CN103954775B - A kind of method detecting biomacromolecule or microbial body - Google Patents
A kind of method detecting biomacromolecule or microbial body Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6875—Nucleoproteins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
Abstract
The present invention relates to a kind of method detecting biomacromolecule or microbial body.Described method comprises the steps: that using the super suitable nanometer magnetic bead of single dispersing of the antibody bag quilt of biomacromolecule or microbial body to contact with detected sample carries out incubation reaction, generates granose super suitable nanometer magnetic bead cluster; Carry out Magneto separate after reaction and obtain aggregation, according to the concentration of biomacromolecule or microbial body in the aggregation extent determination detected sample of described aggregation.Immune magnetic enrichment and Visual retrieval are integrated in one by method of the present invention, simple to operate, and whole testing process required time is short, does not need special instrument and equipment, and the purity requirement of method antagonist of the present invention is not high, greatly can reduce testing cost.
Description
Technical field
The present invention relates to technical field of immune assay, particularly relate to a kind of quick, sensitive and Visual retrieval biomacromolecule of low cost or the method for microbial body.
Background technology
The diagnosis and detection of quick, sensitive, the low cost of the biomarkers such as the protein in infectiousness animals and plants pathogenic bacteria, virus and clinical sample to protection human health and life security, guarantee environment and national security, maintain social stability significant.The detection method of current comparative maturity mainly comprises the methods such as cultivating detection, immunology detection and molecular biology that is separated.Isolated culture method is simple, easy, but due to low, the consuming time length of its detection sensitivity, cannot reach on-the-spot, requirement fast.Immunological method mainly comprises the method for enzyme-linked immuno assay and immuno-chromatographic test paper strip, enzyme-linked immunoassay method has simply, remolding sensitivity is higher, low cost and other advantages, be suitable for extensive sample examination, but it needs multistep to wash, relatively take time and effort, therefore its use is also subject to certain restrictions.The advantages such as colloidal gold immuno-chromatography test paper strip has fast, simple, low cost, but its sensitivity is relatively low, can not realize quantitative detection.The analytical approach of molecular biology method mainly PCR-based, has detection sensitivity high, the advantages such as specificity is good, but needs expensive instrument and higher professional and technical personnel operation, and its application is subject to certain restrictions.Therefore, build the analytical approach of a kind of applicable scene, quick, highly sensitive and tailored diagnostics, realize having great importance to the detection of pathogenic bacteria in environment, food and biological sample, virus and protein.
Excellent magnetics and the optical properties such as super suitable nanometer magnetic bead has that saturation magnetization is strong, monodispersity good, size tunable and multivariate color, therefore there is based on the immunomagnetic isolation technology of super suitable nanometer magnetic bead quick, the simple and efficient advantage of immunoreactive high degree of specificity and magnetic resolution, be current most widely used separation, beneficiation technologies, be widely used in separation and the enrichment of the biomacromolecules such as pathogenic bacteria, virus, albumen, tumour cell or microbial body in recent years.In immune magnetic enrichment reaction, the identification of immunomagnetic beads and object is carried out in aaerosol solution, it is a kind of immune response pattern of homogeneous phase, be conducive to the abundant reaction of antibody-antigene, the speed of fast response can be added, therefore can get up with other a lot of combine with technique, build the analytical approach of series of new, the methods such as such as Magneto separate-fluorescence immune analysis method, Magneto separate-chemiluminescence analysis method, enrichment-microfluidic chip technology, Magneto separate-surface plasma resonance biological sensor, the micro-balance biology sensor of magnetic enrichment-quartz-crystal.Due to the development of immune magnetic separation technique, greatly facilitate the development of Correlation Analysis Technique, its application potential is constantly excavated out.
Summary of the invention
For the deficiencies in the prior art, we detect immunomagnetic beads and further investigate, find that the granose super suitable nanometer magnetic bead cluster formed by immune response is different from the state of aggregation of the super suitable nanometer magnetic bead of single dispersing in magnetic field, and the being proportionate property of content of object in the aggregation extent of immune super suitable nanometer magnetic bead and sample.
Therefore, the object of the present invention is to provide a kind of quick, sensitive and Visual retrieval biomacromolecule of low cost or the method for microbial body, in described method, the super suitable nanometer magnetic bead of immunity is both as the carrier of Magneto separate, enrichment; Due to it, there is color simultaneously, and granose super suitable nanometer magnetic bead cluster is different from the super suitable nanometer magnetic bead of single dispersing aggregation extent in magnetic field, can be used as visual signal reporting flag, Magneto separate in immune response, enrichment and detection are combined in a step complete, realize to object in sample quick, detect delicately.
For realizing object of the present invention, the invention provides following technical scheme:
A kind of method detecting biomacromolecule or microbial body, comprise the steps: that using the super suitable nanometer magnetic bead of single dispersing of the antibody bag quilt of biomacromolecule or microbial body to contact with detected sample carries out incubation reaction, generates granose super suitable nanometer magnetic bead cluster; Carry out Magneto separate after reaction and obtain aggregation, according to the concentration of biomacromolecule or microbial body in the aggregation extent determination detected sample of described aggregation.
The antibody of corresponding biomacromolecule or microbial body in the surperficial coupling of super suitable nanometer magnetic bead, is prepared into the super suitable nanometer magnetic bead of immunity.In immune response, biomacromolecule or microbial body have multiple antigenic determinant, by the specific recognition between antibody-antigene, can connect the super suitable nanometer magnetic bead of immunity of multiple monodispersity.The super suitable nanometer magnetic bead of immunity of monodispersity by " bridge " connection function of this antigen, the granose super suitable nanometer magnetic bead cluster of turn agglomerate tufted.Then under the effect of externally-applied magnetic field, become the super suitable nanometer magnetic bead cluster (aggregation) of many particles of aggregative state, because the super suitable nanometer magnetic bead of single dispersing is different from the disperse state of the super suitable nanometer magnetic bead of many particles of gathering in magnetic field, cause aggregation extent different, and the content positive correlation of object (biomacromolecule or microbial body) in the aggregation extent of the super suitable nanometer magnetic bead of the immunity of monodispersity and sample, based on this, can according to the concentration of biomacromolecule or microbial body in the aggregation extent determination detected sample of aggregation, thus build the visual analysis method detecting biomacromolecule or microbial body.
As preferred version of the present invention, the aggregation extent of described aggregation and the concentration of described biomacromolecule or microbial body are proportionate.The concentration of described biomacromolecule or microbial body is higher, then the color of described aggregation is darker; Therefore the curve relation figure between the concentration of standard model structure biomacromolecule or microbial body and the aggregation extent of aggregation can be used in advance, can determine the concentration of biomacromolecule corresponding to a particular color degree of depth or microbial body according to this curve relation figure, the concentration quantitative realizing biomacromolecule or microbial body in sample is determined.
As preferred version of the present invention, described biomacromolecule comprises protein, nucleic acid and polysaccharide, preferred protein.Because protein, nucleic acid and polysaccharide all may have corresponding epitope, combine for corresponding antibodies, realize the generation of granose super suitable nanometer magnetic bead cluster, therefore all can be used as biomacromolecule detection object of the present invention.
Preferably, described protein comprises lipoprotein, glycoprotein, nucleoprotein and biomarker.
As preferred version of the present invention, described microbial body comprises bacterium, fungi and virus.Because the albumen of bacterium, fungi and virus surface has epitope, can be combined with corresponding antibodies, realize the generation of granose super suitable nanometer magnetic bead cluster, therefore all can be used as microbial body detected object of the present invention.Mentioned microorganism body can be pathogenic bacteria or non-pathogenic bacteria, but the present invention gets rid of the situation of medical diagnosis on disease.
As preferred version of the present invention, described detected sample is the sample of environment, biology or food sources.The sample deriving from environment can be such as the sample deriving from polluted-water, and wherein containing biomacromolecule to be detected or microbial body, the method for the application of the invention, carries out quantitatively, realizing the assessment of pollution situation to these materials.The sample deriving from food can be such as derive from the prepared food of artificial or the sample of packaged food, and wherein containing biomacromolecule to be detected or microbial body, the method for the application of the invention, carries out quantitatively to these materials, realizes food security assessment.
As preferred version of the present invention, described antibody is monoclonal antibody and/or polyclonal antibody, preferred polyclonal antibody.Because the biomacromolecule in detected sample or microbial body surface may have multiple antigenic determinant, use monoclonal antibody also can realize " bridge " connection function when there being same or similar antigenic determinant; Polyclonal antibody, owing to having the binding site of various antigenic determinant, can be easy to realize " bridge " connection function.Certainly, the present invention also can use the mixing of monoclonal antibody and polyclonal antibody, and the present invention is not high to the purity requirement of monoclonal antibody, even if containing impurity, also can ignore for impact of the present invention.
As preferred version of the present invention, described biomacromolecule or microbial body have multiple antigenic determinant.The antibody on multiple antigenic determinants of same biomacromolecule or microbial body suitable nanometer magnetic bead surface super from different immunity respectively combines, and realizes " bridge " connection function, generates super suitable nanometer magnetic bead cluster.
As preferred version of the present invention, the described incubation reaction time is 5-60min, such as 5min, 6min, 10min, 12min, 15min, 20min, 25min, 30min, 40min, 45min, 50min, 55min, 58min, 10-40min, 10-30min, 5-20min, 15-30min, preferred 15min.
As preferred version of the present invention, the time of described Magneto separate is 0.5-5min, such as 0.5min, 1min, 1.5min, 2min, 2.5min, 3min, 4min, 4.5min, 4.8min, preferred 1min.
As preferred version of the present invention, according to the concentration of biomacromolecule or microbial body in the aggregation extent determination detected sample of described aggregation, be specially:
Obtain the photo of described aggregation, quantitatively determined the concentration of biomacromolecule or microbial body in detected sample by comparison film gray-scale value.
Compared to existing technology, beneficial effect of the present invention is:
The method of detection biomacromolecule of the present invention or microbial body, based on excellent magnetics and the optical property of super suitable nanometer magnetic bead, immune magnetic enrichment and Visual retrieval are integrated in one, simple to operate, whole testing process required time is short, do not need special instrument and equipment, only need a kind of antibody, avoid the needs of traditional immune detection to two kinds of antibody (capture antibody and detection antibody), and the purity requirement of method antagonist of the present invention is not high, greatly can reduce testing cost.In addition, method of the present invention has general applicability, can bacterial detection, fungi, virus and protein etc., be applicable to Site Detection, have great application prospect.
Accompanying drawing explanation
Fig. 1 is the reaction principle schematic diagram that the method for visualizing assembled based on the super suitable nanometer magnetic bead of immunity of the present invention detects biomacromolecule or microbial body.Wherein, (1) represent single dispersing immunity super suitable nanometer magnetic bead and sample in biomacromolecule or microbial body between immune response; (2) the super suitable nanometer magnetic bead cluster of granose immunity formed by " bridge " effect of antigen is represented; (3) represent that the super suitable nanometer magnetic bead cluster of granose immunity is under the effect of externally-applied magnetic field, form the super suitable nanometer magnetic bead aggregation of immunity assembled; In sample, object (biomacromolecule or microbial body) is more, and the aggregation extent of the super suitable nanometer magnetic bead of immunity is larger, and its color relation is darker.
Fig. 2 is the mechanism figure that the super suitable nanometer magnetic bead magnetic of immunity of the present invention is assembled.Wherein, (a) represents that the super suitable nanometer magnetic bead of immunity is together with sample mix, after reaction 15min, does not have the constitutional diagram under externally-applied magnetic field; B () represents that the super suitable nanometer magnetic bead of immunity is together with sample mix, after reaction 15min, and the constitutional diagram under magnetic field; C externally-applied magnetic field removes by () expression, by resuspended for super for the immunity of state of aggregation suitable nanometer magnetic bead, be then placed on and do not have on the test tube rack of externally-applied magnetic field, the constitutional diagram of the super suitable nanometer magnetic bead of described immunity under gravity independent role.The concentration (cfu/mL) of numeral biomacromolecule or microbial body.
Fig. 3 is that method of the present invention detects colibacillary result figure.Wherein, (a) represents that the super suitable nanometer magnetic bead of immunity and E. coli SampLes mix, after reaction, and the constitutional diagram under magnetic field; In sample, e. coli concentration (cfu/mL) is higher, and the aggregation extent of the super suitable nanometer magnetic bead of immunity is larger, and its color relation is darker; B () to represent in aggregation photo gray-scale value and sample that (in figure, 1-6 represents e. coli concentration 10 to e. coli concentration gradient respectively
7, 10
6, 10
5, 10
4, 10
3, 0cfu/mL) graph of a relation; E. coli concentration is higher, and gray-scale value is larger.
Fig. 4 is the result figure that method of the present invention detects tire cancer protein (CEA).Wherein, (a) represents that the super suitable nanometer magnetic bead of immunity is together with CEA sample mix, after reaction, and the constitutional diagram under magnetic field; In sample, CEA concentration (ng/mL) is higher, and the aggregation extent of the super suitable nanometer magnetic bead of immunity is larger, and its color relation is darker; B () represents the graph of a relation of CEA concentration (ng/mL) in aggregation photo gray-scale value and sample; CEA concentration is higher, and gray-scale value is larger.
Fig. 5 is the result figure that method of the present invention detects alpha-fetoprotein (AFP).Wherein 1-6 represents that AFP concentration is respectively 40,20,10,5,2.5 and the sample of 0ng/mL and the super suitable nanometer magnetic bead of immunity and mixes respectively, after reaction, and the constitutional diagram under magnetic field; In sample, AFP concentration (ng/mL) is higher, and the aggregation extent of the super suitable nanometer magnetic bead of immunity is larger, and its color relation is darker.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail.It will be understood to those of skill in the art that following examples are only the preferred embodiments of the present invention, so that understand the present invention better, thus should not be considered as limiting scope of the present invention.
Experimental technique in following embodiment, if no special instructions, is conventional method; Experiment material used, if no special instructions, is and is purchased available from routine biochemistry chemical reagent work.
Reaction principle schematic diagram of the present invention as shown in Figure 1, (1) represent single dispersing immunity super suitable nanometer magnetic bead and sample in biomacromolecule or microbial body between immune response; (2) the super suitable nanometer magnetic bead cluster of granose immunity formed by " bridge " effect of antigen is represented; (3) represent that the super suitable nanometer magnetic bead cluster of granose immunity is under the effect of externally-applied magnetic field, form the super suitable nanometer magnetic bead aggregation of immunity assembled; In sample, object (biomacromolecule or microbial body) is more, and the aggregation extent of the super suitable nanometer magnetic bead of immunity is larger, and its color relation is darker.
Reagent instrument and equipment source used in the following example is as follows: Escherichia coli purchased from American Culture Collection, Escherichia coli antibody (resisting) purchased from American Genway more; Alpha-fetoprotein, tire cancer protein and associated antibodies thereof are purchased from Beijing Hotgen Biotechnology Co., Ltd.; Magneto separate frame is purchased from profit micro-nano new material company limited of Shanghai Austria; Eddy oscillating device is purchased from German IKA company.
Embodiment 1
Preparation and the magnetic of the super suitable nanometer magnetic bead of immunity assemble visual immune detection, and concrete steps are as follows:
(1) preparation of the super suitable nanometer magnetic bead of immunity:
The super suitable nanometer magnetic bead (0.2-0.5mg) of carboxyl a certain amount of finishing is had to be dispersed in (pH=5.6 in the MES buffer solution of certain volume, 0.01M), then a certain amount of carbodiimide (EDC is added, 0.01-0.1mg) with N-hydroxy-succinamide (NHS, 0.01-0.1mg), oscillating reactions 0.5h lightly on eddy oscillating device, then Magneto separate, use the PBS damping fluid of pH=7.4 resuspended again, then a certain amount of antibody (0.1-0.5mg) is added, reaction 2h, add 5%BSA (100-500 μ L) again and react 0.5h, last Magneto separate, remove supernatant, then the resuspended super suitable nanometer magnetic bead of a certain amount of PBST cleansing solution is added, and then Magneto separate, remove supernatant, repeat to wash 3 times, the super suitable nanometer magnetic bead of the immunity finally obtained, resuspended with PBS damping fluid, at 4 DEG C of Refrigerator stores.
(2) magnetic assembles visual immune detection step:
The biomacromolecule (as protein) of super for a certain amount of immunity suitable nanometer magnetic bead and a series of concentration or microbial body (as bacterium, fungi or virus) are mixed, react 10-20min on the oscillator, then the test tube that the super suitable nanometer magnetic bead of immunity is housed is placed on Magneto separate frame, after 1min, observes the state of aggregation of the super suitable nanometer magnetic bead of immunity.
As shown in Figure 2, (a) represents that the super suitable nanometer magnetic bead of immunity is together with sample mix, after reaction 15min, does not have the constitutional diagram under externally-applied magnetic field to result after above-mentioned steps; B () represents that the super suitable nanometer magnetic bead of immunity is together with sample mix, after reaction 15min, and the constitutional diagram under magnetic field; C externally-applied magnetic field removes by () expression, by resuspended for super for the immunity of state of aggregation suitable nanometer magnetic bead, be then placed on and do not have on the test tube rack of externally-applied magnetic field, the constitutional diagram of the super suitable nanometer magnetic bead of described immunity under gravity independent role.Visible, in sample, object (as Escherichia coli) is more, and the aggregation extent of the super suitable nanometer magnetic bead of immunity is larger, and its color relation is darker; After resuspended, the settling velocity under gravity independent role and degree larger.
Embodiment 2
Detect the Escherichia coli in water sample, specific experiment step is as follows:
(1) by the Escherichia coli (10 of a series of variable concentrations
7, 10
6, 10
5, 10
4, 10
3and 0cfu/mL) each 900 μ L and a certain amount of super suitable nanometer magnetic bead (100 μ L) mix, oscillating reactions 15min on eddy oscillating device;
(2) immune potpourri complete for above-mentioned reaction is placed on Magneto separate frame, Magneto separate 1min;
(3) state of the super suitable nanometer magnetic bead of visual inspection, in visible sample, e. coli concentration (cfu/mL) is higher, and the aggregation extent of the super suitable nanometer magnetic bead of immunity is larger, and its color relation is darker (Fig. 3 a); Take pictures with camera, and carry out quantitatively with relevant PaintShop to its gray-scale value, result shows: e. coli concentration is higher, gray-scale value larger (Fig. 3 b).
Embodiment 3
Detect the Avian pneumo-encephalitis virus in chicken blastochyle, specific experiment step is as follows:
(1) by the Avian pneumo-encephalitis virus (10 of a series of variable concentrations
7, 10
6, 10
5, 10
4, 10
3and 0copy/mL) each 900 μ L and a certain amount of super suitable nanometer magnetic bead (100 μ L) mix, oscillating reactions 15min on eddy oscillating device;
(2) immune potpourri complete for above-mentioned reaction is placed on Magneto separate frame, Magneto separate 1min;
(3) state of the super suitable nanometer magnetic bead of visual inspection, takes pictures with camera, and carries out quantitatively its gray-scale value with relevant PaintShop.Result is similar to Fig. 3.
Embodiment 4
Detect the content of the tire cancer protein (CEA) in urine, specific experiment step is as follows:
(1) by the CEA of a series of variable concentrations (20,10,5,2,1,0ng/mL) each 900 μ L and a certain amount of super suitable nanometer magnetic bead (100 μ L) mix, oscillating reactions 15min on eddy oscillating device;
(2) immune potpourri complete for above-mentioned reaction is placed on Magneto separate frame, Magneto separate 1min;
(3) state of the super suitable nanometer magnetic bead of visual inspection, in visible sample, CEA concentration (ng/mL) is higher, and the aggregation extent of the super suitable nanometer magnetic bead of immunity is larger, and its color relation is darker (Fig. 4 a); Take pictures with camera, and carry out quantitatively with relevant PaintShop to its gray-scale value, result shows: CEA concentration is higher, gray-scale value larger (Fig. 4 b).
Embodiment 5
Detect the content of the alpha-fetoprotein (AFP) in urine, specific experiment step is as follows:
(1) AFP of a series of variable concentrations (40,20,10,5,2.5 and 0ng/mL) each 900 μ L and a certain amount of super suitable nanometer magnetic bead (100 μ L) are mixed, oscillating reactions 15min on eddy oscillating device;
(2) immune potpourri complete for above-mentioned reaction is placed on Magneto separate frame, Magneto separate 1min;
(3) state of the super suitable nanometer magnetic bead of visual inspection, in sample, AFP concentration (ng/mL) is higher, and the aggregation extent of the super suitable nanometer magnetic bead of immunity is larger, its color relation darker (Fig. 5).Take pictures with camera, and carry out quantitatively with relevant PaintShop to its gray-scale value, display gray shade value and AFP concentration are proportionate.
Applicant states, the present invention illustrates detailed features of the present invention and method detailed by above-described embodiment, but the present invention is not limited to above-mentioned detailed features and method detailed, namely do not mean that the present invention must rely on above-mentioned detailed features and method detailed could be implemented.Person of ordinary skill in the field should understand, any improvement in the present invention, to equivalence replacement and the interpolation of auxiliary element, concrete way choice etc. that the present invention selects component, all drops within protection scope of the present invention and open scope.
Claims (14)
1. one kind is detected the method for biomacromolecule or microbial body, comprise the steps: that using the super suitable nanometer magnetic bead of single dispersing of the antibody bag quilt of biomacromolecule or microbial body to contact with detected sample carries out incubation reaction, generates granose super suitable nanometer magnetic bead cluster; Carry out Magneto separate 0.5-5min after reaction and obtain aggregation, according to the concentration of biomacromolecule or microbial body in the aggregation extent determination detected sample of described aggregation.
2. method according to claim 1, is characterized in that, the aggregation extent of described aggregation and the concentration of described biomacromolecule or microbial body are proportionate.
3. method according to claim 1 and 2, is characterized in that, described biomacromolecule is protein, nucleic acid or polysaccharide.
4. method according to claim 3, is characterized in that, described biomacromolecule is protein.
5. method according to claim 4, is characterized in that, described protein is lipoprotein, glycoprotein, nucleoprotein or biomarker.
6. method according to claim 1, is characterized in that, described microbial body is bacterium, fungi or virus.
7. method according to claim 1, is characterized in that, described detected sample is the sample of environment, biology or food sources.
8. method according to claim 1, is characterized in that, described antibody is monoclonal antibody and/or polyclonal antibody.
9. method according to claim 8, is characterized in that, described antibody is polyclonal antibody.
10. method according to claim 1, is characterized in that, described biomacromolecule or microbial body have multiple antigenic determinant.
11. methods according to claim 1, is characterized in that, the described incubation reaction time is 5-60min.
12. methods according to claim 11, is characterized in that, the described incubation reaction time is 15min.
13. methods according to claim 1, is characterized in that, the time of described Magneto separate is 1min.
14. methods according to claim 1, is characterized in that, according to the concentration of biomacromolecule or microbial body in the aggregation extent determination detected sample of described aggregation, are specially:
Obtain the photo of described aggregation, quantitatively determined the concentration of biomacromolecule or microbial body in detected sample by comparison film gray-scale value.
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| CN102426229A (en) * | 2011-09-26 | 2012-04-25 | 广东海洋大学 | Preparation method of human IgG immunomagnetic bead for staphylococcus aureus enrichment |
| CN103278521A (en) * | 2012-11-30 | 2013-09-04 | 中国检验检疫科学研究院 | Magnetic resonance immune sensing method for detecting biomacromolecule |
| CN103529225A (en) * | 2013-11-04 | 2014-01-22 | 武汉华美生物工程有限公司 | Liver fatty acid binding protein content detection kit and preparation method thereof |
| CN103713134A (en) * | 2013-12-17 | 2014-04-09 | 武汉大学 | Detection kit for visibly detecting virus and virus detection method |
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2014
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| WO2004092732A1 (en) * | 2003-04-16 | 2004-10-28 | Sekisui Chemical Co., Ltd. | Particle having magnetic material incorporated therein, process for producing the same, particle for immunoassay and method of immunoassay |
| CN101865984A (en) * | 2010-06-03 | 2010-10-20 | 复旦大学 | Magnetic relaxation switch based on Fe3O4 at Au and preparation method thereof |
| CN102323408A (en) * | 2011-05-31 | 2012-01-18 | 上海师范大学 | Method for rapid detection of enterobacter sakazakii |
| CN102426229A (en) * | 2011-09-26 | 2012-04-25 | 广东海洋大学 | Preparation method of human IgG immunomagnetic bead for staphylococcus aureus enrichment |
| CN103278521A (en) * | 2012-11-30 | 2013-09-04 | 中国检验检疫科学研究院 | Magnetic resonance immune sensing method for detecting biomacromolecule |
| CN103529225A (en) * | 2013-11-04 | 2014-01-22 | 武汉华美生物工程有限公司 | Liver fatty acid binding protein content detection kit and preparation method thereof |
| CN103713134A (en) * | 2013-12-17 | 2014-04-09 | 武汉大学 | Detection kit for visibly detecting virus and virus detection method |
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