CN103969445B - The preparation of heme-manganese dioxide composites and the method for detecting human IgG thereof - Google Patents
The preparation of heme-manganese dioxide composites and the method for detecting human IgG thereof Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 239000000243 solution Substances 0.000 claims abstract description 36
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 21
- 241000283973 Oryctolagus cuniculus Species 0.000 claims abstract description 17
- 230000003647 oxidation Effects 0.000 claims abstract description 12
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 12
- 238000006243 chemical reaction Methods 0.000 claims abstract description 9
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 8
- 239000008366 buffered solution Substances 0.000 claims abstract description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims abstract description 8
- 239000010452 phosphate Substances 0.000 claims abstract description 8
- 150000003278 haem Chemical class 0.000 claims abstract description 6
- 235000011114 ammonium hydroxide Nutrition 0.000 claims abstract description 4
- 241000283707 Capra Species 0.000 claims description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 8
- 229940098773 bovine serum albumin Drugs 0.000 claims description 8
- 239000000047 product Substances 0.000 claims description 8
- 238000002835 absorbance Methods 0.000 claims description 7
- 239000004793 Polystyrene Substances 0.000 claims description 6
- 229920002223 polystyrene Polymers 0.000 claims description 6
- 229920001661 Chitosan Polymers 0.000 claims description 5
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 5
- 239000007979 citrate buffer Substances 0.000 claims description 5
- 238000011534 incubation Methods 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 238000004737 colorimetric analysis Methods 0.000 claims description 4
- 239000012467 final product Substances 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 11
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 abstract description 10
- 108060003951 Immunoglobulin Proteins 0.000 abstract description 4
- 239000000427 antigen Substances 0.000 abstract description 4
- 102000036639 antigens Human genes 0.000 abstract description 4
- 108091007433 antigens Proteins 0.000 abstract description 4
- 238000006555 catalytic reaction Methods 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 4
- 238000003018 immunoassay Methods 0.000 abstract description 4
- 102000018358 immunoglobulin Human genes 0.000 abstract description 4
- 238000004458 analytical method Methods 0.000 abstract description 3
- 108040007629 peroxidase activity proteins Proteins 0.000 abstract description 3
- 102000003992 Peroxidases Human genes 0.000 abstract 1
- 239000007853 buffer solution Substances 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- BTIJJDXEELBZFS-QDUVMHSLSA-K hemin Chemical compound CC1=C(CCC(O)=O)C(C=C2C(CCC(O)=O)=C(C)\C(N2[Fe](Cl)N23)=C\4)=N\C1=C/C2=C(C)C(C=C)=C3\C=C/1C(C)=C(C=C)C/4=N\1 BTIJJDXEELBZFS-QDUVMHSLSA-K 0.000 description 3
- 229940025294 hemin Drugs 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000013016 damping Methods 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000003547 immunosorbent Substances 0.000 description 2
- 102000013415 peroxidase activity proteins Human genes 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000007812 electrochemical assay Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Chemical & Material Sciences (AREA)
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Abstract
The invention discloses the preparation of heme-manganese dioxide composites and the method for detecting human IgG thereof.Heme is dissolved in phosphate buffered solution by the present invention under ammoniacal liquor effect, then adds MnAc successively
2solution and NaOH solution reaction, obtained heme-manganese dioxide composites (Hemin-MnO
2); Hemin-MnO
2there is peroxidase activity, can it be made to become blueness from colourless, by traditional " sandwich " sandwich structure method, with Hemin-MnO by catalysis hydrogen peroxide oxidation TMB
2as rabbit anti-human igg's label, carry out the detection of human immunoglobulin IgG antigen, use Hemin-MnO
2colorimetric immunoassay method detect there is the advantages such as good stability, highly sensitive, selectivity good, reappearance is excellent, analysis speed is fast; This immuno-sensing method is simple, cheap, and naked eyes and the change of its color of observable, be with a wide range of applications.
Description
Technical field
The present invention relates to the preparation of heme-manganese dioxide composites and the method for detecting human IgG thereof, belonging to bio-sensing field.
Background technology
In recent years, the construction and application development of new bio sensing technology rapidly, is widely applied in fields such as clinical early diagnosis, environmental monitoring, food inspection.And a lot of method for sensing and application thereof cannot be popularized in many developing countries by the restriction of instrument and correlation technique professional, so build a kind of simple, economical and without the need to becoming scientific research hot topic research field by the method for sensing of precision instrument and equipment.Realize the biological immune sensing method detected based on color change, because of its be swift in response, the convenient feature such as economic, simple to operate and be subject to numerous researcher parent and look at.
The method of existing quantitative detection human IgG comprises electrochemical assay, fluorescence detection, chemoluminescence method etc.But these methods depend on certain instrument and equipment, and can not same time detecting several samples, spent time is longer.
Summary of the invention
The object of the invention is the preparation method being to provide a kind of Stability Analysis of Structures, having the heme-manganese dioxide composites of peroxidase activity, this preparation method is simple, cost is low.
Another object of the present invention there are provided a kind of method based on the detection human IgG of heme-manganese dioxide composites, the method utilizes heme-manganese dioxide composites catalysis hydrogen peroxide oxidation 3, 3 ', 5, 5 '-tetramethyl benzidine (TMB) produces color change, achieve human IgG to detect, the euzymelinked immunosorbent assay (ELISA) that the method is relatively traditional is compared, do not need to use enzyme as antibody labeling thing, there is good stability, simply, highly sensitive, selectivity is good, the advantage such as cheap, the high flux achieving Immunoglobulin IgG in human body detects.
The invention provides a kind of preparation method of heme-manganese dioxide composites, this preparation method is after being mixed with phosphate buffered solution by heme, adds ammoniacal liquor and impels heme to dissolve, then add excessive MnAc successively in mixed solution
2solution and excessive NaOH solution, stir, then after centrifugal treating, the MnAc that removing unreacted is complete
2and NaOH, to obtain final product.
The preparation method of heme-manganese dioxide composites of the present invention comprises following preferred version:
Described centrifugal treating is centrifugal 15 ~ 25min under the speed of 8000 ~ 10000rpm.
Described phosphate buffered solution pH is 7.4.
Obtained heme-manganese dioxide composites PBS buffer solution cleaning 2 ~ 3 times, stores for future use under being not more than the environment of 4 DEG C.
Present invention also offers a kind of method based on the detection human IgG of heme-manganese dioxide composites, the method first fixes goat anti-human igg at the basal surface in each hole of polystyrene 96 orifice plate, and close with the basal surface of bovine serum albumin(BSA) by each hole described; After basal surface again in each hole that described bovine serum albumin(BSA) was closed assembles the human IgG of different molar weight respectively, assemble heme-manganese dioxide composites further and modify rabbit anti-human igg; After having assembled, what in each hole, add equivalent contains H
2o
2react with the citrate buffer solution of TMB, after having reacted, measure the absorbance of oxidation product at 652nm of TMB in each hole by microplate reader, carry out colorimetric analysis.
The method of the detection human IgG based on heme-manganese dioxide composites of the present invention also comprises following preferred version:
Preferred method in each hole of polystyrene 96 orifice plate, first adds goat anti-human igg leave standstill 8 ~ 12h, removes unnecessary goat anti-human igg, then in each described hole, add the standing 2 ~ 3h of bovine serum albumin(BSA), removes unnecessary bovine serum albumin(BSA); The human IgG solution of equal-volume variable concentrations is added again respectively in each hole, incubation 0.8 ~ 1.2h at 37 DEG C, remove the residue human IgG solution in each hole, in each hole, add heme-manganese dioxide composites again modify rabbit anti-human igg, incubation 1.2 ~ 1.7h at 37 DEG C, the residue heme-manganese dioxide composites removed in each hole modifies rabbit anti-human igg; What in each hole, finally add equivalent contains H
2o
2after carrying out reaction 10 ~ 20min with the citrate buffer solution of TMB, measure the absorbance of oxidation product at 652nm of TMB in each hole by microplate reader, carry out colorimetric analysis.
Described heme-manganese dioxide composites is modified rabbit anti-human igg and is prepared by the following method, described heme-manganese dioxide composites is placed in successively chitosan solution, glutaraldehyde solution and rabbit anti-human igg reaction, centrifugal treating, to obtain final product.
Described heme-manganese dioxide composites is placed in the chitosan solution reaction 1.8 ~ 2.2h of 0.3 ~ 0.8wt% successively, centrifugal 3 ~ 7min, 0.4 ~ 0.6h, centrifugal 3 ~ 7min is reacted, in 40 ~ 60 μ g mL in the glutaraldehyde solution of 0.2 ~ 0.3wt%
-1rabbit anti-human igg in 0.8 ~ 1.2h, after centrifugal treating, centrifugal thing is distributed in phosphate buffered solution, obtains heme-manganese dioxide composites and modify rabbit anti-human igg's solution.Described phosphate buffered solution pH value is preferably pH=7.4.It is 1mg mL that described heme-manganese dioxide composites modifies rabbit anti-human igg's solution concentration
-1.
Beneficial effect of the present invention: the simple method of first passage of the present invention has synthesized heme-manganese dioxide composites (Hemin-MnO
2), find that it has peroxidase activity, can catalysis hydrogen peroxide oxidation TMB (TMB) make it become blueness from colourless; Heme-manganese dioxide composites is applied to the detection of human IgG by the present patent application further as antibody labeling thing, have higher sensitivity, with the naked eye can distinguish that its color changes.The present invention adopts traditional " sandwich " sandwich structure method, polystyrene 96 hole microtiter plate fixes goat anti-human igg, with synthesized Hemin-MnO
2compound is as rabbit anti-human igg's label, carry out the detection of human immunoglobulin IgG antigen, find along with antigen concentration increases, also there is regular change (colourless-light blue-dark blue) in the coloured product of final oxidation, the absorbance that can measure by microplate reader realizes the quantitative test of human IgG.Use Hemin-MnO
2the colorimetric immunoassay method of compound detects euzymelinked immunosorbent assay (ELISA) traditional in hinge structure, does not need to use enzyme as antibody labeling thing, has the advantages such as good stability, highly sensitive, selectivity good, reappearance is excellent, analysis speed is fast.This immuno-sensing method is simple, cheap, and naked eyes and the change of its color of observable, have potential application prospect at biotechnology, biologicall test and biomedical aspect.
Accompanying drawing explanation
The Hemin-MnO that [Fig. 1] is prepared for the embodiment of the present invention 1
2the transmission electron microscope image of compound.
The Hemin-MnO that [Fig. 2] is prepared for the embodiment of the present invention 1
2the energy dispersion X-ray spectrogram of compound.
The Hemin-MnO that [Fig. 3] is prepared for the embodiment of the present invention 1
2compound and Hemin, MnO
2with colour developing variation diagram and the corresponding absorbance-wavelength curve figure of TMB catalysis hydrogen peroxide oxidation TMB; 1 is TMB; 2 is MnO
2, 3 is Hemin, and 4 is Hemin-MnO
2compound; Colour developing variation diagram is corresponding in turn to curve 4,3,2 and 1 from top to bottom.
Hemin-MnO prepared by the embodiment of the present invention 1 that [Fig. 4] is variable concentrations
2the colour developing variation diagram of complex catalysts oxidation equivalent TMB and corresponding absorbance-wavelength curve figure; Curve 9,8,7,6 and 5 is corresponding in turn to Hemin-MnO
2complex concentration is to from high to low, and colour developing variation diagram is corresponding in turn to 9,8,7,6 and 5 from top to bottom.
[Fig. 5] is for being Hemin-MnO prepared by the embodiment of the present invention 1
2immunosensor after compound adopts double-antibody method to modify measures the canonical plotting of variable concentrations human IgG antigen gained.
Embodiment
Following examples are intended to further illustrate content of the present invention, instead of limit the scope of the invention.
Embodiment 1
Novel heme-manganese dioxide composites (Hemin-MnO
2) synthesis: by the PBS buffer solution of Hemin and the 10mL of 15mg pH=7.4 mix after, add the ammoniacal liquor of 50 μ L, stirring and dissolving, then add the MnAc of 1.6mL100mM while stirring
2solution, stirred at ambient temperature 5min, then adds the NaOH solution of 100 μ L1M, continues stirring 6 ~ 8h, finally obtains Hemin-MnO
2compound, then centrifugal 20min under 9000rpm, remove more than MnAc
2and NaOH, then with PBS buffer solution cleaning 2 ~ 3 times, finally that the centrifugal product storage obtained is for subsequent use in the refrigerator of 4 DEG C.
Heme of the present invention-manganese dioxide composites modifies rabbit anti-human igg's synthetic method: by the Hemin-MnO of synthesis
2compound is distributed in the chitosan solution of 0.5wt%, reaction 2h, centrifugal 5min; Again with glutaraldehyde dispersion 0.5h, the centrifugal 5min of 0.25wt%; Then 50 μ g mL are used
-1rabbit anti-human igg react 1h with it, centrifugal; Finally be distributed in the PBS of pH=7.4, obtained 1mg mL
-1solution, be stored in the refrigerator of 4 DEG C for subsequent use.
Heme-manganese dioxide composites of the present invention is used for the detection method of immunoassay detection to Immunoglobulin IgG: 150 μ L goat anti-human iggs (are diluted to 10 μ g mL with damping fluid
-1) add in the microtiter plate of polystyrene 96 hole, at 4 DEG C, hatch a whole night, antibody is fixed in hole.Loose goat anti-human igg's solution rinses three times with 300 μ L lavation buffer solutions and washes away.Then at 37 DEG C, add 250 μ L Block buffer, incubation 2.5h, reduce the non-specific adsorption of human IgG, excessive BSA then rinses three times with the lavation buffer solution of 300 μ L.IgG solution (0,1,2,5,10,50,100,500, the 1000pg mL of variable concentrations is prepared respectively with the PBS solution of pH=7.4
-1), then respectively by 150 μ L join variable concentrations human IgG solution add in hand-hole, hatch 1h, then rinse three times.Add 150 μ LHemin-MnO subsequently
2modify rabbit anti-human igg, hatch 1.5h.Four times are being rinsed with the lavation buffer solution of 300 μ L.Finally, add the 2.5mM TMB that 150 μ L prepare containing the citrate buffer solution of 4.3mM hydrogen peroxide in each hole, reaction 15min, then measure the absorbance of TMB oxidation product by microplate reader, obtained typical curve as shown in Figure 5.
Embodiment 2
Heme-manganese dioxide composites is utilized to be used for the mensuration of clinical immunoassays detection to the human IgG recovery in human serum: by the PBS damping fluid stepwise dilution of human serum sample pH=7.4 to 10
-10doubly, the 0pg mL of variable concentrations is then added
-1, 10pg mL
-1, 50pg mL
-1, 100pg mL
-1and 500pgmL
-1human IgG solution.To add 0pg mL
-1the pure serum solution of human IgG is blank reference solution, the absorbance of TMB oxidation product is measured according to the concrete operations of embodiment 1, the obtained recovery is as table 1, and the human IgG recovery is high as can be seen from Table 1, can prove feasibility and the accuracy of the detection method of embodiment 1.
Mensuration (the pg mL of the human IgG recovery in table 1. human serum
-1)
。
Claims (6)
1. detect the method for human IgG based on heme-manganese dioxide composites, it is characterized in that, first fix goat anti-human igg at the basal surface in each hole of polystyrene 96 orifice plate, and close with the basal surface of bovine serum albumin(BSA) by each hole described; After basal surface again in each hole that described bovine serum albumin(BSA) was closed assembles the human IgG of different molar weight respectively, assemble heme-manganese dioxide composites further and modify rabbit anti-human igg; After having assembled, what in each hole, add equivalent contains H
2o
2react with the citrate buffer solution of TMB, after having reacted, measure the absorbance of oxidation product at 652nm of TMB in each hole by microplate reader, carry out colorimetric analysis;
Described heme-manganese dioxide composites is obtained by following preparation method: after being mixed with phosphate buffered solution by heme, adds ammoniacal liquor and impels heme to dissolve, then add excessive MnAc successively in mixed solution
2solution and excessive NaOH solution, stir, then after centrifugal treating, the MnAc that removing unreacted is complete
2and NaOH, to obtain final product.
2. the method for claim 1, is characterized in that, described centrifugal treating is centrifugal 15 ~ 25min under the speed of 8000 ~ 10000rpm.
3. the method for claim 1, is characterized in that, described phosphate buffered solution pH is 7.4.
4. the method for claim 1, it is characterized in that, in each hole of polystyrene 96 orifice plate, first add goat anti-human igg leave standstill 8 ~ 12h, remove unnecessary goat anti-human igg, in each described hole, add bovine serum albumin(BSA) again leave standstill 2 ~ 3h, remove unnecessary bovine serum albumin(BSA); The human IgG solution of equal-volume variable concentrations is added again respectively in each hole, incubation 0.8 ~ 1.2h at 37 DEG C, remove the residue human IgG solution in each hole, in each hole, add heme-manganese dioxide composites again modify rabbit anti-human igg, incubation 1.2 ~ 1.7h at 37 DEG C, the residue heme-manganese dioxide composites removed in each hole modifies rabbit anti-human igg; What in each hole, finally add equivalent contains H
2o
2after carrying out reaction 10 ~ 20min with the citrate buffer solution of TMB, measure the absorbance of oxidation product at 652nm of TMB in each hole by microplate reader, carry out colorimetric analysis.
5. the method for claim 1, it is characterized in that, described heme-manganese dioxide composites is modified rabbit anti-human igg and is prepared by the following method, described heme-manganese dioxide composites is placed in successively chitosan solution, glutaraldehyde solution and rabbit anti-human igg reaction, centrifugal treating, to obtain final product.
6. the method for claim 1, it is characterized in that, described heme-manganese dioxide composites is placed in the chitosan solution reaction 1.8 ~ 2.2h of 0.3 ~ 0.8wt% successively, centrifugal 3 ~ 7min, 0.4 ~ 0.6h is reacted in the glutaraldehyde solution of 0.2 ~ 0.3wt%, centrifugal 3 ~ 7min, in 40 ~ 60 μ g mL
-1rabbit anti-human igg in 0.8 ~ 1.2h, after centrifugal treating, centrifugal thing is distributed in phosphate buffered solution, obtains heme-manganese dioxide composites and modify rabbit anti-human igg's solution.
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