CN115326763A - A novel hollow-core anti-resonant optical fiber sensor combined with noble metal nanoparticles and its detection method - Google Patents

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

A novel hollow-core anti-resonance optical fiber sensor combined with noble metal nanoparticles and detection test method

technical field

The invention relates to a sensor composed of noble metal nanoparticles combined with a novel anti-resonance hollow-core optical fiber and a corresponding detection method, which can be applied to the detection of trace substances in the biochemical field, and belongs to the field of optical fiber sensing in biomedical photonics.

Background technique

Microstructured optical fiber (MOF) has become an effective tool for modern digital communication due to its broadband and low loss characteristics. In addition to being an excellent optical communication medium, MOFs also play a key role in fluid detection, trace gas detection, internal damage detection of infrastructure, and safety monitoring of bridges, oil and gas pipelines.

Among them, the antiresonant hollow core fiber (HARF) is a new type of microstructured fiber. Different from common solid-core optical fibers, its light guiding mechanism is based on the ARROW principle of planar waveguides. The cladding quartz tube in HARF is equivalent to a Fabry-Perot (FP) resonant cavity. The overly high index of refraction of the quartz tube layer leaks out, while light at the antiresonant wavelength is reflected back into the fiber core. Since the FP cavity in the HARF is destructively interfering, the leakage is significantly limited, so most of the light will be reflected to the core. HARF has a wide light window in the visible light range, and has the advantages of single-mode transmission, near zero dispersion, high laser damage threshold and low nonlinear effects, especially its special geometric structure makes the light strictly confined to the core, This greatly enhances the interaction between light and matter. Therefore, HARF is particularly suitable for applications in the field of biosensing and detection. In addition, the existence of fiber-clad small tubes and hollow cores also facilitates the integration with specific functional materials to develop new functional sensors.

Noble metal nanoparticles (such as gold and silver) can quench the fluorescence of ultra-close-range fluorescent substances by the fluorescence resonance energy transfer (FRET) effect, and because of their wide absorption spectrum and large absorption coefficient, they are a kind of fluorescence quencher with superior performance . It provides a promising direction for novel functionalized fiber optic sensors. At present, some new detection methods use the FRET effect of noble metal nanoparticles to quench fluorescence, but most of them use electrostatic adsorption to combine fluorescent reporter molecules with noble metal nanoparticles. This connection method is easily affected by the detection environment, resulting in stable detection. Sexuality and specificity are not high.

Contents of the invention

In view of this, the present invention provides a novel hollow-core antiresonant optical fiber sensor combined with noble metal nanoparticles and a detection method. Reporter molecules) conjugates are immobilized on the inner wall of the new anti-resonance hollow-core fiber, based on the principle of fluorescence quenching recovery, combined with the ability of the new anti-resonance hollow-core fiber to bind light waves for detection, which solves the problem of low stability in the detection of biochemical substances, greatly The detection limit of biochemical substances is reduced to the greatest extent, and it also has high sensitivity and specificity.

The specific technical scheme is as follows:

A new type of hollow-core anti-resonance optical fiber sensor combined with noble metal nanoparticles, including a light source part, a collimation part, a sensing part and a signal collection part. The light emitted by the light source part is coupled into the sensing part through the collimation part, and the signal collection part collects The light signal in the sensing part enables detection. The sensing part adopts functionalized HARF (6). Functionalized HARF (6) refers to an anti-resonant hollow-core optical fiber with a biological probe fixed on the inner wall. The biological probe refers to noble metal nanoparticles combined with double-stranded DNA. Wherein, the double-stranded DNA is hybridized with a nucleic acid aptamer matching the target analyte and a fluorescent reporter molecule, the fluorescent reporter molecule includes the complementary strand of the nucleic acid aptamer and a fluorescent dye, and the absorption of the noble metal nanoparticles The spectrum overlaps with the emission spectrum of fluorescent dyes, and an anti-resonant hollow-core fiber with a wide transmission passband and very low light attenuation is used to enhance the interaction between light and matter.

In the novel hollow-core anti-resonant fiber sensor combined with noble metal nanoparticles, the light source part adopts a continuous laser (1); the collimation part includes sequentially connected discontinuous attenuation sheets (2), mirrors (3), two A color mirror (4) and a first plano-convex lens (5); the signal acquisition part includes a sequentially connected filter (7), a second plano-convex lens (8), a large-aperture multimode fiber (9) and a spectrometer (10) The laser light emitted by the light source part is coupled into the sensing part through the collimating part, and the signal collecting part is used to collect the light signal in the sensing part passing through the first plano-convex lens (5) and the dichroic mirror (4) in sequence.

The wavelength of the light source in the novel hollow-core anti-resonant fiber sensor combined with noble metal nanoparticles matches the excitation wavelength of the fluorescent dye.

A biochemical detection method incorporating noble metal nanoparticles. It hybridizes the nucleic acid aptamer matching the target analyte and the fluorescent reporter molecule to form double-stranded DNA, and then modifies the double-stranded DNA onto the noble metal nanoparticles. Finally, the complex of double-stranded DNA and noble metal nanoparticles is fixed on the inner wall of the anti-resonant hollow-core optical fiber. Wherein, the fluorescent reporter molecule includes the complementary chain of the nucleic acid aptamer and the fluorescent dye; the light source enters the anti-resonant hollow-core optical fiber after being collimated, and the absorption spectrum of the noble metal nanoparticles overlaps with the emission spectrum of the fluorescent dye, and because the noble metal nanoparticles and the fluorescent dye The FRET effect will occur when the distance of the fluorescent dye is close and the fluorescence of the fluorescent dye will be quenched;

Fill the anti-resonant hollow-core fiber with target analyte, the target analyte binds to its matching nucleic acid aptamer, and the fluorescent reporter molecule is displaced and separated away from the noble metal nanoparticles. At this time, the FRET effect will not occur and will be excited by the light source. Fluorescence;

The fluorescence recovery value calculated according to the two fluorescence values before and after filling has a linear relationship with the concentration of the target analyte, thereby realizing the detection of the target analyte.

The fabrication of HARF sensors is divided into two steps: (1) Fabrication of biological probes: first, nucleic acid aptamers are hybridized with fluorescent reporter molecules to form double-stranded DNA; then, double-stranded DNA is immobilized on the surface of noble metal nanoparticles. At this time, because the fluorescent dye in the fluorescent reporter molecule is close to the noble metal nanoparticles, its fluorescence will be quenched due to the FRET effect; (2) HARF biofunctionalization: the biological probe is modified on the HARF by using the covalent bond at one end of the nucleic acid aptamer When the low-concentration target analyte solution is filled into the optical fiber, its matching nucleic acid aptamer will specifically capture the target analyte and bind to it, at this time the double-stranded DNA will be untied and a fluorescent reporter molecule will be released. The fluorescence recovery value calculated according to the two fluorescence values before and after filling has a linear relationship with the concentration of the target analyte, thereby realizing the detection of the target analyte.

The present invention combines the ability of the anti-resonant hollow-core fiber core to amplify the interaction between light and matter, the quenching ability of noble metal nanoparticles, and the specificity of aptamers. Compared with conventional methods, the effect is significantly enhanced, and it has high specificity, strong detection sensitivity, It has the advantages of simple structure and less sample required.

The optical fiber used in the present invention includes various types of anti-resonant hollow-core optical fibers; the noble metal nanoparticles used include various metal nanoparticles with strong FRET effects, such as gold, silver or some alloy nanoparticles or the like; The needle is mainly the aptamer of the analyte; the sensor can detect many substances, including microorganisms, cells, exosomes, inorganic or organic substances such as residual drugs and antibiotics in the biochemical field.

Several elements to evaluate the performance of the sensor mainly include: the time required for detection, the required sample volume to be tested, specificity, detection limit, and stability.

Compared with the prior art, the present invention has the following advantages:

1. The detection time is short. After the analyte is filled into the optical fiber, it will completely react with its aptamer in a short period of time. When the laser is coupled into the HARF, it will instantly excite the detached fluorescent reporter molecules to generate fluorescence, and the whole process takes a short time.

2. The required sample volume is small. HARF generally has a micron-scale fiber core, so only microliters of the sample to be tested are needed to complete the detection, which is very important for the practical application of biochemical detection.

3. Highly specific. The nucleic acid aptamer sequence used by the sensor is obtained by SELEX (systematic evolution of ligands by exponential enrichment) technology. Using this technology, the nucleic acid aptamer with high affinity to the test sample can be screened from the random single-stranded nucleic acid sequence library, and its binding to the test sample has extremely high specificity.

4. Low detection limit. The fluorescence reaction takes place inside the HARF with little interference from the surrounding environment. Functionalized noble metal nanoparticles coat the inner surface, which can fully react with the sample to be tested. Due to the extremely low loss of HARF, most of the laser is limited to the core transmission, which greatly enhances the interaction between light and matter, and greatly reduces the detection limit.

5. High stability. Biological probes are composed of noble metal nanoparticles and stable double-stranded DNA (one strand is a nucleic acid aptamer, and the other strand is a fluorescent reporter molecule). The stable structure of double-stranded DNA makes the sensor less susceptible to non-specific effects of environmental substances. Small, high stability.

In general, the anti-resonant hollow-core fiber provides an ideal platform for biochemical sensing. The sensor made by it can use the micron-scale fiber core to enhance the interaction between light and matter, and has a long reaction path. It still has low loss and is one of the most promising platforms in the field of sensing.

Description of drawings:

Figure 1(a) is used as a schematic diagram of the cross-sectional structure of a novel anti-resonant hollow-core fiber;

Fig. 1 (b) is used as the schematic diagram of the cross-sectional structure of the core of the novel anti-resonant hollow-core fiber;

Fig. 2 The transmission frequency band diagram of the novel anti-resonant hollow-core fiber;

Figure 3 TEM image of noble metal nanoparticles combined with aptamers;

Figure 4 TEM image of functionalized noble metal particles fixed on the inner wall of the fiber core;

Fig. 5(a) Schematic diagram of the novel anti-resonant hollow-core fiber sensor;

Figure 5(b) Schematic diagram of the reaction mechanism of the novel anti-resonant hollow-core fiber sensor;

Fig. 6 Schematic diagram of the structure of the new optical fiber sensing system.

Detailed ways

The present invention will be further described below in conjunction with embodiment, but the present invention is not limited to following embodiment.

Implementation Case 1

In this experiment, an anti-resonant hollow-core fiber sensor was used to detect breast cancer exosomes. The surface of breast cancer cell exosomes has an excess of MUC1 protein compared to ordinary cell exosomes, so the bioprobe of the sensor uses a nucleic acid aptamer that can bind to MUC1 protein with high specificity, and the precious metal nanoparticles use gold nanoparticles with a diameter of 15nm (AuNPs), the fluorescent dye is FAM dye whose emission spectrum overlaps with the absorption spectrum of 15nm AuNPs. When the breast cancer cell exosomes are filled into the sensor, the fluorescent reporter molecule will be separated from the noble metal nanoparticles due to the specific combination with the MUC1 protein nucleic acid aptamer, and the fluorescent signal will change. The fluorescence recovery value calculated according to the two fluorescence values before and after filling has a linear relationship with the concentration of breast cancer cell exosomes, thereby realizing the detection of breast cancer cell exosomes. The fluorescence recovery value before and after filling breast cancer cell exosomes will be much higher than that of ordinary cell exosomes, so as to distinguish normal cells. The experiment combines the ability of anti-resonant hollow-core fiber to enhance the interaction between light and matter, and cooperates with the fluorescence quenching ability of AuNPs, the specificity of aptamers, and the stability of double-stranded DNA to carry out ultra-sensitive, ultra-low concentration, high-specificity, and stable Detection of exosomes in breast cancer cells.

The experiment is divided into several steps. (1) Synthesis of double-stranded DNA: one strand of the double-stranded DNA is the nucleic acid aptamer of the MUC1 protein, and one end of it is modified with a SH bond for binding to AuNPs. The other chain is a reporter molecule modified with a FAM fluorescent dye. (2) Synthesis of AuNPs-double-stranded DNA: AuNPs and double-stranded DNA were combined by the commonly used salt aging method, and a certain concentration of salt solution was gradually dropped into the mixed solution of AuNPs and double-stranded DNA. Increased salt concentration can shield the negative charge between AuNPs and double-stranded DNA, and gradual dripping can prevent the aggregation of AuNPs. At this time, the SH bond modified at one end of the nucleic acid aptamer in double-stranded DNA can bind to AuNPs. After the formation of AuNPs-double-stranded DNA, due to the close distance between AuNPs and FAM fluorescent dye, and the absorption spectrum of AuNPs with a diameter of 15nm overlaps with the emission spectrum of FAM fluorescent dye, FRET effect will occur between them, which can effectively quench the fluorescence of FAM fluorescent dye . (3) Biological functionalization of HARF: The HARF used in the experiment is a nodeless anti-resonant hollow-core seven-hole fiber. The outer diameter of HARF is about 200 μm, the diameter of the seven cladding tubes that are not in contact with each other is about 13 μm, the thickness of the tube wall is about 400 nm, and the diameter of the inscribed circle surrounded by the seven thin-layer tubes is about 31 μm. This kind of HARF has a wide transmission passband, and the ability to transmit in the visible light 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. A silane coupling agent (APTES) was introduced into the HARF to modify the amino group, and then the AuNPs-double-stranded DNA complex could be combined with the amino group and fixed on the inner wall of the HARF. (4) Sensing detection: The experiment put the biofunctionalized HARF in the sensing system. The experimental setup is shown in Figure 6. To excite the fluorescence of the FAM fluorescent dye, a continuous laser with a wavelength of 473 nm 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 turn, and finally is coupled into the HARF through the plano-convex lens (5). The 490nm long-pass dichroic mirror can pass through the FAM fluorescence and intercept part of the light source laser. The power at the fiber front end was controlled at about 100 μW. When the light source enters the HARF and excites the fluorescent reporter molecule, the fluorescent signal returns to the dichroic mirror through the original path, and then passes through the 500nm long-wave filter (7) to completely filter the light source laser light, and then uses another plano-convex lens (8) to couple to the receiving fluorescent light. In the large-aperture multimode optical fiber (9) of the signal, the fluorescence signal is finally received by the QE65 Pro spectrometer (10), whose detection wavelength range is 185-1100 nm, and the optical resolution is 0.14-7.7 nm. Firstly, pure water was filled in the biofunctionalized HARF as a control. At this time, the fluorescence signal was recorded due to the FRET effect between AuNPs and FAM fluorescent dye. Then when the breast cancer cell exosomes rich in MUC1 protein are filled with biologically functionalized HARF, the exosomes will specifically bind to their matching nucleic acid aptamers, thereby destroying the double-stranded structure of double-stranded DNA. As a result, the fluorescent reporter molecule with the FAM fluorescent dye is released, the fluorescence gradually recovers, and the fluorescent signal at this time is recorded. Finally, the fluorescence recovery value calculated based on the two fluorescence values has a linear relationship with the concentration of breast cancer cell exosomes, thereby realizing the detection of breast cancer cell exosomes. This method takes full advantage of the advantages of HARF to achieve ultra-low concentration, high specificity, rapid and stable detection of exosomes containing MUC1 protein.

Implementation Case 2

In this experiment, an anti-resonance hollow fiber sensor was used to detect Hg ²⁺ . Hg ²⁺ is a heavy metal ion that is extremely harmful to the human body. The ability to detect trace amounts of Hg2 ⁺ in environmental samples is therefore of great interest.

The principle is the same as Case 1, one strand of double-stranded DNA is the nucleic acid aptamer of Hg ²⁺ , and the other strand is the fluorescent reporter molecule connected with HEX fluorescent dye. In order to ensure the FRET effect, gold nanoparticles with a diameter of 80nm were used to quench the fluorescence. The HARF is also treated with APTES to modify the surface of the fiber core with amino functional groups, which is conducive to the effective combination with the AuNPs-double-stranded DNA complex. After the combination, the detection system is in the initial state, and a 532nm wavelength laser that can excite the HEX fluorescent dye is used as the light source. Due to the strong ability of AuNPs to quench the HEX fluorescent dye at this time, the detected background fluorescence value is low. Then pass through the solution containing Hg ²⁺ in the biofunctionalized HARF, at this time, Hg ²⁺ will combine with its nucleic acid aptamer to form an aptamer-mercury ion complex, and the fluorescent reporter molecule will break away from the nucleic acid aptamer and enter the fiber core. dissociated, leading to recovery of fluorescence. The fluorescence recovery value calculated according to the two fluorescence values before and after filling with Hg ²⁺ has a good linear relationship with the concentration of Hg ²⁺ within a certain range, so the concentration of Hg ²⁺ can be analyzed and quantified.

The invention cooperates with the ability of the novel anti-resonant hollow-core optical fiber to bind light waves, the quenching ability of noble metal nanoparticles, and the specificity of aptamers to detect, which greatly reduces the detection limit of biochemical substances compared with traditional detection methods, and also has relatively high High sensitivity and specificity. Compared with other new sensing technologies, the stable structure of double-stranded DNA firmly combines fluorescent reporter molecules with noble metal nanoparticles, effectively solving the problem of low stability in the detection of biochemical substances.

Claims (4)

1. A new type of hollow-core anti-resonance optical fiber sensor combined with noble metal nanoparticles, including a light source part, a collimation part, a sensing part and a signal acquisition part. The light emitted by the light source part is coupled into the sensing part through the collimation part, and the signal acquisition part The detection is realized by partially collecting the optical signal in the sensing part, which is characterized in that: the sensing part adopts a biofunctionalized novel hollow-core anti-resonant optical fiber (HARF) (6), and the biofunctionalized HARF (6) refers to an inner wall fixed HARF with biological probes, the biological probes refer to noble metal nanoparticles combined with double-stranded DNA, wherein the double-stranded DNA is hybridized by a nucleic acid aptamer matching the target analyte and a fluorescent reporter molecule, The fluorescent reporter molecule includes the complementary chain of the nucleic acid aptamer and the fluorescent dye, the absorption spectrum of the noble metal nanoparticles overlaps with the emission spectrum of the fluorescent dye, and an anti-resonant hollow core with a wide transmission passband and very low light attenuation Optical fibers are used to enhance the interaction of light with matter. 2. A novel hollow-core anti-resonance optical fiber sensor combined with noble metal nanoparticles according to right 1, characterized in that: the light source part adopts a continuous laser (1); the collimation part includes sequentially connected discontinuous attenuation sheets (2) , reflector (3), dichroic mirror (4) and the first plano-convex lens (5); the signal acquisition part includes sequentially connected filters (7), the second plano-convex lens (8), large-aperture multimode fiber ( 9) and a spectrometer (10), the laser light emitted by the light source part is coupled into the sensing part through the collimation part, and the signal acquisition part is used to collect the transmitted light passing through the first plano-convex lens (5) and the dichroic mirror (4) successively light signal in the sensing part. 3. A novel hollow-core anti-resonance optical fiber sensor combined with noble metal nanoparticles according to claim 1 or 2, characterized in that: the wavelength of the light source part matches the excitation wavelength of the fluorescent dye. 4. A biochemical detection method in conjunction with noble metal nanoparticles, characterized in that: a nucleic acid aptamer matched with the target analyte and a fluorescent reporter molecule are hybridized to form double-stranded DNA, and then the double-stranded DNA is modified onto the noble metal nanoparticles, Finally, the double-stranded DNA and noble metal nanoparticle complex is immobilized on the inner wall of the HARF, wherein the fluorescent reporter molecule includes the complementary strand of the nucleic acid aptamer and the fluorescent dye; the light source enters the HARF after being collimated, and the absorption spectrum of the noble metal nanoparticle It overlaps with the emission spectrum of the fluorescent dye, and due to the close distance between the noble metal nanoparticles and the fluorescent dye, the FRET effect occurs and the fluorescence of the fluorescent dye is quenched; The target analyte is filled into the HARF, the target analyte binds to its matching nucleic acid aptamer, and the fluorescent reporter molecule is displaced and separated away from the noble metal nanoparticles. At this time, there will be no FRET effect and the fluorescence will be excited by the light source; There is a linear relationship between the fluorescence recovery value calculated according to the previous two fluorescence values and the concentration of the target analyte, thereby realizing the detection of the target analyte.

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