OFDR type esophageal squamous cell carcinoma marker detection device and functionalization and inspection method

Abstract

The application discloses an OFDR type esophageal squamous cell carcinoma marker detection device and a functionalization and inspection method, relates to the field of distributed optical fiber biochemical sensing, and comprises a light splitting assembly, an interference assembly and a demodulation processing assembly. The light splitting assembly is used for splitting a frequency linearly modulated laser into auxiliary interferometer light and main interferometer light, and splitting the main interferometer light into reference light and measurement light. The demodulation processing assembly is used for collecting interference signals, and combining a cross-correlation algorithm and a reference RBS signal to calculate wavelength drift results of the stepped taper optical fiber in various refractive index environments. The main interferometer comprises a stepped taper optical fiber used for receiving measurement light and generating back Rayleigh scattering light. The stepped taper optical fiber comprises a first target taper region of a middle thin taper region part and a second target taper region located on both sides of the first target taper region. The application can realize high-precision detection of the Cyfra21-1 protein concentration in the serum of an esophageal squamous cell carcinoma patient.

Description

Technical Field

[0001] This application relates to the field of distributed optical fiber biochemical sensing, specifically to an OFDR-type esophageal squamous cell carcinoma marker detection device and its functionalization and examination method. Background Technology

[0002] Esophageal squamous cell carcinoma (ESCC) is a prevalent malignant tumor with persistently high incidence and mortality rates, primarily attributed to its insidious early symptoms, lack of specific diagnostic methods, and poor treatment outcomes in advanced stages. While endoscopic biopsy and imaging are considered the "gold standard" for diagnosis, their invasiveness, high cost, and reliance on operator experience limit large-scale screening applications. Therefore, developing non-invasive and efficient serum tumor marker detection technologies has become a key focus of clinical research.

[0003] The significance of serum biomarkers in ESCC management is mainly reflected in three aspects: early screening, dynamic assessment of treatment efficacy, and prognostic stratification. By detecting abnormally elevated biomarkers in serum, such as cytokeratin 19 (Cyfra21-1) and squamous cell carcinoma antigen (SCC-Ag), early identification of high-risk groups such as long-term smokers, drinkers, and patients with esophageal epithelial dysplasia can be aided, thereby significantly improving prognosis.

[0004] At the level of detection technology, hospitals currently mainly rely on chemiluminescence immunoassay (CLIA) and electrochemiluminescence immunoassay (ECLIA) platforms to detect Cyfra21-1. CLIA testing typically takes 30 to 120 minutes, while ECLIA, through electrochemiluminescence, can shorten the testing time to within 30 minutes. Both technologies utilize the specific binding of labeled antibodies to the target antigen and quantitative analysis using luminescent signals, offering advantages such as high automation and sensitivity. However, they also face challenges related to high instrument costs and expensive reagents. Furthermore, some primary care hospitals still use enzyme-linked immunosorbent assay (ELISA), which is cumbersome to operate, has low throughput, and is susceptible to human error.

[0005] In comparison, fiber optic biosensors, with their advantages such as resistance to electromagnetic interference, low background noise, stable signal transmission, and multi-parameter detection capabilities, are gradually becoming the preferred platform for high-precision biomolecular detection. Their unique advantages in sensitivity, integration, and adaptability to various scenarios have provided revolutionary impetus for the development of early disease detection technologies, enabling rapid, highly sensitive, and low-cost clinical testing using Cyfra21-1.

[0006] The primary challenge in the clinical translation of fiber optic detection technology lies in non-specific interference from complex biological matrices: heterogeneous proteins such as albumin and immunoglobulins in serum readily adsorb non-specifically onto the surface of fiber optic probes, leading to increased background signal, especially significantly reducing the signal-to-noise ratio in the detection of low-concentration markers. Furthermore, multiple studies have confirmed that the concentration level of Cyfra21-1 in the serum of ESCC patients is closely related to tumor stage, metastasis, and prognosis. For example, the median concentration of Cyfra21-1 in stage III-IV lung cancer patients can reach 6.5 ng / mL, while that in healthy controls is typically below 3.3 ng / mL. Therefore, a low detection limit is required for the sensor. However, the detection limit (LOD) of fiber optic sensing is limited by the sensitivity of the optical system. Traditional optical fibers, due to their limited evanescent field attenuation length, can only detect molecular binding events within a few hundred nanometers of the surface, making it difficult to capture low-abundance markers. Summary of the Invention

[0007] This application provides an OFDR-type esophageal squamous cell carcinoma marker detection device and its functionalization and examination method, which can achieve high-precision detection of Cyfra21-1 protein concentration in the serum of esophageal squamous cell carcinoma patients.

[0008] In a first aspect, embodiments of this application provide an OFDR-type esophageal squamous cell carcinoma marker detection device, the OFDR-type esophageal squamous cell carcinoma marker detection device comprising:

[0009] A beam splitting assembly is used to split a frequency-linearly tuned laser into an auxiliary interferometer beam and a main interferometer beam, and to split the main interferometer beam into a reference beam and a measurement beam.

[0010] Interference assembly, comprising an auxiliary interferometer for outputting an interference signal by realizing optical interference of an auxiliary interferometer, and a main interferometer for outputting an interference signal by realizing interference of the s-component and p-component of a reference beam and a measurement beam;

[0011] The demodulation processing component is used to acquire interference signals and, in combination with the cross-correlation algorithm and the reference RBS signal, calculate the wavelength drift results of the stepped tapered fiber under various refractive index environments.

[0012] The main interferometer includes a stepped tapered optical fiber for receiving measurement light and generating backscattered Rayleigh light. The stepped tapered optical fiber includes a first target cone region with a middle fine cone region and a second target cone region located on both sides of the first target cone region.

[0013] In conjunction with the first aspect, in one implementation method,

[0014] The beam splitting assembly includes a tunable light source for emitting frequency linearly tuned laser light, a first optical coupler for splitting the frequency linearly tuned laser light into auxiliary interferometer light and main interferometer light, and a second optical coupler for splitting the main interferometer light into reference light and measurement light.

[0015] The demodulation processing component includes a data acquisition card and a processing computer connected to the data acquisition card.

[0016] In conjunction with the first aspect, in one implementation method,

[0017] The auxiliary interferometer includes a time-delay fiber and a third optical coupler and a fourth optical coupler disposed at both ends of the time-delay fiber, and a first photodetector is disposed at the rear end of the fourth optical coupler;

[0018] The main interferometer includes a reference optical path assembly and a measurement optical path assembly;

[0019] The reference optical path assembly includes a polarization controller, a first polarization beam splitter, a fifth optical coupler, and a second photodetector arranged sequentially.

[0020] The measurement optical path assembly includes an optical ring type, a stepped tapered optical fiber connected to the optical ring type, and a second polarizing beam splitter, a sixth optical coupler, and a third photodetector that are sequentially arranged and connected to the optical ring type.

[0021] In conjunction with the first aspect, in one implementation method,

[0022] The signal expression of the frequency-linearly tuned laser for:

[0023]

[0024] in, This represents the normalized amplitude of a frequency-linearly tuned laser. Represents the natural constant. This indicates the initial frequency of the tunable light source. Indicates the sampling time. This represents the linear sweep rate of the tunable light source. This represents the phase noise of a tunable light source. Indicates the imaginary part;

[0025] The expression for the RBS signal at each location in the stepped tapered optical fiber. for:

[0026]

[0027] in, The Rayleigh scattering factor represents the total attenuation during light transmission. , This represents the reflection coefficient at the current position in a stepped tapered optical fiber. This represents the attenuation factor of the optical fiber. This represents the delay time at the current position in the stepped tapered optical fiber. This represents the speed of light in a vacuum. Indicates the refractive index of the optical fiber. Indicates a point in time Phase noise;

[0028] In the stepped tapered optical fiber, at different positions and A beat frequency signal is generated at the photodetector, wherein the photodetector detects the signal. The expression is:

[0029]

[0030] in, This indicates the beat frequency at the current position in the stepped tapered optical fiber. , This indicates the length of the stepped tapered optical fiber.

[0031] Secondly, embodiments of this application provide a method for functionalizing a stepped tapered optical fiber, used to functionalize the stepped tapered optical fiber of the OFDR-type esophageal squamous cell carcinoma marker detection device described above. The method for functionalizing the stepped tapered optical fiber includes:

[0032] Fix both ends of the stepped tapered optical fiber, keep the first target cone region suspended, and place a glass slide below the first target cone region;

[0033] Based on the functionalization requirements, functionalized droplets are added and replaced on the glass slide, and the immersion operation is repeated until the functionalization is completed.

[0034] The immersion operation involves adding functionalized liquid droplets to a glass slide, then controlling the slide to rise so that the functionalized liquid droplets envelop the first target cone region. After the functionalization reaction is completed, the slide is controlled to fall so that the functionalized liquid droplets separate from the first target cone region.

[0035] Thirdly, embodiments of this application provide a method for detecting esophageal squamous cell carcinoma markers, based on the aforementioned OFDR-type esophageal squamous cell carcinoma marker detection device, the method comprising:

[0036] The refractive index sensitivity of the stepped tapered optical fiber was obtained by calibrating it with target solutions of different concentrations, and the first target cone region was functionalized to immobilize Cyfra21-1 specific antibody on the first target cone region.

[0037] The protein wavelength drift sensitivity was obtained by using PBS solution and Cyfra21-1 protein calibration with a ladder-shaped tapered optical fiber.

[0038] The stepped tapered optical fiber is immersed in the serum solution to be tested to calculate the serum wavelength drift of the first and second target cone regions;

[0039] By combining the serum wavelength drift, refractive index sensitivity, and protein wavelength drift sensitivity, the concentration of Cyfra21-1 protein in the serum solution to be tested was calculated.

[0040] In conjunction with the second aspect, in one embodiment, the calibration of the stepped tapered optical fiber using target solutions of different concentrations to obtain the refractive index sensitivity specifically includes:

[0041] The stepped tapered optical fiber of the OFDR-type esophageal squamous cell carcinoma marker detection device was immersed in a target solution of a specific concentration, and the RBS signal was detected as a reference signal.

[0042] Stepped tapered optical fibers were immersed in target solutions of different concentrations to simulate different external refractive index environments, and RBS signals under target solutions of different concentrations were detected.

[0043] Based on the RBS signal of the target solution at the current concentration and combined with the reference signal, the wavelength drift of the first target cone region and the wavelength drift of the second target cone region at the current concentration of the target solution are calculated using a cross-correlation algorithm.

[0044] Linear fitting was performed on the wavelength drift of the first target cone region under different concentrations of target solution to obtain the refractive index sensitivity of the first target cone region. Linear fitting was also performed on the wavelength drift of the second target cone region under different concentrations of target solution to obtain the refractive index sensitivity of the second target cone region.

[0045] In conjunction with the second aspect, in one embodiment, the method of using PBS solution in conjunction with Cyfra21-1 protein calibration using a ladder-shaped tapered optical fiber to obtain protein wavelength shift sensitivity specifically includes:

[0046] The stepped tapered optical fiber, after functionalization of the first target tapered region, was immersed in PBS solution, and the RBS signal was detected and used as the first reference signal.

[0047] Cyfra21-1 protein was added dropwise to PBS solution to obtain PBS solutions with different concentrations of Cyfra21-1 protein, and the RBS signal of PBS solutions with different concentrations of Cyfra21-1 protein was detected.

[0048] Based on the RBS signal of the PBS solution with the current Cyfra21-1 protein concentration and combined with the first reference signal, the wavelength drift of the first target cone region and the wavelength drift of the second target cone region under the current Cyfra21-1 protein concentration in the PBS solution are calculated using a cross-correlation algorithm.

[0049] The wavelength drift of the first target cone region under different Cyfra21-1 protein concentrations in PBS solutions was linearly fitted to obtain the protein wavelength drift sensitivity of the first target cone region. The wavelength drift of the second target cone region under different Cyfra21-1 protein concentrations in PBS solutions was linearly fitted to obtain the protein wavelength drift sensitivity of the second target cone region.

[0050] In conjunction with the second aspect, in one embodiment, the step of immersing the stepped tapered optical fiber in the serum solution to be tested to calculate the serum wavelength shift of the first and second target tapered regions specifically includes:

[0051] The stepped tapered optical fiber, after functionalization of the first target tapered region, was immersed in PBS solution, and the RBS signal was detected and used as the first reference signal.

[0052] The stepped tapered optical fiber, after functionalization of the first target cone region, was immersed in the serum solution to be tested, and the RBS signal under the serum solution to be tested was detected.

[0053] Based on the RBS signal of the serum solution to be tested and combined with the first reference signal, the wavelength drift of the first target cone region under the serum solution to be tested is calculated using a cross-correlation algorithm, denoted as the serum wavelength drift of the first target cone region, and the wavelength drift of the second target cone region is denoted as the serum wavelength drift of the second target cone region.

[0054] In conjunction with the second aspect, in one embodiment, the concentration of Cyfra21-1 protein in the serum solution to be detected is calculated by combining the serum wavelength drift, refractive index sensitivity, and protein wavelength drift sensitivity. The specific calculation method is as follows:

[0055]

[0056] in, Indicates the refractive index sensitivity of the first target cone region. Indicates the refractive index sensitivity of the second target cone region. This indicates the protein wavelength shift sensitivity in the first target cone region. This indicates the protein wavelength shift sensitivity in the second target cone region. This indicates the serum wavelength shift in the first target cone region. This indicates the serum wavelength shift in the second target cone region. This represents the change in volume refractive index, specifically the difference in refractive index between the serum solution and the PBS solution being tested. This indicates the concentration of Cyfra21-1 protein in the serum solution to be tested.

[0057] The beneficial effects of the technical solutions provided in this application include:

[0058] By employing a stepped tapered fiber structure, sensing sensitivity in two regions on a single fiber can be obtained under OFDR detection conditions, significantly improving miniaturized integration and distributed sensing. For longer OFDR sensing regions, the proposed droplet coating method effectively achieves functionalization of only the target region, providing technical support for subsequent noise compensation. This application uses a noise compensation mechanism for target protein detection in the functionalized region and noise compensation in the non-functionalized region, effectively solving the problems of non-specific adsorption of proteins in different serums and noise interference in the clinical detection of Cyfra21-1 biomarkers, improving detection accuracy, and enabling high-precision detection of Cyfra21-1 protein concentration in the serum of esophageal squamous cell carcinoma patients. Attached Figure Description

[0059] Figure 1 This is a schematic diagram of the OFDR-type esophageal squamous cell carcinoma marker detection device of this application;

[0060] Figure 2 This is a schematic diagram of the stepped tapered optical fiber of this application;

[0061] Figure 3 A schematic diagram of the functionalization of the first target cone region in a stepped tapered optical fiber;

[0062] Figure 4 This is a flowchart illustrating the method for detecting esophageal squamous cell carcinoma markers in this application;

[0063] Figure 5 This is a schematic diagram illustrating the calculation principle of wavelength drift in this application;

[0064] Figure 6 The experimental results of converting the time-domain signal received by the OFDR-type esophageal squamous cell carcinoma marker detection device into a distance-domain signal are shown in the figure.

[0065] Figure 7 The time-domain signal received by the OFDR-type esophageal squamous cell carcinoma marker detection device is converted into a distance-domain signal, and the magnified image of the reflection peak in the cone region of the stepped tapered optical fiber is shown.

[0066] Figure 8 This is a graph showing the demodulation results of the OFDR-type esophageal squamous cell carcinoma marker detection device on the external refractive index change of a stepped tapered optical fiber. Detailed Implementation

[0067] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present application.

[0068] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be described in further detail below with reference to the accompanying drawings.

[0069] In the first aspect, embodiments of this application provide an OFDR-type esophageal squamous cell carcinoma marker detection device, which can achieve external refractive index and non-specific adsorption compensation, effectively improving detection accuracy, and is used for high-precision detection of Cyfra21-1 in the serum of esophageal squamous cell carcinoma patients.

[0070] In one embodiment, reference is made to Figure 1 , Figure 1 This is a schematic diagram of the OFDR-type esophageal squamous cell carcinoma marker detection device of this application. Figure 1 As shown, the OFDR-type esophageal squamous cell carcinoma marker detection device includes: a spectrometer, an interference unit, and a demodulation processing unit.

[0071] The beam splitting component is used to split the frequency linearly tuned laser into auxiliary interferometer light and main interferometer light, and to split the main interferometer light into reference light and measurement light; the interference component includes an auxiliary interferometer for outputting an interference signal by realizing interference of the auxiliary interferometer light, and a main interferometer for outputting an interference signal by realizing interference of the s-component and p-component of the reference light and the measurement light; the demodulation processing component is used to acquire the interference signal and, in combination with the cross-correlation algorithm and the reference RBS signal, calculate the wavelength drift result of the stepped tapered fiber under various refractive index environments; wherein, the main interferometer includes a stepped tapered fiber (fiber optic sensor) for receiving the measurement light and generating back-scattered Rayleigh light, and the stepped tapered fiber includes a first target tapered region in the middle thin tapered region and a second target tapered region located on both sides of the first target tapered region.

[0072] Specifically, the beam splitting assembly includes a tunable light source for emitting linearly tuned laser light, a first optical coupler that splits the linearly tuned laser light into auxiliary interferometer light and main interferometer light, and a second optical coupler that splits the main interferometer light into reference light and measurement light. The demodulation processing assembly includes a data acquisition card and a processing computer connected to the data acquisition card, with a demodulation processing system deployed on the processing computer.

[0073] Specifically, the auxiliary interferometer includes a time-delay fiber and a third and a fourth optical coupler located at both ends of the time-delay fiber, with a first photodetector located at the rear end of the fourth optical coupler; the main interferometer includes a reference light optical path assembly and a measurement light optical path assembly; the reference light optical path assembly includes a polarization controller, a first polarization beam splitter, a fifth optical coupler, and a second photodetector arranged sequentially; the measurement light optical path assembly includes an optical ring type and a stepped tapered fiber connected to the optical ring type, as well as a second polarization beam splitter, a sixth optical coupler, and a third photodetector arranged sequentially and connected to the optical ring type.

[0074] Figure 1 In this diagram, TLS Laser represents a tunable light source, C1 represents the first optical coupler, C2 represents the second optical coupler, C3 represents the third optical coupler, C4 represents the fourth optical coupler, C5 represents the fifth optical coupler, C6 represents the sixth optical coupler, Delay fiber represents a time-delay fiber, PD1 represents the first photodetector, PC represents a polarization controller, PBS1 represents the first polarization beam splitter, PD2 represents the second photodetector, DAQ represents a data acquisition card, Computer represents a processing computer, Circulation represents an optical ring type, L1 represents a stepped tapered fiber, PBS2 represents the second polarization beam splitter, and PD3 represents the third photodetector.

[0075] The optical path routing in the OFDR-type esophageal squamous cell carcinoma marker detection device described in this application is explained below.

[0076] A tunable light source emits linearly tuned laser light, which is split into auxiliary interferometer light and main interferometer light by a first optical coupler. The auxiliary interferometer light enters a Mach-Zehnder interferometer with a time-delay fiber and interferes, generating an interference signal after being detected by a first photodetector. The main interferometer light is split into reference light and measurement light by a second optical coupler. The reference light is split into s-components and p-components after passing through a polarization controller and a first polarization beam splitter. The measurement light enters an optical ring and is output into a stepped tapered fiber, generating backscattered Rayleigh light, which is then split into s-components and p-components by a second polarization beam splitter. The s-components of the reference light and measurement light interfere and are detected by a sixth optical coupler, while the p-components interfere and are detected by a fifth optical coupler. All output interference signals are acquired by a data acquisition card and sent to the demodulation processing system of a processing computer. After cross-correlation algorithm data processing, the wavelength drift results of the first and second target cone regions caused by changes in external refractive index are obtained.

[0077] For stepped tapered optical fibers, the frequency shift caused by changes in external refractive index measured by the OFDR-type esophageal squamous cell carcinoma marker detection device can be expressed as:

[0078]

[0079]

[0080] in, This represents the cross-correlation frequency shift caused by changes in the external refractive index. This represents the ratio of the local RBS offset to the change in RI due to the external environment. Indicates the external refractive index, Indicates the initial refractive index. This represents the speed of light in a vacuum. This refers to the frequency of the light wave, specifically the frequency of a linearly tuned laser. This represents the refractive index of the cladding of a stepped tapered optical fiber. This represents the waist radius of a stepped tapered optical fiber. This represents the effective refractive index. It can be seen that... and Inversely proportional to this, in order to obtain high sensitivity, the waist radius of the stepped tapered fiber needs to be reduced.

[0081] The OFDR-type esophageal squamous cell carcinoma marker detection device of this application achieves distributed measurement by detecting the frequency shift of the Rayleigh backscattered (RBS) signal. The measurement process is as follows: a tunable light source performs a linear frequency scan, and the output light is split into two paths by a first optical coupler. One path of the main interferometer enters a stepped tapered optical fiber, and the resulting backscattered light interferes with the other reference light of the main interferometer. An auxiliary interferometer provides an external clock signal to ensure that the data acquisition card samples at equal intervals in the optical frequency domain to compensate for the nonlinearity of the laser sweep frequency. Data from three photodetectors is input into the data acquisition card, which then sends the data to a processing computer for processing.

[0082] In the OFDR-type esophageal squamous cell carcinoma marker detection device, the signal expression of the frequency-linearly tuned laser. for:

[0083]

[0084] in, This represents the normalized amplitude of a frequency-linearly tuned laser. Represents the natural constant. This indicates the initial frequency of the tunable light source. Indicates the sampling time. This represents the linear sweep rate of the tunable light source. This represents the phase noise of a tunable light source. Let represent the imaginary part; for stepped tapered optical fibers, i.e., in the first and second target tapered regions, each scattering point will generate a Rayleigh scattering basis (RBS), which will also cause attenuation during light transmission. Therefore, the total Rayleigh scattering factor considering attenuation is:

[0085] ;

[0086] Substituting the total Rayleigh scattering factor into the optical field expression, we can obtain the expression for the RBS signal at each location in the stepped tapered fiber:

[0087]

[0088] in, The Rayleigh scattering factor represents the total attenuation during light transmission. , This represents the reflection coefficient at the current position in a stepped tapered optical fiber. This represents the attenuation factor of the optical fiber. This represents the delay time at the current position in the stepped tapered optical fiber. This represents the speed of light in a vacuum. Indicates the refractive index of the optical fiber. Indicates a point in time Phase noise;

[0089] In a stepped tapered optical fiber, at different positions and A beat frequency signal is generated at the photodetector, wherein the photodetector detects the signal. The expression is:

[0090]

[0091] in, This indicates the beat frequency at the current position in the stepped tapered optical fiber. , This indicates the length of the stepped tapered optical fiber. The signals from the three photodetectors are acquired by a data acquisition card and transmitted to a processing computer for demodulation, reconstructing the location of the refractive index change and the magnitude of the wavelength shift.

[0092] The following is a detailed description of the fabrication process of the stepped tapered optical fiber in this application.

[0093] The OFDR-type esophageal squamous cell carcinoma marker detection device of this application is based on the demodulation and calibration of the Rayleigh scattering signal in the fiber optic sensing area to obtain the concentration of the external target protein. Therefore, this application selects a thin-core fiber with higher Rayleigh scattering intensity as the sensing fiber (stepped tapered fiber) to improve the detection signal-to-noise ratio and detection stability.

[0094] The stepped tapered optical fiber was fabricated using a fiber fusion tapering machine (FFTM). The original thin-core fiber had a cladding diameter of 125 mm. The mode field diameter is smaller than that of standard single-mode fiber. The thin-core fiber is fixed to the grooved platform of the FFTM by magnets and moves towards both ends at a constant speed. A central flame probe scans back and forth on the fiber at a constant speed to obtain a uniform initial tapered region. After reaching the target length, the same steps are used to perform a secondary drawing on the central portion of the fiber tapered region. The flame probe scans the central section of the fiber tapered region at a constant speed to obtain a uniform second target tapered region. The target length of the second target tapered region is set to 40mm. After reaching the target length, the same steps are used to perform a secondary drawing on the central portion of the fiber tapered region. The flame probe scans the central section of the fiber tapered region at a constant speed to obtain a uniform first target tapered region. The target drawing length is set to 15mm. To increase the sensing length of the stepped tapered fiber, the FFTM uses a slow and uniform drawing method. The total drawn length of the stepped tapered fiber is 55mm. The structure of the obtained stepped tapered fiber is as follows: Figure 2 As shown.

[0095] The stepped tapered optical fiber of this application has two sensing regions with different diameters. Since the sensing of the external refractive index of the stepped tapered optical fiber depends on the evanescent field intensity, and the evanescent field intensity is inversely proportional to the tapered diameter within a certain range, the stepped tapered optical fiber has two different refractive index sensing sensitivities. Based on the distributed sensing characteristics of OFDR (Optical Frequency Domain Reflectometer), the sensing effects of these two parts can be independently and accurately distinguished, realizing the integration of single-taper dual sensitivity.

[0096] Secondly, this application also provides a method for functionalizing a stepped tapered optical fiber, used to functionalize the stepped tapered optical fiber of the OFDR-type esophageal squamous cell carcinoma marker detection device described above. The method for functionalizing the stepped tapered optical fiber includes:

[0097] S11: Fix both ends of the stepped tapered optical fiber, keep the first target cone region suspended, and place a glass slide below the first target cone region;

[0098] S12: Based on the functionalization requirements, add and replace functionalized droplets on the glass slide, and repeat the immersion operation until functionalization is completed;

[0099] The immersion operation involves adding functionalized liquid droplets to a glass slide, then controlling the slide to rise so that the functionalized liquid droplets envelop the first target cone region. After the functionalization reaction is completed, the slide is controlled to fall so that the functionalized liquid droplets separate from the first target cone region.

[0100] It should be noted that the tapered region of the stepped tapered optical fiber is relatively long. The functionalization method is to use droplet coating to immerse the first target tapered region separately into a functionalized droplet, thereby achieving functionalization of only the first target tapered region.

[0101] See also Figure 3 As shown, the coated areas at both ends of the stepped tapered optical fiber are fixed on horizontal displacement stages 1 and 2, 60 mm apart, respectively. The tapered portion is suspended and kept as taut as possible. A glass slide supported by displacement stage 3 is placed below the suspended first target tapered region. A functionalized droplet is dropped onto the glass slide. Displacement stage 3 is slowly raised until the droplet covers the first target tapered region. The reaction is allowed to proceed, and after the reaction, displacement stage 3 is slowly lowered to separate the droplet from the target tapered region 1. After drying, the chemical droplet is replaced, and the above operation is repeated until functionalization is complete. The functionalization method of this application can avoid the contamination of non-target areas and solution waste caused by the large-area functionalization of traditional immersion methods, and can effectively avoid the problem of optical fiber breakage during the functionalization process.

[0102] Thirdly, this application also provides a method for detecting esophageal squamous cell carcinoma markers, based on the OFDR-type esophageal squamous cell carcinoma marker detection device described above.

[0103] In one embodiment, reference is made to Figure 4 , Figure 4 This is a flowchart illustrating the esophageal squamous cell carcinoma marker detection method of this application. Figure 4 As shown, the methods for detecting markers of esophageal squamous cell carcinoma include:

[0104] S21: The refractive index sensitivity of the stepped tapered optical fiber was obtained by calibrating the target solution with different concentrations, and the first target cone region was functionalized to fix Cyfra21-1 specific antibody on the first target cone region;

[0105] S22: Protein wavelength drift sensitivity was obtained by using PBS solution in conjunction with Cyfra21-1 protein calibration using a ladder-shaped tapered optical fiber; PBS stands for Phosphate Buffer Saline.

[0106] S23: Immerse the stepped tapered optical fiber into the serum solution to be tested to calculate the serum wavelength drift of the first and second target tapered regions;

[0107] S24: Combining the serum wavelength drift, refractive index sensitivity, and protein wavelength drift sensitivity, the concentration of Cyfra21-1 protein in the serum solution to be tested is calculated.

[0108] Furthermore, in one embodiment, the refractive index sensitivity of the stepped tapered optical fiber is obtained by calibrating it with target solutions of different concentrations, specifically including:

[0109] S2101: The stepped tapered optical fiber of the OFDR-type esophageal squamous cell carcinoma marker detection device is immersed in a target solution of a specific concentration, and the RBS signal is detected as a reference signal; in one possible implementation, the target solution is a sucrose solution;

[0110] Specifically, the stepped tapered optical fiber of the OFDR-type esophageal squamous cell carcinoma marker detection device is immersed in a target solution of a specific concentration, and the RBS signal at this time is obtained through computer processing.

[0111] S2102: The stepped tapered optical fiber was immersed in target solutions of different concentrations to simulate different external refractive index environments, and the RBS signal under target solutions of different concentrations was detected.

[0112] Specifically, the stepped tapered optical fiber was immersed in target solutions of different concentrations, and the RBS signal was detected at each immersion in the target solution of different concentrations.

[0113] S2103: Based on the RBS signal of the target solution at the current concentration and combined with the reference signal, the wavelength drift of the first target cone region and the wavelength drift of the second target cone region at the current concentration of the target solution are calculated using a cross-correlation algorithm.

[0114] Specifically, for the RBS signal at the current concentration of the target solution, using the reference signal as a reference, the wavelength shift of the first target cone region in the stepped tapered fiber at the current concentration of the target solution (i.e., the wavelength shift compared to the reference signal) can be calculated by using FFT windowing and combining it with a cross-correlation algorithm. Similarly, the wavelength shift of the second target cone region in the stepped tapered fiber at the current concentration of the target solution can be calculated. The principle of wavelength shift calculation is as follows: Figure 5 As shown;

[0115] Specifically, based on the Rayleigh backscattering spectra of the target solution at a specific concentration and the target solution at the current concentration, the spectrum is transformed into the spatial domain by Fourier transform and then segmented. Cross-correlation calculation is performed on each local spectrum segment to extract the peak wavelength shift.

[0116] S2104: Linearly fit the wavelength drift of the first target cone region under different concentrations of target solution to obtain the refractive index sensitivity of the first target cone region; linearly fit the wavelength drift of the second target cone region under different concentrations of target solution to obtain the refractive index sensitivity of the second target cone region.

[0117] Specifically, the wavelength drift of the first target cone region under different concentrations of target solution is obtained, and the wavelength drift data under different concentrations of target solution are linearly fitted. The slope of the fitted curve is the refractive index sensitivity of the first target cone region. The refractive index sensitivity of the second target cone region is obtained in the same way.

[0118] The following uses a sucrose solution as an example to illustrate the process of obtaining the refractive index sensitivity of this application. It should be noted that the stepped tapered optical fiber is used as the sensing fiber; the central tapered region with a smaller diameter is the first target tapered region, and the noise compensation tapered regions on both sides are the second target tapered regions.

[0119] First, the sensing areas, namely the first and second target cone areas, are immersed in 50 ml of a 1% sucrose solution. The OFDR esophageal squamous cell carcinoma marker detection device detects the RBS signal as a reference signal. By adding purified water to the original sucrose solution to form sucrose solutions with concentrations of 0.9%, 0.8%, 0.7%, and 0.6% (in practical applications, more sucrose solutions of different concentrations are formed to improve the accuracy of subsequent refractive index sensitivity calculations), different external refractive index environments are simulated. The RBS signal is detected for each concentration of sucrose solution formed. Combined with the reference signal, a cross-correlation algorithm is used to obtain the wavelength drift of the first and second target cone areas under each concentration of sucrose solution. By linearly fitting the wavelength drift at different concentrations (under different external refractive index environments), the refractive index sensitivity of the first and second target cone areas can be obtained.

[0120] Furthermore, in one embodiment, the functionalization of the first target cone region to immobilize the Cyfra21-1 specific antibody on the first target cone region is described in detail below.

[0121] The functionalization of the first target cone region specifically includes the following steps:

[0122] S2111: Immerse the stepped tapered optical fiber in a KOH standard solution (0.1 mol / L) for 1 hour, and then rinse it with deionized water 2-3 times;

[0123] S2112: Prepare a 5% silanized ethanol reagent using 98% silanizing (3(-3-triethoxysilylpropyl)oxolane-2,5-dione) reagent and 99% ethanol solution in the laboratory. Immerse the first target cone region in the prepared 5% silanized ethanol reagent for 4 hours to generate carboxyl functional groups on the surface of the first target cone region.

[0124] S2113: Weigh appropriate amounts of EDC and NHSS powder using an analytical balance, prepare a mixed solution of 0.8 mg / mL EDC and 1.2 mg / mL NHSS using PBS solution, wash the first target cone region with PBS buffer (pH=6), and then immerse it in freshly prepared mixed solution of EDC and NHSS (pH=5.6) for 1 hour to react and generate NHS active ester;

[0125] S2114: Immediately immerse the first target cone region after the above treatment in Cyfra21-1 antibody solution for 4 hours. The solvent used is PBS buffer to couple the antibody to the sensor surface.

[0126] S2115: Wash the first target cone region three times with PBS buffer, immerse it in 1% BSA solution to block the remaining unbound carboxyl sites, and then wash it with PBS buffer to complete the functionalization.

[0127] Furthermore, in one embodiment, the protein wavelength shift sensitivity is obtained by using PBS solution in conjunction with Cyfra21-1 protein calibration using a ladder-shaped tapered optical fiber, specifically including:

[0128] S2201: The stepped tapered optical fiber after functionalization of the first target tapered region is immersed in PBS solution (phosphate buffer solution), and the RBS signal is detected as the first reference signal;

[0129] S2202: Cyfra21-1 protein is added dropwise to PBS solution sequentially to obtain PBS solutions with different Cyfra21-1 protein concentrations. The RBS signal of the PBS solutions with different Cyfra21-1 protein concentrations is then detected. That is, each time Cyfra21-1 protein is added dropwise to PBS solution, a PBS solution with a Cyfra21-1 protein concentration is obtained, and the RBS signal is detected at this time. Then, the addition of Cyfra21-1 protein is continued (the amount of Cyfra21-1 protein added each time is flexibly set according to actual needs), and the RBS signal is detected again. This process is repeated until the set number of times is reached.

[0130] S2203: Based on the RBS signal of the PBS solution with the current Cyfra21-1 protein concentration and combined with the first reference signal, the wavelength drift of the first target cone region and the wavelength drift of the second target cone region under the current Cyfra21-1 protein concentration in the PBS solution are calculated using a cross-correlation algorithm.

[0131] S2204: Linear fitting was performed on the wavelength drift of the first target cone region under PBS solutions with different Cyfra21-1 protein concentrations to obtain the protein wavelength drift sensitivity of the first target cone region. Linear fitting was performed on the wavelength drift of the second target cone region under PBS solutions with different Cyfra21-1 protein concentrations to obtain the protein wavelength drift sensitivity of the second target cone region.

[0132] The calculation principle for protein wavelength drift sensitivity is similar to that for refractive index sensitivity, except that different concentrations of sucrose solution are replaced with different concentrations of Cyfra21-1 protein in PBS solution.

[0133] Furthermore, in one embodiment, immersing a stepped tapered optical fiber in the serum solution to be tested to calculate the serum wavelength shift in the first and second target tapered regions specifically includes:

[0134] S2301: The stepped tapered optical fiber after functionalization of the first target cone region is immersed in PBS solution, and the RBS signal is detected as the first reference signal; it should be noted that the functionalization of the first target cone region mentioned in this application refers to the functionalization of the first target cone region to fix Cyfra21-1 specific antibody on the first target cone region.

[0135] S2302: The stepped tapered optical fiber, after functionalization of the first target tapered region, is immersed in the serum solution to be tested, and the RBS signal under the serum solution to be tested is detected.

[0136] S2303: Based on the RBS signal under the serum solution to be tested and combined with the first reference signal, the wavelength drift of the first target cone region under the serum solution to be tested is calculated by the cross-correlation algorithm and denoted as the serum wavelength drift of the first target cone region, and the wavelength drift of the second target cone region is denoted as the serum wavelength drift of the second target cone region.

[0137] Furthermore, in one embodiment, the concentration of Cyfra21-1 protein in the serum solution to be tested is calculated by combining the serum wavelength drift, refractive index sensitivity, and protein wavelength drift sensitivity. The specific calculation method is as follows:

[0138]

[0139] in, Indicates the refractive index sensitivity of the first target cone region. Indicates the refractive index sensitivity of the second target cone region. This indicates the protein wavelength shift sensitivity in the first target cone region. This indicates the protein wavelength shift sensitivity in the second target cone region. This indicates the serum wavelength shift in the first target cone region. This indicates the serum wavelength shift in the second target cone region. This represents the change in volume refractive index, specifically the difference in refractive index between the serum solution and the PBS solution being tested. This indicates the concentration of Cyfra21-1 protein in the serum solution to be tested.

[0140] The following explains the principle behind the calculation of Cyfra21-1 protein concentration in the serum solution to be tested.

[0141] After functionalization, Cyfra21-1 specific antibodies are immobilized on the first target cone region of the stepped tapered optical fiber. When the stepped tapered optical fiber is immersed in a solution, the overall background refractive index of the solution is called the volume refractive index (BRI), and the refractive index change caused by particles adsorbed on the surface of the stepped tapered optical fiber is called the surface refractive index (SRI). When the stepped tapered optical fiber is immersed in PBS solutions with different concentrations of Cyfra21-1 protein, the first target cone region, due to the immobilized Cyfra21-1 specific antibody, specifically binds to the Cyfra21-1 protein in the solution, thereby causing Cyfra21-1 protein to specifically adsorb onto the surface of the first target cone region, resulting in a change in the surface BRI of the first target cone region. This wavelength shift reflected in the first target cone region is detected by an OFDR-type esophageal squamous cell carcinoma marker detection device.

[0142] Since the second target cone region has not been functionalized with Cyfra21-1 specific antibodies, it is not sensitive to changes in the concentration of Cyfra21-1 protein in the external environment. The only factors causing wavelength drift in the second target cone region are non-specific adsorption and changes in the overall refractive index of the solution, i.e., SRI.

[0143] After the stepped tapered optical fiber was calibrated in PBS solution (i.e., the stepped tapered optical fiber functionalized with the first target tapered region was immersed in PBS solution, and the RBS signal was detected as the first reference signal), when detected in the serum solution to be tested, there was a large amount of non-specific adsorption of non-target proteins and changes in SRI. The compensation method is as follows:

[0144] The first target cone region exhibits both specific binding to Cyfra21-1 protein and non-specific binding to non-Cyfra21-1 protein. Its wavelength drift, influenced by changes in SRI and BRI, is affected by these two factors, leading to measurement biases. The second target cone region is also affected by these two factors, with its wavelength drift being more physically influenced by noise. By fitting the spectral drift of the first and second target cone regions under different Cyfra21-1 protein concentrations in PBS solutions, the aforementioned decoupling matrix operation was established to obtain the compensated wavelength drift for each concentration. This compensated wavelength drift represents the accurate wavelength drift after noise compensation, thus providing an accurate determination of the Cyfra21-1 protein concentration in the serum solution being tested.

[0145] Figure 6 The image shows the experimental results of converting the time-domain signal received by the OFDR-type esophageal squamous cell carcinoma marker detection device of this application into a distance-domain signal. The spike at the end of the image represents a strong reflection peak at the end of the optical fiber, which is consistent with the measured length of the stepped tapered optical fiber, demonstrating the accuracy of the distance-domain measurement of the OFDR-type esophageal squamous cell carcinoma marker detection device of this application. Figure 7 This is a magnified image of the reflection peak in the tapered region of a stepped tapered optical fiber. The observation length is approximately 55 mm, which matches the tapered length. The slight error is due to the fact that the thicker part of the transition region does not react strongly with the external refractive index.

[0146] Figure 8 This study demonstrates the wavelength drift response of two sensing regions (the first target cone region and the second target cone region) of a stepped tapered optical fiber in solutions with different refractive indices. First, the stepped tapered optical fiber was immersed in 50 ml of a 1% sucrose solution. An OFDR-type esophageal squamous cell carcinoma marker detection device was used to detect the RBS signal as a reference group. Different external refractive index environments were simulated by adding purified water to the original solution to create 0.9%, 0.8%, 0.7%, and 0.6% sucrose solutions. A cross-correlation algorithm was used to obtain the wavelength drift of the first and second target cone regions at each concentration. It is evident that as the external refractive index increases, the first and second target cone regions exhibit different sensitivities to changes in external refractive index concentration, showing a certain linear relationship, thus proving the feasibility of this application.

[0147] The terms "comprising" and "having," and any variations thereof, in the specification, claims, and accompanying drawings of this application are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or apparatus that includes a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to such process, method, product, or apparatus. The terms "first," "second," and "third," etc., are used to distinguish different objects, etc., and do not indicate a sequence, nor do they limit "first," "second," and "third" to different types.

[0148] In the description of the embodiments of this application, terms such as "exemplary," "for example," or "for instance" are used to indicate examples, illustrations, or explanations. Any embodiment or design described as "exemplary," "for example," or "for instance" in the embodiments of this application should not be construed as being more preferred or advantageous than other embodiments or designs. Specifically, the use of terms such as "exemplary," "for example," or "for instance" is intended to present the relevant concepts in a concrete manner.

[0149] In the description of the embodiments of this application, unless otherwise stated, " / " means "or". For example, A / B can mean A or B. The "and / or" in the text is merely a description of the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, A and B exist simultaneously, and B exists alone. In addition, in the description of the embodiments of this application, "multiple" means two or more.

[0150] In some processes described in the embodiments of this application, multiple operations or steps are included in a specific order. However, it should be understood that these operations or steps may not be executed in the order they appear in the embodiments of this application, or they may be executed in parallel. The sequence number of the operation is only used to distinguish different operations, and the sequence number itself does not represent any execution order. In addition, these processes may include more or fewer operations, and these operations or steps may be executed sequentially or in parallel, and these operations or steps may be combined.

[0151] Through the above description of the embodiments, those skilled in the art can clearly understand that the methods of the above embodiments can be implemented by means of software plus necessary general-purpose hardware platforms. Of course, they can also be implemented by hardware, but in many cases the former is a better implementation method. Based on this understanding, the technical solution of this application, in essence, or the part that contributes to the prior art, can be embodied in the form of a software product. This computer software product is stored in a storage medium (such as ROM / RAM, magnetic disk, optical disk) as described above, and includes several instructions to cause a terminal device to execute the methods described in the various embodiments of this application.

[0152] The above are merely preferred embodiments of this application and do not limit the patent scope of this application. Any equivalent structural or procedural transformations made using the content of this application's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of this application.

Claims

1. A method for detecting esophageal squamous cell carcinoma markers, based on an OFDR-type esophageal squamous cell carcinoma marker detection device, wherein the OFDR-type esophageal squamous cell carcinoma marker detection device includes a beam splitting component, an interferometer component, and a demodulation processing component. The beam splitting component is used to split a linearly tuned laser into an auxiliary interferometer beam and a main interferometer beam, and to split the main interferometer beam into a reference beam and a measurement beam. The interferometer component includes an auxiliary interferometer for outputting an interference signal by realizing interference of the auxiliary interferometer beam, and a main interferometer for outputting an interference signal by realizing interference of the s-component and p-component of the reference beam and the measurement beam. The demodulation processing component is used to acquire the interference signal and, in conjunction with a cross-correlation algorithm and a reference RBS signal, calculate the wavelength drift result of a stepped tapered optical fiber under various refractive index environments. The main interferometer includes a stepped tapered optical fiber for receiving the measurement beam and generating back-scattered Rayleigh light. The stepped tapered optical fiber includes a first target tapered region with a central thin tapered region and second target tapered regions located on both sides of the first target tapered region. The first target tapered region is obtained by secondary drawing of the second target tapered region. The method for detecting esophageal squamous cell carcinoma markers includes: The refractive index sensitivity of the stepped tapered optical fiber was obtained by calibrating it with target solutions of different concentrations, and the first target cone region was functionalized to immobilize Cyfra21-1 specific antibody on the first target cone region. The protein wavelength drift sensitivity was obtained by using PBS solution and Cyfra21-1 protein calibration with a ladder-shaped tapered optical fiber. The stepped tapered optical fiber is immersed in the serum solution to be tested to calculate the serum wavelength drift of the first and second target cone regions; By combining the serum wavelength drift, refractive index sensitivity, and protein wavelength drift sensitivity, the concentration of Cyfra21-1 protein in the serum solution to be tested was calculated. The concentration of Cyfra21-1 protein in the serum solution to be tested is calculated by combining the serum wavelength drift, refractive index sensitivity, and protein wavelength drift sensitivity. The specific calculation method is as follows: in, Indicates the refractive index sensitivity of the first target cone region. Indicates the refractive index sensitivity of the second target cone region. This indicates the protein wavelength shift sensitivity in the first target cone region. This indicates the protein wavelength shift sensitivity in the second target cone region. This indicates the serum wavelength shift in the first target cone region. This indicates the serum wavelength shift in the second target cone region. This represents the change in volume refractive index, specifically the difference in refractive index between the serum solution and the PBS solution being tested. This indicates the concentration of Cyfra21-1 protein in the serum solution to be tested.

2. The method for detecting esophageal squamous cell carcinoma markers as described in claim 1, characterized in that, The calibration of the stepped tapered optical fiber using target solutions of different concentrations to obtain the refractive index sensitivity specifically includes: The stepped tapered optical fiber of the OFDR-type esophageal squamous cell carcinoma marker detection device was immersed in a target solution of a specific concentration, and the RBS signal was detected as a reference signal. Stepped tapered optical fibers were immersed in target solutions of different concentrations to simulate different external refractive index environments, and RBS signals under target solutions of different concentrations were detected. Based on the RBS signal of the target solution at the current concentration and combined with the reference signal, the wavelength drift of the first target cone region and the wavelength drift of the second target cone region at the current concentration of the target solution are calculated using a cross-correlation algorithm. Linear fitting was performed on the wavelength drift of the first target cone region under different concentrations of target solution to obtain the refractive index sensitivity of the first target cone region. Linear fitting was also performed on the wavelength drift of the second target cone region under different concentrations of target solution to obtain the refractive index sensitivity of the second target cone region.

3. The method for detecting esophageal squamous cell carcinoma markers as described in claim 1, characterized in that, The method of using PBS solution in conjunction with Cyfra21-1 protein calibration using a stepped tapered optical fiber to obtain protein wavelength shift sensitivity specifically includes: The stepped tapered optical fiber, after functionalization of the first target tapered region, was immersed in PBS solution, and the RBS signal was detected and used as the first reference signal. Cyfra21-1 protein was added dropwise to PBS solution to obtain PBS solutions with different concentrations of Cyfra21-1 protein, and the RBS signal of PBS solutions with different concentrations of Cyfra21-1 protein was detected. Based on the RBS signal of the PBS solution with the current Cyfra21-1 protein concentration and combined with the first reference signal, the wavelength drift of the first target cone region and the wavelength drift of the second target cone region under the current Cyfra21-1 protein concentration in the PBS solution are calculated using a cross-correlation algorithm. The wavelength drift of the first target cone region under different Cyfra21-1 protein concentrations in PBS solutions was linearly fitted to obtain the protein wavelength drift sensitivity of the first target cone region. The wavelength drift of the second target cone region under different Cyfra21-1 protein concentrations in PBS solutions was linearly fitted to obtain the protein wavelength drift sensitivity of the second target cone region.

4. The method for detecting esophageal squamous cell carcinoma markers as described in claim 1, characterized in that, The step of immersing a stepped tapered optical fiber in the serum solution to be tested to calculate the serum wavelength shift in the first and second target tapered regions specifically includes: The stepped tapered optical fiber, after functionalization of the first target tapered region, was immersed in PBS solution, and the RBS signal was detected and used as the first reference signal. The stepped tapered optical fiber, after functionalization of the first target cone region, was immersed in the serum solution to be tested, and the RBS signal under the serum solution to be tested was detected. Based on the RBS signal of the serum solution to be tested and combined with the first reference signal, the wavelength drift of the first target cone region under the serum solution to be tested is calculated using a cross-correlation algorithm, denoted as the serum wavelength drift of the first target cone region, and the wavelength drift of the second target cone region is denoted as the serum wavelength drift of the second target cone region.

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