A kind of tilted fiber grating material of gold particle composite silver film and its application as a biosensor

Abstract

The present invention provides a material of a gold particle composite silver film on an inclined fiber grating and its application as a biosensor. The present invention relates to a method for uniformly growing two-dimensional materials on a fiber structure by optical deposition and a method for using the "particle-film" composite fiber grating plasmonic biosensor prepared by this method for PD-L1 molecule detection. Based on the multi-propagation modes of the tilted fiber grating (TFBG), the surface plasmon resonance effect (SPR) of the silver film, and the local surface plasmon resonance effect (LSPR) of the gold particles, multiple coupling modes act together to generate a high-precision comb-shaped sensing spectrum, realizing real-time, in-situ monitoring and analysis of biomolecule modification in a liquid environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of SPR biosensors, and particularly relates to a material of an inclined fiber grating with gold particles composite silver film and the application of the material as a biosensor, and also relates to a biosensor for PD-L1 detection and a detection method. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Malignant tumor is one of the diseases that seriously threaten human health and life at present. Immunotherapy with immune checkpoint blockade has become the most popular immunotherapy today, and the PD-1 / PD-L1 immune blockade therapy is a typical representative. Programmed death receptor 1 (PD-1) is an important immunosuppressive molecule, which can down-regulate the immune system response to avoid autoimmune diseases. While programmed death ligand (PD-L1) on the surface of tumor cells is the ligand of PD-1, and it enables tumor cells to achieve immune escape by binding to PD-1 on the surface of immune cells. The inherent PD-1 of tumor cells can be used as a potential biomarker for patients to select immune checkpoint therapy. Studying and implementing an effective biomarker sensing and detection method with rapidity, high sensitivity and good specificity is an important guarantee for realizing the precise treatment of immune checkpoint blockade therapy.

[0004] Optical fiber SPR-based biosensors are convenient for integration and coupling, enabling high-throughput and highly sensitive detection of biomolecules. By combining the surface plasmon mode of the plasma structure with the strong evanescent field of highly sensitive micro-nano optical fibers, the local surface plasmon enhancement effect can be excited, enhancing the interaction with the sample to be measured. Common plasma structures are gold and silver coatings, but they have defects such as strong confinement effects and weak biological affinity. MXene is an ideal two-dimensional material that has recently emerged and can improve the application scenarios of optical fiber SPR sensors. Its main features are: having a unique accordion-like structure, which can increase the specific surface area and promote the adsorption of molecules; a large number of functional groups are convenient for the modification of biomolecules; excellent plasma properties. Theoretical calculations show that the sensitivity of the SPR sensor can be significantly improved by optimizing the number of MXene layers. Effective SPR enhancement was achieved using relatively thick Ti3C2-MXene nanosheets deposited on the fiber by photodeposition. The thickness of this coating is 5 μm, which is not a two-dimensional material in the traditional sense. However, the hydrophilic functional groups of this material promote complete contact between the analyte and the sensor surface, thus increasing the effective sensing area. However, at present, continuous and uniform single-layer or few-layer MXene has not been obtained experimentally, which limits the development and application of the sensor. The preparation limitation of uniform and large-area two-dimensional materials is the primary problem in the development of optical fiber SPR biosensors.

[0005] Meanwhile, it should be noted that the penetration depth of SPR is very important. The penetration depth of surface plasmon polaritons based on metal films in the medium is about 100 nanometers. The 100-nanometer region on the metal surface is the SPR hot spot region. Once beyond this range, the SPR intensity will decay exponentially. For local surface plasmon polaritons based on metal particles, although the SPR intensity can be enhanced by controlling the gap between two particles, it is a challenge to manipulate the molecular distribution at the gap. These single plasmon modes with simple structures lead to inefficient utilization of the hot spot region, which limits the detection of biological macromolecules such as proteins and long-chain DNA. The characteristic lengths of these macromolecules reach several hundred nanometers, and the binding conditions of molecules distributed outside the sensitive region cannot be effectively detected. Therefore, new plasma structures need to be developed to enhance the ability to capture and collect signals for macromolecules. Summary of the Invention

[0006] The present invention proposes an inclined fiber Bragg grating / silver film / Ta2C-MXene / gold particle composite plasmonic structure. This invention solves the problem of difficult preparation of MXene two-dimensional materials on fiber optic sensors and improves the effective sensing area of the sensors; furthermore, the present invention adopts a novel silver film / Ta2C-MXene / gold particle plasmonic composite structure to change the previous single excitation mode, realizing the effective localization and signal amplification of macromolecules. The proposed modification method provides a new way for the development of highly sensitive, highly specific, rapid and label-free PD-L1 analysis and detection, and early prediction of the efficacy of immune checkpoint inhibitor therapy.

[0007] Based on the above technical effects, the present invention provides the following technical solutions:

[0008] In the first aspect of the present invention, there is provided an inclined fiber Bragg grating material with gold particle composite silver film. The main body of the material is an inclined fiber Bragg grating, the surface of the optical fiber has a silver film, and the silver film is covered with Ta2C-MXene / gold particles.

[0009] In the inclined fiber Bragg grating material with gold particle composite silver film designed by the present invention, first, the surface plasmon polaritons and localized surface plasmon polaritons supported by the "particle-film" composite structure formed by gold particles and silver film generate coupling, expanding the resonance sensitive region to the outside of the particles, thus facilitating the amplification of molecular signals. In addition, the two metal elements of gold and silver form a bimetallic plasmonic coupling structure, which can also expand the plasmonic excitation band.

[0010] In the above material, the inclined fiber Bragg grating as the main body of the material is a single-mode silica optical fiber, which has a Bragg grating structure by writing. In one embodiment of the present invention, the grating is obtained by the phase mask technique: the laser beam, the mask template, and the grating region are kept on the same axis, and different orders of diffraction fringes are generated by irradiating the mask template with ultraviolet laser, generating a permanent refractive index modulation in the fiber core, and then rotating the mask template by a certain angle along the axial direction to obtain an inclined grating structure. The parameters such as the inclination angle and distance of the fiber grating can be adjusted according to the expected effective refractive index interval factor.

[0011] In addition, the above inclined fiber Bragg grating should also have certain light-sensitive characteristics. In one embodiment provided by the present invention, in order to enhance the light-sensitive characteristics of the above optical fiber material, the above optical fiber material needs to be hydrogen-loaded, and annealed at 95-105 °C for 40-50 h after writing to remove hydrogen.

[0012] Preferably, the silver film covers the grating region of the optical fiber.

[0013] Preferably, the thickness of the silver film is 40-60 nm; in order to achieve good coupling between the optical fiber and the silver film, the silver film can be vacuum-evaporated by multiple depositions. Further, the vacuum degree of evaporation should be lower than 7×10 -5 Pa, and the deposition rate is

[0014] Preferably, the Ta2C-MXene / gold particles are deposited on the surface of the silver film by photoinduced deposition. The specific steps are as follows: Drop the solution containing Ta2C-MXene / gold particles on the surface of the silver film and perform deposition by laser irradiation. The wavelength of the laser is 1300-1700 nm, and the deposition time is 100-120 min.

[0015] Since the growth area of Ta2C-Mxene can be reflected by the change of the spectrum, further, the present invention also provides a method for controllable growth of Ta2C-MXene / gold particles on the surface of the silver film, that is, connecting the optical fiber with a silver film coating to a spectrometer, then connecting a laser light source, applying a solution containing Ta2C-MXene / gold particles on the surface of the silver film for induced deposition, and stopping the deposition process when the wavelength signal in the spectrum no longer increases.

[0016] The above controllable growth method provided by the present invention overcomes the technical defect that the existing fiber optic SPR biosensor cannot prepare a uniform and large-area Mxene. Not only a continuous, uniform and high-quality Ta2C-MXene thin film is obtained by photodeposition, but also the three-dimensional MXene structure increases the specific surface area of the sensing area and provides binding sites for biomolecules. After the biomolecules bind, they are naturally located in the gaps between the particles and the hot spot areas between the particles and the film, directly increasing the modification efficiency of the molecules.

[0017] Furthermore, the concentration of the solution of Ta2C-MXene / gold particles is 1.5-2.5 mg / mL, and the diameter of the gold particles is 18-22 nm; in a specific embodiment of the present invention, the above Ta2C-MXene / gold particles are synthesized based on the chemical reduction method: Add a mixed solution of NaBH4 and NaOH as a reducing agent to the Ta2C-MXene dispersion, and then add HAuC14 solution to prepare the Ta2C-MXene / gold particles by ultrasonic dispersion.

[0018] In the second aspect of the present invention, an application of the tilted fiber Bragg grating material of the gold particle composite silver film described in the first aspect as a biosensor is provided.

[0019] In the above-mentioned tilted fiber grating material of gold particle composite silver film, the Ta2C-Mxene two-dimensional material has a uniform large surface, can have a good binding effect with biomolecules, and can identify and bind to the target to be detected in the environment by connecting biological probes; the biological probes are nucleic acids, proteins or polypeptides that can specifically bind or capture the detection target. In an implementation manner of the above second aspect application, it is designed to be used for the capture of PD-L1 exosomes.

[0020] Therefore, in the third aspect of the present invention, a biosensor for PD-L1 detection is provided. The biosensor uses the silver film-covered area in the tilted fiber grating material of the gold particle composite silver film described in the first aspect as the sensing area. The sensing area binds and captures the multifunctional peptide chain of PD-L1 exosomes through the Ta2C-Mxene two-dimensional material. The probe design includes the following functional domains: anti-fouling domain (SS), assembly domain (IMVTESSD), binding domain (YSSY), and recognition domain (FHYQRDTPKSYN). The above functional domains are connected by amide bonds or have a linker before, and are connected to Ta2C-Mxene through π-π bonds. In a specific implementation manner, the amino acid sequence of the probe is as follows: SSIMVTESSDYSSYFHYQRDTPKSYN.

[0021] Preferably, the biosensor is constructed as follows: The surface of the Ta2C-Mxene two-dimensional material in the tilted fiber grating material of the gold particle composite silver film described in the first aspect is cleaned, and a buffer solution containing the probe is applied to the surface of the two-dimensional material.

[0022] Further, the concentration of the probe in the buffer solution is 10 4 ~10 6 Particles / mL.

[0023] In the fourth aspect of the present invention, a method for detecting PD-L1 exosomes is provided. The detection method includes capturing PD-L1 exosomes in a sample to be tested by using the biosensor described in the third aspect, and detecting the concentration of PD-L1 exosomes by detecting the degree of SPR signal shift of the fiber grating material.

[0024] Preferably, the specific steps of the detection method are as follows: A sample containing PD-L1 exosomes to be tested is passed through the surface of the sensing area in the sensor, and the sensor captures the PD-L1 exosomes; further, the flow rate of the sample to be tested is 4-6 μL / min.

[0025] The beneficial effects of the above one or more technical solutions are:

[0026] In addition, the present invention provides a biosensor based on a gold particle composite silver film inclined fiber grating material, which mainly operates in the near-infrared light wave band. This optical frequency range not only has natural biocompatibility with the organism to be detected (compared with visible light detection, the near-infrared band where the fiber grating operates causes less damage to biological tissues and has stronger penetration), but also can inherit modern communication and display technologies and related devices (light sources, modulators, detectors, etc.). Micron-level optical fibers can be packaged and integrated into probe-type sensors for real-time in-situ detection in organisms, with great practical application value. Brief Description of the Drawings

[0027] The schematic diagrams in the specification forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0028] Figure 1 is the spectral change of the resonance wavelength during the growth process of Ta2C-MXene;

[0029] Figure 2 is a schematic diagram of the sensor optical path and the sensing area;

[0030] Figure 3 is the detection spectrum and linear relationship of different concentrations of PD-L1 molecules. Detailed Description of the Embodiments

[0031] It should be noted that the following detailed descriptions are all illustrative and are intended to provide further explanations of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0032] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0033] In order to enable those skilled in the art to more clearly understand the technical solutions of the present invention, the technical solutions of the present invention will be described in detail below in combination with specific embodiments.

[0034] Example 1

[0035] This example provides a preparation method for a gold particle / Ta2C-MXene / silver film composite plasmonic fiber grating biosensor and a method for detecting PD-L1 molecules. The implementation steps are as follows:

[0036] 1. Inclined fiber Bragg grating inscription

[0037] The optical fiber used for inscription is a single-mode silica optical fiber, with the following parameters: core diameter: 8.2 μm, refractive index 1.45000; cladding diameter 125 μm, refractive index 1.47650.

[0038] The optical fiber is subjected to hydrogen loading treatment to enhance the photosensitive characteristics of the core. Take an optical fiber with a length of 60 - 80 cm, and remove the cladding area with a length of 1.5 - 2 cm at the center position for grating structure inscription. After wiping it clean with alcohol, fix the optical fiber on the inscription platform. The grating structure is obtained by the phase mask technique. Keep the laser beam, mask template, and grating area on the same axis. Use ultraviolet laser with a wavelength of 248 nm to irradiate the mask template to generate diffraction fringes of different orders, and produce permanent refractive index modulation in the core. Rotate the mask template by a certain angle along the axial direction to obtain an inclined grating structure. The specific parameters of the phase mask template are as follows: center spacing = 1099.05 nm, mask length = 10 mm, mask height = 15 mm, substrate length = 17.17 mm, substrate height = 25.4 mm, inclination angle 12°. During the inscription process, pay attention to keeping the optical fiber stable and mark the grating inclination direction for subsequent film deposition. The inscription time for the entire Bragg grating is 25 - 30 minutes. After inscription, the TFBG needs to be annealed at 100 °C for 48 hours to remove hydrogen.

[0039] 2. Silver film deposition

[0040] The silver film is obtained by vacuum thermal evaporation coating. In order to ensure good coupling between the cladding mode of the TFBG and the SPR of the silver film, a two-step deposition method is used. The deposited film layers are respectively coated on the upper and lower reflection directions of the grating to obtain the maximum optical field energy coupling. The specific implementation method is as follows:

[0041] Fix the optical fiber on the substrate, and then put the optical fiber, tungsten evaporation boat, and silver target (purity 99.999%) into the evaporation chamber. Wait until the vacuum degree is lower than 7×10 -5 Pa and then start coating. Gradually increase the current to 40 A, and silver starts to pre-evaporate. Gradually adjust the current size so that the deposition rate is After the rate is stable, open the substrate baffle to start evaporation coating. The film thickness is monitored by a quartz crystal film thickness monitor. Stop evaporation coating when the thickness reaches 50 nanometers to complete one silver film deposition. After taking out the optical fiber, rotate it 180° along the axial direction and repeat the above steps to evaporate again to obtain a TFBG uniformly wrapped with a uniform silver film.

[0042] 3. Ta2C-MXene / gold particle assembly

[0043] Disperse 20 mg of Ta2C-MXene powder in 10 mL of deionized water and ultrasonicate for 2 hours. After standing for 10 minutes, remove the precipitate to obtain few-layer Ta2C-MXene nanosheets. Then, Ta2C-MXene / gold particles are synthesized using a chemical reduction method. First, take 8 mL of the Ta2C-MXene dispersion and add 25 μL of a mixed solution of 0.16 M NaBH4 and 1.6 M NaOH as a reducing agent. Then, add 30 μL of the HAuC14 solution (0.1 g / mL). The whole process is carried out under magnetic stirring and the mixture is stirred at a speed of 1000 rpm for 30 minutes. The resulting solution is centrifuged at a speed of 12000 rpm for 5 minutes. After collecting the precipitate, add deionized water and ultrasonicate for 20 minutes to redisperse. Repeat the above centrifugation-ultrasonication redispersion process three times to obtain Ta2C-MXene / gold particles. The diameter of the obtained gold particles is between 18 - 22 nanometers.

[0044] Through the photoinduced deposition method, the assembly of silver-coated TFBG and Ta2C-MXene / gold particles is achieved. Under the irradiation of the input laser, the evanescent field generated in the sensing region adsorbs Ta2C-MXene nanosheets, making them adhere uniformly to the sensing region. During this process, the optical fiber is connected to a composite light source (ASE-CL-30-M and SLD-1470-10-B). Subsequently, use a syringe to drop 500 μL of the Ta2C-MXene nanosheet dispersion with a concentration of 2 mg / mL into the sensing region. The optical fiber is connected to a spectrometer (Yokogawa AQ63700) for signal recording and analysis. The increase in the growth area of the two-dimensional material is accompanied by a red shift of the SPR wavelength, while the increase in the material thickness leads to an increase in the signal peak amplitude. Therefore, the growth condition of Ta2C-MXene can be reflected by the change in the spectrum. As Figure 1 shown, as the growth time increases, the resonance peak gradually shifts towards the direction of larger wavelengths, indicating that the deposition area of Ta2C-MXene nanosheets gradually increases. At the same time, the resonance peak amplitude also increases with time, representing the increase in the thickness of Ta2C-MXene nanosheets. When the growth time reaches 100 - 120 minutes, the resonance wavelength of the spectrum basically no longer changes, only the amplitude increases. This phenomenon indicates that Ta2C-MXene has completely covered the silver film surface. Continuing to increase the deposition time will only increase the thickness of Ta2C-MXene. Using this method, the controllable growth of Ta2C-MXene nanosheets on the optical fiber can be achieved.

[0045] 4. Probe molecule modification

[0046] Fix the sensor as Figure 2The detection light path shown. The channel parameters of the microfluidic chip are as follows: width: 2μm, height: 2μm, length: 3.8cm. Then, 300μL of 100μg / mL polypeptide chains with anti-pollution function and specific PD-L1 exosome capture function flowed through the fiber grating area at a flow rate of 5μL / min, and the polypeptide chains were fixed on the Ta2C-MXene surface through π-π bond stacking. After that, the buffer solution was circulated in the microfluidic chip for 10 minutes to remove excess polypeptide chains. The buffer solution used in the modification process was deionized water, and the entire modification process was carried out under the monitoring of a spectrometer.

[0047] 5. PD-L1 molecular detection

[0048] First, the microfluidic chip sensing area was cleaned with deionized water at a flow rate of 5 μL / min. Then, 300 μL of PD-L1 exosomes with different concentrations were flowed through the sensor sensing area at a flow rate of 5 μL / min. During the process, PD-L1 molecules gradually combined with polypeptide chains, changing the resonance frequency of the plasma structure, which was reflected as a shift in the SPR signal in the spectrum.

[0049] Figure 3 The spectra and linear relationships of PD-L1 molecules at different concentrations are shown. During the modification process, the binding of molecules to the Ta2C-MXene surface causes a change in the surface refractive index, which in turn affects the shift of the SPR wavelength in the spectrum. When PD-L1 exosomes of different concentrations are modified to the surface, the number of bound molecules affects the slight change in the SPR wavelength. At this time, the slight shift of the SPR wavelength can be measured by measuring the change in amplitude. Figure 3 The fitting curve on the right shows an excellent linear relationship between concentration and signal amplitude. The specific relationship is y = 0.61433 × Log (C) + 17.36779, where y represents the signal intensity and C is the solution concentration. 4 to 1×10 6 Correlation of phenomena within the concentration range R 2 The detection limit is 6.36×10 3 particles per ml.

[0050] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A tilted fiber grating material of gold particle composite silver film, characterized in that, The material body is an inclined fiber grating, the surface of the optical fiber has a silver film, and the Ta2C-MXene / gold particles are covered at the silver film; The Ta2C-MXene / gold particles are synthesized based on a chemical reduction method: a mixed solution of NaBH4 and NaOH is added to the Ta2C-MXene dispersion liquid as a reducing agent, and then an HAuC14 solution is added and dispersed by ultrasonic waves to prepare the Ta2C-MXene / gold particles.

2. The inclined fiber Bragg grating material of the gold particle composite silver film according to claim 1, wherein The inclined fiber grating is a single-mode quartz optical fiber and has a Bragg grating structure by inscription.

3. The tilted fiber Bragg grating material of the gold particle composite silver film according to claim 2, characterized in that, The grating is obtained by a phase mask technique: the laser beam, the mask template, and the grating area are kept on the same axis. Different orders of diffraction fringes are generated by irradiating the mask template with ultraviolet laser, and permanent refractive index modulation is generated in the fiber core. Then, the mask template is rotated by a certain angle along the axis to obtain an inclined grating structure.

4. The tilted fiber grating material of the gold particle composite silver film according to claim 3, characterized in that, The inclined fiber grating also needs to be hydrogen-loaded and annealed at 95-105 °C for 40-50 h after inscription to remove hydrogen.

5. The tilted fiber grating material of the gold particle composite silver film according to claim 1, characterized in that, The silver film covers the grating area of the optical fiber.

6. The inclined fiber Bragg grating material of the gold particle composite silver film according to claim 5, characterized in that The thickness of the silver film is 40-60 nm; the silver film is deposited by vacuum evaporation.

7. The tilted fiber grating material of the gold particle composite silver film according to claim 6, characterized in that, The degree of vacuum in the vacuum evaporation is lower than 7×10 -5 Pa, and the deposition rate is 8. The tilted fiber grating material of the gold particle composite silver film according to claim 1, characterized in that, The Ta2C-MXene / gold particles are deposited on the surface of the silver film by photoinduced deposition. The specific steps are as follows: a solution containing Ta2C-MXene / gold particles is dropped on the surface of the silver film and deposited by laser irradiation. The wavelength of the laser is 1300-1700 nm, and the deposition time is 100-120 min.

9. The tilted fiber Bragg grating material of the gold particle composite silver film according to claim 8, characterized in that, The specific steps of the photoinduced Ta2C-MXene / gold particles are as follows: the optical fiber with a silver film coating is connected to a spectrometer, and then a laser light source is connected. A solution with Ta2C-MXene / gold particles is applied to the surface of the silver film for induced deposition. When the resonance wavelength in the spectrum no longer shifts, the deposition process is stopped.

10. The tilted fiber grating material of the gold particle composite silver film according to claim 9, characterized in that, The concentration of the solution of Ta2C-MXene / gold particles is 1.5-2.5 mg / mL, and the diameter of the gold particles is 18-22 nanometers.

11. Application of the inclined fiber grating material with gold particle composite silver film according to any one of claims 1-10 as a biosensor.

12. A biosensor for PD-L1 detection, characterized in that, The biosensor uses the silver film-covered area in the inclined fiber grating material with gold particle composite silver film according to any one of claims 1-10 as a sensing area, and the sensing area binds and captures a multifunctional peptide chain of PD-L1 exosomes through the Ta2C-Mxene two-dimensional material.

13. The biosensor for PD-L1 detection according to claim 12, wherein The construction method of the biosensor is as follows: the surface of the Ta2C-Mxene two-dimensional material in the inclined fiber grating material with gold particle composite silver film is cleaned, and a buffer solution containing a probe is applied to the surface of the two-dimensional material.

14. The biosensor for PD-L1 detection according to claim 13, wherein The concentration of the probe in the buffer is 10 4 ~10 6 Particles / mL.

15. A method for detecting PD-L1 exosomes, characterized in that, The detection method includes capturing PD-L1 exosomes in a sample to be measured by using the biosensor according to claim 12, 13, or 14, and detecting the concentration of PD-L1 exosomes by detecting the degree of SPR signal shift of the fiber grating material.

16. The detection method of the PD-L1 exosome according to claim 15, wherein The specific steps of the detection method are as follows: a sample containing PD-L1 exosomes to be measured is flowed through the surface of the sensing area in the sensor, and the sensor captures the PD-L1 exosomes.

17. The detection method of PD-L1 exosomes according to claim 16, wherein The flow rate of the sample to be measured is 4 - 6 μL / min.

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