CN102854304A - Pathogen detection method based on micro-fluidic chip - Google Patents
Pathogen detection method based on micro-fluidic chip Download PDFInfo
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Abstract
The invention relates to a pathogen detection method based on a micro-fluidic chip. The method adopts a micro-fluidic chip integrated with a micro-magnetic field as a reaction container, adopts magnetic balls as solid phase carriers, and adopts quantum dots modified by streptavidin (SA-QDs) as fluorescent markers; under the action of the micro-magnetic field, magnetic balls are captured at specific parts in a chip channel, and thus a micro-reaction zone is formed; pathogens are captured through a sandwich immunization reaction; with the interaction between biotin and streptavidin, the immunofluorescence quantitative analysis of the pathogens is realized. The detection method combines the micro-fluidic chip, magnetic immunoassay, and fluorescence detection, has the advantages of rapidness, high efficiency, and few sample using amount for the micro-fluidic chip, high specificity and strong manoeuvrability for magnetic immunoassay, and excellent optical properties for quantum dots, and is a multi-objective real-time pathogen detection method.
Description
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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, break out continuously SARS, bird flu, hand-foot-and-mouth disease, Influenza A H1N1.Popular society, the economy of all giving of serious communicable disease has caused huge loss each time, has brought great threat for people's life.Pathogen detection method commonly used has PCR technology, culture technique, immunoenzyme technics (EIA), enzyme linked immunosorbent assay (ELISA) etc. at present.These technology have been brought into play huge effect in clinical diagnosis, but still have some shortcomings, and mostly more consuming time, complex disposal process and cost are high.Set up a kind of fast, the method for sensitive, special detection pathogen is the important guarantee of prevention and control infectious disease.
The micro-fluidic chip technology is chip structure chemistry or the biology laboratory at more than square centimeters, to say the various sample preparation that relate in chemistry and the Bioexperiment, reaction, separate, detect to wait to be integrated together, have that microminiaturization, robotization, sample consumption are little, the detection efficiency advantages of higher.Magnetic particle, separates in targeting substance identification as a solid phase carrier, the control administration, and the aspects such as sorting cells have widely purposes.On micro-fluidic chip, be one of hot issue of current research in conjunction with the magnetic bead technology.
Because quantum dot has unique again superior optical property, large such as extinction coefficient, quantum yield is high, the exciting light spectrum width, emission spectrum is narrow, the high 10-100 of the fluorescent dye that brightness ratio is traditional doubly, light stability has been widely used in biological fluorescent labelling at present than the high 100-1000 of the traditional fluorescent dye characteristics such as doubly.
We combine micro-fluidic chip, magnetic immunoassay and fluoroscopic examination, set up a kind of fast, efficient, low consumption, high specific, high sensitivity, real-time pathogen detection method are for the Site Detection of pathogen provides strong instrument.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of detection method of fast and efficiently pathogen.
With the micro-fluidic chip in the integrated little magnetic field container as reaction, the magnetic ball is as solid phase carrier, the quantum dot (SA-QDs) that streptavidin is modified is as fluorescent marker, under the effect in little magnetic field, privileged site is caught the magnetic ball in chip channel, has formed little reaction zone, catches pathogen by the sandwich immunoassay reaction, by the interaction of biotin and streptavidin, realize the fluorescence immunoassay quantitative test to pathogen.Separation, enrichment, immune response, the analyzing and testing of pathogen have been realized at chip.
Concrete technical scheme comprises the steps:
(1) making of the micro-fluidic chip in integrated little magnetic field: the formpiston that makes micro-fluidic chip with the method for soft etching; Two magnetic conductive rods are fixed in the both sides of formpiston microchannel, the angle between the magnetic conductive rod is 0～180
o, the angle between the plane of the formed plane of magnetic conductive rod and microchip is 90
oAdopt the reaction in-situ method of forming, liquid prepolymer is poured on the formpiston of fixing magnetic conductive rod, after the reaction curing, stripping forming is beaten sample holes and sample outlet hole, then with the cover plate bonding, namely obtains the micro-fluidic chip (as shown in Figure 1, 2) in integrated little magnetic field;
(2) be modified with the magnetic ball preparation of anti-special pathogen surface protein monoclonal antibody: the carboxyl that activates magnetic ball surface with ethyl-[3-(dimethylamino) propyl group] carbodiimide (EDAC) and N-hydroxy-succinamide (NHS) potpourri, then the monoclonal antibody that adds anti-pathogen surface protein to be measured, after will be connected with antibody the magnetic ball be dispersed in the PBS(pH 7.4 that contains sodium azide, BSA) in the damping fluid, save backup;
(3) preparation of biotinylated antipathogen surface protein monoclonal antibody: adopt biotinylated kit modified biological element on the HA monoclonal antibody, carry out purifying with desalting column;
(4) carry out magnetic holder heart Immunofluorescence Reactions at chip and detect pathogen: at first with BSA solution sealing chip channel, on the magnetic conductive rod of chip, respectively put simultaneously a block permanent magnet, then in passage, inject magnetic ball, special pathogen, the biotinylated quantum dot (SA-QDs) that resists this pathogen surface protein monoclonal antibody, streptavidin to modify that is modified with anti-special pathogen surface protein monoclonal antibody successively, all use PBS damping fluid irrigation channel after per step reaction, wash unreacted medicine off, carry out fluoroscopic examination by fluorescent microscope and fiber spectrometer at last.
In the such scheme: the material of preparation micro-fluidic chip is elastomer polymer such as dimethyl silicone polymer (PDMS) and thermoplastic such as polymethylmethacrylate (PMMA), polycarbonate (PC), polystyrene (PS) etc. in the step (1); The formpiston that the making micro-fluidic chip is used 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 has set up a kind of magnetic fluorescence immunologic detection method that detects pathogen based on the Simple fast of micro-fluidic chip, magnetic ball, quantum dot.Micro-fluidic chip, magnetic immunoassay and fluoroscopic examination are combined, quick, the efficient and amount of samples that has had micro-fluidic chip concurrently is little, the high specific of magnetic immunoassay, strong navigability, and the advantages such as optical property of quantum dot excellence, be a kind of simple, quick, efficient, portable, method of being suitable for Site Detection.The present invention can be applied even more extensively in fields such as biological and medical sciences.
Description of drawings
Fig. 1 is the microfluidic chip structure schematic diagram among the embodiment 1,1. micro-fluidic chip substrates among the figure, 2. microchannel, 3. magnetic conductive rod, 4. permanent magnet, 5. cover plate.
Fig. 2 is the vertical section structure schematic diagram among the embodiment 1,1. micro-fluidic chip substrates among the figure, 2. microchannel, 3. magnetic conductive rod, 4. permanent magnet, 5. cover plate.
Fig. 3 is the schematic flow sheet of magnetic fluorescence immune detection pathogen on the chip among the embodiment 1.
Fig. 4 is the fluorescence picture (a) of the variable concentrations H9N2 magnetic fluorescence immune detection among the embodiment 2, with corresponding spectrogram (b).
Fig. 5 is variable concentrations H9N2 among the embodiment 2 and the changing trend diagram (a) of fluorescence intensity, and is with canonical plotting (b), corresponding with Fig. 4.
Fig. 6 is the specificity analyses of the sandwich immunofluorescent detection method of detection H9N2 magnetic among the embodiment 2, comprises the 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 among the embodiment 2, comprises the histogram of reagent blank and negative control, and is corresponding with Fig. 6.
Fig. 8 is the fluorescence picture (a) of the variable concentrations NDV magnetic fluorescence immune detection among the embodiment 3, with corresponding typical curve (b).
Fig. 9 is the specificity analyses of the sandwich immunofluorescent detection method of detection NDV magnetic among the embodiment 3, comprises the fluorescence picture (a) of reagent blank and negative control, with corresponding histogram (b).
Embodiment
(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 smooth silicon chip surface of cleaning, obtain the thickness about 40 μ m, under the covering of the film mask of printing, use ultraviolet exposure, develop, obtain the mask that protrudes from silicon chip surface of a standard.Two autonomous guidance bar magnets are fixed in the both sides of formpiston microchannel, the angle between the magnetic conductive rod is 0～180
o, the angle between the plane of the formed plane of magnetic conductive rod and microchip is 90
oThe prepolymer mass ratio of mediation dimethyl silicone polymer (PDMS) is the 10:1(mass ratio), vacuum is got rid of bubble, be poured on the mask, then in baking oven 75 ℃ the baking 3-4 hour, after the curing, cut with scalpel, uncover above mask, pattern just copies on the PDMS, throws sample and outlet into the perforating needle of a tack; With the microslide of cleaning with have the PDMS of fluid passage, be put in and process 70s activation PDMS surface in the oxygen plasma, produce the great amount of hydroxy group free radical, take out, up two sides is buckled together, 75 ℃ of bakings are 10 minutes in baking oven, realize permanent bonding.
(2) be modified with magnetic ball (Master Beads Carboxylic Acid 0215, the 500 nm diameter) preparation of anti-special pathogen surface protein monoclonal antibody
(at first the PBS with 0.01 mol/L pH 7.4 washes three times the superparamagnetism ball that roll into a ball for carboxyl function on 10 μ L surface.Then the magnetic ball is dispersed in the PBS solution of the 0.01 mol/L pH 7.4 that contains 0.10 mol/L EDAC and 0.1 mol/L NHS 37
oOscillating reactions 30 min on the C shaking table, the carboxyl on activation magnetic ball surface.After then washing three times with the PBS of 0.01 mol/L pH 7.4, it is 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 that adds 20 μ L, 0.59 mg/mL, oscillating reactions 2 h on the shaking table.PBS with 0.01 mol/L pH 7.4 after the reaction washes three times, removes responseless Streptavidin, then it is dispersed among the BSA of 1 mL 2%, seals 30 min.PBS with 0.01 mol/L pH 7.4 washes three times at last, it is dispersed among 0.01 mol/L pH, 7.4 PBS that contain 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), react by the amino with this pathogen surface protein monoclonal antibody, biotin is connected on the antibody.Concrete operations are as follows: get 0.1mg biotinylation reagent Sulfo-NHS-LC-Biotin, join in the 80 μ L ultrapure waters, and then add anti-this pathogen surface protein monoclonal antibody solution of 20 μ L, 0.59 mg/mL, room temperature shaking table reaction 2h.Unnecessary biotinylation reagent utilizes NAP-5(GE Healthcare) desalting column remove, obtaining volume is good anti-these pathogen surface protein monoclonal antibody solution of 500 μ L marks.
(4) carry out magnetic holder heart Immunofluorescence Reactions at chip and detect pathogen.
Experimental program as shown in Figure 3, at first use BSA solution closed channel, behind the 30min, wash 1min with the PBS damping fluid, on the magnetic conductive rod of integrated chip, respectively put simultaneously a block permanent magnet (" NS " pole pair), then injecting 2 μ L is modified with the magnetic ball that anti-special pathogen surface protein monoclonal antibody modifies (Fig. 3-a) has formed little reaction zone (Fig. 3-b) under the effect in little magnetic field; After the PBS damping fluid was washed, (Fig. 3-c) arrheaed and hatches 5min(Fig. 3-d) this pathogen particle of 2 μ L that reinjects; Then the PBS damping fluid is washed passage, and (Fig. 3-e) arrheas and hatches 5min(Fig. 3-f) to inject the monoclonal antibody of biotinylated anti-this pathogen surface protein of 2 μ L; The PBS damping fluid is washed passage, and (Fig. 3-g) arrheas and hatches 5min(Fig. 3-h) then to inject the quantum dot that 2 μ L streptavidins modify; Wash the SA-QDs that does not have in the reaction off with the PBS damping fluid at last, carry out fluoroscopic examination by fluorescent microscope and fiber spectrometer.The flow velocity of pump is 1 μ L/min in the whole experimentation.
This technology can fast, efficiently realize the detection to a lot of pathogen.Such as H9N2(embodiment 1), ewcastle disease (Newcastle Disease Virus NDV) (embodiment 2), H1N1, the virions such as H5N1.
Embodiment 2:
Utilize the method for embodiment 1 to make integrated chip, prepared the magnetic ball and the 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 carry out magnetic holder heart Immunofluorescence Reactions at chip and detect H9N2.Fig. 4 is the 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, and is with canonical plotting (b), corresponding with Fig. 4.Fig. 6 is for detecting the specificity analyses of the sandwich immunofluorescent detection method of H9N2 magnetic, and fluorescence picture (a) is with corresponding spectrogram (b).Fig. 7 comprises the histogram of reagent blank and negative control for detecting the specificity analyses of the sandwich immunofluorescent detection method of H9N2 magnetic, and is corresponding with Fig. 6.
Embodiment 3:
Utilize the method for embodiment 1 to make integrated chip, prepared the magnetic ball and the 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 carry out magnetic holder heart Immunofluorescence Reactions at chip and detect H9N2.Fig. 8 is the fluorogram of variable concentrations NDV magnetic fluorescence immune detection, spectrogram, typical curve.Fig. 9 comprises the fluorogram of reagent blank and negative control, spectrogram, histogram for detecting the specificity analyses of the sandwich immunofluorescent detection method of NDV magnetic.
Claims (4)
1. the pathogen detection method based on micro-fluidic chip is characterized in that, comprises the steps:
(1) making of the micro-fluidic chip in integrated little magnetic field: the formpiston that makes micro-fluidic chip with the method for soft etching; Two magnetic conductive rods are fixed in the both sides of formpiston microchannel, the angle between the magnetic conductive rod is 0～180
o, the angle between the plane of the formed plane of magnetic conductive rod and microchip is 90
oAdopt the reaction in-situ method of forming, liquid prepolymer is poured on the formpiston of fixing magnetic conductive rod, after the reaction curing, stripping forming is beaten sample holes and sample outlet hole, then with the cover plate bonding, namely obtains the micro-fluidic chip in integrated little magnetic field;
(2) be modified with the magnetic ball preparation of anti-special pathogen surface protein monoclonal antibody: with the carboxyl on ethyl-[3-(dimethylamino) propyl group] carbodiimide and N-hydroxy-succinamide potpourri activation magnetic ball surface, then the monoclonal antibody that adds anti-pathogen surface protein to be measured, after will be connected with antibody the magnetic ball be dispersed in the PBS damping fluid that contains sodium azide, BSA, save backup;
(3) preparation of biotinylated antipathogen surface protein monoclonal antibody: adopt biotinylated kit modified biological element on the HA monoclonal antibody, carry out purifying with desalting column;
(4) carry out magnetic holder heart Immunofluorescence Reactions at chip and detect pathogen: at first with BSA solution sealing chip channel, on the magnetic conductive rod of chip, respectively put simultaneously a block permanent magnet, then in passage, inject magnetic ball, special pathogen, the biotinylated quantum dot that resists this pathogen surface protein monoclonal antibody, streptavidin to modify that is modified with anti-special pathogen surface protein monoclonal antibody successively, all use PBS damping fluid irrigation channel after per step reaction, wash unreacted medicine off, carry out fluoroscopic examination by fluorescent microscope and fiber spectrometer at last.
2. method according to claim 1 is characterized in that, the material of preparation 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 that the making micro-fluidic chip is used is Metal Substrate formpiston, silicon mold or glass mold plungers.
4. method according to claim 1 and 2 is characterized in that, making magnetic conduction Bang material is iron oxide or silicon steel class soft magnetic material.
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