CN1811384A - Biomolecule interaction analyzing method based on solid phase surface intensified light scattering - Google Patents
Biomolecule interaction analyzing method based on solid phase surface intensified light scattering Download PDFInfo
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- CN1811384A CN1811384A CN 200610054066 CN200610054066A CN1811384A CN 1811384 A CN1811384 A CN 1811384A CN 200610054066 CN200610054066 CN 200610054066 CN 200610054066 A CN200610054066 A CN 200610054066A CN 1811384 A CN1811384 A CN 1811384A
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Abstract
The present invention relates to an analysis method capable of utilizing interaction produced by bioprobe molecule and biotarget molecule on the slide surface to intensify light-scattering signal to implement qualitative and quantitative determinations and intermolecular interaction character analysis. Said method includes the following processes: pretreatment of concave slide, fixation of probe molecule, combination of bioprobe and biotarget molecule, adopting synchronous scanning mode on the general spectrofluorometer to obtain scattering spectrum, utilizing said scattering spectrum to make qualitative and quantitative determinations of biotarget molecule and analysis of intermolecular intraction character and regeneration of bioprobe.
Description
Technical field
The invention belongs to light analysis technology, be specifically related to biomolecule and cause the surface light scattered signal to strengthen and carry out the qualitative and quantitative measurement of biomolecule and the analytical approach of bio-molecular interaction specificity analysis, can be widely used in specific transactional analysis between the quantitative measurement of high sensitivity, high selectivity of protein, nucleic acid, enzyme, cell and bacterium, biochemical drug etc. and protein-protein (as antigen and antibody, to body and acceptor, protein and enzyme etc.) DNA and DNA, DNA and the protein (comprising enzyme) etc. in the specific gathering of solid phase surface.
Background technology
Light scattering phenomenon extensively is present in the mechanism of light and particle.Existing result of study shows that identification, assembling and the gathering of biomacromolecule cause strong resonant light scattering signal (RLS) to strengthen, and the signal enhancing is directly proportional with the concentration of aggregation system (scattering particle).In recent years, scattering technology has been applied to the analysis of biomacromolecule, biochemical drug, surfactant, nano particle and inorganic metal ion.Yet, be based upon the scattering analysis technology on the solution body basis, though highly sensitive, the instrument of use is simple, and is easy and simple to handle, has fatal defective.1, selectivity is bad, and antijamming capability is relatively poor.At bulk solution, all can all can bring big interference to the measured object analysis to the material that light produces scattering.2, the scattering technology that adopts at present all is to measure in aqueous solution, then can not carry out for the analysis that need carry out in oil-soluble reagent, thereby make the scope of its analysis be subjected to certain limitation.3, many owing to interference component in bulk solution, for the analysis of carrying out bio-molecular interaction bigger difficulty is arranged also.
For similar surface plasma analysis of the present invention (SPR) technology, though it also utilizes between the biomolecule specificity to interact and carries out assay determination, but it need be fixed on biomolecule special gold surface, and utilize be total internal reflection the time evanescent wave influence, carry out assay determination by the variation of measuring reflection angle and energy of reflection light.The principle that it adopts and the present invention are inequality fully, and need expensive professional instrument and equipment (SPR instrument) to finish.
Summary of the invention
Of the present invention at the prior art above shortcomings, a kind of biomolecule interaction analyzing method based on solid phase surface intensified light scattering is proposed, scattering technology is incorporated into solid phase surface, and combine with the bio-molecular interaction of high selectivity, obtain a kind of new light analytical approach thereby make up, fully inherit the advantage of scattering technology, overcome the limitation that it only can be used and selectivity is low in bulk solution, for the assay of biomolecule and the sign of bio-molecular interaction mechanism provide a kind of easy, quick, sensitive technology platform.
Technical scheme of the present invention is as follows:
Biomolecule interaction analyzing method based on solid phase surface intensified light scattering, total method is earlier the bioprobe molecule to be fixed on surface of glass slide, utilize probe and bio-target molecule to interact again in surface of glass slide, thereby cause that light scattering signal strengthens, and then implement the qualitative and quantitative analysis of bio-target molecule is measured and intermolecular interaction characteristic (as affinity characteristic, dynamics etc.) analysis.Specifically may further comprise the steps:
1, elder generation is according to the characteristic of different biological molecules probe, select the concave slide (as common slide, quartzy slide etc.) of suitable material, use appropriate chemical modifier (as amino silane reagent, hydrosulphonyl silane reagent, glutaraldehyde etc.), carry out finishing and handle.
2, by covalent bonding mode stationary probe molecule: can adopt respectively in the practical operation that physisorption, chemical bonding, physisorption combine with chemical bonding, bifunctional reagent is crosslinked, nano material (as golden nanometer particle), special adhesion protein (as G albumen, A albumen etc.) etc. carry out probe biomolecule fixing on surface of glass slide.
3, utilize the specificity interaction between the biomolecule that bioprobe is combined with bio-target molecule.
4, on common fluorophotometer, adopt the synchronous scanning mode to obtain scattering spectrum, and change the qualitative and quantitative measurement of implementing bio-target molecule by spectral intensity before and after the biomolecule action, and the analysis of intermolecular interaction characteristic.When adopting common fluorophotometer to detect, incident light and slide angle at 45 implement the detection of scattered signal in 90 ° of angular direction of incident light, and employing excites the synchronous scanning pattern of identical with emission wavelength (being Δ λ=0) to carry out.
5, bioprobe is regenerated, make probe can continue on for the detection of target molecule.
Advantage of the present invention and good effect:
1, the present invention is incorporated into solid phase surface with scattering technology, has overcome scattering technology uses suffered coexisting substances in bulk solution interference, the sensitivity for analysis height, and improved the selectivity of analytical approach greatly.
2, overcome the assay determination that scattering technology can only apply to the material of water as solvent, expanded the utilization scope of scattering technology.
2, the present invention can not only carry out the qualitative and quantitative measurement to bio-target molecule, and can also carry out biomolecule interphase interaction characteristic (as compatibility, dynamics) analysis.
3, the technology of the present invention only needs common instrument, and instrument is simple, and analysis cost is cheap.
Description of drawings:
Fig. 1: the fixed sturcture synoptic diagram that is slide.
Embodiment
Below be that example describes implementation procedure of the present invention in detail to adopt analytical approach of the present invention to antigen-antibody analysis:
(1) select common concave slide for use, its specification is 25mm * 70mm, and the diameter of concave surface is 10mm;
(2) after slide is fully cleaned through concentrated sodium hydroxide, the concentrated sulphuric acid, distilled water, with aminopropyl trimethoxysilane reagent the slide concave surface is carried out silanization and handle;
(3) with 5% glutaraldehyde as coupling agent,----goat anti-human igg (antibody) is fixed on the slide concave surface with probe biomolecule;
(4) splash into bio-target molecule----human IgG (antigen) solution at the slide concave surface, target molecule is adsorbed on surface of glass slide by specific reaction between probe and the target molecule (being the specific reaction of antigen-antibody);
(5) slide 1 is put into pedestal 2, as shown in Figure 1, insert again in the common fluorophotometer, adopt the synchronous scanning mode to scan and obtain scattering spectrum, and pass through qualitative, the quantitative measurement of the variation enforcement of biomolecule action front and back spectral intensity, and the analysis of intermolecular interaction characteristic to bio-target molecule;
(6) the urea solution wash-out that adds 0.1mol/l at the slide concave surface is adsorbed on the target molecule on the probe, thus the regeneration probe, so that it carries out the detection of target molecule again.
Experimental result shows, adopts analytical approach of the present invention, fixedly goat anti-human igg's (antibody) to human IgG (antigen) when target molecule is measured, the related coefficient of analysis can reach 0.993 as probe, minimal detectable concentration can reach 10ng/ml.
Claims (4)
1, based on the biomolecule interaction analyzing method of solid phase surface intensified light scattering, may further comprise the steps
(1) earlier according to the characteristic of different biological molecules probe, concave slide is carried out pre-service;
(2) stationary probe molecule;
(3) utilize the specificity interaction between the biomolecule that bioprobe is combined with bio-target molecule;
(4) on common fluorophotometer, adopt the synchronous scanning mode to obtain scattering spectrum, and do by biomolecule
Implement qualitative, the quantitative measurement to bio-target molecule and the analysis of intermolecular interaction characteristic with the variation of front and back spectral intensity.
(5) bioprobe is regenerated, make probe can continue on for the detection of target molecule.
2, method according to claim 1 is characterized in that: the pre-service in the step (1) is to adopt chemical modifiers such as amino silane reagent, hydrosulphonyl silane reagent and glutaraldehyde that slide is carried out finishing to handle.
3, method according to claim 1, it is characterized in that: step (2) is that mode has by covalent bonding mode stationary probe molecule: physisorption, chemical bonding, physisorption combine with chemical bonding, bifunctional reagent is crosslinked, nano material or special adhesion protein combination.
4, method according to claim 1, it is characterized in that: when adopting common fluorospectrophotometer to detect in the step (4), incident light and slide angle at 45, implement the detection of scattered signal in 90 ° of angular direction of incident light, and employing excites the synchronous scanning pattern identical with emission wavelength to carry out.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100540664A CN100491976C (en) | 2006-01-28 | 2006-01-28 | Biomolecule interaction analyzing method based on solid phase surface intensified light scattering |
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2006100540664A CN100491976C (en) | 2006-01-28 | 2006-01-28 | Biomolecule interaction analyzing method based on solid phase surface intensified light scattering |
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| Publication Number | Publication Date |
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| CN1811384A true CN1811384A (en) | 2006-08-02 |
| CN100491976C CN100491976C (en) | 2009-05-27 |
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| CNB2006100540664A Expired - Fee Related CN100491976C (en) | 2006-01-28 | 2006-01-28 | Biomolecule interaction analyzing method based on solid phase surface intensified light scattering |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101178360B (en) * | 2007-09-24 | 2010-12-15 | 重庆医科大学 | Homogeneous phase affinity analyzing novel methods based on integrating sphere reinforced light scattering testing technology |
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2006
- 2006-01-28 CN CNB2006100540664A patent/CN100491976C/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101178360B (en) * | 2007-09-24 | 2010-12-15 | 重庆医科大学 | Homogeneous phase affinity analyzing novel methods based on integrating sphere reinforced light scattering testing technology |
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| CN100491976C (en) | 2009-05-27 |
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Granted publication date: 20090527 Termination date: 20120128 |