CN102661927A - Biosensing method for composite nano-composite material quantitative detecting virus and enzyme based on surface-active agent decorated connecting utricle glycolipin - Google Patents
Biosensing method for composite nano-composite material quantitative detecting virus and enzyme based on surface-active agent decorated connecting utricle glycolipin Download PDFInfo
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- CN102661927A CN102661927A CN201210116143XA CN201210116143A CN102661927A CN 102661927 A CN102661927 A CN 102661927A CN 201210116143X A CN201210116143X A CN 201210116143XA CN 201210116143 A CN201210116143 A CN 201210116143A CN 102661927 A CN102661927 A CN 102661927A
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Abstract
The invention discloses a biosensing method for quantitatively analyzing cholera enterotoxin. A gold nanoparticle probe decorated by raman dye and wrapped with a phosphatide bilayer membrane is prepared, functionalized phosphatide (namely phosphatide with a hydrosulfuryl decorated head end) is selected, and the functionalized phosphatide is in covalent bonding to the surfaces of gold nanoparticles to improve the stability of the gold nanoparticle probe. Cholera enterotoxin B subunit (CTB) and a plurality of acceptor GM1 molecules are in specific binding, which causes the gold nanoparticles wrapped with the GM 1 molecules to reunite, surface plasmas cause a coupling effect, and high-sensitive SERS (Surface Enhanced Raman Scattering) detection is achieved. Meanwhile, the method can cause the change of optical absorption property, and the detection also can be performed through colorimetry. The operating step is simple and fast, any marks to substrate is not required, the cost is low, the sensitivity is high, the specificity is excellent, the method is expected to be a useful technological means for detecting and identifying the cholera enterotoxin, and is significant to food safety monitoring and control as well as prevention of foodborne diseases.
Description
Technical field
The invention belongs to a kind of bioassay technique of reuniting based on the nm of gold of surfactant modified connecting utricle glycolipid, it is specifically related to the method for modifying based on surfactant modified nanogold particle connecting utricle glycolipid quasi-molecule; The idiosyncrasy of glycosyl sphingolipid class decorated nanometer gold grain and correlated virus and enzyme; Idiosyncrasy causes nanogold particle reunion reaction, the variation that produces the UV, visible light absorbance, and quantitative test detects correlated virus and enzyme, and the application in actual analysis.
Background technology
The fast detecting of virus and enzyme is extremely important for fields such as biological study, drug screenings.Detection technique commonly used at present is based on the detection of accessory substance in virus or the enzymic catalytic reaction and directly detects modified outcome, and detection method mainly contains the fluorescence labeling tracer analysis, immuning adsorpting analysis, PCR method and mass spectroscopy etc.Not high, the poor reliability of these detection technique sensitivity, and need the instrument of large-scale precision, can not satisfy the demand of accurate fast detecting.
Summary of the invention
The technical matters that the present invention will solve is that the deficiency to prior art exists has proposed a kind of based on the composite nano materials detection by quantitative virus of surfactant modified connecting utricle glycolipid and the bio-sensing method of enzyme.The method can directly act on the glycosyl sphingolipid class nm of gold surface of modification of surfaces activating agent, with the idiosyncrasy of its correlated virus and enzyme after, the variation through UV, visible light absorbing light intensity realizes detecting.The method is simple to operate, quick, highly versatile, and is highly sensitive.
Technical scheme of the present invention is; The glycosyl sphingolipid quasi-molecule is directly acted on the nm of gold surface of modification of surfaces activating agent; Utilize a plurality of monomers in the viruses molecule to combine with the nanogold particle generation specificity of a plurality of finishing glycosyl sphingolipid quasi-molecules simultaneously; Cause nm of gold reunion reaction, can cause that the variation of ultra-violet absorption spectrum is used for its correlated virus of quantitative test detection; And when enzyme acts on the glycosyl fragment of glycosyl sphingolipid quasi-molecule; Can make the fragment fracture, the monomer of virus then can not be had an effect with the glycosyl sphingolipid quasi-molecule again, causes the variation of corresponding ultra-violet absorption spectrum equally; Quantitative test detects its relevant enzyme; That is, do the time spent, cause the reaction of reuniting when nanogold particle and its correlated virus of finishing glycosyl sphingolipid quasi-molecule; And when adding relevant enzyme, then blocked the effect of virus with the glycosyl sphingolipid quasi-molecule, nm of gold can not be reunited.This moment, reacting final product can both produce corresponding UV, visible light absorbance variation, but quantitative test detects correlated virus and enzyme.
Said based on the composite nano materials detection by quantitative virus of surfactant modified connecting utricle glycolipid and the bio-sensing method of enzyme, comprise the steps:
(1) glycosyl sphingolipid quasi-molecule decorated nanometer gold grain;
(2) idiosyncrasy of glycosyl sphingolipid class decorated nanometer gold grain and its correlated virus and enzyme;
(3) idiosyncrasy causes nanogold particle reunion reaction, the variation that produces the UV, visible light absorbance, and quantitative test detects its correlated virus and enzyme.
Wherein, described nanogold particle is the nanogold particle that is modified with surfactant;
Wherein, correlated virus and enzyme are meant the virus and the enzyme (as: cholera toxin, beta-glycosidase etc.) that can combine with the nanogold particle specificity that is modified with the glycosyl sphingolipid quasi-molecule.
Below the present invention made further specify.
1, glycosyl sphingolipid quasi-molecule decorated nanometer gold grain
Freshly prepared 0.1M NaBH in a, the ice-water bath
4Under stirring, join 4%HAuCl
4In 0.01M sodium citrate potpourri, it is subsequent use to be prepared into seed liquor.4%HAuCl
4Add surfactant in the solution, stirring heating is down processed growth-promoting media.Seed liquor is joined in the growth-promoting media, mix, be prepared into the nano-Au solution of the finishing surfactant of suitable dimension.With synthetic good nano-Au solution and PB (50mM; PH=7.4) mix after, in the hypervelocity refrigerated centrifuge, concentrate (4 ℃ of centrifuge washings three times; 10000r/min; 15min).
B, glycosyl sphingolipid class solution is joined in a liquid, continue to stir (25 ℃; 120r/min), make the glycosyl sphingolipid quasi-molecule directly modify (lucifuge, subsequent use) on the nanogold particle.
2, the idiosyncrasy of glycosyl sphingolipid quasi-molecule decorated nanometer gold grain and its correlated virus and enzyme
A, nano-Au solution that the glycosyl sphingolipid quasi-molecule is modified and react buffer and mix, (4 ℃ of twice of centrifuge washings; 10000r/min; 15min), add its correlated virus and react 10min down in 37 ℃ of waters bath with thermostatic control.
B, glycosyl sphingolipid quasi-molecule decorated nanometer gold solution is mixed (4 ℃ of twice of centrifuge washings with reaction buffer; 10000r/min; 15min), add relevant enzyme and react 10min down in 37 ℃ of waters bath with thermostatic control.Afterwards, add correlated virus again and react 10min down in 37 ℃ of waters bath with thermostatic control.
3, idiosyncrasy causes nanogold particle reunion reaction, the variation that produces the UV, visible light absorbance, and quantitative test detects correlated virus and enzyme
The quantitative test of a, virus detects step
With glycosyl sphingolipid quasi-molecule decorated nanometer gold solution and correlated virus reaction, final product (60 μ L) is transferred in the micro-quartz colorimetric utensil, puts into ultraviolet spectrometer (UVS), and monitoring 400nm is to interior its uv absorption intensity of 800nm wavelength coverage.
The quantitative test of b, enzyme detects step
After glycosyl sphingolipid quasi-molecule decorated nanometer gold solution and relevant enzyme reaction, add the correlated virus reaction again, final product (60 μ L) is transferred in the micro-quartz colorimetric utensil, puts into ultraviolet spectrometer (UVS), and monitoring 400nm is to interior its uv absorption intensity of 800nm wavelength coverage.
Embodiment
Embodiment 1: the bio-sensing method of the composite nano materials detection by quantitative cholera toxin of modifying based on gangliosides (GM1) (CTB)
1) based on gangliosides (GM1) decorated nanometer gold grain method:
Freshly prepared 60uL 0.1MNaBH in a, the ice-water bath
4Under stirring, join 2mL4%HAuCl
4In 0.01M sodium citrate potpourri, it is subsequent use to be prepared into seed liquor.4%HAuCl
4Add cetyl trimethyl ammonium bromide (CTAB) in the solution, stirring heating is down processed growth-promoting media.Seed liquor is joined in the growth-promoting media, mix, be prepared into a footpath and be about the nano-Au solution about 10nm.With synthetic good nano-Au solution and PB (50mM; PH=7.4) mix after, concentrate (4 ℃ of centrifuge washings three times; 10000r/min; 15min).
B, 10ul 0.64mM GM1 is joined in the nano-Au solution that CTAB modifies, continue to stir (25 ℃; 120r), GM1 is directly modified on the nanogold particle.
2) the idiosyncrasy step of GM1 decorated nanometer gold grain and cholera toxin (CTB):
The nano-Au solution of modifying at 20ul GM1 adds variable concentrations CTB (60pM～220nM), react 10min down in 37 ℃ of waters bath with thermostatic control.
3) idiosyncrasy causes nanogold particle reunion reaction, the variation that produces the UV, visible light absorbance, and quantitative test detects the step of cholera toxin (CTB):
With 20ul gangliosides (GM1) decorated nanometer gold solution and 5ul variable concentrations cholera toxin (CTB) (60pM～220nM) react final product (ultrapure water constant volume 60 μ L) to transfer in the micro-quartz colorimetric utensil; Put into ultraviolet spectrometer (UVS), monitoring 400nm is to interior its uv absorption intensity of 800nm wavelength coverage.
Embodiment 2: modify the composite nano materials detection by quantitative beta-glycosidase (bio-sensing method of β-galactosidase) based on gangliosides (GM1)
1) based on gangliosides (GM1) decorated nanometer gold grain method:
Freshly prepared 60uL 0.1MNaBH in a, the ice-water bath
4Under stirring, join 2mL4%HAuCl
4In 0.01M sodium citrate potpourri, it is subsequent use to be prepared into seed liquor.4%HAuCl
4Add cetyl trimethyl ammonium bromide (CTAB) in the solution, stirring heating is down processed growth-promoting media.Seed liquor is joined in the growth-promoting media, mix, be prepared into a footpath and be about the nano-Au solution about 10nm.With synthetic good nano-Au solution and PB (50mM; PH=7.4) mix after, concentrate (4 ℃ of centrifuge washings three times; 10000r/min; 15min).
B, 10ul 0.64mM GM1 is joined in the nano-Au solution that CTAB modifies, continue to stir (25 ℃; 120r), GM1 is directly modified on the nanogold particle.
2) the idiosyncrasy step of GM1 decorated nanometer gold grain and beta-glycosidase and cholera toxin (CTB):
A, ((1U～510U) is in 37 ℃ of waters bath with thermostatic control reaction 10min down for β-galactosidase) to add different activity beta-glycosidases in the nano-Au solution of modifying at 20ul GM1.
B, add 10ul cholera toxin (CTB) in the above-mentioned a liquid after reaction is accomplished again, continue at and react 10min 37 ℃ of waters bath with thermostatic control under.
3) idiosyncrasy causes the nanogold particle reaction of reuniting, the variation that produces the UV, visible light absorbance, quantitative test detect beta-glycosidase (step of β-galactosidase):
(β-galactosidase) is (after the reaction of 1U～510U) with the different activity beta-glycosidases with 5ul of 20ul gangliosides (GM1) decorated nanometer gold solution; React with 10ul CTB again; Its final product (ultrapure water constant volume 60 μ L) is transferred in the micro-quartz colorimetric utensil; Put into ultraviolet spectrometer (UVS), monitoring 400nm is to interior its uv absorption intensity of 800nm wavelength coverage.
Claims (4)
1. bio-sensing method based on the cholera toxin of the nanoparticle agglomerates of phospholipid bilayer film parcel may further comprise the steps:
(1) the gold nano grain probe of preparation phospholipid bilayer film parcel, wherein, the phospholipid bilayer film is by GM
1Molecule and phospholipid molecule form jointly;
(2) CTB and acceptor GM
1The specificity association reaction;
(3) detection of product solution.
2. bio-sensing method according to claim 1 is characterized in that, comprises the phosphatide that head end is modified sulfydryl in the composition of the described phospholipid bilayer film of step (1).
3. bio-sensing method according to claim 1 is characterized in that, the described detection method of step (3) is to utilize UV, visible light light absorption analysis technology that product solution is detected.
4. bio-sensing method according to claim 1 is characterized in that, the described nanoparticle probes of step (1) is the nanoparticle probes that is modified with the Raman dyestuff; The described detection method of step (3) is to utilize the SERS analytical technology that product solution is detected.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102608311A (en) * | 2012-04-19 | 2012-07-25 | 湖南大学 | Biological sensing method for cholera toxin based on agglomeration of phospholipid bilayer membrane covered nanoparticles |
| CN103063597A (en) * | 2013-01-09 | 2013-04-24 | 南京工业大学 | Method for detecting lipase activity |
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| EA000409B1 (en) * | 1995-02-13 | 1999-06-24 | Дзе Регентс Оф Дзе Юниверсити Оф Калифорния | Three-dimensional colorimetric assay assemblies |
| CN101718708A (en) * | 2009-11-25 | 2010-06-02 | 福州大学 | Method for quickly detecting melamine in milk sample based on nanogold |
| CN101858881A (en) * | 2010-06-28 | 2010-10-13 | 华中科技大学 | Sensor for detecting penicillin in liquid |
| US7959861B2 (en) * | 2007-09-19 | 2011-06-14 | Stc.Unm | Integrated affinity microcolumns and affinity capillary electrophoresis |
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| EA000409B1 (en) * | 1995-02-13 | 1999-06-24 | Дзе Регентс Оф Дзе Юниверсити Оф Калифорния | Three-dimensional colorimetric assay assemblies |
| US7959861B2 (en) * | 2007-09-19 | 2011-06-14 | Stc.Unm | Integrated affinity microcolumns and affinity capillary electrophoresis |
| CN101718708A (en) * | 2009-11-25 | 2010-06-02 | 福州大学 | Method for quickly detecting melamine in milk sample based on nanogold |
| CN101858881A (en) * | 2010-06-28 | 2010-10-13 | 华中科技大学 | Sensor for detecting penicillin in liquid |
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| CLAIRE L. SCHOFIELD, ET. AL.: "Glyconanoparticles for the Colorimetric Detection of Cholera Toxin", 《ANAL. CHEM.》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102608311A (en) * | 2012-04-19 | 2012-07-25 | 湖南大学 | Biological sensing method for cholera toxin based on agglomeration of phospholipid bilayer membrane covered nanoparticles |
| CN103063597A (en) * | 2013-01-09 | 2013-04-24 | 南京工业大学 | Method for detecting lipase activity |
| CN103063597B (en) * | 2013-01-09 | 2014-12-31 | 南京工业大学 | Method for detecting lipase activity |
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Application publication date: 20120912 |