectively with equilibrium liquid;
Get 100 μ L loadings to sample addition zone, and start detection equipment, timing 15 minutes, detects linear dependence and each concentration coefficient of variation respectively.
5) test 10 times at identical conditions, the statistics obtaining linear dependence and the coefficient of variation is as follows:
The MYO antigen linear dependence of table 2 variable concentrations and the statistics of the coefficient of variation
embodiment 3
The present embodiment is described for c reactive protein.
1) be coated with and the immunomagnetic beads making of the antibody that CRP protein-specific is combined can comprise in blood: immunomagnetic beads balances through MES damping fluid, to pH value of solution to 4.5-5.5 (being preferably 5), ionic strength 0.1M;
Then add EDC and NHS respectively, the final concentration to EDC and NHS is 1mg/mL, reacts after 20 minutes.Centrifugally abandon supernatant, and the sediment after abandoning supernatant with the redissolution of described PBS damping fluid (before balance), add antibody CRP135 (Cat.:4C28, Hytest) ultimate density to CRP135 is 0.5mg/mL, mixing bag is by 1h, the ultimate density adding BSA to BSA is 1%, cessation reaction.
2) mark for CRP antigenic mark marker collaurum (self-control): 40nm colloidal gold solution is with K
2cO
3solution equilibria is 9 (in 8.5-9.5) to pH, adds antibody CRP36 (Cat.:4C28, Hytest), marks 1 hour, and adding BSA to described BSA ultimate density is in the solution 1% cessation reaction, centrifugal after cessation reaction;
To take ionic strength as 0.1M, pH be 8.0 PBS damping fluid redissolve centrifugal after sediment, until when the liquor capacity after redissolving is cessation reaction 0.1 times.
3) immune micro-fluidic chip is with implementing 1.Prepared by micro-fluidic chip: PMMA (polymethylmethacrylate) through injection moulding, impression, modification, function input, bonding is shaping, wherein function input comprises, wrap by CRP135 immunomagnetic beads input hemofiltration system, mark CRP36 marks collaurum and inputs immune labeled module.
4) sample detection: CRP antigen (Cat.:8C72, hytest) 5ng/mL ~ 250ng/mL is diluted as, mix with equilibrium liquid respectively, get 100 μ L loadings to sample addition zone, and start inspection vehicle device, timing 15 minutes, detects linear dependence and each concentration coefficient of variation respectively.
5) test 10 times at identical conditions, the statistics obtaining linear dependence and the coefficient of variation is as follows:
The CRP antigen linear dependence of table 3 variable concentrations and the statistics of the coefficient of variation
embodiment 4
The present embodiment illustrates for H-FABP (FABP).
1) be coated with to make with the immunomagnetic beads of the antibody of FABP specific binding in blood and comprise:
Immunomagnetic beads, through MES damping fluid balance, is 4-5 (being preferably 4.5), ionic strength 0.1M to pH value of solution;
Add EDC and NHS respectively in solution after balance, the concentration to EDC and NHS is 1mg/mL, reacts after 20 minutes.Centrifugally abandon supernatant, sediment after abandoning supernatant is redissolved with described MES damping fluid (before balance), add antibody FABP5B5 (Cat.:4F29, Hytest) to FABP5B5 concentration be 1.0mg/mL, mixing bag was by 1 hour, adding BSA to BSA concentration is 1%, cessation reaction.
2) quantum dot-labeled for FABP antigenic mark marker: to comprise: PBS solution to the quantum dot final concentration that 1mg quantum dot balance pH is 7.4, concentration is 0.01M is 0.1mg/mL;
Then add EDC and NHS respectively, the final concentration to EDC and NHS is 0.5mg/mL, activates after 30 minutes, centrifugally abandons supernatant.Sediment after abandoning supernatant is redissolved it with described PBS solution (before balance), adds antibody FABP28 (Cat.:4F29, Hytest), mark 20 minutes, adding BSA to BSA concentration is 1% cessation reaction.
3) immune micro-fluidic chip is with implementing 1.Prepared by micro-fluidic chip: PMMA through injection moulding, impression, modification, function input, bonding is shaping, wherein function input comprises, and wrap by FABP5B5 immunomagnetic beads input hemofiltration system, flag F ABP28 marks collaurum and inputs immune labeled module.
4) sample detection: FABP antigen (Cat.:8F65, hytest) 2.5ng/mL ~ 50ng/mL is diluted as, mix with equilibrium liquid respectively, get 100 μ L loadings to sample addition zone, and start inspection vehicle device, timing 15 minutes, detects linear dependence and each concentration coefficient of variation respectively.
5) test 10 times at identical conditions, the statistics obtaining linear dependence and the coefficient of variation is as follows:
The FABP antigen linear dependence of table 4 variable concentrations and the statistics of the coefficient of variation
embodiment 5
The present embodiment for N akrencephalon pro-BNP (NT-proBNP) for example illustrates.
1) be coated with and the immunomagnetic beads making of the antibody that NT-proBNP protein-specific is combined can comprise in blood:
Immunomagnetic beads balance PBS damping fluid is 6-7 (preferred PH is chosen as 6.5) to pH, ionic strength 0.01M;
Add EDC and NHS respectively in solution after balance, the concentration to described EDC and NHS is 1mg/mL, reacts centrifugal after 20 minutes and abandons supernatant;
By centrifugal remove supernatant after sediment redissolve with described PBS damping fluid (before balance), antibody 18H5 (Cat.:4NT1 is added in solution after redissolving, Hytest) to 18H5 concentration be 1mg/mL, mixing bag was by 1 hour, adding BSA to (BSA) concentration is 1%, cessation reaction.
2) marker is marked for NT-proBNP quantum dot-labeled: 1mg quantum dot balance is the PBS solution of 7.5-8.5 (being preferably 8.0), concentration 0.01M to pH, be 0.1mg/mL to quantum dot final concentration, EDC and NHS is added respectively after balance, concentration to EDC and NHS is 0.5mg/mL, activate after 30 minutes, centrifugally abandon supernatant.Redissolve with described PBS solution to the sediment after abandoning supernatant, add antibody 15F11 (Cat.:4NT1, Hytest), mark 30 minutes, the concentration adding BSA to BSA is 1%, cessation reaction.
3) immune micro-fluidic chip is with implementing 1.Prepared by micro-fluidic chip: PMMA through injection moulding, impression, modification, function input, bonding is shaping, wherein function input comprises, and wrap by 18H5 immunomagnetic beads input hemofiltration system, mark 15F11 quantum dot inputs immune labeled module.
4) sample detection: NT-proBNP antigen (Cat.:8NT2, hytest) 150pg/mL ~ 9000pg/mL is diluted as, NT-proBNP antigen is mixed with equilibrium liquid, get 100 μ L loadings to sample addition zone, and start inspection vehicle device, timing 15 minutes, detects linear dependence and each concentration coefficient of variation respectively.
5) test 10 times at identical conditions, the statistics obtaining linear dependence and the coefficient of variation is as follows:
The NT-proBNP antigen linear dependence of table 5 variable concentrations and the statistics of the coefficient of variation
embodiment 6
The present embodiment for myeloperoxidase (MPO) for example illustrates.
1) be coated with and the immunomagnetic beads making of the antibody that MPO protein-specific is combined can comprise in blood:
Immunomagnetic beads balance PB damping fluid is 7.5-8.5 to pH, and ionic strength is 0.01M;
In solution after balance, add EDC and NHS respectively, until the concentration of EDC and NHS is 1mg/mL, reacting centrifugal after 20 minutes and abandon supernatant, to going the remaining sediment of supernatant to redissolve with described PB damping fluid, in the solution that redissolution obtains, adding antibody 19H2 (Cat.:4M43, Hytest) concentration to antibody 19H2 is 0.5mg/mL, mixing bag was by 1 hour, and then adding BSA to BSA concentration is 1%, cessation reaction.
2) for MPO antigenic mark marker fluorescent microsphere: 1mg fluorescent microsphere balance pH is 6.5-7.5 (preferred PH can select 7), the PBS solution of concentration 0.1M is 0.1mg/mL to the concentration of described fluorescent microsphere, then the concentration adding EDC and NHS to described EDC and NHS is respectively 0.5mg/mL, activate after 30 minutes, centrifugally abandon supernatant.
Redissolve abandoning the sediment after supernatant with described PBS solution (PBS solution before balance), in the solution that redissolution obtains, add antibody 18B7 (Cat.:4M43, Hytest), mark 20 minutes, then the concentration 1% of BSA to BSA is added, cessation reaction.
3) immune micro-fluidic chip is with implementing 1.Prepared by micro-fluidic chip: PMMA through injection moulding, impression, modification, function input, bonding is shaping, wherein function input comprises, and wrap by 19H2 immunomagnetic beads input hemofiltration module, mark 18B7 fluorescent microsphere inputs immune labeled module.
4) sample detection: MPO antigen (Cat.:8M80, hytest) being diluted as concentration range is 25ng/mL ~ 500ng/mL, antigen after dilution mixes with equilibrium liquid respectively, get 100 μ L loadings to sample addition zone, and start inspection vehicle device, timing 15 minutes, detects linear dependence and each concentration coefficient of variation respectively.
5) test 10 times at identical conditions, the statistics obtaining linear dependence and the coefficient of variation is as follows:
The MPO antigen linear dependence of table 6 variable concentrations and the statistics of the coefficient of variation
In the present invention, the magnetic bead of sample and coated antibody is mixed into microfluidic channel, be combined with the immune marking thing of contraposition antibody after passage buffering and form " antigen-antibody sandwich structure " (shown in figure 2, wherein labelled antibody is contraposition antibody), enter strong magnetic room to be magnetized by kicker magnet, detect target in sample and be captured, native system is based on capillary force effect, rely on fluid to drive and enrichment with magnetic bead process is provided, complete the detection to target.The present invention can provide unique binding sit based on covalent bond for coated antibody, controls the carrying out of reaction in high sensitivity, provides precisely instant detection scheme.
Above embodiment only in order to technical scheme of the present invention to be described, is not intended to limit; Although with reference to previous embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that: it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature; And these amendments or replacement, do not make the essence of appropriate technical solution depart from the spirit and scope of various embodiments of the present invention technical scheme.
Claims (22)
1. a micro-fluidic chip, is characterized in that, comprising:
Sample addition zone, y-type structure passage and recovery room;
Y-type structure passage is divided into the funnel channel on top and the cylindrical passageway of bottom; The bottom of described funnel channel is immune labeled module; Immune labeled module top is hemofiltration module;
The cylindrical passageway bottom fork of bottom is the immune passage walked abreast for a pair; Each immune channel end connects one and reclaims room;
Cylindrical passageway arranges upper and lower two chambers, and upper chamber is strong magnetic room, and lower chamber is Quality Control room.
2. micro-fluidic chip according to claim 1, is characterized in that, described hemofiltration module is glass or nonwoven cloth material.
3. micro-fluidic chip according to claim 1, is characterized in that, described hemofiltration module is with rabbit anti-human erythrocyte albumen and agglutinin 2:1 process in mass ratio, and freeze-drying obtains.
4. the micro-fluidic chip according to any one of claim 1-3, is characterized in that, described hemofiltration module middle part also comprises the immunomagnetic beads of binding antibody, and described immunomagnetic beads activates through activator.
5. micro-fluidic chip according to claim 1, is characterized in that, the bottom at described micro-fluidic chip strong magnetic room place is embedded with strong magnetic piece.
6. catch a method for Applications of Cardiac Markers, it is characterized in that, described method is applied to the micro-fluidic chip described in any one of claim 1-5, and described method comprises:
Make be coated with can with the immunomagnetic beads of the antibody of Blood Center flesh mark specific binding;
To the contraposition antibody labeling immune marking thing of described Applications of Cardiac Markers;
The immunomagnetic beads of coated antibody is inputted the hemofiltration module of described chip, the immune marking thing of mark contraposition antibody is inputted immune labeled module;
The mixed liquor of a certain amount of cardiac marker sample and equilibrium liquid is added to sample addition zone;
Start detection equipment, reaction Preset Time, detects predetermined detection project result.
7. method according to claim 6, is characterized in that, different Applications of Cardiac Markers has different magnetic bead bags by system.
8. method according to claim 6, is characterized in that, different Applications of Cardiac Markers has different markers;
Described marker comprises: quantum dot, fluorescent microsphere and collaurum.
9. the method according to any one of claim 6-8, it is characterized in that, described Applications of Cardiac Markers comprises: cardiac troponin cTnI, myoglobins MYO, c reactive protein CRP, H-FABP FABP, N akrencephalon pro-BNP NT-proBNP, myeloperoxidase MPO.
10. method according to claim 8, is characterized in that, when described Applications of Cardiac Markers is cardiac troponin cTnI, the preparation of the immunomagnetic beads of coated antibody comprises:
Immunomagnetic beads balance MES damping fluid is 4 ~ 5 to the pH of described MES damping fluid, and ionic strength is 0.1M;
In solution after balance PH, add EDC and NHS respectively, be 1mg/mL to EDC and NHS concentration in the solution, react centrifugal after 20 minutes and abandon supernatant;
The sediment after supernatant is abandoned with the MES damping fluid redissolution before described balance;
The concentration adding antibody 8E10 to described antibody 8E10 in the solution that redissolution obtains is 0.5mg/mL;
Mixing bag was by 1 hour, and the concentration adding BSA to described BSA is 1% cessation reaction.
11. methods according to claim 8, is characterized in that, when described Applications of Cardiac Markers is cardiac troponin cTnI, described Applications of Cardiac Markers labelled immune marker comprises:
1mg quantum dot balance pH is 7.5 ~ 8.5, the PBS damping fluid of concentration 0.01M is 0.1mg/mL to the concentration of described quantum dot;
In solution after balance, add EDC and NHS respectively, be 0.5mg/mL to the concentration of described EDC and NHS, activate and centrifugally after 30 minutes abandon supernatant;
The sediment after supernatant is abandoned with the PBS damping fluid redissolution before described balance;
In solution after redissolution, add antibody 7G2, mark 20 minutes, the concentration adding BSA to described BSA is 1% cessation reaction.
12. methods according to claim 8, is characterized in that, when described Applications of Cardiac Markers is myoglobins MYO, the preparation of the immunomagnetic beads of coated antibody comprises:
Immunomagnetic beads balance PB damping fluid is 7 ~ 8 to the pH of damping fluid, and ionic strength is 0.1M;
Add EDC and NHS respectively in solution after balance, the concentration to described EDC and NHS is 1mg/mL, reacts centrifugal after 20 minutes and abandons supernatant;
Redissolve with the PB damping fluid before described balance and abandon supernatant postprecipitation thing;
In the solution that redissolution obtains, the concentration adding antibody 4E2 to described antibody 4E2 is 0.5mg/mL, and mixing bag was by 1 hour, and the concentration adding BSA to described BSA is 1% cessation reaction.
13. methods according to claim 8, is characterized in that, when described Applications of Cardiac Markers is myoglobins MYO, described Applications of Cardiac Markers labelled immune marker comprises:
1mg fluorescent microsphere balance pH is 7 ~ 9, the PBS damping fluid of concentration 0.01M is 0.1mg/mL to the concentration of fluorescent microsphere;
Add EDC and NHS respectively in solution after balance, be 0.5mg/mL to the concentration of described EDC and NHS, activate and centrifugally after 30 minutes abandon supernatant;
The sediment after supernatant is abandoned with the PBS damping fluid redissolution before described balance;
In solution after redissolution, add antibody MYO, mark 20 minutes, the concentration adding BSA to described BSA is 1% cessation reaction.
14. methods according to claim 8, is characterized in that, when described Applications of Cardiac Markers is c reactive protein CRP, the preparation of the immunomagnetic beads of coated antibody comprises:
Immunomagnetic beads balance MES damping fluid is 4.5 ~ 5.5 to pH, and ionic strength is 0.1M;
Add EDC and NHS respectively in solution after balance, the concentration to described EDC and NHS is 1mg/mL, reacts centrifugal after 20 minutes and abandons supernatant;
Abandon the sediment after supernatant with the MES damping fluid redissolution before described balance, the concentration added in antibody CRP135 to the solution of described antibody CRP135 after redissolution is 0.5mg/mL;
Mixing bag was by 1 hour, and the concentration added in BSA to the solution of described BSA after redissolution is 1% cessation reaction.
15. methods according to claim 8, is characterized in that, when described Applications of Cardiac Markers is c reactive protein CRP, described Applications of Cardiac Markers labelled immune marker comprises:
40nm colloidal gold solution is with K
2cO
3solution equilibria is 8.5-9.5 to colloidal gold solution pH;
Add antibody CRP36 in solution after balance, mark 1 hour;
Mark after 1 hour, the concentration adding BSA to described BSA is in the solution 1% cessation reaction;
Centrifugal after cessation reaction, to take ionic strength as 0.1M, pH be 7 ~ 9 PBS damping fluid 0.1 times of redissolving to liquor capacity during cessation reaction.
16. methods according to claim 8, is characterized in that, when described mark is H-FABP FABP, the preparation of the immunomagnetic beads of coated antibody comprises:
Immunomagnetic beads balance MES damping fluid is 4 ~ 5, ionic strength 0.1M to pH of buffer;
Add EDC and NHS respectively in solution after balance, the concentration to described EDC and NHS is 1mg/mL, reacts centrifugal after 20 minutes and abandons supernatant;
Abandon the sediment after supernatant with the MES damping fluid redissolution before described balance, adding antibody FABP5B5 in the solution after redissolution to concentration is 1.0mg/mL, and mixing bag was by 1 hour, and adding BSA to BSA concentration is 1% cessation reaction.
17. methods according to claim 8, is characterized in that, when described mark is H-FABP FABP, described Applications of Cardiac Markers labelled immune marker comprises:
PBS solution to the quantum dot concentration that 1mg quantum dot balance pH is 7 ~ 8, concentration is 0.01M is 0.1mg/mL;
Add EDC and NHS respectively in solution after balance, be 0.5mg/mL to EDC and NHS concentration in the solution, activate after 30 minutes, centrifugally abandon supernatant;
Sediment after abandoning supernatant is redissolved with the PBS solution before described balance, adds antibody FABP28 and mark 20 minutes;
Adding BSA to described BSA concentration is 1% cessation reaction.
18. methods according to claim 8, is characterized in that, when described Applications of Cardiac Markers is N akrencephalon pro-BNP NT-proBNP, the preparation of the immunomagnetic beads of coated antibody comprises:
Immunomagnetic beads balance PBS damping fluid is 6 ~ 7 to the pH of damping fluid, and ionic strength is 0.01M;
Add EDC and NHS respectively in solution after balance, be 1mg/mL to described EDC and NHS concentration in the solution, react centrifugal after 20 minutes and abandon supernatant;
Abandon the sediment after supernatant with the PBS damping fluid redissolution before described balance, the concentration added in antibody 18H5 to the solution of described antibody 18H5 after redissolution is 1mg/mL;
Mixing bag was by 1 hour, and the concentration added in the solution after BSA to described BSA redissolution is 1% cessation reaction.
19. methods according to claim 8, is characterized in that, when described Applications of Cardiac Markers is N akrencephalon pro-BNP NT-proBNP, described Applications of Cardiac Markers labelled immune marker comprises:
The concentration 0.1mg/mL of PBS damping fluid to quantum dot that 1mg quantum dot balance pH is 7.5-8.5, concentration is 0.01M;
Add EDC and NHS respectively in solution after balance, be respectively 0.5mg/mL to the concentration of described EDC and NHS, activate and centrifugally after 30 minutes abandon supernatant;
Sediment after abandoning supernatant with the PBS damping fluid redissolution before described balance, add antibody 15F11, mark 30 minutes, the concentration added in BSA to the solution of described BSA after redissolution is 1% cessation reaction.
20. methods according to claim 8, is characterized in that, when described Applications of Cardiac Markers is myeloperoxidase MPO, the preparation of the immunomagnetic beads of coated antibody comprises:
Immunomagnetic beads balance PB damping fluid is 7.5 ~ 8.5, ionic strength 0.01M to pH value of solution;
Add EDC and NHS respectively in solution after balance, be 1mg/mL to EDC and NHS concentration in the solution, react centrifugal after 20 minutes and abandon supernatant;
Abandon the sediment after supernatant with the PB damping fluid redissolution before described balance, the concentration adding antibody 19H2 to described antibody 19H2 in the solution after redissolution is 0.5mg/mL;
Mixing bag, by 1 hour, adds concentration 1% cessation reaction of BSA to described BSA.
21. methods according to claim 8, is characterized in that, when described Applications of Cardiac Markers is myeloperoxidase MPO, described Applications of Cardiac Markers labelled immune marker comprises:
The PBS solution that 1mg fluorescent microsphere balance pH is 6.5 ~ 7.5, concentration is 0.1M is 0.1mg/mL to the concentration of fluorescent microsphere;
Add EDC and NHS respectively in solution after balance, be 0.5mg/mL to EDC and NHS concentration in the solution, activate after 30 minutes, centrifugally abandon supernatant;
Abandon the sediment after supernatant with the PBS solution redissolution before described balance, add antibody 18B7 in the solution after redissolution, mark 20 minutes, the concentration adding BSA to described BSA is 1% cessation reaction.
22. 1 kinds of methods of catching Applications of Cardiac Markers, it is characterized in that, described method is applied to the micro-fluidic chip described in any one of claim 1-5, and described method comprises:
Make be coated with can with the immunomagnetic beads of the antibody of Blood Center flesh mark specific binding;
To the contraposition antibody labeling immune marking thing of described Applications of Cardiac Markers;
The immune marking thing of mark contraposition antibody is inputted immune labeled module;
The mixed liquor of a certain amount of cardiac marker sample and equilibrium liquid is added to sample addition zone, in described equilibrium liquid, comprises the immunomagnetic beads of described coated antibody;
Start detection equipment, reaction Preset Time, detects result.
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Address after: 102200, Beijing, Changping District, super Road, No. 3, building 37 Patentee after: Beijing Lepu Diagnostic Technology Co., Ltd Address before: 102200, Beijing, Changping District, super Road, No. 3, building 37 Patentee before: BEIJING LEPU MEDICAL TECHNOLOGY Co.,Ltd. |