CN110455754A - A kind of DNA probe and production method - Google Patents
A kind of DNA probe and production method Download PDFInfo
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- CN110455754A CN110455754A CN201910634567.7A CN201910634567A CN110455754A CN 110455754 A CN110455754 A CN 110455754A CN 201910634567 A CN201910634567 A CN 201910634567A CN 110455754 A CN110455754 A CN 110455754A
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- dna probe
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- conical fiber
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The present invention is suitable for sensory field of optic fibre, provide a kind of conical fiber Raman DNA probe structure and preparation method thereof, the probe structure includes the DNA probe sensitive layer 3 of pyramid optical fiber substrate 1, one layer of metal nanoparticle enhancing substrate 2 and a kind of hairpin structure of a roughing in surface, the present invention also provides the production methods of this conical fiber Raman DNA probe, comprising the following steps: first with the pyramid optical fiber 1 of femtosecond laser parallel micromachining technology production roughing in surface；Using Electrostatic Absorption or light-induce growth method, modification and growth metal nanoparticle 2 on the surface of conical fiber；Finally by assembling hairpin structure DNA probe sensitive layer 3, this highly sensitive optical fiber DNA probe is realized, conical fiber Raman DNA probe structure provided by the invention is simple, high sensitivity, and highly sensitive, the specific detection of DNA target fragment may be implemented.
Description
Technical field
The invention belongs to sensory field of optic fibre more particularly to a kind of conical fiber Raman DNA probe and production methods.
Background technique
Carrier of the gene as hereditary information can control the character of bion by instructing albumen synthesis, by multiple
Hereditary information is passed to offspring by system, thus the analysis detection of gene has important answer in medical diagnosis and prenatal and postnatal care field
With value.The disease for seriously affecting human health has the symptom of more than half related to gene；It is thereby achieved that the base that causes a disease
The clinical detection of cause is of great significance, so far, there are many ways to genetic test, such as high-flux sequence, melting curve
Analysis, denaturation liquid-phase chromatographic analysis etc., but these methods, process is cumbersome or is difficult to realize clinical detection, in contrast, optical fiber
DNA probe then has small structure, light-weight, high sensitivity, can go deep into the advantages that organization internal, remote sensing, living in clinic
The application potential of physical examination cls gene.
Fiber Raman technology is identified for DNA molecular can be divided into label detection method and label-free detection by detection mode
Method.And the obvious increasing for leading to Surface enhanced Raman scattering (SERS) signal by DNA hybridization can also be divided by marking detection method to be divided into
By force, and the decline of SERS signal intensity caused by hybridization identifies two methods of target gene.Wherein using Raman signal decline come
The method of identification target gene will usually be coupled a upper molecular beacon in Raman substrate.In the shape not hybridized with target dna
Under state, molecular beacon is in hairpin structure, and the dye molecule on molecular beacon is because close to SERS substrate, signal is significantly enhanced.
After molecular beacon hybridizes with target gene, hairpin structure is opened, therefore dye molecule reduces far from SERS substrate, signal.Than
Play other marking type optical fiber SERS-DNA detection methods for being reported, this method is used in conjunction with without a variety of DNA probe sequences, also without
It need to carry out amplification instrument.Using molecular beacon as DNA probe, the process for preparing optical fiber SERS gene probe is relatively simple, at present
Temporarily without such DNA probe and production method.
Summary of the invention
The technical problems to be solved by the present invention are: providing a kind of conical fiber Raman DNA probe, realize to cell, people
The identification and detection of body tissue and low concentration intentional DNA.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention are as follows: provide a kind of DNA probe, feature exists
In: including conical fiber, metal nanoparticle substrate, the probe acute layer along optical path direction.
Further, the conical fiber includes integrally formed cylindrical section and pyramid section, and the pyramid can be trigone
Cone, rectangular pyramid or multi-panel pyramid.
Further, the conical fiber is common refractive index gradient type, graded refractive index single mode optical fiber, multimode light
One of the silica fibre that fine, core size is 10 microns to 600 microns.
Further, the metal nanoparticle substrate, material are gold or silver.
Further, the probe acute layer includes the single strand dna of one section of both ends complementation, and the molecule one is terminal modified
There are sulfenyl or mercapto functional group, the other end is modified with fluorescent molecule.
The present invention also provides a kind of DNA probe production methods, comprising:
S1, processing conical fiber；
S2, metal nanoparticle substrate fabrication is made on the conical fiber surface；
S3, in the metal nanoparticle substrate surface assembled dna probe acute layer.
Further, the processing conical fiber is that the optical fiber for cutting flat with one end is placed in below the femto-second laser of focusing
It is processed into conical fiber.
Further, the metal nanoparticle substrate fabrication is that manufactured conical fiber is inserted into reducing solution, laser
Irradiation, makes metal particles deposition on the conical fiber surface.
Further, the optical maser wavelength is 785nm, 633nm or 532nm.
Further, the assembled dna probe acute layer includes that will grow the conical fiber of metal nanoparticle substrate
Probe is put into the solution containing probe acute layer, in such a way that chemical bond couples, probe acute layer is fixed on metal and is received
Rice grain surface.
The beneficial effects of the present invention are: this sensor production process is simple, effect is good, can produce in batches, and senses
Device structure is small, light-weight, high sensitivity, can go deep into organization internal, have the advantages that remote sensing, has in clinical In vivo detection base
The application potential of cause.
Detailed description of the invention
Specific structure of the invention is described in detail with reference to the accompanying drawing:
Fig. 1 is a kind of conical fiber Raman DNA probe structural schematic diagram provided in an embodiment of the present invention；
Fig. 2 is hairpin structure DNA sensitive layer enlarged diagram；
Fig. 3 is a kind of femtosecond laser production method of triangular pyramid optical fiber provided in an embodiment of the present invention；
Fig. 4 is the production method of triangular pyramid fiber Raman metal nanoparticle substrate provided in an embodiment of the present invention；
Fig. 5 is a kind of method of modifying of conical fiber Raman DNA probe provided in an embodiment of the present invention；
Fig. 6 is the schematic diagram that a kind of conical fiber Raman DNA probe provided in an embodiment of the present invention carries out DNA detection；
Wherein: 1- optical fiber；2- metal nanoparticle substrate；3- probe acute layer；301- single strand dna；302- function
Group；303- fluorescent molecule；4- femtosecond laser beam；5- laser focus point run trace；6- laser；7- reducing solution；8- probe acute
Layer solution；9- Raman signal；10- prepare liquid；11- target dna molecule；
Specific embodiment
In order to describe the technical content, the structural feature, the achieved object and the effect of this invention in detail, below in conjunction with embodiment
And attached drawing is cooperated to be explained in detail.
Refering to fig. 1-6, bright in order to understand technical problems, technical solutions and advantages to be solved more
White, the present invention is further described in detail below with reference to the accompanying drawings and embodiments；It should be appreciated that described herein specific
Embodiment is only used to explain the present invention, is not intended to limit the present invention.
A kind of conical fiber Raman DNA probe structure provided in an embodiment of the present invention, if Fig. 1 includes one kind along optical path direction
Triangular pyramid optical fiber 1, the pyramid silica fibre SERS probe with microsize tip can invade single cell organisms, detection life
Object molecule, and Raman signal is acquired, evanescent field strength is big, it helps enhancing Raman signal intensity；Metal nanoparticle substrate
2, forming Raman enhances substrate, expands Raman reinforcing effect；Probe acute layer 3 is used for and target dna molecule Complementary hybridization, production
Raw Raman signal variation.
Embodiment one
The optical fiber 1 is common refractive index gradient type, graded refractive index single mode optical fiber, multimode fibre, fibre core ruler
Very little one of the silica fibre for being 10 microns to 600 microns, preferably 200 μm of core size of silica fibre, the gold
Metal nano-particle substrate 2, including but not limited to spherical shape, triangle, sheet, cube metallic particles, in the present embodiment metal
Nano-particle material is silver, and the probe acute layer 3 includes that the single strand dna 301 of one section of both ends complementation, one is terminal modified
There is the functional group 302 of sulfenyl (- S) or sulfydryl (- SH), one end is modified with fluorescent molecule 303, and the fluorescent molecule 303 can be selected
FAM, Cy5, ROX etc., this kind of DNA molecular are curled into hairpin structure in a free state.
The production of following steps method can be used in above-mentioned conical fiber Raman DNA probe:
As shown in Figure 3, optical fiber processing end is placed in the femtosecond laser beam 4 of focusing in the following, process control laser focuses
Point run trace 5, is processed into conical fiber 1 by the way of progressively scanning, processing layer by layer, is processed into triangular pyramid in the present embodiment
Shape optical fiber, the triangular pyramid angle of taper is 3 degree to 120 degree, and cleans the two of optical fiber 1 in ethanol with supersonic wave cleaning machine
End removes the optical fiber clast generated in femtosecond laser process and the micropollutants for being adsorbed on optical fiber both ends of the surface, nitrogen
Drying.
Manufactured conical fiber 1 is inserted into reducing solution 7 referring to Fig. 4, and laser 6 is passed through optical fiber 1, controls exciting light function
Rate and/or light application time make the metal nanoparticle substrate 2 reach required size；In the present embodiment, the reducing solution 7
For concentration be 1mM silver nitrate and concentration is that 1mM sodium citrate is mixed in the ratio of 1:1；Preferably, used in the present embodiment
Laser be optical maser wavelength be 785nm, the semiconductor laser that continuously runs, injecting power 20mW.
Ag nanostructure can be divided into three steps in the growth course of optical fiber surface: firstly, since electrostatic adsorption, silver
Ion A g+ is adsorbed on the surface of fiber end face；Second step, the citrate quilt under laser irradiation, near fiber end face
An electronics is decomposed and generates, the silver ion Ag+ on optical fiber surface is reduced to silver atoms Ag0 after obtaining electronics；Third step, silver
The positively charged dimeric structure Ag2+ that atom and the silver ion Ag+ of surrounding will form, subsequent citrate and positively charged
Silver-colored dimer is complexed, and is formed complex [Ag2+.. (Citrate) -], and citrate therein is released because of laser irradiation decomposition
The electron injection complex of releasing forms small Silver Clusters, while the chemical activity on cluster surface is increased, so around cluster
Silver ion Ag+ be easier to be reduced, the silver atoms of formation are attached on Silver Clusters；In this way, nano-Ag particles are just in 1 table of optical fiber
Face is constantly grown, and the conical fiber probe for being assembled with silver metal nanoparticles is cleaned with deionized water, is dried with nitrogen.
Power meter, which is placed, in conical fiber cone when induced with laser metal nanoparticle is grown is used for detection light watt level,
Metal nanoparticle growth, optical power reduce, according to the size of detection optical power come the indirect size for inferring metallic particles with it is close
Degree, experiment can find optimum power according to experiment effect and reduce degree, and laser is closed when optical power drops to most suitable
Device makes metal nanoparticle particle stop growing.
Manufactured enhancing Raman fiber probe is put into the solution containing probe acute layer by Fig. 5, and probe acute layer includes:
The single strand dna 301 of one section of both ends complementation, one end are modified with functional group 302, and one end is modified with fluorescent molecule 303, passing through
The mode that key couples is learned, DNA probe molecule is fixed on 1 surface of metal nanoparticle, the present embodiment is using functional group as sulfydryl base
For group (- SH), mercapto groups (- SH) can be reacted with silver atoms generates stable chemical bond Ag-S key, will be modified with probe
The optical fiber probe of sensitive layer 3 PBS buffer solution repeated flushing, is dried with nitrogen.
If the conical fiber Raman DNA probe being fabricated to is put into the prepare liquid 10 containing target dna molecule 11 by Fig. 6,
Laser 6 is passed through on spread fiber to optical fiber cone, and metallic particles can enhance light field signal, and the light field signal of enhancing can enhance gold again
The scattering of the fluorescent molecule of metal particles surface link, probe acute layer 3 and target dna molecule 11 will carry out Complementary hybridization, and work as mesh
In the presence of marking DNA molecular 11, after hybridization, probe acute layer 3 is opened under the action of stress, and fluorescent molecule 303 is separate
Metal nano substrate 2, electromagnetic field locating for fluorescent molecule 303 weaken, and the fluorescent molecule Raman signal 9 being collected into weakens rapidly,
Therefore the presence or absence of target dna molecule 11 can be judged according to the intensity of Raman signal, and can carry out low concentration DNA solution
Detection and measurement.
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair
Equivalent structure or equivalent flow shift made by bright specification and accompanying drawing content is applied directly or indirectly in other relevant skills
Art field, is included within the scope of the present invention.
Claims (10)
1. a kind of DNA probe, it is characterised in that: quick including conical fiber, metal nanoparticle substrate, the probe along optical path direction
Feel layer.
2. DNA probe as described in claim 1, which is characterized in that the conical fiber include integrally formed cylindrical section and
Pyramid section；The pyramid can be triangular pyramid, rectangular pyramid or multi-panel pyramid.
3. the DNA probe as described in claims 1 or 2 any one, which is characterized in that the conical fiber is refractive index gradient
One of type, graded refractive index single mode optical fiber, multimode fibre, silica fibre that core size is 10 microns to 600 microns.
4. DNA probe as described in claim 1, which is characterized in that the metal nanoparticle substrate, material are gold or silver.
5. DNA probe as described in claim 1, which is characterized in that the probe acute layer includes the list of one section of both ends complementation
Ssdna molecule, the DNA molecular one end are modified with sulfenyl or mercapto functional group, and the other end is modified with fluorescent molecule.
6. a kind of DNA probe production method characterized by comprising
S1, processing conical fiber；
S2, metal nanoparticle substrate is made on the conical fiber surface；
S3, in the metal nanoparticle substrate surface assembled dna probe acute layer.
7. DNA probe production method as claimed in claim 6, which is characterized in that the processing conical fiber is to cut one end
Flat optical fiber, which is placed in below the femto-second laser of focusing, is processed into conical fiber.
8. DNA probe production method as claimed in claim 6, which is characterized in that the metal nanoparticle substrate fabrication is
Manufactured conical fiber is inserted into reducing solution, laser irradiation makes metal particles deposition on the conical fiber surface.
9. DNA probe production method as claimed in claim 8, which is characterized in that the optical maser wavelength be common 785nm,
633nm or 532nm.
10. DNA probe production method as claimed in claim 6, which is characterized in that the assembled dna probe acute layer includes
The conical fiber probe for having grown metal nanoparticle substrate is put into the solution containing probe acute layer, chemical bond coupling is passed through
Probe acute layer is fixed on metal nanoparticle surface by the mode of connection.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910634567.7A CN110455754B (en) | 2019-07-15 | 2019-07-15 | DNA probe and manufacturing method thereof |
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| CN201910634567.7A CN110455754B (en) | 2019-07-15 | 2019-07-15 | DNA probe and manufacturing method thereof |
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| CN110455754B CN110455754B (en) | 2022-03-18 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111537492A (en) * | 2020-04-30 | 2020-08-14 | 东南大学 | Preparation method of uniform high-sensitivity surface-enhanced Raman spectrum probe, probe prepared by same and application of probe |
| CN113219581A (en) * | 2020-11-24 | 2021-08-06 | 桂林电子科技大学 | Single-fiber-core beak-shaped optical fiber tweezers with sorting function and preparation method thereof |
| CN115046981A (en) * | 2022-05-27 | 2022-09-13 | 燕山大学 | Tongue-shaped optical fiber probe based on surface enhanced Raman scattering and preparation method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111537492A (en) * | 2020-04-30 | 2020-08-14 | 东南大学 | Preparation method of uniform high-sensitivity surface-enhanced Raman spectrum probe, probe prepared by same and application of probe |
| CN113219581A (en) * | 2020-11-24 | 2021-08-06 | 桂林电子科技大学 | Single-fiber-core beak-shaped optical fiber tweezers with sorting function and preparation method thereof |
| CN115046981A (en) * | 2022-05-27 | 2022-09-13 | 燕山大学 | Tongue-shaped optical fiber probe based on surface enhanced Raman scattering and preparation method thereof |
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| CN110455754B (en) | 2022-03-18 |
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