CN112680503A - Biosensor for nucleic acid detection and preparation method thereof - Google Patents
Biosensor for nucleic acid detection and preparation method thereof Download PDFInfo
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- CN112680503A CN112680503A CN202011631325.1A CN202011631325A CN112680503A CN 112680503 A CN112680503 A CN 112680503A CN 202011631325 A CN202011631325 A CN 202011631325A CN 112680503 A CN112680503 A CN 112680503A
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Abstract
The invention relates to the field of biomedicine, in particular to a biosensor for nucleic acid detection and a preparation method thereof. The biosensor comprises two micro-nano optical fibers which are connected together in a melting way and probe single-stranded DNA fixed on the surfaces of the two micro-nano optical fibers; the two micro-nano optical fibers comprise an even waist region and tapered transition regions positioned on two sides of the even waist region, optical power is coupled and transmitted between the two micro-nano optical fibers, and the shape of the tapered transition regions and the diameter of the even waist region inhibit high-order mode transmission. The biosensor has the advantages of simple structure, strong operability, low cost, small volume, high efficiency, electromagnetic interference resistance and the like, has extremely high sensitivity near the turning point, and has wide application prospect in the field of biochemical sensing.
Description
Technical Field
The invention relates to a method and a structure for detecting nucleic acid, in particular to a biosensor for detecting nucleic acid and a preparation method thereof.
Background
Deoxyribonucleic acid (DNA) is a biological macromolecule indispensable for life phenomena, controls protein synthesis by being a carrier of genetic information, has a great influence on organisms due to structural changes thereof, and is associated with causes of various genetic diseases. Biological gene sequences are determined more and more along with the implementation of human genome plans, and the gene detection technology for rapidly sequencing and accurately identifying chaotic sequences causes the research enthusiasm of researchers in various countries. Therefore, the search for a novel, efficient and accurate DNA detection technique is very important for the analysis of genetic information.
With the completion of human genome project and the deep research of functional genes, gene diagnosis has become an important research field in molecular biology and biomedicine, and the detection of specific sequences plays an important role in the fields of gene analysis, disease diagnosis, food pollution and the like. The nucleic acid molecule hybridization refers to a process that two nucleic acid single strands with complementary sequences form a double strand under a certain condition according to the base pairing principle, and mainly comprises a probe and nucleic acid to be detected. The nucleic acid molecular hybridization technique is one of the most widely applied techniques in the field of life science research at present, can realize qualitative or quantitative detection of specific DNA or RNA sequence fragments, can screen specific clones in a library or a genome library to obtain a certain recombinant, and can determine the nucleotide homologous sequence of a specific region on the genome DNA. As a method for identifying by utilizing the nucleic acid base pairing principle, the DNA biosensor can realize continuous, rapid, sensitive and selective detection on specific series of gene fragments, so that how to realize high sensitivity, high flux, low cost and the like is an important target for the development of DNA detection methods.
Disclosure of Invention
The present invention has been made to solve the above problems, and provides a biosensor for nucleic acid detection and a method for preparing the same.
In order to achieve the purpose, the invention adopts the following specific technical scheme:
the invention provides a biosensor for nucleic acid detection, which comprises two micro-nano optical fibers and probe single-stranded DNA (deoxyribonucleic acid), wherein the two micro-nano optical fibers are connected together in a melting way; the two micro-nano optical fibers comprise an even waist region and tapered transition regions positioned on two sides of the even waist region, optical power is coupled and transmitted between the two micro-nano optical fibers, and the shape of the tapered transition regions and the diameter of the even waist region inhibit high-order mode transmission.
In the biosensor for nucleic acid detection provided by the invention, the probe single-stranded DNA and the target single-stranded DNA are hybridized to cause the change of the refractive index of the surface of the optical fiber.
In the biosensor for nucleic acid detection provided by the invention, the two micro-nano optical fibers work in the area near the turning point; the turning point means that the effective group refractive indexes of the two micro-nano optical fibers are equal.
In the biosensor for nucleic acid detection provided by the invention, the effective group refractive indexes of the two micro-nano optical fibers are equal.
The biosensor for nucleic acid detection provided by the invention further comprises an input port and an output port, wherein the input port and the output port are arranged at two ends of the two micro-nano optical fibers; incident light of TE/TM polarization irradiates the input port, and the output port observes interference spectral lines; and detecting the change of the refractive index of the surface of the optical fiber by detecting the shift of the wave crest or the wave trough of the interference spectral line.
In the biosensor for nucleic acid detection provided by the present invention, the input ports are P1 and P2, and the output ports are P3 and P4; TE/TM polarized incident light irradiates P1 and/or P2, and an even mode and an odd mode can be simultaneously excited in the two micro-nano optical fibers; energy exchange occurs when the even mode and the odd mode are transmitted along the two micro-nano optical fibers, and the interference spectral lines are observed at P3 and P4.
In the biosensor for nucleic acid detection provided by the invention, the fixation of the probe single-stranded nucleic acid on the surfaces of the two micro-nano optical fibers is realized by a polylysine adsorption method.
In another aspect, the present invention also provides a method for preparing a biosensor for nucleic acid detection, comprising the steps of:
s1, calculating sensitivity curves near the down-conversion points of different coupler fiber diameters and working wavelengths by taking the refractive index of the solvent as reference;
s2, stretching the two optical fibers by using a hot melting stretching method, and obtaining the optical fiber coupler with the required size by controlling process parameters; the two optical fibers comprise a uniform waist region and tapered transition regions positioned at two sides of the uniform waist region, optical power is coupled and transmitted between the optical fiber couplers, and the shape of the tapered transition regions and the diameter of the uniform waist region inhibit high-order mode transmission;
s3, fixing the optical fiber coupler on a sample cell, attaching a layer of surface modification group on the surface of the optical fiber through biological modification, and then fixing the probe single-stranded nucleic acid.
In the method for producing a biosensor for nucleic acid detection provided by the present invention, in step S2, the hot-melt drawing method specifically includes the steps of:
s201, taking two single-mode optical fibers, stripping a section of protective layer in the middle of each optical fiber to expose an optical fiber cladding, and cleaning the outer surface of the optical fiber cladding;
s201, fixing the processed optical fibers into movable V-shaped grooves respectively, and knotting the two optical fibers together;
s201, heating the bare part of the optical fiber, and applying axial tension to the optical fiber through the V-shaped groove to realize drawing of the optical fiber coupler.
In the method of manufacturing a biosensor for nucleic acid detection provided by the present invention, the uniform waist region of the optical fiber coupler has a diameter of about 1 μm; the solvent has a refractive index of about 1.33.
Compared with the prior art, the invention has the following beneficial technical effects:
(1) the system has the advantages of simple structure, strong operability, low cost, small volume, high efficiency, electromagnetic interference resistance and the like;
(2) the fiber coupler DNA biosensor has extremely high sensitivity near the turning point and has wide application prospect in the field of biochemical sensing;
(3) the real-time monitoring of the DNA hybridization process has very important significance for the dynamic research of the DNA hybridization process in the future;
(4) the sensor has good stability and repeatability, and can realize high-specificity monitoring on DNA.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a schematic diagram of a micro-nano fiber coupler sensor;
FIG. 2 is a schematic view of surface modification of an optical fiber DNA sensor;
FIG. 3 is a theoretical refractive index curve of a micro-nano optical fiber coupler sensor, mainly showing sensitivities corresponding to different coupler diameters;
FIG. 4 is a graph showing wavelength shifts corresponding to different concentrations of DNA solutions, which show different wavelength peaks and undergo red shifts with increasing concentrations.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not to be construed as limiting the invention.
The invention provides a high-sensitivity biosensor based on a turning point of a micro-nano optical fiber coupler, aiming at solving the problems of low sensitivity and the like in the existing nucleic acid biosensor.
The optical fiber coupler realizes the coupling transmission of optical power between two optical fiber waveguides by high-temperature melting and stretching after the two optical fibers are mutually wound, and can realize the inhibition of high-order mode transmission by controlling the shape of a transition region and the diameter of a waist region, thereby controlling the number of conduction modes in the coupler.
Fig. 1 is a schematic structural diagram of a micro-nano fiber coupler DNA sensor provided by the present invention, which includes a uniform waist region, two tapered transition regions, input ports P1 and P2, and output ports P3 and P4. Energy exchange occurs when the even and odd modes are transmitted along the optical fiber coupler, so that interference spectral lines can be observed at the output end, the interference spectral lines depend on the change of the external refractive index, and the change of the external refractive index can be detected by detecting the deviation of wave crests or wave troughs. According to the overlapping integral theory, two micro-nano optical fibers fused and connected together in a coupler can be regarded as a new waveguide. When TE/TM polarized incident light strikes an input port P1 or P2, which excites both the even and odd modes in the waveguide, the energy at the output port can be expressed as follows:
in the formula
Representing the phase difference between the two modes in the TE/TM polarization coupling region, the phase difference generated in the transition region is much lower than that generated in the waist region due to the geometrical reasons, and thus can be approximately expressed as:
βeven，βoddand
the propagation constants and effective indices for the even and odd modes, respectively, and L and λ represent the length and wavelength of the coupling region, respectively. The N-order trough in the transmission line satisfies the following condition:
the sensitivity generated by the external refractive index is obtained by using the formula (4) as follows:
Δneffrepresenting the difference in effective indices of even and odd modes,
the effective group refractive index difference is defined, and when G is 0, that is, the inflection point position, the sensitivity theory is infinite, so in order to obtain a relatively high sensitivity, the sensing region is usually selected near the inflection point.
The invention comprises the following steps:
calculating sensitivity curves near turning points under different coupler optical fiber diameters and working wavelengths by using a method of combining numerical simulation software and analytical solution according to requirements and taking a solvent refractive index as a reference;
step two, stretching the two optical fibers by using a hot melting stretching method, and obtaining the optical fiber coupler with the required diameter by controlling parameters such as gas flow, motor speed and the like;
fixing the optical fiber coupler on a sample cell, attaching a layer of surface modification group on the surface of the optical fiber through biological modification, and then fixing the probe single-stranded DNA.
And (3) filling target single-stranded DNA solutions or target RNA solutions with different concentrations into a sample cell, determining the concentration of the solution by the peak wavelength shift by using the optical fiber coupler prepared by the method, and observing the DNA or RNA hybridization process in real time.
FIG. 2 is a schematic diagram of surface modification of an optical fiber DNA sensor, in which 1 is polylysine PLL, 2 is probe single-stranded ssDNA, and 3 is target DNA; the surface functionalized biological sensitive film of the micro-nano optical fiber coupler has specific identification capability, can react with biomolecules such as absorption and adsorption to cause the change of the refractive index of the surface of the sensor, and can detect the information in a required biological sample by measuring the peak wavelength shift. The patent adopts polylysine (Poly-L-Lysine, PLL) adsorption method to realize the biological sensing experiment of the micro-nano optical fiber coupler. PLL is a viscous molecule rich in amino (-NH2) with isoelectric point of 10.5, and the amino is easily protonated and has positive charge, and can be attracted to anion through electrostatic interaction. DNA molecules contain a large number of phosphate groups, and when the environmental pH is lower than the isoelectric point, the phosphate groups ionize H < + > to make DNA negatively charged. And because the surface of the optical fiber is negatively charged, the micro-nano optical fiber coupler sensor is directly soaked in a polylysine solution (0.1% w/v) for 1 hour, is washed with ionized water for 2-3 times, is then soaked in a probe single-stranded DNA solution with the concentration of 100nM for 1 hour, and is then hybridized with a target single-stranded DNA. Similarly, the method of the present embodiment can also be applied to the detection and observation of RNA.
The invention has the following advantages:
the arrayed biological detection method based on the optical fiber coupler and the optical fiber wavelength division multiplexer utilizes the characteristics of multipath characteristics of the optical fiber coupler, multi-wavelength shunt processing capability of the optical fiber wavelength division multiplexer, optical fiber arraying assembly and easy surface treatment of the end face of the optical fiber, and can realize simultaneous detection of multiple meals with high sensitivity and high specificity. The sensing unit is arrayed, and the excitation unit and the detection unit are integrated, so that the disadvantages of large volume and device recycling of the array detection system can be effectively reduced, and the in-situ and in-vivo detection capability of the detection system can be improved.
Examples
The probe ssDNA used in this example was:
5’-CGCTGAGGAACGCATAACGTT-3’，
the target ssDNA is: 5'-AACGTTATGCGTTCCTCAGCG-3' are provided.
1. Manufacture of micro-nano optical fiber coupler
The experimental curve of sensitivity at different diameters and wavelengths calculated by COMSOL simulation using water (refractive index n ═ 1.33) as the solvent is shown in fig. 3, and the fiber coupler diameter is selected to be 1 μm.
Taking two single-mode optical fibers, stripping a 30mm protective layer in the middle of each optical fiber by using an optical fiber stripper, and cleaning an optical fiber cladding by using alcohol cotton; respectively fixing the processed optical fibers into a movable V-shaped groove, knotting the two optical fibers together, heating the optical fibers by using oxyhydrogen flame, applying axial tension to the optical fibers through the V-shaped groove, and drawing the optical fiber coupler with the diameter of 1 micrometer by controlling parameters such as hydrogen flow, motor speed, flame head size and the like; and fixing the drawn optical fiber coupler on a sample cell for later use.
Experiment of DNA biosensor
The sensor is fixed on a PMMA sample cell, light generated by a wide-spectrum light source enters an input port P1 or P2 of a micro optical fiber coupler, an output port P3 or P4 of the coupler is connected with a spectrometer, the effective refractive index of the surface of the optical fiber sensor is changed by changing DNA solutions with different concentrations, the resonance wavelength of the coupler is shifted, and therefore detection of the DNA solutions with different concentrations is achieved by detecting wavelength movement of the spectrometer.
The experimental procedure was as follows:
1) directly soaking the micro-nano optical fiber coupler in a Polylysine (PLL) (0.1% w/v, deionized water) solution for 1 hour, and then cleaning the micro-nano optical fiber coupler with the deionized water for 2 to 3 times;
2) soaking the probe single-stranded DNA solution with the concentration of 100nM for 1 hour, and washing for 2-3 times;
3) injecting target DNA solutions with different concentrations into a sample cell, and monitoring a spectral curve in real time;
4) in a repeated experiment, the optical fiber coupler sensor can be repeatedly used by repeatedly washing for 3 times by using a mixed solution (0.1% w/v, 95 ℃) of Phosphate Buffered Saline (PBS) and Sodium Dodecyl Sulfate (SDS) and then washing by using deionized water.
FIG. 4 is a graph showing wavelength shifts corresponding to different concentrations of DNA solutions, which show different wavelength peaks and undergo red shifts with increasing concentrations.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it should be understood that the above embodiments are exemplary and should not be taken as limiting the invention. Variations, modifications, substitutions and alterations of the above-described embodiments may be made by those of ordinary skill in the art without departing from the scope of the present invention.
The above embodiments of the present invention should not be construed as limiting the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. A biosensor for nucleic acid detection is characterized by comprising two micro-nano optical fibers which are connected together in a melting way and probe single-stranded DNA fixed on the surfaces of the two micro-nano optical fibers; the two micro-nano optical fibers comprise an even waist region and tapered transition regions positioned on two sides of the even waist region, optical power is coupled and transmitted between the two micro-nano optical fibers, and the shape of the tapered transition regions and the diameter of the even waist region inhibit high-order mode transmission.
2. The biosensor as claimed in claim 1, wherein the probe single-stranded DNA hybridizes to the target single-stranded DNA to cause a change in refractive index of the surface of the optical fiber.
3. The biosensor as claimed in claim 1, wherein the two micro-nano optical fibers work in the region near the turning point; the turning point means that the effective group refractive indexes of the two micro-nano optical fibers are equal.
4. The biosensor of claim 1, wherein the effective group refractive indices of the two micro-nanofiber fibers are equal.
5. The biosensor of claim 1, further comprising an input port and an output port, wherein the input port and the output port are disposed at two ends of the two micro-nano optical fibers; incident light of TE/TM polarization irradiates the input port, and the output port observes interference spectral lines; and detecting the change of the refractive index of the surface of the optical fiber by detecting the shift of the wave crest or the wave trough of the interference spectral line.
6. The biosensor of claim 5, wherein the input ports are P1 and P2, and the output ports are P3 and P4; TE/TM polarized incident light irradiates P1 and/or P2, and an even mode and an odd mode can be simultaneously excited in the two micro-nano optical fibers; energy exchange occurs when the even mode and the odd mode are transmitted along the two micro-nano optical fibers, and the interference spectral lines are observed at P3 and P4.
7. The biosensor of claim 1, wherein the probe single-stranded nucleic acid is immobilized on the surfaces of the two micro-nano optical fibers by a polylysine adsorption method.
8. A method for preparing a biosensor for nucleic acid detection, comprising the steps of:
s1, calculating sensitivity curves near the down-conversion points of different coupler fiber diameters and working wavelengths by taking the refractive index of the solvent as reference;
s2, stretching the two optical fibers by using a hot melting stretching method, and obtaining the optical fiber coupler with the required size by controlling process parameters; the two optical fibers comprise a uniform waist region and tapered transition regions positioned at two sides of the uniform waist region, optical power is coupled and transmitted between the optical fiber couplers, and the shape of the tapered transition regions and the diameter of the uniform waist region inhibit high-order mode transmission;
s3, fixing the optical fiber coupler on a sample cell, attaching a layer of surface modification group on the surface of the optical fiber through biological modification, and then fixing the probe single-stranded nucleic acid.
9. The method for manufacturing a biosensor in accordance with claim 8, wherein in step S2, the hot melt drawing method comprises the following steps:
s201, taking two single-mode optical fibers, stripping a section of protective layer in the middle of each optical fiber to expose an optical fiber cladding, and cleaning the outer surface of the optical fiber cladding;
s201, fixing the processed optical fibers into movable V-shaped grooves respectively, and knotting the two optical fibers together;
s201, heating the bare part of the optical fiber, and applying axial tension to the optical fiber through the V-shaped groove to realize drawing of the optical fiber coupler.
10. The method of claim 8, wherein the uniform waist region of the fiber coupler has a diameter of about 1 μm; the solvent has a refractive index of about 1.33.
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Citations (3)
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|---|---|---|---|---|
| US20090304551A1 (en) * | 2006-01-31 | 2009-12-10 | Drexel University | Ultra Sensitive Tapered Fiber Optic Biosensor For Pathogens, Proteins, and DNA |
| CN110132896A (en) * | 2019-05-06 | 2019-08-16 | 山西大学 | The micro optical fiber biosensor of markers for breast cancer in a kind of quick detection serum |
| CN110160960A (en) * | 2019-06-22 | 2019-08-23 | 南昌航空大学 | Micro-nano fiber biosensor and preparation method thereof based on coupler structure |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090304551A1 (en) * | 2006-01-31 | 2009-12-10 | Drexel University | Ultra Sensitive Tapered Fiber Optic Biosensor For Pathogens, Proteins, and DNA |
| CN110132896A (en) * | 2019-05-06 | 2019-08-16 | 山西大学 | The micro optical fiber biosensor of markers for breast cancer in a kind of quick detection serum |
| CN110160960A (en) * | 2019-06-22 | 2019-08-23 | 南昌航空大学 | Micro-nano fiber biosensor and preparation method thereof based on coupler structure |
Non-Patent Citations (3)
| Title |
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| DANDAN SUN ET AL: ""Label-Free Thrombin Detection Using a Tapered Fiber-Optic Interferometric Aptasensor"", 《JOURNAL OF LIGHTWAVE TECHNOLOGY》 * |
| KAIWEI LI ET AL: ""Spectral Characteristics and Ultrahigh Sensitivities Near the Dispersion Turning Point of Optical Microfiber Couplers"", 《JOURNAL OF LIGHTWAVE TECHNOLOGY》 * |
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