CN101368207A - Long period optical fiber optical grating nucleic acid sensor system and detection method for detecting mononucleotide polymorphism - Google Patents
Long period optical fiber optical grating nucleic acid sensor system and detection method for detecting mononucleotide polymorphism Download PDFInfo
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Abstract
The invention relates to a distribution type long period fiber grating nucleic acid sensor system and a method for detecting the polymorphism of a single nucleotide which belong to the testing field of a biosensor. The system consists of a Broad Band light source (1), a tree type fiber coupler (2),a sensing fiber (4), a computer (7), a spectrograph and a terminal reflection type long period fiber grating nucleic acid sensor probe (5) or a distribution type long period fiber grating nucleic acid sensor probe. In the method, the SNP of the DNA in a sample to be detected is detected through measuring the change of a resonance peak caused by the change of a dielectric constant in the test liquid of the sample to be detected. The distribution type long period fiber grating nucleic acid sensor system and a method for detecting the polymorphism of a single nucleotide have the characteristics of high sensitivity, the realization of distributed measurement and real-time high pass detection.
Description
Technical field
The present invention relates to a kind of novel optical fiber DNA sensing system that long period fiber grating specific refractory power susceptibility and optical fiber detection technique are combined, it can be applicable to human genome research single nucleotide polymorphism (SNP).
Background technology
Long period fiber grating (hereinafter to be referred as LPG) is a kind of sensitivities grating based on fibre core guided mode and cladding mode coupled wave theory, and it is very responsive to the variations in refractive index of environment of living in around it, and then can obtain the concentration of particle in the solution.Biosensor technique based on this coupled wave theory has been widely used in a plurality of Biochemical Research fields such as antigenic content measurement, proteinaceous substances discriminating, drug screening in the blood.
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 thymus nucleic 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 specificity.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 hybridization 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 fluorescent mark 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 LPG determination techniques belongs to a kind of characteristic of spike biomolecules from the teeth outwards and interactional method, have in real time, need not mark, to advantages such as the own not damageds of sample, and can obtain the more accurate data of more many additive methods.Over the past two years, some study group had begun to attempt to use the LPG technology and had carried out biological detection both at home and abroad.
Aspect the LPG biosensor, 2000, people such as Pilevar utilize LPG and the combination of Bragg grating to make antibody one antigen biosensor at The 14th OFS work literary composition " EvanecentWave Antibody-Antigen Biosensor Based on Long Period Fiber Gratings. ", are used for antigenic having or not in the detection of biological solution.2006, in the laser biology journal, Zhuan Qiren introduced a kind of LPG transmitter of measuring microbial film thickness in " long period fiber grating microbial film transmitter ", can be used for determining whether there is antigen in the blood.
What research and develop both at home and abroad at present is in order to detect antibody one antigenic LPG biosensor, that is: to utilize specific microbial film technology of preparing mostly, on exposed LPG surface that the bio-sensing element is fixing to form one deck sensitive thin film.According to the difference (as protein, antigen etc.) of object to be checked, the heat transfer agent primary element can be phase paired enzyme, antibody etc. with it.Because biosensor (as enzyme) commonly used has very high catalytic efficiency and specificity mostly, so being fixed in the active film on exposed LPG surface can do very thinly, and even be that unit molecule is arranged, so can think still that grating is the naked state of no covering this moment.
During measurement, LPG is immersed testing liquid, if wherein there is material to be detected, as certain protein the substrate of enzyme (promptly corresponding to), to be attended by corresponding biochemical reaction generation and have specific mixture to produce, the mixture that generates is adsorbed on the LPG surface, thereby has formed one deck plated film in its surface, and this film can be considered the new covering of naked grating.According to the difference of detected object, the LPG cladding index changes, thereby, can differentiate whether there is certain antibody-antigen.Domestic a lot of research institution is studied in the application of aspects such as biology, chemistry and biochemistry detection this transmitter, and has obtained certain achievement.
Along with deepening continuously of research work, detect antibody-antigenic LPG biosensor and can not satisfy the demand of people gradually for aspects such as volume, sensitivity.Thereupon, many improvement projects such as enhanced sensitivity film, unidirectional detection have appearred, as: 2005, Guan Shouhua in the sensing technology journal, deliver " based on the concentration sensor of Long-Period Fiber Grating with Single Head "; " research of long period fiber grating thin film sensor " that Xu Yanping delivers in Shanghai University of Science and Technology's newspaper etc.We make and can realize accurate localized long period optical fiber optical grating nucleic acid sensor in that above novel optical fiber sensing arrangement is carried out further introducing distributed frame on the basis of big quantity research.
The method in extensive studies SNP, discovery mutational site mainly is the nucleotide sequencing that relies on gene, i.e. Ce Xu method the most at present.Because these methods all need fluorescent mark 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.
Summary of the invention
The objective of the invention is to overcome shortcomings such as the precision of original Nucleotide in detecting is lower.Provide a kind of highly sensitive, can realize distributed measurement, realize to determine the distributed long period optical fiber optical grating nucleic acid sensor system and the detection method of the detection single nucleotide polymorphism of the concrete location of SNP and feature.Simultaneously owing to adopted single-ended detection, so the test section volume that it can avoid the LPG sensor-based system to show again is bigger, is not suitable for the shortcoming of extraneous factors influences such as long-haul telemetry occasion, light source fluctuation.
A kind of distributed long period optical fiber optical grating nucleic acid sensor system that detects single nucleotide polymorphism, it is characterized in that: composed as follows: wideband light source links to each other with the input terminus of tree type fiber coupler, the output terminal of tree type fiber coupler is connected in sensor fibre by the coupling mechanism joint, this sensor fibre end is an end reflection formula long period optical fiber optical grating nucleic acid sensor probe, or distributed long period optical fiber optical grating nucleic acid sensor probe, the reflection end of coupling mechanism is connected in the spectrograph that links to each other with computer; Described end reflection formula long period optical fiber optical grating nucleic acid sensor probe comprises the fibre core that is provided with LPG sensing grid region, the reflecting metallic film that is positioned at the fibre core end face; Described distributed long period optical fiber optical grating nucleic acid sensor probe, be concrete the composition, the multifiber parallel connection, and be carved with LPG sensing grid region at the different positions of each root fiber core, terminal at every optical fiber is provided with reflecting metallic film, the LPG transmitter resonance peak difference of different root optical fiber.
The detection method of the long period optical fiber optical grating nucleic acid sensor system of above-mentioned detection single nucleotide polymorphism, it is characterized in that: utilize the sensitive natur of LPG sensing grid region grid region surrounding environment change in dielectric constant, by measuring because the change of the resonance peak that the change in dielectric constant of sample to be tested causes in the liquid phase, thereby realize identification to SNP.Change of refractive is just measured to grid region surrounding environment change in dielectric constant in said measurement LPG sensing grid region.
End reflection formula long period optical fiber optical grating nucleic acid sensor probe mainly is the other end metal-coated membrane at grating among the present invention, such as the gold and silver film, effectively improves the reflectivity of light at the single-ended internal surface of optical fiber, guarantees the measuring accuracy of loss peak peak value.The peak loss of LPG is much larger than 3dB, therefore can drop to the right self-interference effect of LPG minimumly, can not produce a plurality of loss peak peak values influences and measure.Simultaneously, single-ended LPG sensing head is convenient to operation and is moved, thereby adapts to the actual measurement condition of various complexity, and can simplify sensor-based system.The composition of distributed long period optical fiber optical grating nucleic acid sensor is: can prepare a plurality of online long period optical fiber optical grating nucleic acid sensor probes on same optical fiber, the end of this optical fiber is set to end reflection formula long period optical fiber optical grating nucleic acid sensor probe, simultaneously, multifiber is by coupling mechanism and link up, in order to realize space division multiplexing.
The invention has the beneficial effects as follows: employing is test organisms molecular characterization and interactional LPG technology from the teeth outwards, utilize LPG to surrounding medium specific refractory power, strength of solution change that resolving power height, response are fast, high-throughput, sensitivity, special, easy, to advantages such as the own not damageds of sample; Simultaneously owing to adopted optical fiber as the sensing matrix, have again anti-electromagnetic interference capability strong, high pressure resistant, corrosion-resistant, can realize advantages such as distributed measurement and long-haul telemetry monitoring; By simplifying the sensor-based system structure and adopting spectrum 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 long period optical fiber optical grating nucleic acid sensor system is formed synoptic diagram.
Fig. 2 is a tree type fiber coupler synoptic diagram.
Fig. 3 is an end reflection formula long period optical fiber optical grating nucleic acid sensor probe synoptic diagram.
Fig. 4 is distributed long period optical fiber optical grating nucleic acid sensor probe synoptic diagram.
Label title among the figure: 1. wideband light source, 2. tree type fiber coupler, 3. coupling mechanism joint, 4. sensor fibre, 5. end reflection formula long period optical fiber optical grating nucleic acid sensor probe, 6. spectrograph, 7. computer, 61.LPG grid region, 62. fibre cores, 63. fibre core end reflection metallic membrane, 64. specificity DNA probing needle, 9. online long period optical fiber optical grating nucleic acid sensor probe, 11. input terminuss, 12. reflection end, 13. output terminals.
Embodiment
As shown in Figure 1, the concrete composition of long period optical fiber optical grating nucleic acid sensor system of the present invention is, wideband light source 1 (can adopt LG150 molded breadth band light source), enter the input terminus of tree type fiber coupler 2 (as shown in Figure 2), propagate into the output terminal of tree type fiber coupler 2, again by coupling mechanism joint 3, propagate into end reflection formula long period optical fiber optical grating nucleic acid sensor probe 5 through sensor fibre 4, interact with dna molecular in the sample to be tested and to produce the coupling effect of fibre core and covering, the formation reflection ray enters spectrograph 6 (can adopt AQ6317 type spectrograph) by the reflection end of tree type fiber coupler 2 after through the reflective metals membrane interaction of the fibre core end face in the probe 10, pass through the relation curve that computer 7 is handled between output reflection light intensities and the photopeak value again, thereby realized the full fiberize of whole measurement light path part.
Fig. 3 is an end reflection formula long period optical fiber optical grating nucleic acid sensor probe synoptic diagram.It comprises specificity DNA probing needle 64, fibre core end reflection metallic membrane (gold or silver) 63 and the anchor 62 on grid region 61, surface, grid region.Anchor 62 is mainly used in the grid region that makes long period fiber grating and keeps straight, and avoiding influences detected result owing to the grid region is crooked, and its two inboardends distance should be greater than grid region length.
Utilize γ-(2 at grating surface by covalent cross-linking method, 3-epoxy third oxygen) propyl trimethoxy silicane (YDH-560) carries out chemically modified, the specificity DNA probing needle that designed being used to is detected SNP is attached to the grating top layer, carry out crossover process with the sample to be tested dna molecular in liquid phase, hybrid combines by the avidin of bag quilt on vitamin H and the magnetic bead and is hunted down.Realize identification by the changing conditions that detects resonance peak to the SNP site of sample DNA molecule.
LPG nucleic acid technology 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 molecular interaction that also can real-time quantitative, even thermodynamical coordinate (enthalpy of reaction).This technology has been utilized physioptial principle, when research two interactions of molecules, a kind of molecule is fixed on LPG sensing probe surface, and the solution stream of another kind of molecule is crossed its surface, the combination of two kinds of molecules can make the specific refractory power on sensing grating surface change, and therefore can detect two intermolecular interactions.
Fig. 4 is distributed long period optical fiber optical grating nucleic acid sensor probe synoptic diagram.Its concrete composition is that the different positions at same fiber core is carved into LPG sensing grid region, or adopts the LPG transmitter of different resonance peaks respectively at different root optical fiber, and plates reflection gold or silverskin in the terminal of every optical fiber.Therefore a plurality of reflective long period optical fiber optical grating nucleic acid sensor probes can be set on same optical fiber simultaneously, and the end of this optical fiber is processed as end reflection formula long period optical fiber optical grating nucleic acid sensor probe.
According to LPG spectrum output characteristic, can realize the distributed long period optical fiber optical grating nucleic acid sensor of multidigit point detection by the LPG of different resonance peaks to SNP.When these LPG sensing sections were in do not coexist test environment or the medium, different hybridization can take place with sensor probe in different dna moleculars to be measured, thereby caused near the variation of the specific inductivity of each LPG transmitter.By detection, can obtain the signal of Distributed Detection to the different resonance peaks of transmitter output LPG spectrographic.
Claims (2)
1. distributed long period optical fiber optical grating nucleic acid sensor system that detects single nucleotide polymorphism is characterized in that:
Composed as follows: wideband light source (1) links to each other with the input terminus of tree type fiber coupler (2), the output terminal of tree type fiber coupler is connected in sensor fibre (4) by coupling mechanism joint (3), this sensor fibre (4) end is an end reflection formula long period optical fiber optical grating nucleic acid sensor probe (5), or distributed long period optical fiber optical grating nucleic acid sensor probe, the reflection end of coupling mechanism is connected in the spectrograph (6) that links to each other with computer (7);
Described end reflection formula long period optical fiber optical grating nucleic acid sensor probe (5) comprises the fibre core that is provided with LPG sensing grid region (61), the reflecting metallic film that is positioned at the fibre core end face, is positioned at the specificity DNA probing needle (64) on surface, LPG sensing grid region;
Described distributed long period optical fiber optical grating nucleic acid sensor probe, concrete composition is, the multifiber parallel connection, and the different positions at each root fiber core (62) is carved with LPG sensing grid region, each surface, root LPG sensing grid region is provided with the specificity DNA probing needle (64) that detects specific liquid phase, terminal at every optical fiber is provided with fibre core end reflection metallic membrane (63), the initial resonance peak difference of different root optical fiber LPG transmitters.
2. the detection method of the long period optical fiber optical grating nucleic acid sensor system of detection single nucleotide polymorphism according to claim 1, it is characterized in that: utilize the sensitive natur of LPG sensing grid region grating grid region surrounding environment variations in refractive index, because the variation of the resonance peak that the variation of sample to be tested test solution medium dielectric constant microwave medium causes, detect the SNP of DNA in the sample to be checked by measuring; That is: when self-assembly when target sequence to be detected reacts in the dna probe of grating surface and liquid phase, the resonance peak that causes by the variation that detects by surface, grid region liquid phase environment medium dielectric constant microwave medium changes, and then realizes the identification to SNP.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| WO2021068482A1 (en) * | 2020-03-12 | 2021-04-15 | 广州中国科学院先进技术研究所 | Probe-style on-line biomass measurement apparatus able to undergo high temperature sterilization |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| WO2021068482A1 (en) * | 2020-03-12 | 2021-04-15 | 广州中国科学院先进技术研究所 | Probe-style on-line biomass measurement apparatus able to undergo high temperature sterilization |
| CN113388488A (en) * | 2020-03-12 | 2021-09-14 | 广州中国科学院先进技术研究所 | Probe type biomass on-line detection device capable of achieving high-temperature sterilization |
| CN113388488B (en) * | 2020-03-12 | 2023-09-29 | 迪必尔智能科技(深圳)有限公司 | Probe type biomass on-line detection device capable of achieving high-temperature sterilization |
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