Exosome quantitative detection method based on TFBG-SPR

Abstract

The invention provides an exosome quantitative detection method based on TFBG-SPR (Total Fiber Bragg Grating-Surface Plasmon Resonance). The detection device is composed of a broadband light source, a single mode fiber, a polarization controller, a TFBG-SPR exosome sensor, a detection sample pool and a spectrum analyzer. The detection principle can be summarized as follows: light emitted by the broadband light source is transmitted to the polarization controller through the single-mode optical fiber, incident light is modulated to be in a P polarization state at the position of the polarization controller, P-state light is transmitted to the TFBG-SPR exosome sensor, the sensor is fixed in the detection sample pool, and finally an output light signal is presented on the spectrum analyzer in a transmission spectrum mode. In the detection process, an exosome is selectively captured by a CD63 antibody layer on the surface of the TFBG-SPR exosome sensor, so that the refractive index of the surface of the TFBG-SPR exosome sensor is changed, and an output transmission spectrum drifts. By comparing the relationship between the concentrations of different exosome solutions and the transmission spectrum drift amount, the portable, rapid and high-sensitivity quantitative detection of the exosome is realized. And meanwhile, directional measurement of different types of exosomes can be realized by changing the types of detection antibodies on the surface of the sensor. The invention provides a novel method which is high in sensitivity, simple to operate and rapid for quantitative detection of the exosome, and has wide application value in the aspects of cancer prediction, diagnosis and prognosis and other biomedical detection.

Description

Technical Field

[0001] This invention relates to the fields of fiber optic sensing technology, surface chemistry and bioconjugation technology, and to a quantitative detection method for exosomes based on TFBG-SPR. Background Technology

[0002] Exosomes are tiny vesicles, 30-150 nm in diameter, produced by cells and carrying a wealth of biological information, such as specific surface proteins, RNA, and DNA. Exosomes are stably present in almost all bodily fluids, such as blood, saliva, and urine. As an important intercellular communication tool, exosomes play a crucial role in both physiological and pathological processes.

[0003] As an important detection substance in liquid biopsy, exosomes are considered promising biomarkers for early cancer diagnosis and prognosis. Simple, reliable, and sensitive cancer exosome biosensors are crucial for early cancer diagnosis and prognosis.

[0004] Traditional exosome detection requires two steps: separation and detection, and suffers from drawbacks such as operational complexity, high cost, and long detection time. Therefore, research on platforms that can directly achieve exosome separation and detection is increasing daily.

[0005] Fiber optic sensors, due to their small size, light weight, and ease of integration, have outstanding advantages among a range of biosensors. Among fiber optic sensors, tilted fiber gratings (TFBGs), as a highly sensitive structure, combined with a plasmon resonance (SPR) platform, have shown significant effectiveness in detecting low concentrations of biomolecules, providing a new solution for highly sensitive quantitative detection of exosomes. Summary of the Invention

[0006] To overcome the shortcomings of traditional detection technologies, this invention provides a quantitative detection method for exosomes based on TFBG-SPR. The detection device consists of a broadband light source, a single-mode optical fiber, a polarization controller, a TFBG-SPR exosome sensor, a sample cell, and a spectrometer. The broadband light source is connected to the left end of the TFBG-SPR exosome sensor via the single-mode optical fiber and the output of the polarization controller, while the right end of the TFBG-SPR exosome sensor is connected to the spectrometer.

[0007] The TFBG-SPR exosome sensor has the following structure: the sensor consists of a photosensitive optical fiber with TFBG etched on it, a single-mode optical fiber for transmission, a gold film layer, and a CD63 antibody layer.

[0008] The fabrication steps of the TFBG-SPR exosome sensor are as follows: Ⅰ) Photosensitive fiber fusion splicing for transmission of single-mode fiber; II) Hydrogen-carrying optical fiber for 14 days; III) Etching TFBG in the core of a hydrogen-loaded photosensitive optical fiber; IV) Annealing of photosensitive optical fibers etched with TFBG; V) Magnetron sputtering of a gold film on the surface of the etched TFBG; VI) A CD63 antibody layer was chemically assembled on a gold film on the surface of TFBG; thus, the TFBG-SPR exosome sensor was prepared.

[0009] Step I) Photosensitive fiber double-end fusion splicing transmission single-mode fiber: The single-mode fusion splicing mode is completed using a fiber optic fusion splicer. The photosensitive fiber used is 20 mm long, with a core diameter of 8 μm and a cladding diameter of 125 μm; the transmission single-mode fiber (2) has a core diameter of 8 μm and a cladding diameter of 125 μm. Step II) Hydrogen loading of photosensitive fiber for 14 days: Using a high-pressure hydrogenation reactor, the pressure value is set to 10-15 MPa and the temperature is 75 ℃. The photosensitive fiber obtained in step I) with double-ended fusion spliced ​​transmission single-mode fiber is placed in the reactor and maintained for 14 days. Step III) Etching TFBG in the hydrogen-loaded photosensitive fiber core: The method for etching TFBG in the photosensitive fiber is the phase mask method, the etching length of TFBG is 10 mm, and the tilt angle is 8°. Step IV) Annealing of photosensitive fiber etched with TFBG: The annealing temperature of the photosensitive fiber etched with TFBG is 120℃ and the time is 12 h. Step V) Magnetron sputtering of gold film on the surface of TFBG: The instrument used for magnetron sputtering of gold film is a high-vacuum magnetron sputtering fiber coating instrument, and the thickness of the gold film is 50 nm. Step VI) Chemical assembly of CD63 antibody layer on gold film on TFBG surface: CD63 antibody layer is assembled on gold film surface by chemical bonds.

[0010] The steps of the TFBG-SPR-based exosome quantitative detection method are as follows: First, the TFBG-SPR exosome sensor (4) is fixed in the detection sample cell (5). The incident light emitted by the broadband light source (1) is transmitted through the single-mode fiber (2) to the polarization controller (3), which adjusts the incident light to the P polarization state. The P polarization state beam is transmitted to the TFBG-SPR exosome sensor (4), and finally the transmission spectrum is obtained on the spectrometer (6). Then, the exosome solution to be tested is added to the sample cell (5), and the CD63 antibody layer (44) realizes the exosome in the solution to be tested. The capture of exosomes causes a change in the surface refractive index of the TFBG-SPR exosome sensor (4); this change in refractive index is sensed by the TFBG-SPR exosome sensor (4) and is ultimately reflected in the change of the transmission spectrum on the spectrometer (6); secondly, before changing the exosome sample solution with different concentrations for detection, the detection sample cell (5) is cleaned with deionized water; finally, the data recorded on the spectrometer (6) of adding exosome solutions of different concentrations to the detection sample cell (5) are fitted to obtain the relationship between the gradient exosome concentration solution and the transmission spectrum drift, so as to realize the quantitative detection of exosomes.

[0011] The theoretical analysis based on the TFBG-SPR exosome sensor is as follows: First, the effective refractive index of each cladding mode of TFBG is related to its resonant wavelength. The relationship between them can be represented as follows: , in , Let i and j represent the effective refractive indices of the i-th cladding mode and the core mode, respectively. It is the grating period. It is the tilt angle between the grating plane and the fiber axis.

[0012] The resonance intensity of the cladding mode resonance in the transmission spectrum can be expressed as, , Where T represents the resonance intensity of the cladding mode. This is the coupling coefficient between the cladding mode and the core mode, and L is the length of the TFBG. Due to the evanescent field of the cladding mode, the effective refractive index... It depends on the surrounding refractive index.

[0013] After depositing a 50 nm thick gold film on the TFBG surface, the TFBG-SPR spectrum can be obtained.

[0014] It is worth noting that only P-polarized core modes can be emitted into the TFBG, and only then can the cladding modes of the TFBG be excited and coupled to the SPR mode. The effective refractive index of the SPR mode can be expressed as, , in It is the relative permittivity of the metal thin film. It is the relative capacitance of the surrounding medium.

[0015] Based on the above theories, the working principle of the TFBG-SPR-based exosome quantitative detection method provided by this invention can be summarized as follows: When incident light is transmitted to the TFBG, it is completely reflected by the fiber sidewall, exciting an evanescent surface plasmon resonance wave on the gold film surface. P-polarized light enters the gold film, and the free electrons of the gold film interact with the P-polarized component of the light, generating a plasma wave. At the interface between the fiber and the gold film, plasma vibration forms a surface plasmon wave. When the horizontal component of the evanescent wave vector matches the wave vector of the surface plasmon wave and energy transfer occurs, the evanescent wave and the surface plasmon wave resonate, producing surface plasmon resonance. Light energy near the resonance wavelength is absorbed, resulting in a resonance trough in the output transmission spectrum. The CD63 antibody on the TFBG surface captures exosomes in the test solution, causing a change in local refractive index, and consequently, changes in the resonance trough and resonance wavelength. Therefore, the concentration of the exosome solution can be inferred by monitoring the shift or intensity change of the resonance wavelength.

[0016] The TFBG-SPR exosome sensor comprises an etched TFBG photosensitive fiber, a single-mode transmission fiber, a gold film layer, and a CD63 antibody layer. The single-mode transmission fiber serves as both the input channel for incident light and the output channel for output light. During detection, the etched TFBG photosensitive fiber region is directly immersed in the exosome sample solution. The CD63 antibody layer then captures the exosomes in the sample, causing a change in the refractive index of the sensor surface. This change in refractive index is sensed by the etched TFBG photosensitive fiber, resulting in a change in the output spectral signal, thus enabling the measurement of exosome sample solutions with different concentrations.

[0017] The beneficial effects of this invention are as follows: The fiber optic TFBG-SPR sensor offers the advantages of label-free, real-time detection, allowing for direct separation and detection of exosomes on the surface of the fiber optic device. Compared to the labeling and washing steps of traditional ELISA or fluorescence methods, this greatly simplifies the detection process. It also significantly improves sensitivity, as the fiber optic TFBG-SPR sensor directly excites surface plasmon resonance on the gold film surface, and the binding of exosomes with the functionalized gold film causes a change in refractive index. Furthermore, it exhibits strong selectivity; changing the type of detection antibody on the sensor surface allows for targeted measurement of different types of exosomes. Simultaneously, the fiber optic TFBG-SPR sensor offers advantages such as miniaturization and remote operation, making it suitable for point-of-care and in vivo monitoring. Its small size, flexibility, and low transmission loss allow for integration with portable spectrometers, enabling point-of-care testing (POCT) or minimally invasive implantable continuous monitoring, overcoming the limitations of traditional SPR instruments that are large and fixed in use. Attached Figure Description

[0018] Figure 1 This is a diagram of a detection system based on the TFBG-SPR exosome sensor.

[0019] Figure 2 Flowchart for the fabrication of the TFBG-SPR exosome sensor.

[0020] Figure 3 This is the original SPR spectrum of the TFBG-SPR sensor.

[0021] Figure 4 This is a spectral response diagram for detecting gradient concentration exosome solutions using this method.

[0022] Figure 5 According to Figure 4 The graph shows the intensity of the six selected envelope patterns (patterns 1-6 from left to right) near the SPR as a function of exosome concentration. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0024] Example 1: Fabrication of TFBG-SPR exosome sensor.

[0025] In this embodiment, see Appendix Figure 1 Chinese illustrations, with Figure 1 The inset shows a TFBG-SPR exosome sensor, comprising: a photosensitive optical fiber (41) for etching TFBG, a single-mode optical fiber for transmission (42), a gold film layer (43), and a CD63 antibody layer (44).

[0026] See appendix Figure 2The present invention provides a method for preparing a TFBG-SPR exosome sensor, comprising the following six steps: I) Photosensitive fiber double-ended fusion splicing for transmission of single-mode fiber; First, take a 20 mm photosensitive fiber, use fiber strippers to strip off the fiber coating, and then use a fiber cleaver to cut its double ends flat; similarly, use a fiber cleaver to cut the end face of the single-mode fiber flat, and then use the single-mode fusion splicing mode of a small fiber fusion splicer to fusion the double ends of the photosensitive fiber with the two single-mode fibers respectively, leaving a length of 150 mm for transmission of single-mode fiber, to obtain photosensitive fiber with double-ended fusion splicing for transmission of single-mode fiber.

[0027] II) Hydrogen-loaded optical fiber for 14 days; using a high-pressure hydrogenation reactor, set the pressure to 15 MPa and the temperature to 75 °C, place the optical fiber obtained in step I) with the double-ended fusion spliced ​​single-mode fiber into the reactor and maintain it for 14 days. Multiple optical fibers can be placed at one time.

[0028] III) Etching TFBG in the core of a hydrogen-loaded photosensitive fiber; the fabrication method is the phase mask method. The hydrogen-loaded and sensitized fiber obtained in II) is placed into the fiber fixture of the etching system, so that the stripes of the phase mask are at an 8° angle to the axis of the photosensitive fiber. The high-energy pulsed ultraviolet laser generated by the frequency-doubled argon ion laser is controlled by the precision displacement stage to etch the TFBG in the fiber structure onto the photosensitive fiber through the phase mask, thus obtaining the photosensitive fiber with TFBG etched in the core.

[0029] IV) Annealing of photosensitive optical fiber after TFBG etching: Place the TFBG-etched optical fiber into a high-temperature furnace, set the temperature to 120℃, and hold for 12 hours to eliminate stress and drive away unbound hydrogen.

[0030] V) Magnetron sputtering of a gold film layer on the etched TFBG surface; using a high-vacuum magnetron sputtering fiber coating instrument, the target material is pure gold with a purity of 99.999%. The TFBG fiber device obtained in step IV) is loaded into a customized fiber mold. The fiber mold platform can realize the revolution and rotation of the fiber, ensuring the uniform formation of the gold film during the sputtering process. The sputtering pressure is 0.5 Pa and the sputtering time is 100 s, resulting in a gold film layer with a thickness of 50 nm.

[0031] VI) Chemically assemble the CD63 antibody layer on the TFBG surface gold film; the following assembly regions are for the TFBG-SPR sensing region.

[0032] Assemble carboxyl groups (-COOH) on the surface of the gold film: First, clean the sensing area with deionized water and ethanol. Next, prepare a 5 mM MUA (mercaptoundecanoic acid) solution with ethanol as the solvent. Immerse the fiber optic sensing area in the MUA solution for 12 h. After the immersion, clean it with ethanol and deionized water for later use.

[0033] Activation of -COOH on the surface of the gold film: Prepare a mixed solution of EDC and NHS (concentrations of 0.4 M and 0.2 M, respectively) and mix them together in a 1:1 volume ratio. Immerse the fiber optic sensing area in the mixed solution for 30 min to activate the -COOH on the surface of the gold film. After the activation, wash with PBS and set aside for later use.

[0034] Assemble the CD63 antibody layer: Prepare a CD63 antibody solution with a concentration of 25 μg / mL using PBS as the solvent. Immerse the activated -COOH fiber sensing region in the CD63 antibody solution for 1.5 h. After immersion, wash with PBS to obtain the TFBG-SPR exosome sensor.

[0035] Example 2: Construction of the detection platform.

[0036] In this embodiment, see Appendix Figure 1 It is a quantitative exosome detection system based on TFBG-SPR, including: a broadband light source (1), a single-mode fiber (2), a polarization controller (3), a TFBG-SPR exosome sensor (4), a detection sample cell (5), and a spectrometer (6). The broadband light source (1) has a wavelength range of 1420~1620 nm and is used to provide the light source in the system. The single-mode fiber (2) is used for the optical path transmission of various optical elements in the platform. The polarization controller (3) is used to obtain a larger stripe contrast. The detection sample cell (5) is used to drop the exosome solution to be tested. The spectrometer (6) is used to monitor and record the spectral changes. When the CD63 antibody modified on the surface of the TFBG-SPR exosome sensor (4) captures the exosomes in the test solution, the refractive index of the sensor surface changes, which is reflected in the drift of the transmission spectrum on the spectrometer (6). By comparing the relationship between different exosome concentrations and the transmission spectrum drift, the quantitative detection of exosomes is realized.

[0037] Example 3: Detection of exosome gradient concentration solutions.

[0038] The specific testing process is as follows: First, 100 μg of commercial exosome lyophilized powder (containing 1×10⁻⁶ exosomes) was tested. 12 Add 100 μL of PBS buffer to the particels to obtain a concentration of 10. 13 Exosome stock solution with particles / mL; then, using PBS buffer as a solvent, 10 μL of exosome stock solution was added to 990 μL of PBS buffer to obtain a concentration of 10. 10 The exosome solution was further serially diluted 10-fold using a pipette to obtain a final concentration of 10. 9 108 10 7 10 6 10 5 A gradient concentration solution of exosomes (particles / mL) was prepared. The TFBG-SPR exosome sensor was then fixed in the sample cell, and the exosome sample solution was added using a pipette. After 30 minutes, the spectral data could be read. After detection, the liquid in the sample cell was drained, and deionized water was introduced to rinse the fiber optic surface and the sample cell. Then, another set of test solutions was added, and the process was repeated to complete the spectral acquisition. The transmission spectra of the gradient concentration exosome solutions were obtained, and this process was repeated to complete the spectral acquisition.

[0039] See appendix Figure 3 The image shows the original SPR spectrum of the TFBG-SPR sensor.

[0040] See appendix Figure 4 The figure shows the spectral response of gradient concentration exosome solutions detected using this method.

[0041] See appendix Figure 5 According to the appendix Figure 4 The graph shows the intensity of the six selected envelope patterns (patterns 1-6 from left to right) near the SPR as a function of exosome concentration.

[0042] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0043] Those skilled in the art will readily understand that, according to the method of the present invention, exosomes secreted by various cells can be detected stably and efficiently, and can be combined and optimized without conflicting technical features.

Claims

1. A quantitative detection method for exosomes based on TFBG-SPR, characterized in that: The detection device consists of a broadband light source (1), a single-mode fiber (2), a polarization controller (3), a TFBG-SPR exosome sensor (4), a detection sample cell (5), and a spectrometer (6); the broadband light source (1) is connected to the input end of the polarization controller (3) through the single-mode fiber (2); the output end of the polarization controller (3) is connected to the left end of the TFBG-SPR exosome sensor (4), and the right end of the TFBG-SPR exosome sensor (4) is connected to the spectrometer (6); The structure of the TFBG-SPR exosome sensor (4) is as follows: the sensor consists of a photosensitive optical fiber (41) with TFBG etched on it, a single-mode optical fiber (42) for transmission, a gold film layer (43), and a CD63 antibody layer (44); The preparation steps of the TFBG-SPR exosome sensor (4) are as follows: First, the photosensitive fiber is fused at both ends to transmit a single-mode fiber; then, the photosensitive fiber is loaded with hydrogen for 14 days; TFBG is etched in the core of the hydrogen-loaded photosensitive fiber; the photosensitive fiber with etched TFBG is annealed; and a gold film layer is deposited on the surface of the etched TFBG by magnetron sputtering. A CD63 antibody layer was chemically assembled on a gold film on the surface of TFBG to obtain a TFBG-SPR exosome sensor. The steps of the TFBG-SPR-based exosome quantitative detection method are as follows: First, the TFBG-SPR exosome sensor (4) is fixed in the detection sample cell (5). The incident light emitted by the broadband light source (1) is transmitted through the single-mode fiber (2) to the polarization controller (3), which adjusts the incident light to the P polarization state. The P polarization state beam is transmitted to the TFBG-SPR exosome sensor (4), and finally the transmission spectrum is obtained on the spectrometer (6). Then, the exosome solution to be tested is added to the sample cell (5), and the CD63 antibody layer (44) realizes the exosome in the solution to be tested. The capture of exosomes causes a change in the surface refractive index of the TFBG-SPR exosome sensor (4); this change in refractive index is sensed by the TFBG-SPR exosome sensor (4) and is ultimately reflected as a change in the transmission spectrum on the spectrometer (6); secondly, before changing to different concentrations of exosome sample solutions for detection, the detection sample cell (5) is cleaned with deionized water; finally, the data recorded on the spectrometer (6) by adding different concentrations of exosome solutions to the detection sample cell (5) are fitted to obtain the relationship between different exosome solution concentrations and transmission spectrum drift, thereby realizing the quantitative detection of exosomes.

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