CN104714011B - A kind of method of gold nanoparticle probe colorimetric determination influenza A virus H3N2 - Google Patents
A kind of method of gold nanoparticle probe colorimetric determination influenza A virus H3N2 Download PDFInfo
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Abstract
A kind of method that the invention discloses gold nanoparticle probe colorimetric determination influenza A virus H3N2, comprises the following steps: 1) the choosing of monoclonal antibody and influenza A virus；2) synthesis of gold nanoparticle probe；3) gold nanoparticle probe and influenza A virus effect；4) observe and record viral solution color situation of change；5) utilize ultraviolet-visual spectrometer and transmission electron microscope that viral solution is detected.The present invention utilizes gold nanoparticle probe after the assembling on influenza A virus cyst membrane surface, and solution is become blue from claret, and color change substantially, therefore may utilize colorimetric determination influenza A virus.The present invention is effectively utilized the characteristic of nano material, it is not necessary to detects by precision instrument, simplifies detection method, significantly reduce Viral diagnosis cost, the advantage that the present invention has low cost, quick, easy, sensitive and specificity is good.
Description
Technical field
The invention belongs to biosensor technique field, a kind of method being specifically related to gold nanoparticle probe colorimetric determination influenza A virus H3N2, specifically refer to monoclonal antibody functional modification to behind golden nanometer particle surface, specific recognition influenza A virus H3N2 is also assembled into its cyst membrane surface, thus causes surface plasma body resonant vibration (the surface plasmon of golden nanometer particle
Resonance, SPR) peak generation red shift, then observe that solution colour changes.
Background technology
Influenza virus, is called for short influenza virus, is that one causes people, Canis familiaris L., horse, pig and birds etc. to suffer from grippal RNA viruses.All over the world, influenza infection is the key factor affecting M & M.When bird flu and human influenza virus infect intermediate host simultaneously, due to gene recombinaton, easily produce novel influenza, harm society.
The main method of traditional influenza virus detection has viral culture of isolated, immunology diagnosis, diagnosis of molecular biology.Virus isolation procedure have passed through test for a long time, testing result is the most reliable, sensitive, very small amount virus can be detected, but exist operation sequence numerous and diverse, costly, laboratory safety rank require high (must carry out) at BSL-3 level laboratory, the detection time about needs 1～3 week, the longest, promoting in epidemic-stricken area large-area applications, there is relatively Multiple Constraints in a large amount of sample detection aspects.Immunology detection technology is mainly based upon what the characteristic of antigen and antibody specific association reaction was set up, and detection object is mainly blood serum sample.But due to the interference of serum blood clotting inhibitive factor, may result in hemagglutination inhibition test result false positive.Diagnosis of molecular biology develops faster having obtained in recent years, and especially real-time fluorescence quantitative PCR diagnostic techniques has become as the national standard of influenza test in laboratory and quick diagnosis.But owing to detection means is loaded down with trivial details, need precision instrument, relatively costly, limit its application clinically.
In recent years except to the development of traditional detection method and perfect, nanotechnology is as a kind of emerging influenza virus detection method, by more concerns of people.Along with the development of science and technology drives making constant progress of nanotechnology, constantly launching with deeply about golden nanometer particle in the research of field of biological detection, achieving great successes and long-range development.Golden nanometer particle has the optical effect of uniqueness, utilize the SPR character of golden nanometer particle, after antigen antibody reaction causes golden nanometer particle to assemble formation network structure, light is absorbed on relatively long wavelength and scatters so that color transition is aubergine finally becomes blue by aggregation.Utilize the change of this optical characteristics, the high sensitivity of binding biomolecules and specificity so that golden nanometer particle has extensive and important application prospect in biological detection.
Therefore a kind of simple, influenza virus new method quickly, accurately, in detection by quantitative clinical sample is developed for controlling the generation of Influenza epidemic situation and spreading significant.
Summary of the invention
Goal of the invention: the technical problem to be solved there is provided a kind of method of gold nanoparticle probe colorimetric determination influenza A virus H3N2.For gold nanoparticle probe optical property generation significant change after virus envelope surface-assembled in the present invention, thus set up colorimetric determination influenza A virus, it has, and principle is simple, experimental period is short, raw materials used cost is relatively low, and experimental result the most just can directly be observed and without any large-scale instrument.
Technical scheme: in order to solve above-mentioned technical problem, the technical solution adopted in the present invention is: a kind of method of gold nanoparticle probe colorimetric determination influenza A virus H3N2, comprises the following steps:
1) the choosing of monoclonal antibody and influenza A virus；
2) synthesis of gold nanoparticle probe；
3) by gold nanoparticle probe and influenza A virus effect；
4) observe and record viral solution color situation of change；
5) utilize ultraviolet-visual spectrometer and transmission electron microscope that viral solution is detected.
Wherein, said monoclonal antibody is influenza A virus H3N2 hemagglutinin mouse monoclonal antibody.
Wherein, above-mentioned steps 2) synthesis step of gold nanoparticle probe is as follows: taking an EP pipe, add 0.5 ~ 1 mL, 2 nmol/L solution of gold nanoparticles, with 0.l mol/L K2CO3The pH value of regulation solution is 8.2 ~ 9.2, add monoclonal antibody 15 ~ 30 μ L of 0.1 mg/mL, on shaking table 120 ~ 300 revs/min, incubation 1 ~ 2 hour at 37 DEG C, is subsequently adding 10 ~ 20 μ L 1%(m/v) BSA, incubation 0.5 ~ 1 hour at 37 DEG C, finally by probe solution at 12200 ~ 13200 rpm, it is centrifuged 20 ~ 30 minutes at 4 DEG C, redissolves in 200 ~ 500 μ L PBS buffer solution after centrifugal three times, 4 DEG C of preservations.
Wherein, above-mentioned steps 3) gold nanoparticle probe and influenza A virus action step as follows: depletion nanoparticle probes joins in influenza A virus solution, at 37 DEG C, incubation is after 1 ~ 2 hour, its solution is recorded and uv-vis spectra detection, take 5 ~ 10 μ L viral solution to be added drop-wise on the copper mesh that carbon film is supported, after ambient temperatare puts 1 ~ 3 minute, surplus solution on copper mesh is sucked with filter paper, then copper mesh is dipped in the Sodium phosphotungstate solution of 1% ~ 2% after 1 ~ 3 minute, take out, naturally drying under room temperature, transmission electron microscope detects.
Wherein, above-mentioned PBS buffer solution is the pH containing NaCl and KCl
The PBS buffer solution of 10 mmol/L of 7.2 ~ 7.4, described NaCl initial concentration is 136.89 mmol/L, and KCl initial concentration is 2.67 mmol/L.
Wherein, a diameter of 13 nanometers of above-mentioned golden nanometer particle.
Wherein, the above-mentioned influenza A virus H3N2 final concentration of 0 ~ 400 HAU in buffer solution.
The present invention is by the hemagglutinin on a kind of gold nanoparticle probe specific recognition influenza A virus H3N2 cyst membrane surface modifying influenza A virus H3N2 hemagglutinin (HA) mouse monoclonal antibody based on surface, golden nanometer particle is assembled into virus envelope surface, then by the SPR character of virus envelope surface golden nanometer particle, colorimetric determination influenza A virus H3N2.It is prepared for the gold nanoparticle probe that monoclonal antibody is modified.Owing to each influenza virus diameter is in 80 ~ 120 nanometers, about 500 hemagglutinins are contained on cyst membrane surface, therefore 8 nanometers it are about between cyst membrane surface hemagglutinin is adjacent, and a diameter of 13 nanometers of golden nanometer particle, more than the adjacent spacing of hemagglutinin, therefore the golden nanometer particle of virus envelope surface-assembled can effectively produce SPR effect, the SPR absworption peak of golden nanometer particle from 525 ran red shifts to 600 ran, and 525 the SPR peak intensity of nanometers weaken, the SPR peak intensity of 600 nanometers increases, increase along with virus concentration, reactant liquor color gradually becomes navy blue from claret.Therefore the present invention utilizes the specificity of monoclonal antibody and the SPR character of golden nanometer particle, it is achieved colorimetric determination influenza A virus H3N2.
Beneficial effect: compared with prior art, the present invention has following characteristic and an advantage:
(1) present invention utilizes gold nanoparticle probe after the assembling on influenza A virus cyst membrane surface, and solution is become blue from claret, and color change substantially, therefore can use colorimetric determination influenza A virus.
(2) present invention is effectively utilized the characteristic of nano material, it is not necessary to detects by precision instrument, simplifies detection method, significantly reduce Viral diagnosis cost, the advantage that the present invention has low cost, quick, easy, sensitive and specificity is good.
Accompanying drawing explanation
Fig. 1 shows the flow chart of the gold nanoparticle probe colorimetric determination influenza A virus H3N2 modifying influenza A virus H3N2 hemagglutinin (HA) mouse monoclonal antibody based on surface；
Fig. 2 A shows the transmission electron microscope picture of golden nanometer particle；As can be seen from the figure gold nanometer particle grain size is homogeneous, good dispersion；Fig. 2 B shows a line: the ultra-violet absorption spectrum of golden nanometer particle；B line: the ultra-violet absorption spectrum of gold nanoparticle probe；It can be seen that gold nanoparticle probe does not change a lot relative to the ultra-violet absorption spectrum of golden nanometer particle, gold nanoparticle probe is stable in properties in buffer solution；
Fig. 3 A shows gold nanoparticle probe and the transmission electron microscope picture of influenza A virus H3N2 assembly, and scale is 50 nanometers；As can be seen from the figure gold nanoparticle probe is successfully assembled into influenza A virus H3N2 cyst membrane surface；Fig. 3 B shows H3N2 line: gold nanoparticle probe and the ultra-violet absorption spectrum of influenza A virus H3N2 assembly；Control line: gold nanoparticle probe ultra-violet absorption spectrum in buffer solution；Illustration is the digital photograph of corresponding solution；It can be seen that when there being influenza A virus H3N2 in solution, the SPR absworption peak of gold nanoparticle probe occurs obvious red shift, corresponding solution color is become blue from claret.
Fig. 4 shows detection influenza A virus H3N2 specific ultraviolet spectra variation diagram and block diagram；A: under different probe effect, the ultraviolet spectrogram obtained (a: gold nanoparticle probe and influenza A virus H3N2 effect；Golden nanometer particle contrast probe and the influenza A virus H3N2 effect of BSA is modified on b: surface；C: gold nanoparticle probe), illustration is the digital photograph of corresponding solution；As can be seen from the figure the gold nanoparticle probe only having monoclonal antibody to modify can be with specific recognition influenza A virus H3N2；Block diagram after B: gold nanoparticle probe and various flows Influenza Virus effect；Illustration is the digital photograph of corresponding solution；As can be seen from the figure gold nanoparticle probe specificity capture influenza A virus H3N2, with other influenza virus without obvious effect.
Fig. 5 shows the ultraviolet spectra variation diagram of detection by quantitative influenza A virus H3N2.A: under different amounts of influenza A virus H3N2 effect, the ultraviolet spectrogram obtained (concentration of influenza A virus H3N2: (a) 0, (b) 10, (c) 20, (d) 40, (e) 60, (f) 80, (g) 100, (h) 200, (i) 400 HAU)；The matched curve to specific strength Yu influenza A virus H3N2 concentration of B: the uv absorption；Illustration: uv absorption is to the linear relationship between specific strength and influenza A virus H3N2 concentration；As can be seen from the figure influenza A virus H3N2 is 10
HAU to 80 HAU is good linear relationship.
Detailed description of the invention
Below by specific embodiment and accompanying drawing, the present invention is further described; it should be pointed out that, for the person of ordinary skill of the art, under the premise without departing from the principles of the invention; can also make some modification and improvement, these also should be regarded as belonging to protection scope of the present invention.
The reagent used in this experiment and instrument:
Influenza A virus (H3N2 IAV, H1N1
IAV, H2N2 IAV), Influenza B virus (Yamagata, Victoria), influenza A virus H3N2 hemagglutinin (HA) mouse monoclonal antibody (mAb), bovine serum albumin (BSA), gold chloride (HAuCl4·3H2O), sodium citrate (Na3C6H5O7·2H2O), Sodium phosphotungstate (PTA), ultraviolet spectrophotometer (Shimadzu UV 2450, Kyoto, Japan), transmission electron microscope (JEM 2010, Hitachi, Japan), mixing vortex instrument (IKA German), centrifuge (Eppendorf German), digital camera (Canon IXUS 115, Japan).
Embodiment 1:
Modify the analysis method of the gold nanoparticle probe colorimetric determination influenza A virus H3N2 of influenza A virus H3N2 hemagglutinin (HA) mouse monoclonal antibody based on surface, detecting step is:
Probe synthesis step: taking an EP pipe, add 1 mL, 2 nmol/L solution of gold nanoparticles, with 0.l mol/L K2CO3The pH value of regulation solution is 8.2, adds the monoclonal antibody 30 μ L of 0.1 mg/mL, on shaking table 120 revs/min, incubation 2 hours at 37 DEG C.It is subsequently adding 20 μ L 1%(m/v) BSA, incubation 30 minutes at 37 DEG C.Finally by probe solution at 13200 rpm, it is centrifuged 20 minutes at 4 DEG C, redissolves in 200 μ L PBS buffer solution after centrifugal three times, 4 DEG C of preservations.This solution is as probe solution.
Take an EP pipe, add 1 mL, 2 nmol/L solution of gold nanoparticles, add 20 μ L 1%(m/v) BSA, incubation 30 minutes at 37 DEG C.Then by probe solution at 13200 rpm, it is centrifuged 20 minutes at 4 DEG C, redissolves in 200 μ L PBS buffer solution after centrifugal three times, 4 DEG C of preservations.This solution probe solution as a comparison.
Proof-Of Principle step: 50 μ L, 10 nmol/L probe solutions join in 400 HAU 200 μ L influenza A virus H3N2 solution, fully after mixing, after reacting 2 hours in 37 DEG C of thermostat water baths, take 5 μ L viral solution and be added drop-wise on the copper mesh that carbon film is supported, after ambient temperatare puts 3 minutes, suck surplus solution on copper mesh with filter paper, then copper mesh is dipped in the Sodium phosphotungstate solution of 1% after 3 minutes, take out, naturally drying under room temperature, transmission electron microscope detects.Experimental result is shown in that Fig. 3 A: gold nanoparticle probe is successfully assembled into the cyst membrane surface of influenza A virus H3N2.Viral solution goes to scan uv-vis spectra simultaneously, and the solution colour before and after contrast virus adds changes.Experimental result is shown in that Fig. 3 B:control line is the ultraviolet absorption spectrum of probe solution, H3N2 line is the ultraviolet absorption spectrum of solution after virus adds, H3N2 line compares the SPR absworption peak of control line from 525 nanometer red shifts to 650 ran, and corresponding solution is become blue from claret simultaneously.Result display gold nanoparticle probe is successfully assembled into the cyst membrane surface of influenza A virus H3N2, thus causes the SPR peak generation red shift of golden nanometer particle, and corresponding solution color is become blue from claret.
Specific detection step: in two EP pipes containing 400 HAU 200 μ L influenza A virus H3N2 solution, is separately added into 50 μ L, 10 nmol/L probe solutions and contrast probe solution.Fully after mixing, react in 37 DEG C of thermostat water baths 30 minutes, then go solution in two EP pipes to scan uv-vis spectra, simultaneously comparative solution color.Experimental result is shown in Fig. 4 A: the uv absorption data containing probe solution compared with contrast probe solution, and the ultraviolet absorption peak of probe solution occurs obvious red shift, and the obvious purpling of solution colour, and contrast probe solution is without significant change.By 50 μ L, 10 nmol/L probe solutions are added separately to various flows Influenza Virus solution (the H2:H3N2 IAV of 400 HAU 200 μ L, H1:H1N1IAV, H3:H2N2 IAV, Yamagata, Victoria) in, fully after mixing, 37 DEG C of thermostat water baths react 30 minutes, then go reaction mixture to scan uv-vis spectra, simultaneously comparative solution color.Experimental result is shown in that Fig. 4 B: gold nanoparticle probe specificity captures influenza A virus H3N2, with other influenza virus without obvious effect.Result display probe solution is fine to the Detection results of influenza A virus H3N2.
Detection by quantitative influenza A virus H3N2 step: by 50 μ L, after 10 nmol/L probe solutions and different amounts of influenza A virus H3N2 (0,10,20,40,60,80,100,200,400 HAU) fully mixing, 37 DEG C of thermostat water baths react 30 minutes, after reaction terminates, go reaction mixture to scan uv-vis spectra.Analysis obtains ultraviolet absorpting spectrum, draws influenza A virus H3N2Linear graph.Experimental result is shown in that Fig. 5: influenza A virus H3N2 is good linear relationship at 10 HAU to 80 HAU, and detection limit is 7.8 HAU.Note: reaction cumulative volume is 250 μ L.
Embodiment 2:
Modify the analysis method of the gold nanoparticle probe colorimetric determination influenza A virus H3N2 of influenza A virus H3N2 hemagglutinin (HA) mouse monoclonal antibody based on surface, detecting step is:
Probe synthesis step: take an EP pipe, add 500 μ L, 2 nmol/L solution of gold nanoparticles, add 10 μ L, 0.l mol/L K2CO3, after fully shaking up, add 15 μ L, the monoclonal antibody of 0.1 mg/mL, on shaking table 300 revs/min, incubation 2 hours at 37 DEG C.It is subsequently adding 10 μ L 1%(m/v) BSA, incubation 1 hour at 37 DEG C.Finally by probe solution at 12200 rpm, it is centrifuged 30 minutes at 4 DEG C, redissolves in 200 μ L PBS buffer solution after centrifugal three times, 4 DEG C of preservations.This solution is as probe solution.
Take an EP pipe, add 500 μ L, 2 nmol/L solution of gold nanoparticles, add 10 μ L 1%(m/v) BSA, incubation 1 hour at 37 DEG C.Then by probe solution at 12200 rpm, it is centrifuged 30 minutes at 4 DEG C, redissolves in 200 μ L PBS buffer solution after centrifugal three times, 4 DEG C of preservations.This solution probe solution as a comparison.
Proof-Of Principle step: 50 μ L, 10 nmol/L probe solutions join in 400 HAU 200 μ L influenza A virus H3N2 solution, fully after mixing, after reacting 1 hour in 37 DEG C of thermostat water baths, take 5 μ L viral solution and be added drop-wise on the copper mesh that carbon film is supported, after ambient temperatare puts 3 minutes, suck surplus solution on copper mesh with filter paper, then copper mesh is dipped in the Sodium phosphotungstate solution of 2% after 1 minute, take out, naturally drying under room temperature, transmission electron microscope detects.Experimental result: gold nanoparticle probe is successfully assembled into the cyst membrane surface of influenza A virus H3N2.Viral solution goes to scan uv-vis spectra simultaneously, and the solution colour before and after contrast virus adds changes.Experimental result: the SPR absworption peak of probe solution is from 525 nanometer red shifts to 650 ran, and corresponding solution is become blue from claret simultaneously.
Specific detection step: in two EP pipes containing 512 HAU 200 μ L influenza A virus H3N2 solution, is separately added into 100 μ L, 5 nmol/L probe solutions and contrast probe solution.Fully after mixing, react in 37 DEG C of thermostat water baths 1 hour, then go solution in two EP pipes to scan uv-vis spectra, simultaneously comparative solution color.Experimental result is shown in Fig. 4 A: the uv absorption data containing probe solution compared with contrast probe solution, and the ultraviolet absorption peak of probe solution occurs obvious red shift, and the obvious purpling of solution colour, and contrast probe solution is without significant change.By 100 μ L, 5 nmol/L probe solutions are added separately to various flows Influenza Virus solution (the H2:H3N2 IAV of 512 HAU 200 μ L, H1:H1N1 IAV, H3:H2N2 IAV, Yamagata, Victoria) in, fully after mixing, 37 DEG C of thermostat water baths react 1 hour, then go reaction mixture to scan uv-vis spectra, simultaneously comparative solution color.Experimental result is shown in that Fig. 4 B: gold nanoparticle probe specificity captures influenza A virus H3N2, with other influenza virus without obvious effect.Result display probe solution is to influenza A virus H3N2Detection results fine.
Detection by quantitative influenza A virus H3N2 step: by 100 μ L, after the influenza A virus H3N2 (0,20,40,80,160,200,300,400 HAU) of 5 nmol/L probe solutions and 200 μ L variable concentrations fully mixing, 37 DEG C of thermostat water baths react 1 hour, after reaction terminates, go reaction mixture to scan uv-vis spectra.Analysis obtains uv absorption spectrogram, draws influenza A virus H3N2 linear graph.Experimental result is shown in that Fig. 5: influenza A virus H3N2 is good linear relationship at 40 HAU to 200 HAU, and detection limit is 37.8 HAU.Note: reaction cumulative volume is 300 μ L.
Embodiment 3:
Modify the analysis method of the gold nanoparticle probe colorimetric determination influenza A virus H3N2 of influenza A virus H3N2 hemagglutinin (HA) mouse monoclonal antibody based on surface, detecting step is:
Probe synthesis step: taking an EP pipe, add 1 mL, 2 nmol/L solution of gold nanoparticles, with 0.l mol/L K2CO3The pH value of regulation solution is 9.2, adds the monoclonal antibody 30 μ L of 0.1 mg/mL, on shaking table 200 revs/min, incubation 2 hours at 37 DEG C.It is subsequently adding 20 μ L 1%(m/v) BSA, incubation 1 hour at 37 DEG C.Finally by probe solution at 13200 rpm, it is centrifuged 20 minutes at 4 DEG C, redissolves in 200 μ L PBS after centrifugal three times, 4 DEG C of preservations.This solution is as probe solution.
Take an EP pipe, add 1 mL, 2 nmol/L solution of gold nanoparticles, add 20 μ L 1%(m/v) BSA, incubation 30 minutes at 37 DEG C.Then by probe solution at 13200 rpm, it is centrifuged 20 minutes at 4 DEG C, redissolves in 200 μ L PBS after centrifugal three times, 4 DEG C of preservations.This solution probe solution as a comparison.
Proof-Of Principle step: 50 μ L, 10 nmol/L probe solutions join in 400 HAU 200 μ L influenza A virus H3N2 solution, fully after mixing, after reacting 1 hour in 37 DEG C of thermostat water baths, take 10 μ L viral solution and be added drop-wise on the copper mesh that carbon film is supported, after ambient temperatare puts 1 minute, suck surplus solution on copper mesh with filter paper, then copper mesh is dipped in the Sodium phosphotungstate solution of 1% after 3 minutes, take out, naturally drying under room temperature, transmission electron microscope detects.Experimental result: gold nanoparticle probe is successfully assembled into the cyst membrane surface of influenza A virus H3N2.Viral solution goes to scan uv-vis spectra simultaneously, and the solution colour before and after contrast virus adds changes.Experimental result: the SPR absworption peak of probe solution is from 525 nanometer red shifts to 650 ran, and corresponding solution is become blue from claret simultaneously.
Specific detection step: in two EP pipes containing 512 HAU 200 μ L influenza A virus H3N2 solution, is separately added into 50 μ L, 10 nmol/L probe solutions and contrast probe solution.Fully after mixing, react in 37 DEG C of thermostat water baths 1 hour, then go solution in two EP pipes to scan uv-vis spectra, simultaneously comparative solution color.Experimental result is shown in Fig. 4 A: the uv absorption data containing probe solution compared with contrast probe solution, and the ultraviolet absorption peak of probe solution occurs obvious red shift, and the obvious purpling of solution colour, and contrast probe solution is without significant change.By 50 μ L, 10 nmol/L probe solutions are added separately to various flows Influenza Virus solution (the H2:H3N2 IAV of 512 HAU 200 μ L, H1:H1N1 IAV, H3:H2N2 IAV, Yamagata, Victoria) in, fully after mixing, 37 DEG C of thermostat water baths react 1 hour, then go reaction mixture to scan uv-vis spectra, simultaneously comparative solution color.Experimental result is shown in that Fig. 4 B: gold nanoparticle probe specificity captures influenza A virus H3N2, with other influenza virus without obvious effect.Result display probe solution is fine to the Detection results of influenza A virus H3N2.
Detection by quantitative influenza A virus H3N2 step: by 100 μ L, after the influenza A virus H3N2 (0,30,60,90,120,150,300,400 HAU) of 5 nmol/L probe solutions and 200 μ L variable concentrations fully mixing, 37 DEG C of thermostat water baths react 1 hour, after reaction terminates, go reaction mixture to scan uv-vis spectra.Analysis obtains uv absorption spectrogram, draws influenza A virus H3N2 linear graph.Experimental result is shown in that Fig. 5: influenza A virus H3N2 is good linear relationship at 30 HAU to 150 HAU, and detection limit is 28.4 HAU.Note: reaction cumulative volume is 250 μ L.
Above are only the preferred embodiment of the invention, be not restricted to the present invention.To those of ordinary skill in the art, change or the variation of other multi-forms can also be made on the basis of the above description.Here without also all of embodiment being illustrated.And within the obvious change that thus scheme is extended out or variation are still in protection scope of the present invention.
Claims (5)
1. the method for the detection influenza A virus H3N2 of a gold nanoparticle probe colorimetry non-diagnostic purpose, it is characterised in that comprise the following steps:
1) the choosing of monoclonal antibody and influenza A virus H3N2 solution；
2) synthesis of gold nanoparticle probe；
3) gold nanoparticle probe and influenza A virus H3N2 solution effects；
4) observe and solution colour situation of change after record effect；
5) utilize ultraviolet-visual spectrometer and transmission electron microscope that solution after effect is detected；
Described monoclonal antibody is influenza A virus H3N2 hemagglutinin mouse monoclonal antibody, a diameter of 13 nanometers of described golden nanometer particle.
The method of the detection influenza A virus H3N2 of gold nanoparticle probe colorimetry non-diagnostic purpose the most according to claim 1, it is characterized in that, described step 3)-step 5) is as follows: depletion nanoparticle probes joins in influenza A virus H3N2 solution, at 37 DEG C, incubation is after 1 ~ 2 hour, solution after incubation is carried out observed and recorded and the uv-vis spectra detection of color change, take the solution after 5 ~ 10 μ L incubations to be added drop-wise on the copper mesh that carbon film is supported, after ambient temperatare puts 1 ~ 3 minute, surplus solution on copper mesh is sucked with filter paper, then copper mesh is dipped in the Sodium phosphotungstate solution of 1% ~ 2% after 1 ~ 3 minute, take out, naturally dry under room temperature, transmission electron microscope detects.
The method of the detection influenza A virus H3N2 of gold nanoparticle probe colorimetry non-diagnostic purpose the most according to claim 1, it is characterized in that, described step 2) synthesis step of gold nanoparticle probe is as follows: take an EP pipe, add 0.5 ~ 1 mL, 2 nmol/L solution of gold nanoparticles, with 0.l mol/L K2CO3The pH value of regulation solution is 8.2 ~ 9.2, add influenza A virus H3N2 hemagglutinin mouse monoclonal antibody 15 ~ 30 μ L of 0.1 mg/mL, 120 ~ 300 revs/min of vibrations on shaking table, incubation 1 ~ 2 hour at 37 DEG C, is subsequently adding 10 ~ 20 μ L 1%(m/v) BSA, incubation 0.5 ~ 1 hour at 37 DEG C, finally by solution at 12200 ~ 13200rpm, it is centrifuged 20 ~ 30 minutes at 4 DEG C, redissolves in 200 ~ 500 μ L PBS buffer solution after centrifugal three times, 4 DEG C of preservations.
The method of the detection influenza A virus H3N2 of gold nanoparticle probe colorimetry non-diagnostic purpose the most according to claim 3, it is characterised in that described PBS buffer solution is the PBS buffer solution of 10 mmol/L of the pH 7.2 ~ 7.4 containing NaCl and KCl.
The method of the detection influenza A virus H3N2 of gold nanoparticle probe colorimetry non-diagnostic purpose the most according to claim 1, it is characterised in that the concentration of described influenza A virus H3N2 solution is 400 HAU.
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| CN103983776A (en) * | 2014-05-27 | 2014-08-13 | 国家纳米科学中心 | Colloidal gold immunochromatography test strip for simultaneously detecting viral antigens and antibodies |
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Non-Patent Citations (2)
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| Antibody Modified Gold Nanoparticles for Fast and Selective, Colorimetric T7 Bacteriophage Detection;Adam Lesniewski 等;《Bioconjugate Chemistry》;20140328;第25卷(第4期);摘要，第645页左栏第2段-第647页右栏第1段 * |
| One-step assay for detecting influenza virus using dynamic light scattering and gold nanoparticles;Jeremy D. Driskell 等;《Analyst》;20110807;第136卷(第15期);摘要，第3085页左栏倒数第1段-右栏第3段 * |
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