IgG concentration detection method based on optical fiber

Abstract

The invention discloses an optical fiber-based IgG protein concentration detection method. A used device comprises a broadband light source, a single-mode optical fiber, a polarization controller, a flow cell, a TFBG-SPR sensor and a spectrum analyzer. Firstly, light with the wavelength range of 1420-1620 nm is emitted by a broadband light source, the polarization state of the light is adjusted to a P state through a polarization controller, then the light is input into an AuNPs / 11-MAA / rabbit anti-human IgG modified sensing probe, and finally a spectrum is displayed on a spectrum analyzer. Rabbit anti-human IgG on the surface of the TFBG can be combined with human IgG to cause change of the refractive index, drifting of a transmission spectrum is shown on a spectrum analyzer, and rapid, accurate and trace detection of the IgG concentration is realized by comparing the relationship between the concentrations of different IgG solutions and the drifting of the transmission spectrum. The invention provides a novel method which is simple in structure, high in accuracy and quick in response for measuring the concentration of IgG, and has great application potential.

Description

Technical Field

[0001] This invention relates to the field of fiber optic sensing technology, and specifically to a method for detecting IgG concentration based on gold-plated TFBG combined with AuNPs / 11-MUA / rabbit anti-human IgG material. Background Technology

[0002] Human immunoglobulin G (human IgG) is an important biomarker that plays a crucial role in the adaptive immune response. It is the most abundant antibody found in the blood, accounting for approximately 75% of all antibodies in the human body. By detecting the concentration of human IgG, a person's health status can be assessed.

[0003] The normal concentration range of IgG in human blood is typically 700-1600 mg / dL, though this may vary slightly depending on age, testing methods, and reference standards. Excessively high IgG concentrations can lead to chronic infections, autoimmune diseases (such as rheumatoid arthritis), liver disease, and multiple myeloma (due to elevated monoclonal IgG levels). Conversely, excessively low IgG concentrations may result in primary / secondary immunodeficiency (such as common variant immunodeficiency) and nephrotic syndrome (due to increased IgG loss).

[0004] Currently, various IgG detection technologies have been developed, including methods such as immunoturbidimetry, ELISA, radioimmunodiffusion (RID), and chemiluminescent immunoassay (CLIA). However, these methods are mostly cumbersome to operate, involve complex sample pretreatment processes, and require laboratory equipment that is bulky, inconvenient to carry, has relatively low detection sensitivity, and long reaction times.

[0005] In recent years, fiber optic biochemical sensors have demonstrated great application potential in many fields such as biomedicine and chemical detection due to their advantages such as strong anti-electromagnetic interference capability, low cost, high sensitivity and corrosion resistance.

[0006] A tilted fiber Bragg grating (TFBG) is a special type of short-period fiber grating that is tilted at a certain angle relative to the fiber axis. By depositing gold on the surface of the TFBG, specific cladding modes can be converted into SPR modes. These modes are extremely sensitive to local refractive index changes around the gold-coated TFBG surface; any minute physicochemical reaction can lead to wavelength shifts and amplitude changes. Meanwhile, the core mode Bragg resonance of the TFBG is unaffected by changes in the surrounding refractive index, which helps to eliminate interference from ambient temperature variations and fluctuations in light source power. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this study developed a highly sensitive IgG concentration sensor based on tilted fiber Bragg grating plasmon resonance (TFBG-SPR) technology. The sensor's detection capability is achieved by modifying the surface of a TFBG with an antibody that specifically binds to human IgG, including the following steps: S1, Preparation of gold-plated TFBG; S2. Preparation and modification of AuNPs / 11-MUA / rabbit anti-human IgG multilayer membranes that can specifically bind to IgG; S3. Connection of broadband light source (1), polarization controller (2), TFBG-SPR sensor (5), and spectrometer (6); S4. Detection of IgG concentration, repeatability, and time response.

[0008] In step S1, after loading hydrogen for 24 days, TFBG was etched into the fiber core using a phase mask method with ultraviolet light, with a length set to 1 cm. The tilt angle between the phase mask (period of 541 nm) and the fiber was set to 8 degrees, and then the fiber was annealed in a 125°C temperature chamber for 12 hours. Finally, a 50 nm thick layer of gold was deposited on the TFBG in a magnetron sputtering deposition chamber.

[0009] In step S2, the gold-plated TFBG surface is modified using gold-sulfur (Au-S) covalent surface modification technology, and functionalized using a multilayer structure composed of 11-mercaptoundecanoic acid (11-MUA), rabbit anti-human IgG antibody, and gold nanoparticles (AuNPs).

[0010] Preferably, the particle size of AuNPs is 20 nm.

[0011] In step S2, the modification of AuNPs / 11-MUA / rabbit anti-human IgG, which specifically binds to IgG, involves repeatedly rinsing the prepared gold-plated TFBG three times in deionized water to remove impurities from the sensor surface. Then, the sensor is immersed in an 11-MUA ethanol solution for 12-24 hours. The thiol group (-SH) at one end of the 11-MUA connects to the gold on the TFBG surface via a gold-sulfur bond (Au-S), forming a self-assembled monolayer on the gold film surface. Next, the 11-MUA-modified TFBG is immersed in an EDC / NHS mixed solution for 1-2 hours to activate its functional groups. Then, the TFBG is immersed in a rabbit anti-human IgG antibody solution for about 1 hour. The amino groups on the rabbit anti-human IgG antibody can bind to the activated carboxyl groups, which are then stably fixed on the TFBG surface. Finally, the TFBG is immersed in a low-concentration bovine serum albumin (BSA) solution to block the specific sites. Finally, the human IgG solution was mixed with gold nanoparticles (AuNPs) in a specific ratio to improve sensitivity. Since the AuNPs surface coated with citrate anions exhibits a negative charge in a solution at pH 6.2, and the pI value of IgG is close to 7.0, IgG should carry a net positive charge. Therefore, when the IgG solution is added to the AuNPs solution, IgG is adsorbed onto the AuNPs surface through ion interactions. At the end of each modification process, the probe surface was rinsed three times with deionized water to remove surface residues.

[0012] In step S3, the broadband light source (1), polarization controller (3), TFBG-SPR sensor (5), and spectrometer (6) are connected as follows: the broadband light source (1) is connected to the left end of the polarization controller (3) through a single-mode optical fiber (2), the polarization controller (3) is connected to the TFBG-SPR sensor (5), the TFBG-SPR sensor (5) is fixed in the flow cell (4), and its right end is connected to the spectrometer (6); the broadband light source (1) is used to provide light source, the polarization controller (3) is used to obtain greater fringe contrast, the flow cell (4) is used to add the IgG solution to be tested, and the spectrometer (6) is used to monitor and record spectral changes; when the rabbit anti-human IgG modified on the surface of TFBG-SPR (5) binds to IgG, the refractive index changes, which is manifested as a shift in the transmission spectrum on the spectrometer (6). By comparing the relationship between different IgG concentrations and the transmission spectrum shift, the concentration of IgG can be detected.

[0013] Preferably, the wavelength of the broadband light source (1) is 1420-1620 nm.

[0014] The IgG concentration detected in step S4 is modulated to be: 100pg / ml, 200pg / ml, 300pg / ml, 400pg / ml, 500pg / ml, 600pg / ml, 700pg / ml, and 800pg / ml.

[0015] The working principle of the gold-plated TFBG-based IgG concentration detection sensor prepared in this invention is as follows: When light waves enter the optical fiber and are completely reflected by the fiber sidewalls, TFBG can also excite evanescent surface plasmon resonance waves on the gold surface. Due to the presence of a gold film on the sensor surface, when the p-polarized component of light enters the gold film, the free electrons of the gold film interact with the p-polarized component of the light, generating plasma. At the interface between the optical fiber and the gold film, plasma vibration forms surface plasmon waves. 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, generating surface plasmon resonance. Light energy near the resonance wavelength is absorbed, resulting in a resonance valley in the output spectrum. When the IgG concentration changes, the local refractive index of the TFBG surface sensitive film changes, and the resonance valley and resonance wavelength also change. Therefore, the IgG concentration can be inferred by monitoring the shift in the resonance wavelength.

[0016] In summary, this invention provides a TFBG-SPR sensor capable of specifically detecting IgG concentration. This sensor is based on the fact that rabbit anti-human IgG antibody can specifically bind to IgG, thereby changing the effective refractive index and causing a shift in the transmission spectrum, thus enabling the detection of IgG. It has the advantages of simple structure, low detection limit, and fast response time.

[0017] The beneficial effects of this invention are as follows: First, a thin gold film is deposited on the surface of TFBG to excite the SPR effect. Then, a functionalized coating of AuNPs / 11-MUA / rabbit anti-human IgG is used as the sensing material for IgG. The addition of AuNPs significantly enhances the SPR signal of TFBG, while the binding of IgG to the antibody causes a change in the refractive index of the sensitive membrane on the surface of the TFBG-SPR sensor. The limit of detection (LOD) of this sensor reaches 30 pg / ml, representing a performance improvement of at least two orders of magnitude compared to existing IgG concentration sensors. Furthermore, this sensor also possesses excellent sensing stability and temperature self-compensation capability, showing broad application prospects in clinical diagnostics and biomedicine. Attached Figure Description

[0018] Figure 1 A schematic diagram of an experimental setup for IgG sensing TFBG-SPR spectral measurement.

[0019] Figure 2 The images show the transmission spectra of the TFBG-SPR sensor in IgG solutions of different concentrations.

[0020] Figure 3 The figure shows the fitted curves of the transmission peak intensity changes of the TFBG-SPR sensor in IgG solutions of different concentrations. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to specific embodiments:

[0022] Example 1: Preparation of gold-plated TFBG.

[0023] In this embodiment, see Figure 1 First, the photosensitive optical fiber (PS1250 / 1500, FIBERCORE) underwent hydrogen loading treatment, a process that lasted for 14 days at a pressure of 15.2 MPa and a temperature of 20 degrees Celsius, to enhance the fiber's photosensitivity. Subsequently, a TFBG (transient fiber optic membrane) was fabricated in the fiber core using UV etching via a phase mask method, with a length set to 1 cm. The tilt angle between the phase mask (period of 541 nm) and the fiber was set to 8 degrees. After etching, the TFBG was annealed at 125 degrees Celsius for 12 hours. Then, the TFBG was immersed in a piranha solution (a mixture of deionized water, hydrogen peroxide, and ammonium hydroxide in an 8:1:1 ratio) for 30 minutes to remove surface contaminants. Finally, it was placed in a magnetron sputtering deposition chamber (PCXT350, PENGCHENG) for gold film deposition.

[0024] Example 2: Preparation and modification of AuNPs / 11-MUA / rabbit anti-human IgG that can specifically bind to IgG.

[0025] In this embodiment, the preparation and modification of AuNPs / 11-MUA / rabbit anti-human IgG that can specifically bind to IgG are specifically carried out through the following steps: a) Dissolve 11-MUA in anhydrous ethanol to prepare a 10 mM 11-MUA solution. Then immerse the gold-plated TFBG in the solution for 12 hours, then remove it and rinse the surface repeatedly with deionized water 3 times. b) Place TFBG in a mixed solution of 0.4M EDC and 0.1M NHS and incubate for 1-2 hours. Then remove it and rinse the surface repeatedly with deionized water 3 times. c) Soak TFBG in 250ug / ml rabbit anti-human IgG solution for 1 hour, then remove it and rinse the surface repeatedly with deionized water 3 times; d) Soak TFBG in a low-concentration BSA solution for 1 hour, then remove it and rinse the surface repeatedly with deionized water 3 times.

[0026] Example 3: Construction of the detection platform.

[0027] In this embodiment, see Figure 1 The detection platform is composed of a single-mode optical fiber (2) connecting a broadband light source (1), a polarization controller (3), a TFBG-SPR sensor (5) placed in a flow cell (4), and a spectrometer (6). The broadband light source (1) has a wavelength range of 1420-1620 nm and is used to provide light. The polarization controller (3) is used to obtain greater stripe contrast. The flow cell (4) is used to add the IgG solution to be tested. The spectrometer (6) is used to monitor and record the spectral changes. When the AuNPs / MPBA / β-CD modified on the gold-plated TFBG surface binds to IgG, the refractive index changes, which is reflected in the drift of the transmission spectrum on the spectrometer (6). By comparing the relationship between different IgG concentrations and the transmission spectrum drift, the concentration of IgG can be detected.

[0028] Example 4: Detection of IgG concentration, repeatability, and time response.

[0029] IgG solution, specificity, and time response assays were performed. All IgG solutions were prepared with anhydrous ethanol. The specific steps are as follows:

[0030] IgG concentration detection: The AuNPs / 11-MUA / rabbit anti-human IgG modified TFBG-SPR sensor was rinsed multiple times with deionized water and anhydrous ethanol and placed in the flow cell (4). Different concentrations of IgG solutions were prepared using anhydrous ethanol and mixed with an equal volume of AuNPs solution to achieve concentrations of 100 pg / ml, 200 pg / ml, 300 pg / ml, 400 pg / ml, 500 pg / ml, 600 pg / ml, 700 pg / ml, and 800 pg / ml. Anhydrous ethanol was added to the flow cell (4), and the initial spectrum was recorded using a spectrometer (6). The solution in the flow cell (4) was rinsed with deionized water, and then the IgG solution was added to the flow cell (4) and allowed to stand. The transmission spectrum was then recorded using a spectrometer (6). Different concentrations of IgG solutions were added sequentially, and the above steps were repeated to obtain the transmission spectra of IgG solutions of different concentrations. Fitting curves were then plotted, as shown in the figure. Figure 2 As shown. (a) Repeatability testing: The used TFBG surface functional film was wiped off with alcohol, and then re-gold plated on the surface before testing. We functionalized a new AuNPs / 11-MUA / rabbit anti-human IgG multilayer membrane. When the sensor was reused, IgG re-binded to the surface antibody. We selected mode 1 (resonant wavelength at 1539.5 nm) of the SPR amplitude change on the right side for observation. Experimental results showed that the sensor exhibited good repeatability. Figure 3 As shown. (b) Time response detection: Prepare an IgG test solution with a concentration of 500 pg / ml. Detect the TFBG-SPR sensor in anhydrous ethyl acetate. Transmission spectra in alcohols were obtained by rinsing the optical fiber multiple times with deionized water and anhydrous ethanol, adding the test solution to the flow cell and allowing it to stand, and then recording the transmission spectrum over time using a spectrometer (6). The above steps were repeated to evaluate the time response of the TFBG-SPR sensor to the analyte.

[0031] The embodiments described above provide a detailed explanation of the technical solution of the present invention, but the scope of protection of this disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of the present invention.

Claims

1. A method for detecting IgG protein concentration based on optical fiber, characterized in that: The detection device consists of a broadband light source (1), a single-mode fiber (2), a polarization controller (3), a flow cell (4), a TFBG-SPR sensor (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 sensor (5), and the right end of the TFBG-SPR sensor (5) is connected to the spectrometer (6); The fabrication steps of the TFBG-SPR sensor (5) are as follows: First, hydrogen is loaded onto the photosensitive fiber (51) and then a tilted fiber grating (52) with a tilt angle of 10 degrees is formed by ultraviolet etching and annealing. Then, the tilted fiber grating (42) is placed in a magnetron sputtering deposition chamber and a gold film with a thickness ranging from 50 nm to 150 nm is deposited on its surface. Finally, 11-MUA and rabbit anti-human IgG are assembled sequentially on the gold film by surface self-assembly.

2. The steps of the fiber-optic IgG concentration detection method are as follows: First, fix the TFBG-SPR sensor (5) in the flow cell (4), then add the IgG test solution to the flow cell (4). The IgG binds to the rabbit anti-human IgG modified on the surface of the TFBG-SPR sensor (5). The light emitted by the broadband light source (1) passes through the TFBG-SPR sensor (5) and the transmission spectrum is recorded on the spectrometer (6). The flow cell (4) is cleaned with anhydrous ethanol for 3 minutes. The data recorded on the spectrometer (6) are fitted with the IgG solutions of different concentrations added to the flow cell (4) to obtain the relationship between different IgG concentrations and the transmission spectrum drift, thereby realizing the detection of IgG concentration.

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