CN1598538A - Optical surface plasma wave nucleic acid sensor system for detecting mononucleotide polymorphism and detecting method - Google Patents
Optical surface plasma wave nucleic acid sensor system for detecting mononucleotide polymorphism and detecting method Download PDFInfo
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- CN1598538A CN1598538A CNA2004100415237A CN200410041523A CN1598538A CN 1598538 A CN1598538 A CN 1598538A CN A2004100415237 A CNA2004100415237 A CN A2004100415237A CN 200410041523 A CN200410041523 A CN 200410041523A CN 1598538 A CN1598538 A CN 1598538A
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Abstract
The invention relates to an optical fiber surface plasma wave nucleic acid sensor and method for detecting polymorphism of nucleotide. The system includes wideband lamp house, P type Polaroid, gamma type optical fiber coupler, coupler interface, sensing optical fiber, detector of terminal reflective optical fiber surface plasma wave resonance biology sensor, spectrum device and computer. The invention uses terminal reflective optical fiber surface plasma wave nucleic sensor detector and distributed optical fiber surface plasma wave nucleic sensor detector to realize the detection of polymorphism of nucleotide. Compared with spectrum detection method, the accurate value of resonance spectrum wavelength of the surface plasma wave can be acquired. The invention needs not to be signed, and it has no damage to the sample.
Description
Technical field:
The present invention relates to a kind of novel optical fiber nucleic acid sensor system that surface plasma wave resonance effects and optical fiber technology are combined, it can be applicable to human genome research single nucleotide polymorphism (SNP).
Background technology:
Surface plasma wave resonance (hereinafter to be referred as SPR) effect is a kind of physical optics phenomenon that occurs on metal and the dielectric interface, and it is to very responsive attached to the dielectric variations in refractive index in metal surface.Biosensor technique based on this effect has been widely used in a plurality of Biochemical Research fields such as immunology, drug screening.
Along with finishing of the accurate sequence chart of human genome, the emphasis of human genome research enters the genome times afterwards comprehensively, one of its main contents are exactly to set up with single nucleotide polymorphism (single nucleotidepolymorphism, SNP) be the DNA (deoxyribonucleic acid) of representative (deoxyribonucleic acid, DNA) system directory of sequence variations.China is making up and is studying Chinese nation's genome single nucleotide polymorphism (SNP) system directory.
Human genome is made up of 3,000,000,000 mononucleotides.Modal variant form is exactly single nucleotide polymorphism (hereinafter to be referred as SNP) in human genome DNA's sequence, and promptly different bases appears in some certain location on its dna sequence dna.SNP is distributed widely in stable polymorphic site in the human full genome, has represented hereditary difference maximum between the Different Individual.SNP gene frequency between different crowd can have sizable difference, some SNP even also present colony's selectivity.The research of SNP and sudden change thereof has been become the research focus of current life science.
At present, the method in the research SNP that is most widely used, discovery mutational site mainly is the nucleotide sequencing that relies on gene, i.e. Ce Xu method.The target gene group is obtained amplified production with polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) method, to the product analysis order-checking again with public database in sequence alignment.Sequencing can only be found a small amount of SNP, and low-frequency SNP often can not find.Along with the development of molecular biology experiment technology, there are many new technologies to be applied to SNP research.With the molecular hyridization basis a lot of relevant technology of having derived, as the application of locus specificity probe, Tagman experiment, molecular beacon (molecularbeacons) technology, Minisequencing or the like.But because these methods need to read fluorescence labeling and special analytical instrument, the feasibility of finding and screening SNP on a large scale is relatively poor.The method of some other detection SNP also can only be found the zone at SNP place, but can not determine concrete location and the feature of SNP.
Because the SPR determination techniques belongs to a kind of characteristic of spike biomolecule from the teeth outwards and interactional method, have in real time, need not mark, the ability of in-situ tracking dynamics surface process, to advantages such as the own not damageds of sample, and can obtain the dynamics data that many additive methods are difficult to obtain.Over the past two years, abroad some seminar began to attempt to use the SPR technology and studied SNP.
U.S. Wilson showed literary composition " Tech Sight.Analyzingbiomolecular interactions " in 2002 at Science, utilized the spr sensor that has probe molecule to detect SNP.U.S. Oldenburg equals to deliver research report at Anal Biochem (analytical biochemistry) in October, 2002, with the SNP of SPR method and microarray hybridization technology for detection human breast carcinoma gene BRCA1.Germany Behrensdorf equals to deliver research report at Nucleic Acids Res (nucleic acids research) in 2002, preparation dna probe chip, with the hybridization of SPR method by fluorescence labeling probe successfully fast detecting the single base mutation site of the different extrons of p53 gene (studying maximum tumor suppressor genes at present), be polymorphism, be suitable for the extensive examination of SNP.Japan Nakatan equals calendar year 2001 and delivers research report at Nat Biotechnol (Nature Biotechnol), detects the G-G single base mismatch of HSP70-2 gene with the SPR method.
The domestic and international at present spr sensor of researching and developing that is based on prism structure mostly.As nineteen eighty-three, the Experiment of Applied Physics chamber Liedberg of the Sweden LINKOPING Institute of Technology etc. at first uses it for the interactional detection of specificity between IgG and its antigen-antibody, and develops spr sensor by BIAcore company.After this research of spr sensor and improvement develops rapidly, spr sensor and be that the commodity spr sensor of representative is also increasingly extensive in bio-medical applications with BIACORE.At home, also there are many units that the prism surface plasma wave sensor is studied.As CAS Electronics Research Institute, Changchun applied chemistry research institute, zooscopy in one's power Tsing-Hua University etc. all this sensor is studied in the application of aspects such as biological, chemistry and biochemistry detection, and obtained certain achievement.
Along with deepening continuously of research work, it is bigger also to show the transducing part volume based on the Kretschmann model of prism-coupled, is not suitable for the shortcoming that long-haul telemetry occasion and spr signal are vulnerable to extraneous factors influences such as physical construction, light source fluctuation.For addressing these problems, R.C.Jorgenson of Washington, DC university in 1993 and Yee propose fiber core as the carrier that excites the SPR effect, adopt golden increasing as metallic diaphragm to overlay on the fibre core surface and developed optical fiber surface plasmon resonance sensors.We are carrying out on the basis of big quantity research this novel optical fiber sensing arrangement, adopt special coating process to increase on optical fiber probe metallic film top layer again and are covered with fixedly film of one deck SNP specific probe, make the Optical Fiber Surface Plasmon nucleic acid sensor.
Summary of the invention:
The method in the research SNP that is most widely used at present, discovery mutational site mainly is the nucleotide sequencing that relies on gene, i.e. Ce Xu method.Because these methods all need fluorescence labeling and special analytical instrument, the feasibility of finding and screening SNP on a large scale is relatively poor, and can only find the zone at SNP place, and can't determine concrete location and the feature of SNP, the objective of the invention is to overcome above shortcoming.Simultaneously owing to adopted fiber core to excite matrix and transducing part as the SPR effect, therefore its transducing part volume that can avoid the SPR sensor-based system of prism structure to show again is bigger, is not suitable for the shortcoming that long-haul telemetry occasion and spr signal are vulnerable to extraneous factors influences such as physical construction, light source fluctuation.
The Optical Fiber Surface Plasmon nucleic acid sensor system that the present invention detects single nucleotide polymorphism comprises: wideband light source links to each other with the input end of y-type optical fiber coupling mechanism through P type polaroid, the output terminal of y-type optical fiber coupling mechanism is connected in sensor fibre by the coupling mechanism joint, this optical fiber connector can be an end reflection formula Optical Fiber Surface Plasmon nucleic acid sensor probe, or distribution type fiber-optic surface plasma wave nucleic acid sensor probe, the reflection end of y-type optical fiber coupling mechanism is connected in the spectrometer that links to each other with computing machine.
End reflection formula Optical Fiber Surface Plasmon nucleic acid sensor probe comprises fibre core, is positioned at the micro-reflector of fiber cores end face, the metal level on fiber cores surface, and the SNP specific probe that is coated in layer on surface of metal is film fixedly.
The composition of distribution type fiber-optic surface plasma wave nucleic acid sensor is: can prepare a plurality of online reflection type optical fiber surface plasma wave nucleic acid sensor probes on same optical fiber, the end of this optical fiber is set to end reflection formula Optical Fiber Surface Plasmon nucleic acid sensor probe.
The present invention adopts the relative spectral detection method, when measuring, at first the input spectrum signal is preserved as reference signal, then Optical Fiber Surface Plasmon nucleic acid sensor probe is placed the sample to be tested test solution, obtain the surface plasma wave resonance spectrum signal of reflected back, again this spectral signal and reference signal are compared, thus the exact value of acquisition surface plasma wave resonance spectrum resonant wavelength.
The invention has the beneficial effects as follows: employing is test organisms molecular characterization and interactional SPR technology from the teeth outwards, utilize that the SPR effect is fast to surrounding medium variations in refractive index resolution height, response, high flux, sensitivity, special, easy, to advantages such as the own not damageds of sample, and can obtain the dynamics data that many additive methods are difficult to obtain; Simultaneously owing to adopted optical fiber, have again that anti-electromagnetic interference capability is strong, high pressure resistant, corrosion-resistant, the sensing probe size is little, can realize advantages such as distributed measurement and long-haul telemetry monitoring as the sensing matrix; By simplifying the sensor-based system structure and adopting the relative spectral detection technique, can improve measuring accuracy, overcome luminous intensity measurement and be subject to the shortcoming that the flashing photographic fixing rings.
Description of drawings:
Fig. 1 is that the Optical Fiber Surface Plasmon nucleic acid sensor system that detects single nucleotide polymorphism is formed synoptic diagram.
Fig. 2 is a y-type optical fiber coupling mechanism synoptic diagram.
Fig. 3 is an end reflection formula Optical Fiber Surface Plasmon nucleic acid sensor probe synoptic diagram.
Fig. 4 is a distribution type fiber-optic surface plasma wave nucleic acid sensor probe synoptic diagram.
Fig. 5 is online reflection type optical fiber surface plasma wave nucleic acid sensor probe synoptic diagram.
Label title among the above-mentioned figure: 1. wideband light source, 2.P type polaroid, 3.Y type fiber coupler, 4. coupling mechanism joint, 5. sensor fibre, 6. end reflection formula Optical Fiber Surface Plasmon nucleic acid sensor probe, 7. spectrometer, 8. computing machine, 9. online reflection type optical fiber surface plasma wave nucleic acid sensor probe, 61. fibre core, 62. metal level, 63. micro-reflectors, the 64.SNP specific probe is film fixedly, 10. covering, 11. coats.
Embodiment:
As shown in Figure 1, the concrete composition that the present invention detects the Optical Fiber Surface Plasmon nucleic acid sensor system of single nucleotide polymorphism is, wideband light source 1 (can adopt LG150 molded breadth band light source) is through P type polaroid 2, with the elimination of S polarized light, only make the P polarized light be coupled into y-type optical fiber coupling mechanism 3 input end of (as shown in Figure 2), propagate into the output terminal of y-type optical fiber coupling mechanism 3, again by coupling mechanism joint 4, propagate into end reflection formula Optical Fiber Surface Plasmon nucleic acid sensing probe 6 through sensor fibre 5, produce the surface plasma wave resonance effects with the nucleic acid molecules interaction in the sample to be tested, the formation reflection ray enters spectrometer 7 (can adopt EPP2000 type spectrometer) by the reflection end of y-type optical fiber coupling mechanism 3 after through the micro-reflector effect of the fibre core end face in the probe 6, pass through the relation curve that computing machine 8 is handled between output reflection light intensities and the optical wavelength again, thereby realized the full fiberize of whole measurement light path part.
Fig. 3 is an end reflection formula Optical Fiber Surface Plasmon nucleic acid sensor probe synoptic diagram.It comprises fixedly film 64 of the metal level 62 on micro-reflector 63, fibre core 61 top layers of fibre core 61, fibre core 61 end faces and metal level 62 outer field SNP specific probes.
Cover aminopropyl triethoxysilane at metal surface with the numerator self-assembly technique chemical modification, the specific target dna probe of designed detected single nucleotide polymorphism is attached to metal surface with bifunctional coupling agent with e, introduce biotin one Avidin amplification system again, realize complementary the combination with the dna molecular in the sample to be tested.Realize identification and detection by the situation of change that detects resonant wavelength to the SNP of dna molecular in the sample.
Variation ion resonance technology such as surface can judge qualitatively whether interaction is arranged between two molecules, interactional power between more a kind of molecule and other molecules, the affinity parameters (equilibrium constant) and the kinetic parameter (rate constant) of mensuration intermolecular interaction that also can real-time quantitative, even thermodynamic parameter (enthalpy of reaction).This technology has been utilized physioptial principle, when research two interactions of molecules, a kind of molecule is fixed on SPR sensing probe surface, and the flow of solution of another kind of molecule is crossed its surface, the combination of two kinds of molecules can make the refraction index changing on vane surface, therefore can detect two intermolecular interactions.
Fig. 4 is a distribution type fiber-optic surface plasma wave nucleic acid sensor probe synoptic diagram.Its concrete composition is the dissimilar metal level 62 of diverse location coating one deck at same fiber core 61, increases in dissimilar metal levels 62 outsides to cover fixedly film 64 compositions online reflection type optical fiber surface plasma wave nucleic acid sensor probe 9 as shown in Figure 5 of one deck SNP specific probe again.Therefore a plurality of online reflection type optical fiber surface plasma wave nucleic acid sensor probes 9 can be set on same optical fiber simultaneously, and the end of this optical fiber is processed as end reflection formula Optical Fiber Surface Plasmon nucleic acid sensor probe 6.
According to SPR spectrum output characteristics, by apply at the diverse location of fiber core dissimilar metal level (gold or silver) and SNP specific probe fixedly the film formation can realize the distribution type fiber-optic surface plasma wave nucleic acid sensor of multidigit point detection to SNP.When these SPR sensing sections are in do not coexist test environment or the medium, different complementation combinations can take place with specific probe in different determined nucleic acid molecules, thereby cause near change of refractive each spr sensor, this will be on same optical fiber many places excitating surface plasma wave-wave resonant effect.By the detection that sensor is exported the different resonant wavelengths of SPR spectrum, can obtain the signal of Distributed Detection.
The relative spectral detection method:
When structure one timing of optical fiber sensing probe, it is that resonant wavelength is directly relevant with the refractive index of metal surface surrounding medium that surface plasma wave resonance optical wavelength constantly takes place.During each the detection,, just can know the situation of measured biomolecule by investigating the situation that resonant wavelength moves.
Spectral method of detection has higher measuring accuracy and resolution than the light intensity detection method, and is not vulnerable to environmental factor as the influence of power supply shakiness etc.
When measuring, at first the input spectrum signal is preserved as reference signal, then the optical fiber sensing probe is placed environment to be measured, obtain the SPR spectral signal of reflected back, again this signal and reference signal are compared, thus the exact value of acquisition SPR spectral resonance wavelength.
Claims (2)
1. Optical Fiber Surface Plasmon nucleic acid sensor system that detects single nucleotide polymorphism, comprise that wideband light source (1) links to each other through the input end of P type polaroid (2) with y-type optical fiber coupling mechanism (3), the output terminal of y-type optical fiber coupling mechanism (3) is connected in sensor fibre (5) by coupling mechanism joint (4), this sensor fibre (5) end can be an end reflection formula Optical Fiber Surface Plasmon nucleic acid sensor probe (6), or distribution type fiber-optic surface plasma wave nucleic acid sensor probe, the reflection end of y-type optical fiber coupling mechanism is connected in the spectrometer (7) that links to each other with computing machine, it is characterized in that described end reflection formula Optical Fiber Surface Plasmon nucleic acid sensor probe is by fibre core (61), be positioned at the micro-reflector (63) of fibre core (61) end face, the SNP specific probe in the metal level (62) on fibre core (61) top layer and metal level (62) outside fixedly film (64) is formed; The concrete composition of described distribution type fiber-optic surface plasma wave nucleic acid sensor probe is, can prepare a plurality of online reflection type optical fiber surface plasma wave nucleic acid sensor probes (9) on same fiber core (61), the end of this optical fiber is set to end reflection formula Optical Fiber Surface Plasmon nucleic acid sensor probe (6).
2. check and analysis method that detects the Optical Fiber Surface Plasmon nucleic acid sensor system of single nucleotide polymorphism, it is characterized in that adopting the relative spectral detection method, promptly when measuring, at first the input spectrum signal is preserved as reference signal, then the Optical Fiber Surface Plasmon nucleic acid sensor is placed the sample to be tested test solution, obtain the surface plasma wave resonance spectrum signal of reflected back, again this spectral signal and reference signal are compared, thus the exact value of acquisition surface plasma wave spectral resonance wavelength.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1329709C (en) * | 2005-05-27 | 2007-08-01 | 哈尔滨工业大学 | Double optical fiber coupling contact type micro measuring force aiming sensor |
| CN100561198C (en) * | 2005-07-01 | 2009-11-18 | 曾祥楷 | Fibre-optical microstructure Michelson interfere type surface plasma resonance chemistry and biology sensor and system |
| CN102095719A (en) * | 2010-12-30 | 2011-06-15 | 浙江工业大学 | Optical fiber type sensor system based on surface plasma resonance and stimulated Raman scattering |
| CN103335985A (en) * | 2013-07-10 | 2013-10-02 | 北京交通大学 | Quasi-distributed fiber grating surface plasma resonance sensor and making method thereof |
| CN105445224A (en) * | 2014-06-06 | 2016-03-30 | 纳米及先进材料研发院有限公司 | Reusable long period microfiber grating for detection of dna hybridization |
| CN108226272A (en) * | 2018-01-12 | 2018-06-29 | 清华大学深圳研究生院 | A kind of ionic migration spectrometer based on optical fiber SPR sensor |
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| JP2004500020A (en) * | 1999-03-30 | 2004-01-08 | ナノゲン・インコーポレイテッド | Discrimination of Single Nucleotide Polymorphisms by Electronic Dot Blot Assay on Semiconductor Microchip |
| JP2003189862A (en) * | 2001-12-25 | 2003-07-08 | Hitachi Software Eng Co Ltd | Method for detecting target, reagent for detecting single nucleotide polymorphism of dna base sequence and method for detecting single nucleotide polymorphism |
| JP2004191251A (en) * | 2002-12-12 | 2004-07-08 | Olympus Corp | Fluorescence spectroscopic analyzer |
| CN1194217C (en) * | 2003-03-28 | 2005-03-23 | 东南大学 | Longitudianl distributive surface plasma wave sensors |
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| CN1329709C (en) * | 2005-05-27 | 2007-08-01 | 哈尔滨工业大学 | Double optical fiber coupling contact type micro measuring force aiming sensor |
| CN100561198C (en) * | 2005-07-01 | 2009-11-18 | 曾祥楷 | Fibre-optical microstructure Michelson interfere type surface plasma resonance chemistry and biology sensor and system |
| CN102095719A (en) * | 2010-12-30 | 2011-06-15 | 浙江工业大学 | Optical fiber type sensor system based on surface plasma resonance and stimulated Raman scattering |
| CN103335985A (en) * | 2013-07-10 | 2013-10-02 | 北京交通大学 | Quasi-distributed fiber grating surface plasma resonance sensor and making method thereof |
| CN103335985B (en) * | 2013-07-10 | 2016-01-13 | 北京交通大学 | Quasi-distributed fiber grating surface plasma resonance sensor and preparation method |
| CN105445224A (en) * | 2014-06-06 | 2016-03-30 | 纳米及先进材料研发院有限公司 | Reusable long period microfiber grating for detection of dna hybridization |
| CN110100171A (en) * | 2017-01-16 | 2019-08-06 | 矢崎总业株式会社 | Highly selective corrosion sensor system |
| CN108226272A (en) * | 2018-01-12 | 2018-06-29 | 清华大学深圳研究生院 | A kind of ionic migration spectrometer based on optical fiber SPR sensor |
| CN108226272B (en) * | 2018-01-12 | 2020-06-23 | 清华大学深圳研究生院 | Ion mobility spectrometer based on optical fiber SPR sensor |
| CN115372317A (en) * | 2022-07-29 | 2022-11-22 | 天津大学 | Portable optical fiber surface plasma resonance spectrometer |
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| CN100362337C (en) | 2008-01-16 |
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