CN115326763A - Novel hollow anti-resonance optical fiber sensor combined with noble metal nanoparticles and detection method - Google Patents
Novel hollow anti-resonance optical fiber sensor combined with noble metal nanoparticles and detection method Download PDFInfo
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Abstract
The invention relates to a novel hollow anti-resonance optical fiber sensor combined with noble metal nano particles and a detection method, which can be applied to the detection of trace substances in the field of biochemistry and belong to the field of optical fiber sensing in biomedical photonics. When the detection is carried out, firstly, a precise injection instrument is utilized to fill a target analyte into a functional anti-resonance hollow-core optical fiber (HARF), and the target analyte is incubated for a period of time to fully react with a functional interface. The laser light emitted by the continuous laser is then focused through a plano-convex lens and coupled into the functionalized HARF. At this point, the fluorescent reporter molecule is dissociated and fluoresced due to the specific binding of the target analyte to its aptamer. The fluorescence recovery value is linear with the concentration of the target analyte. And finally, filtering the light source part of the light scattered back from the HARF by a filter plate, and collecting, analyzing and quantifying by using a spectrometer.
Description
Technical Field
The invention relates to a sensor formed by combining noble metal nano particles with a novel antiresonant hollow fiber and a corresponding detection method, which can be applied to the detection of trace substances in the field of biochemistry and belong to the field of fiber sensing in biomedical photonics.
Background
Microstructured Optical Fibers (MOFs) have become an effective tool for modern digital communications due to their broadband and low loss characteristics. In addition to being an excellent optical communication medium, MOFs play a key role in fluid detection, trace gas detection, infrastructure internal damage detection, and security monitoring of bridges, oil and gas pipelines.
Among them, anti-resonant hollow core fiber (HARF) is a new type of micro-structured fiber. Unlike common solid-core optical fiber, the light guiding mechanism of the solid-core optical fiber is based on ARROW principle of planar waveguide, a cladding quartz tube in HARF is equivalent to a Fabry-Perot (FP) resonant cavity, light with wavelength meeting the resonance condition can be leaked out through the quartz tube layer with high refractive index, and light with anti-resonance wavelength can be reflected to a fiber core. Since the FP cavity in the HARF is destructive interference, leakage is significantly limited and most of the light will be reflected to the core. HARF has a wide light-passing window in the visible range and has the advantages of single-mode transmission, near-zero dispersion, high laser damage threshold and low non-linear effects, especially its special geometry that causes the light to be tightly confined in the core, which greatly enhances the interaction between the light and the substance. Therefore, HARF is particularly suited for applications in the field of biosensing detection. In addition, the presence of the fiber-clad tubules and the hollow core also facilitates integration with specific functional materials to develop new functionalized sensors.
The noble metal nanoparticles (such as gold and silver) can quench the fluorescence of the ultra-close fluorescent substance by the Fluorescence Resonance Energy Transfer (FRET) effect, and are a fluorescence quenching agent with excellent performance due to wide absorption spectrum and large absorption coefficient. It provides a promising direction for new functionalized fiber sensors. At present, a part of novel detection means uses FRET effect of noble metal nanoparticles to quench fluorescence, but most of the novel detection means combines a fluorescence reporter molecule with the noble metal nanoparticles by using electrostatic adsorption force, and the connection mode is easily influenced by detection environment, so that the detection stability and specificity are not high.
Disclosure of Invention
In view of the above, the invention provides a novel hollow anti-resonance optical fiber sensor and a detection method combining with precious metal nanoparticles, wherein a combination of precious metal nanoparticles and stable double-stranded DNA (one strand of which is aptamer and the other strand of which is fluorescence reporter molecule) is fixed on the inner wall of the novel anti-resonance hollow optical fiber, and based on the fluorescence quenching recovery principle, the combination is used for detecting the ability of the novel anti-resonance hollow optical fiber to bind light waves, so that the problem of low stability in biochemical substance detection is solved, the detection limit of biochemical substances is greatly reduced, and meanwhile, the sensitivity and specificity are higher.
The specific technical scheme is as follows:
the utility model provides a combine noble metal nanoparticle's novel hollow antiresonance optical fiber sensor, includes light source part, collimation portion, sensing part and signal acquisition part, and the light that light source part launched passes through collimation portion coupling and enters sensing part, and the light signal that the sensing part was gathered to the signal acquisition part realizes detecting. The sensing part adopts functionalized HARF (6), the functionalized HARF (6) refers to an antiresonant hollow-core optical fiber of which the inner wall is fixed with a biological probe, the biological probe refers to noble metal nanoparticles combined with double-strand DNA, the double-strand DNA is formed by hybridizing nucleic acid aptamers matched with a target analyte and a fluorescent reporter molecule, the fluorescent reporter molecule comprises a complementary strand of the nucleic acid aptamers and a fluorescent dye, the absorption spectrum of the noble metal nanoparticles is overlapped with the emission spectrum of the fluorescent dye, and the antiresonant hollow-core optical fiber with a very wide transmission passband and very low light attenuation is used for enhancing the interaction of light and substances.
In the novel hollow anti-resonance optical fiber sensor combined with the noble metal nano particles, a continuous laser (1) is adopted in a light source part; the collimating part comprises a discontinuous attenuation sheet (2), a reflector (3), a dichroic mirror (4) and a first plano-convex lens (5) which are connected in sequence; the signal acquisition part comprises a filter (7), a second plano-convex lens (8), a large-aperture multimode optical fiber (9) and a spectrometer (10) which are sequentially connected, laser emitted by the light source part is coupled into the sensing part through the collimation part, and the signal acquisition part is used for acquiring optical signals in the sensing part sequentially passing through the first plano-convex lens (5) and the dichroic mirror (4).
The wavelength of the light source part in the novel hollow anti-resonance optical fiber sensor combined with the noble metal nano particles is matched with the excitation wavelength of the fluorescent dye.
A biochemical detection method combining noble metal nano particles. The nucleic acid aptamer matched with a target analyte and a fluorescent reporter molecule are hybridized to form double-stranded DNA, and then the double-stranded DNA is modified on the noble metal nano-particles. And finally fixing the double-stranded DNA and noble metal nano particle compound on the inner wall of the anti-resonance hollow optical fiber. Wherein, the fluorescent reporter molecule comprises a complementary strand of the aptamer and a fluorescent dye; the light source enters the anti-resonance hollow-core optical fiber after being collimated, the absorption spectrum of the noble metal nano-particles is overlapped with the emission spectrum of the fluorescent dye, and FRET effect can be generated and the fluorescence of the fluorescent dye can be quenched due to the close distance between the noble metal nano-particles and the fluorescent dye;
filling a target analyte into the anti-resonance hollow-core optical fiber, combining the target analyte with the matched nucleic acid aptamer, and displacing and separating the fluorescent reporter molecule out of the anti-resonance hollow-core optical fiber so as to keep away from the noble metal nano-particles, wherein FRET effect cannot occur and fluorescence can be excited by light source light;
the fluorescence recovery value calculated according to the fluorescence values of the two times before and after the filling is in a linear relation with the concentration of the target analyte, so that the target analyte is detected.
The manufacture of the HARF sensor is divided into two steps: (1) preparation of a bioprobe: firstly, hybridizing a nucleic acid aptamer and a fluorescent reporter molecule to form double-stranded DNA; the double stranded DNA is then immobilized on the surface of the noble metal nanoparticle. At the moment, because the fluorescent dye in the fluorescent reporter molecule is close to the noble metal nano-particles, the fluorescence of the fluorescent reporter molecule is quenched due to FRET effect; (2) HARF biofunctionalization: modifying a biological probe on the inner wall of the HARF by utilizing a covalent bond at one end of the aptamer; when a low-concentration target analyte solution is filled into the optical fiber, the matched aptamer can specifically capture and combine with the target analyte, and at the moment, the double-stranded DNA is untwisted to release a fluorescent reporter molecule. The fluorescence recovery value calculated according to the fluorescence values of the two times before and after the filling is in a linear relation with the concentration of the target analyte, so that the target analyte is detected.
The invention combines the capability of amplifying the interaction between light and substances of the anti-resonance hollow fiber core, the quenching capability of the noble metal nano particles and the specificity of the aptamer, has obviously enhanced effect compared with the conventional method, and has the advantages of high specificity, strong detection sensitivity, simple structure, less required samples and the like.
The optical fiber used in the present invention includes various types of antiresonant hollow-core optical fibers; the noble metal nanoparticles used include various metal nanoparticles having a strong FRET effect, such as gold, silver or some alloy nanoparticles, and the like; the used biological probe is mainly an aptamer of an object to be detected; the sensor can detect many substances including microorganisms, cells, exosomes, inorganic or organic substances such as residual drugs, antibiotics, etc. in the biochemical field.
Several factors that evaluate sensor performance are mainly: the time required for detection, the volume of the sample to be detected, the specificity, the detection limit and the stability.
Compared with the prior art, the invention has the following advantages:
1. the detection time is short. After the optical fiber is filled in the substance to be detected, the substance to be detected can completely react with the aptamer in a short time. When laser is coupled into the HARF, the detached fluorescent reporter molecule can be instantly excited to generate fluorescence, and the whole process is short in time consumption.
2. The required sample volume is small. The HARF generally has a micron-sized fiber core, so that only a micro-liter-sized sample to be detected is needed to complete the detection, which is very important for the practical application of biochemical detection.
3. High specificity. The aptamer sequence adopted by the sensor is obtained by SELEX (systematic evolution of ligands by exponential enrichment) technology. By utilizing the technology, the aptamer with high affinity to the sample to be detected can be screened from the random single-stranded nucleic acid sequence library, and the binding of the aptamer and the sample to be detected has extremely high specificity.
4. The detection limit is low. The fluorescence reaction occurs inside the HARF with minimal interference from the surrounding environment. The functionalized noble metal nano particles are coated on the inner surface and can fully react with a sample to be detected. Due to the extremely low loss of the HARF, most of laser is limited to be transmitted in the fiber core, the interaction between light and substances is greatly enhanced, and the detection limit is greatly reduced.
5. The stability is high. The biological probe is formed by combining precious metal nanoparticles and stable double-stranded DNA (one strand of the nucleic acid aptamer and the other strand of the nucleic acid aptamer and the fluorescent reporter molecule), and the stable structure of the double-stranded DNA enables the sensor to be less affected by non-specificity of environmental substances and to have high stability.
In general, the antiresonant hollow-core fiber provides a very ideal platform for biochemical sensing, and a sensor manufactured by using the antiresonant hollow-core fiber can enhance the interaction between light and substances by using a micron-sized fiber core, has low loss when a reaction path is long, and is one of the most promising platforms in the sensing field.
Description of the drawings:
FIG. 1 (a) is a schematic cross-sectional structure of a novel antiresonant hollow-core fiber;
FIG. 1 (b) is a schematic cross-sectional view of a core for use as a novel antiresonant hollow-core fiber;
FIG. 2 is a transmission band diagram of the novel antiresonant hollow-core fiber;
FIG. 3 TEM image of noble metal nanoparticles after binding to the aptamer;
FIG. 4 is a TEM image of functionalized noble metal particles fixed on the inner wall of the fiber core;
FIG. 5 (a) is a schematic diagram of a novel antiresonant hollow-core fiber sensor;
FIG. 5 (b) is a schematic diagram of the reaction mechanism of the novel antiresonant hollow-core fiber sensor;
fig. 6 is a schematic structural diagram of the novel optical fiber sensing system.
Detailed Description
The present invention will be further illustrated with reference to the following examples, but the present invention is not limited to the following examples.
Example 1
The experiment utilizes an anti-resonance hollow-core optical fiber sensor to detect breast cancer exosomes. The surface of the breast cancer cell exosome is provided with MUC1 protein which is excessive relative to a common cell exosome, so that the aptamer which can be combined with the MUC1 protein in high specificity is selected in the biological probe of the sensor, the gold nanoparticles (AuNPs) with the diameter of 15nm are selected as the noble metal nanoparticles, and FAM dye with the emission spectrum overlapped with the absorption spectrum of the 15nm AuNPs is selected as the fluorescent dye. When the breast cancer cell exosome is filled into the sensor, the fluorescent reporter molecule is separated from the noble metal nano-particles due to the specific combination with the MUC1 protein aptamer, and the fluorescent signal changes. And the fluorescence recovery value calculated according to the fluorescence values of the two times before and after the filling is in a linear relation with the concentration of the breast cancer cell exosomes, so that the detection of the breast cancer cell exosomes is realized. The fluorescence recovery value before and after the breast cancer cell exosomes are filled is far higher than that of common cell exosomes, so that normal cells are distinguished. The experiment combines the capability of enhancing the interaction of light and substances by using the anti-resonance hollow optical fiber, and is coordinated with the fluorescence quenching capability of AuNPs, the specificity of aptamers and the stability of double-stranded DNA (deoxyribonucleic acid) to carry out the ultrasensitive, ultralow-concentration, high-specificity and stable breast cancer cell exosome detection.
The experiment was divided into several steps. (1) Synthesis of double-stranded DNA: one strand of the double-stranded DNA is the aptamer of the MUC1 protein, and one end of the aptamer is modified with an SH bond for combining AuNPs. The other strand is a reporter molecule that modifies the FAM fluorochrome. (2) Synthesis of AuNPs-double-stranded DNA: combining AuNPs and double-stranded DNA by a common salt aging method, and gradually dropping a salt solution with a certain concentration into a mixed solution of the AuNPs and the double-stranded DNA. The salt concentration is increased, the electronegativity of AuNPs and double-stranded DNA can be shielded, the aggregation of AuNPs can be prevented by gradually dropping, and the SH bond modified at one end of the aptamer in the double-stranded DNA can be combined with the AuNPs. After AuNPs-double-stranded DNA is formed, because the AuNPs are close to the FAM fluorescent dye, and the absorption spectrum of the AuNPs with the diameter of 15nm is overlapped with the emission spectrum of the FAM fluorescent dye, FRET effect can occur between the AuNPs and the FAM fluorescent dye, and the fluorescence of the FAM fluorescent dye can be effectively quenched. (3) HARF biofunctionalization: the HARF adopted in the experiment is a node-free anti-resonance hollow-core seven-hole optical fiber. The HARF outer diameter is about 200 μm, the diameter of seven non-contacting cladding tubules is about 13 μm, the thickness of the tube wall is about 400nm, and the diameter of an inner circle surrounded by 7 thin-layer tubes is about 31 μm. Such HARFs have a wide transmission passband and the ability to transmit in the visible band is very beneficial for biomedical applications. It also has very low transmission and bending losses, a higher damage threshold, and optical attenuation measured as low as about 0.1dB/m. Silane coupling Agent (APTES) is introduced into HARF to modify amino, and AuNPs-double-stranded DNA complex can be combined with amino and fixed on the inner wall of HARF. And (4) sensing and detecting: experiments biologically functionalized HARF was placed in the sensing system. The experimental setup is shown in fig. 6. To excite fluorescence of FAM fluorescent dye, a continuous laser with a wavelength of 473nm was used as the light source. The light source passes through the attenuation sheet, the reflector and the 490nm long-pass dichroic mirror (4) in sequence and is finally coupled into the HARF through the plano-convex lens (5). FAM fluorescence can be passed and part of the light source laser can be intercepted using a 490nm long pass dichroic mirror. The power at the front end of the fiber was controlled at about 100 μ W. A fluorescence signal generated by exciting a fluorescence reporter molecule when a light source enters HARF returns to a dichroic mirror through a primary path, the light source laser is completely filtered through a 500nm long-wave filter (7) and then coupled into a large-aperture multimode optical fiber (9) for receiving the fluorescence signal through another plano-convex lens (8), the fluorescence signal is finally received by a QE65 Pro spectrometer (10), the detection wavelength range is 185-1100 nm, and the optical resolution is 0.14-7.7 nm. Pure water was first charged to the biofunctionalized HARF as a control, at which time fluorescence quenching was caused due to FRET effect occurred between AuNPs and FAM fluorescent dye, and at which time the fluorescent signal was recorded. Then when the MUC1 protein-rich breast cancer cell exosome is filled with biologically-functionalized HARF, the exosome is specifically combined with the matched nucleic acid aptamer, so that the double-chain structure of double-chain DNA is damaged. As a result, the fluorescent reporter molecule with the FAM fluorochrome is released and the fluorescence gradually recovers, and the fluorescence signal at this time is recorded. And finally, the fluorescence recovery value calculated according to the two fluorescence values and the concentration of the breast cancer cell exosome form a linear relation, so that the detection of the breast cancer cell exosome is realized. The method fully utilizes the advantages of HARF, and realizes the ultra-low concentration, high specificity, rapid and stable detection of the MUC1 protein-containing exosome.
Example 2
The experiment utilizes the anti-resonance hollow fiber sensor to detect Hg 2+ 。Hg 2+ Is a heavy metal ion, and has great harm to human body. Thus detecting trace amount of Hg in environmental sample 2+ The ability to do so is of great significance.
In principle, in the same case as in case 1, one strand of the double-stranded DNA is Hg 2+ The other strand of the aptamer of (4) is a fluorescent reporter molecule linked to a HEX fluorescent dye. To ensure FRET effect, gold nanoparticles 80nm in diameter were used for quenchingFluorescence. And the HARF is treated by APTES, so that the surface of the fiber core of the optical fiber is modified with amino functional groups, and the optical fiber is favorable for effective combination with the AuNPs-double-stranded DNA compound. After combination, the detection system is in an initial state, and a 532nm wavelength laser capable of exciting HEX fluorescent dye is used as a light source. At this time, the quenching capability of AuNPs to the HEX fluorescent dye is stronger, and the detected background fluorescence value is lower. Then passing through the biologically functionalized HARF containing Hg 2+ At this time Hg 2+ Will combine with its aptamer to form an aptamer-mercury ion complex, and the fluorescent reporter molecule will be separated from the aptamer and free in the core, resulting in fluorescence recovery. According to the filling of Hg 2+ Fluorescence recovery value and Hg obtained by calculating fluorescence values of two times of the previous and the next 2+ The concentration in a certain range shows a good linear relationship and can thus be used for Hg 2+ The concentration was analyzed and quantified.
The method provided by the invention is used for detecting in cooperation with the light wave binding capability of the novel anti-resonance hollow fiber, the quenching capability of the noble metal nano-particles and the aptamer specificity, so that the detection limit of biochemical substances is greatly reduced compared with the traditional detection method, and the method also has higher sensitivity and specificity. Compared with other novel sensing technologies, the stable structure of the double-stranded DNA firmly combines the fluorescent reporter molecule and the noble metal nano-particles, and the problem of low stability in biochemical substance detection is effectively solved.
Claims (4)
1. The utility model provides a combine noble metal nanoparticle's novel hollow antiresonance optical fiber sensor, includes light source part, collimation portion, sensing part and signal acquisition part, and the light of light source part transmission passes through collimation portion coupling and gets into sensing part, and the light signal in the sensing part is gathered to the signal acquisition part realizes detecting its characterized in that: the sensing part adopts a biological functionalized novel hollow anti-resonance optical fiber (HARF) (6), the biological functionalized HARF (6) refers to HARF with a biological probe fixed on the inner wall, the biological probe refers to noble metal nanoparticles combined with double-stranded DNA, the double-stranded DNA is formed by hybridizing a nucleic acid aptamer matched with a target analyte and a fluorescent reporter molecule, the fluorescent reporter molecule comprises a complementary strand of the nucleic acid aptamer and a fluorescent dye, the absorption spectrum of the noble metal nanoparticles is overlapped with the emission spectrum of the fluorescent dye, and the anti-resonance hollow optical fiber with a very wide transmission passband and very low light attenuation is used for enhancing the interaction of light and substances.
2. The novel hollow-core antiresonant optical fiber sensor combined with noble metal nanoparticles as claimed in claim 1, wherein: the light source part adopts a continuous laser (1); the collimating part comprises a discontinuous attenuation sheet (2), a reflector (3), a dichroic mirror (4) and a first plano-convex lens (5) which are connected in sequence; the signal acquisition part comprises a filter (7), a second plano-convex lens (8), a large-aperture multimode optical fiber (9) and a spectrometer (10) which are connected in sequence, laser emitted by the light source part is coupled into the sensing part through the collimating part, and the signal acquisition part is used for acquiring optical signals in the sensing part which sequentially pass through the first plano-convex lens (5) and the dichroic mirror (4).
3. A novel hollow core antiresonant optical fiber sensor incorporating noble metal nanoparticles as claimed in claim 1 or 2, wherein: the wavelength of the light source portion is matched to the fluorescent dye excitation wavelength.
4. A biochemical detection method combining noble metal nano-particles is characterized in that: hybridizing a nucleic acid aptamer matched with a target analyte and a fluorescent reporter molecule to form double-stranded DNA, then modifying the double-stranded DNA onto a noble metal nanoparticle, and finally fixing a double-stranded DNA and noble metal nanoparticle compound on the inner wall of the HARF, wherein the fluorescent reporter molecule comprises a complementary strand of the nucleic acid aptamer and a fluorescent dye; the light source enters HARF after being collimated, the absorption spectrum of the noble metal nano-particles is overlapped with the emission spectrum of the fluorescent dye, and FRET effect is generated and the fluorescence of the fluorescent dye is quenched because the noble metal nano-particles are closer to the fluorescent dye;
filling a target analyte into the HARF, combining the target analyte with the matched aptamer, and displacing and separating the fluorescent reporter molecule out of the HARF so as to be far away from the noble metal nanoparticle, wherein the FRET effect cannot occur and the fluorescent reporter molecule can be excited to emit fluorescence by light source light;
the fluorescence recovery value calculated according to the fluorescence values of the previous and the next two times has a linear relation with the concentration of the target analyte, so that the target analyte is detected.
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| CN110174380A (en) * | 2019-05-10 | 2019-08-27 | 北京工业大学 | Biochemical sensor based on hollow antiresonance optical fiber |
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| CN113030035A (en) * | 2020-12-28 | 2021-06-25 | 北京工业大学 | Biochemical sensor based on hollow microstructure optical fiber and specific aptamer and detection method thereof |
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| US20060098927A1 (en) * | 2003-06-16 | 2006-05-11 | The Regents Of The University Of California | Integrated sensor with electrical and optical single molecule sensitivity |
| EP1801560A1 (en) * | 2005-12-22 | 2007-06-27 | Palo Alto Research Center Incorporated | Fluorescence reader based on anti-resonant waveguide excitation |
| US20140134609A1 (en) * | 2012-08-17 | 2014-05-15 | Agency For Science, Technology And Research | Enzymatic metal nanoparticle sensor for detecting dna binders |
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