CN102854304B - A kind of pathogen detection method based on micro-fluidic chip - Google Patents
A kind of pathogen detection method based on micro-fluidic chip Download PDFInfo
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Abstract
This patent relates to a kind of pathogen detection method based on micro-fluidic chip.The container of the method using the micro-fluidic chip in integrated micro-magnetic field as reaction, magnetic ball is as solid phase carrier, the quantum dot (SA-QDs) that streptavidin is modified is as fluorescent marker, under the effect in micro-magnetic field, in chip channel, privileged site catches magnetic ball, defines micro-reaction zone, catches pathogen by sandwich immunoassay reaction, by the interaction of biotin and streptavidin, realize the fluorescence immunoassay quantitative test to pathogen.Micro-fluidic chip, magnetic immunoassay and fluoroscopic examination combine by this detection method, had the quick, efficient of micro-fluidic chip concurrently and amount of samples little, the high specific of magnetic immunoassay, strong navigability, and the advantage such as the optical property of quantum dot excellence, a kind of multiobject, real-time pathogen detection method.
Description
Technical field
The present invention relates to a kind of pathogen detection method based on micro-fluidic chip, belong to chemistry and biomedical sector.
Background technology
In recent years, SARS, bird flu, hand-foot-and-mouth disease, Influenza A H1N1 is broken out continuously.Serious infectious disease each time popular all gives society, economy causes huge loss, brings great threat to the life of people.Pathogen detection method conventional at present has PCR technology, culture technique, immunoenzyme technics (EIA), enzyme linked immunosorbent assay (ELISA) etc.These technology have played huge effect in clinical diagnosis, but still there are some shortcomings, mostly more consuming time, complex disposal process and cost is high.A kind of method setting up quick, sensitive, special detection pathogen is the important guarantee of prevention and corntrol infectious disease.
Microfluidic chip technology builds chemistry or biology laboratory on the chip of a piece several square centimeters, the various sample preparation related to are said by chemistry and Bioexperiment, reaction, be separated, to detect etc. and be integrated together, have that microminiaturization, robotization, sample consumption are little, detection efficiency advantages of higher.Magnetic particle is as a solid phase carrier, and in targeting substance identification, be separated, control administration, the aspect tools such as sorting cells have been widely used.It micro-fluidic chip is one of hot issue of current research in conjunction with magnetic bead technology.
Because quantum dot has unique optical property superior again, as extinction coefficient is large, quantum yield is high, exciting light spectrum width, emission spectrum is narrow, doubly, light stability, than traditional fluorescent dye height 100-1000 feature such as doubly, is widely used in biological fluorescent labelling to the traditional fluorescent dye height 10-100 of brightness ratio at present.
Micro-fluidic chip, magnetic immunoassay and fluoroscopic examination combine by we, and establish a kind of quick, efficient, low consumption, high specific, high sensitivity, real-time pathogen detection method, the Site Detection for pathogen provides strong instrument.
Summary of the invention
Technical matters to be solved by this invention is the detection method providing a kind of pathogen fast and efficiently.
Container using the micro-fluidic chip in integrated micro-magnetic field as reaction, magnetic ball is as solid phase carrier, the quantum dot (SA-QDs) that streptavidin is modified is as fluorescent marker, under the effect in micro-magnetic field, in chip channel, privileged site catches magnetic ball, defines micro-reaction zone, catches pathogen by sandwich immunoassay reaction, by the interaction of biotin and streptavidin, realize the fluorescence immunoassay quantitative test to pathogen.Chip achieves the separation of pathogen, enrichment, immune response, analysis detection.
Concrete technical scheme comprises the steps:
(1) making of the micro-fluidic chip in integrated micro-magnetic field: the formpiston obtaining micro-fluidic chip by the method for Soft lithograph; Two magnetic conductive rods are fixed on the both sides of formpiston microchannel, the angle between magnetic conductive rod is 0 ~ 180
o, the angle between the plane that magnetic conductive rod is formed and the plane of microchip is 90
o; Adopt the reaction in-situ method of forming, liquid prepolymer be poured on the formpiston of fixing magnetic conductive rod, after reaction solidification, stripping forming, beats sample holes and sample outlet hole, then with cover plate bonding, namely obtains the micro-fluidic chip (as shown in Figure 1, 2) in integrated micro-magnetic field;
(2) the magnetic ball preparation of anti-special pathogen surface protein monoclonal antibody is modified with: the carboxyl activating magnetic ball surface with ethyl-[3-(dimethylamino) propyl group] carbodiimide (EDAC) and N-hydroxy-succinamide (NHS) potpourri, then the monoclonal antibody of anti-pathogen surface protein to be measured is added, after the magnetic ball being connected with antibody is dispersed in PBS(pH 7.4 containing sodium azide, BSA) in damping fluid, save backup;
(3) preparation of biotinylated antipathogen surface protein monoclonal antibody: adopt biotinylated kit modified biological element on HA monoclonal antibody, carry out purifying with desalting column;
(4) on chip, carry out magnetic holder heart Immunofluorescence Reactions detect pathogen: first close chip channel with BSA solution, on the magnetic conductive rod of chip, respectively put a block permanent magnet simultaneously, then in passage, inject the magnetic ball, special pathogen, the biotinylated quantum dot (SA-QDs) resisting this pathogen surface protein monoclonal antibody, streptavidin to modify that are modified with anti-special pathogen surface protein monoclonal antibody successively, often all use PBS wash buffer passage after step reaction, wash unreacted medicine off, carry out fluoroscopic examination finally by fluorescent microscope and fiber spectrometer.
In such scheme: the material preparing micro-fluidic chip in step (1) is that elastomer polymer is if dimethyl silicone polymer (PDMS) and thermoplastic are as polymethylmethacrylate (PMMA), polycarbonate (PC), polystyrene (PS) etc.; The formpiston making micro-fluidic chip is Metal Substrate formpiston, silicon mold or glass mold plungers; Making magnetic conduction Bang material is iron oxide or silicon steel class soft magnetic material.
The present invention establishes a kind of magnetic fluorescence immunologic detection method of the simple and quick detection pathogen based on micro-fluidic chip, magnetic ball, quantum dot.Micro-fluidic chip, magnetic immunoassay and fluoroscopic examination are combined, had the quick, efficient of micro-fluidic chip concurrently and amount of samples little, the high specific of magnetic immunoassay, strong navigability, and the advantage such as the optical property of quantum dot excellence, be a kind of simple, quick, efficient, portable, method of being suitable for Site Detection.The present invention is more broadly applicable to the fields such as biological and medical science.
Accompanying drawing explanation
Fig. 1 is the microfluidic chip structure schematic diagram in embodiment 1,1. micro-fluidic chip substrates, 2. microchannel, 3. magnetic conductive rod, 4. permanent magnet, 5. cover plate in figure.
Fig. 2 is the vertical section structure schematic diagram in embodiment 1,1. micro-fluidic chip substrates, 2. microchannel, 3. magnetic conductive rod, 4. permanent magnet, 5. cover plate in figure.
Fig. 3 is the schematic flow sheet of magnetic fluorescence immune detection pathogen on the chip in embodiment 1.
Fig. 4 is fluorescence picture (a) of the variable concentrations H9N2 magnetic fluorescence immune detection in embodiment 2, with corresponding spectrogram (b).
Fig. 5 is the changing trend diagram (a) of variable concentrations H9N2 in embodiment 2 and fluorescence intensity, with canonical plotting (b), corresponding with Fig. 4.
Fig. 6 is the specificity analyses of the sandwich immunofluorescent detection method of detection H9N2 magnetic in embodiment 2, comprises fluorescence picture (a) of reagent blank and negative control, with corresponding spectrogram (b).
Fig. 7 is the specificity analyses of the sandwich immunofluorescent detection method of detection H9N2 magnetic in embodiment 2, comprises the histogram of reagent blank and negative control, corresponding with Fig. 6.
Fig. 8 is fluorescence picture (a) of the variable concentrations NDV magnetic fluorescence immune detection in embodiment 3, with corresponding typical curve (b).
Fig. 9 is the specificity analyses of the sandwich immunofluorescent detection method of detection NDV magnetic in embodiment 3, comprises fluorescence picture (a) of reagent blank and negative control, with corresponding histogram (b).
Embodiment
Embodiment 1
(1) method for making of integrated chip
The soft lithographic manufacturing technology of utilization standard, get rid of the photoresist of last layer AZ50XT at the silicon chip surface that cleaning is smooth, obtain the thickness of one about 40 μm, with ultraviolet exposure under the covering of the film mask printed, development, obtains the mask protruding from silicon chip surface of a standard.Two autonomous guidance bar magnets are fixed on the both sides of formpiston microchannel, the angle between magnetic conductive rod is 0 ~ 180
o, the angle between the plane that magnetic conductive rod is formed and the plane of microchip is 90
othe prepolymer mass ratio of mediation dimethyl silicone polymer (PDMS) is 10:1(mass ratio), vacuum gets rid of bubble, be poured on mask, then in an oven 75 DEG C dry 3-4 hour, after solidification, cut with scalpel, uncover above mask, pattern just copies on PDMS, throws sample and outlet into the perforating needle of a tack; By the microslide of cleaning and the PDMS with fluid passage, be put in oxygen plasma and process 70s activation PDMS surface, produce great amount of hydroxy group free radical, take out, be buckled together on two sides upward, 75 DEG C are dried 10 minutes in an oven, realize permanent bonding.
(2) magnetic ball (Master Beads Carboxylic Acid 0215, the 500 nm diameter) preparation of anti-special pathogen surface protein monoclonal antibody is modified with
The superparamagnetism ball that 10 μ L surfaces are rolled into a ball for carboxyl function (first washes three times with the PBS of 0.01 mol/L pH 7.4.Then magnetic ball is dispersed in the PBS solution containing the 0.01 mol/L pH 7.4 of 0.10 mol/L EDAC and 0.1 mol/L NHS, 37
ooscillating reactions 30 min on C shaking table, the carboxyl on activation magnetic ball surface.Then after washing three times with the PBS of 0.01 mol/L pH 7.4, be dispersed in the PBS solution of 180 μ L 0.01 mol/L pH 7.4, then the anti-special pathogen surface protein monoclonal antibody solution of 20 μ L 0.59 mg/mL is added, oscillating reactions 2 h on shaking table.Wash three times with the PBS of 0.01 mol/L pH 7.4 after reaction, remove responseless Streptavidin, be then dispersed in the BSA of 1 mL 2%, close 30 min.Finally wash three times with the PBS of 0.01 mol/L pH 7.4, be dispersed in 0.01 mol/L pH 7.4 PBS containing 0.05 % sodium azide 2% BSA, 4
oc stores for future use.
(3) preparation of biotinylated anti-special pathogen surface protein monoclonal antibody
Utilize biotinylation reagent Sulfo-NHS-LC-Biotin(Thermo), by reacting with the amino of this pathogen surface protein monoclonal antibody, biotin is connected on antibody.Concrete operations are as follows: get 0.1mg biotinylation reagent Sulfo-NHS-LC-Biotin, join in 80 μ L ultrapure waters, and then add this pathogen surface protein monoclonal antibody solution anti-of 20 μ L 0.59 mg/mL, room temperature shaker reaction 2h.Unnecessary biotinylation reagent utilizes NAP-5(GE Healthcare) desalting column remove, obtaining volume is this pathogen surface protein monoclonal antibody solution anti-that 500 μ L mark.
(4) on chip, carry out magnetic holder heart Immunofluorescence Reactions detect pathogen.
Experimental program as shown in Figure 3, first BSA solution closed channel is used, after 30min, 1min is washed with PBS damping fluid, on the magnetic conductive rod of integrated chip, respectively put a block permanent magnet (" NS " pole pair) simultaneously, then inject the magnetic ball (Fig. 3-a) that 2 μ L are modified with the modification of anti-special pathogen surface protein monoclonal antibody, under the effect in micro-magnetic field, define micro-reaction zone (Fig. 3-b); After PBS damping fluid is washed, reinject this pathogen particle of 2 μ L (Fig. 3-c), arrheas and hatch 5min(Fig. 3-d); Then passage washed by PBS damping fluid, injects the monoclonal antibody (Fig. 3-e) of biotinylated this pathogen surface protein anti-of 2 μ L, arrheas and hatch 5min(Fig. 3-f); Passage washed by PBS damping fluid, then injects the quantum dot (Fig. 3-g) that 2 μ L streptavidins are modified, arrheas and hatch 5min(Fig. 3-h); Finally wash the SA-QDs not having to react with PBS damping fluid off, carry out fluoroscopic examination by fluorescent microscope and fiber spectrometer.In whole experimentation, the flow velocity of pump is 1 μ L/min.
This technology can fast, effective implemention is to the detection of a lot of pathogen.As H9N2(embodiment 1), ewcastle disease (Newcastle Disease Virus NDV) (embodiment 2), the virions such as H1N1, H5N1.
Embodiment 2:
Utilize the method for embodiment 1 to make integrated chip, prepare the magnetic ball and biotinylated anti-H9N2 avian influenza virus surface protein HA monoclonal antibody that are modified with anti-H9N2 avian influenza virus surface protein HA monoclonal antibody.Then on chip, carry out magnetic holder heart Immunofluorescence Reactions detect H9N2.Fig. 4 is fluorescence picture (a) of variable concentrations H9N2 magnetic fluorescence immune detection, with corresponding spectrogram (b).Fig. 5 is the changing trend diagram (a) of variable concentrations H9N2 and fluorescence intensity, with canonical plotting (b), corresponding with Fig. 4.Fig. 6 is the specificity analyses detecting the sandwich immunofluorescent detection method of H9N2 magnetic, and fluorescence picture (a), with corresponding spectrogram (b).Fig. 7 is the specificity analyses detecting the sandwich immunofluorescent detection method of H9N2 magnetic, comprises the histogram of reagent blank and negative control, corresponding with Fig. 6.
Embodiment 3:
Utilize the method for embodiment 1 to make integrated chip, prepare the magnetic ball and biotinylated anti-new castle disease virus surface protein HA monoclonal antibody that are modified with anti-new castle disease virus (NDV) surface protein HA monoclonal antibody.Then on chip, carry out magnetic holder heart Immunofluorescence Reactions detect H9N2.Fig. 8 is the fluorogram of variable concentrations NDV magnetic fluorescence immune detection, spectrogram, typical curve.Fig. 9 is the specificity analyses detecting the sandwich immunofluorescent detection method of NDV magnetic, comprises the fluorogram of reagent blank and negative control, spectrogram, histogram.
Claims (4)
1. based on a pathogen detection method for micro-fluidic chip, it is characterized in that, comprise the steps:
(1) making of the micro-fluidic chip in integrated micro-magnetic field: the formpiston obtaining micro-fluidic chip by the method for Soft lithograph; Two magnetic conductive rods are fixed on the both sides of formpiston microchannel, the angle between magnetic conductive rod is 0 ~ 180
o, the angle between the plane that magnetic conductive rod is formed and the plane of microchip is 90
o; Adopt the reaction in-situ method of forming, liquid prepolymer be poured on the formpiston of fixing magnetic conductive rod, after reaction solidification, stripping forming, beats sample holes and sample outlet hole, then with cover plate bonding, namely obtains the micro-fluidic chip in integrated micro-magnetic field;
(2) the magnetic ball preparation of anti-special pathogen surface protein monoclonal antibody is modified with: the carboxyl activating magnetic ball surface with ethyl-[3-(dimethylamino) propyl group] carbodiimide and N-hydroxy-succinamide potpourri, then the monoclonal antibody of anti-pathogen surface protein to be measured is added, after the magnetic ball being connected with antibody is dispersed in the PBS damping fluid containing sodium azide, BSA, save backup;
(3) preparation of biotinylated antipathogen surface protein monoclonal antibody: adopt biotinylated kit modified biological element on antipathogen surface protein monoclonal antibody, carry out purifying with desalting column;
(4) on chip, carry out magnetic holder heart Immunofluorescence Reactions detect pathogen: first close chip channel with BSA solution, on the magnetic conductive rod of chip, respectively put a block permanent magnet simultaneously, then in passage, inject the magnetic ball, special pathogen, the biotinylated quantum dot resisting this pathogen surface protein monoclonal antibody, streptavidin to modify that are modified with anti-special pathogen surface protein monoclonal antibody successively, often all use PBS wash buffer passage after step reaction, wash unreacted medicine off, carry out fluoroscopic examination finally by fluorescent microscope and fiber spectrometer.
2. method according to claim 1, is characterized in that, the material preparing micro-fluidic chip is dimethyl silicone polymer or polymethylmethacrylate, polycarbonate, polystyrene.
3. method according to claim 1 and 2, is characterized in that, the formpiston making micro-fluidic chip is Metal Substrate formpiston, silicon mold or glass mold plungers.
4. method according to claim 1 and 2, is characterized in that, making magnetic conductive rod material is iron oxide or silicon steel class soft magnetic material.
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