Preparation method of functionalized tapered thin-core micro-nano fiber, product thereof and method for detecting toxoplasma antibody

Abstract

The application discloses a preparation method of a functionalized taper thin-core micro-nano optical fiber, a product of the functionalized taper thin-core micro-nano optical fiber and a method for detecting toxoplasma antibodies. The functionalized taper thin-core micro-nano optical fiber is hydroxylated and silanized in a sensing area, and then is modified by MoS2. The MoS2 has the advantages of high electron mobility, a large surface-to-volume ratio and existing self-thiol groups. More to-be-detected substances can be fixed, and the functionalized taper thin-core micro-nano optical fiber is widely applied in biosensors. The application establishes an antibody detection method with high sensitivity and strong specificity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of detection, in particular to a preparation method of a functionalized tapered thin-core micro-nano fiber, and further relates to a functionalized tapered thin-core micro-nano fiber and a method for detecting Toxoplasma antibodies. BACKGROUND

[0002] Toxoplasmosis is a parasitic disease caused by Toxoplasma gondii (TG), which can seriously endanger human health. The distribution of Toxoplasmosis is extremely wide, and there are related reports of Toxoplasma infection in various regions of China. Data shows that the average infection rate in China is about 10% to 47.3%. In recent years, Xu Bin et al. conducted antibody monitoring of Toxoplasma infection on 865 dog and cat sera in Chongqing, and the results showed that the antibody positive rate of Toxoplasma infection was as high as 18.96%, indicating that the infection of dogs and cats in our city is serious, and the source pollution is serious. Pregnant women infected with Toxoplasmosis can transmit the infection to infants through the placenta and cause congenital Toxoplasmosis, which can easily cause fetal malformation and even miscarriage and stillbirth. For tumor patients, AIDS and other immunodeficient people, infection with Toxoplasma can easily cause other important complications. Cats are the only final host of Toxoplasma transmission, and with the gradual increase in the number of pet animals in recent years, the risk of Toxoplasma transmission from pets to humans and poultry has increased, which has caused great threat to human health and the livestock industry. At present, there is no effective treatment drug and vaccine for chronic infection of Toxoplasma, therefore, early diagnosis of Toxoplasma is the key to prevention and control of Toxoplasmosis. At present, the main detection techniques for Toxoplasmosis include pathogen isolation and identification, enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) method. The pathogen isolation and identification diagnosis is relatively accurate, but it takes a long time and is complicated to operate, which is not conducive to rapid detection; the ELISA method is simple to operate, suitable for large-scale serological investigation, and has great application prospect, but it needs repeated washing, has limited detection sensitivity, and requires a certain amount of sample; the PCR method can directly detect the gene of the virus, and has high detection sensitivity, but has high detection cost, is easy to be contaminated, and has high technical requirements, which is only suitable for laboratory diagnosis and is not suitable for rapid diagnosis of clinical infection. Therefore, it is of great significance to explore efficient, specific, accurate and simple detection methods for the prevention and control of Toxoplasmosis.

[0003] The micro-nano fiber-based biosensor can detect biomolecules with high sensitivity. The detection of the fiber sensor is achieved by introducing a sensing element that only reacts with the target object. Chemical etching is usually used to expose the core to form a micro-nano fiber area as a sensing area. Then the sensing element is modified to the sensing area. The specific reaction with the target object will cause the refractive index of the fiber to change. The change of the optical signal is observed by a spectrometer to achieve the detection purpose. Compared with ordinary optical fibers, the diameter of micro-nano fiber is below micron level, and the refractive index difference of the core and cladding is larger. With the decrease of the diameter of the micro-nano fiber, when the light is transmitted in the micro-nano fiber, the energy is distributed in the form of evanescent field on the surface of the fiber, so that the micro-nano fiber has the characteristics of strong light field constraint, high sensitivity and the like. Although the micro-nano fiber has high refractive index sensitivity, it lacks corresponding biocompatibility. In order to realize the high sensitivity detection of specific biomolecules, the micro-nano fiber also needs special material modification to distinguish and capture the target molecules in the external environment. So far, there is no report on the method for detecting Toxoplasma SAG1 antibody based on the tapered thin-core micro-nano fiber. Therefore, it is necessary to develop a method for detecting Toxoplasma SAG1 based on the tapered thin-core micro-nano fiber. SUMMARY

[0004] Therefore, one of the purposes of the present application is to provide a preparation method of functionalized tapered thin-core micro-nano fiber; the second purpose of the present application is to provide the functionalized tapered thin-core micro-nano fiber prepared by the preparation method; the third purpose of the present application is to provide a sensor containing the functionalized tapered thin-core micro-nano fiber; and the fourth purpose of the present application is to provide a method for detecting antibody by using the sensor of the functionalized tapered thin-core micro-nano fiber.

[0005] In order to achieve the above purposes, the present application provides the following technical solutions.

[0006] 1. A preparation method of functionalized tapered thin-core micro-nano fiber, comprising the following steps:

[0007] (1) treating the waist-tapered region of the cleaned tapered thin-core micro-nano fiber with NaOH to activate the surface hydroxyl group of the waist-tapered region of the tapered thin-core micro-nano fiber;

[0008] (2) treating the waist-tapered region of the tapered thin-core micro-nano fiber activated in step (1) with methoxypropyltrimethoxysilane ethanol solution to make the surface of the waist-tapered region of the tapered thin-core micro-nano fiber have mercapto group;

[0009] (3) coating MoS2 on the surface of the waist-tapered region of the tapered thin-core micro-nano fiber treated in step (2), and combining the defect sites formed by the loss of sulfur atoms on the surface of MoS2 with the mercapto group on the surface of the fiber to modify MoS2 to the surface of the waist-tapered region of the tapered thin-core micro-nano fiber;

[0010] (4) Then the detection antigen is modified on the MoS2 modified tapered core micro-nano fiber waist cone region surface;

[0011] (5) Blocking the blank binding sites.

[0012] Preferably, the step (1) is to immerse the cleaned tapered core micro-nano fiber waist cone region in a 2 mol / L NaOH solution for at least 30 min, and then wash with water to obtain an activated tapered core micro-nano fiber.

[0013] Preferably, the step (2) is to immerse the activated tapered core micro-nano fiber waist cone region in a 5% methoxypropyltrimethoxysilane ethanol solution by mass fraction for 10 min, so that the tapered core micro-nano fiber surface is provided with a mercapto group, and then wash with water to obtain a silanized tapered core micro-nano fiber.

[0014] Preferably, the step (3) is to immerse the tapered core micro-nano fiber waist cone region treated in the step (2) in a 2 mg / mL MoS2 solution at room temperature for 40 s, repeat 6 times, then wash the tapered core micro-nano fiber waist cone region with a PBS solution to obtain a MoS2 modified tapered core micro-nano fiber.

[0015] Preferably, the step (4) is to immerse the MoS2 modified tapered core micro-nano fiber waist cone region in an antigen solution with a concentration of 50 μg / mL at room temperature for 1 h, then wash the tapered core micro-nano fiber waist cone region with a PBS solution to remove unbound antigen molecules, to obtain an antigen modified tapered core micro-nano fiber.

[0016] Preferably, the step (5) is to immerse the antigen modified tapered core micro-nano fiber waist cone region in a skimmed milk powder blocking solution at room temperature for 1 h to block unbound blank sites, then wash the tapered core micro-nano fiber waist cone region with a PBS solution to obtain a functionalized tapered core micro-nano fiber.

[0017] Preferably, the detection antigen is a Toxoplasma SAG1 antigen.

[0018] 2. The functionalized tapered core micro-nano fiber prepared by the preparation method.

[0019] 3. A sensor containing the functionalized tapered core micro-nano fiber, comprising a broadband light source, a spectrum analyzer and the functionalized tapered core micro-nano fiber according to any one of claims 1-7.

[0020] 4. A method for detecting Toxoplasma antibodies by using the sensor of the functionalized tapered core micro-nano fiber, wherein the tapered core micro-nano fiber waist region is immersed in a to-be-detected antibody, the antibody is combined with the antigen to react, then the fiber surface is washed with a PBS solution, and the spectrum data is recorded in real time.

[0021] The application has the advantages that the functionalized taper thin-core micro-nano optical fiber is hydroxylated and silanized by a sensing area, and then is modified by MoS2, MoS2 has high electron mobility, large surface volume ratio and self-thiol group, and the like; more to-be-detected substances can be fixed, and the functionalized taper thin-core micro-nano optical fiber is widely applied in biosensors, and is taken to fix the Toxoplasma SAG1 antigen as an example, the result shows that the wavelength is red-shifted with the increase of the antibody concentration, and the detection range of the sensor is 1 pg / mL-100 ng / mL, and the saturation point is about 100 ng / mL. When the specificity of the sensor is researched, the Toxoplasma SAG1 antigen is fixed on the surface of the optical fiber, and the pseudorabies antibody solution and the rabies antibody solution are sequentially added, and the experimental result shows that the sensor only has a slight red shift, and has good specificity. When the sensor is clinically researched, 5 groups of Toxoplasma positive serum and 3 groups of Toxoplasma negative serum are taken, and 3 parallel experiments are set for each group, and the experimental result shows that the biosensor has a strong specific reaction with the Toxoplasma positive serum, and is suitable for clinical detection. Therefore, the application establishes a Toxoplasma antibody detection method with high sensitivity and strong specificity. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to make the purpose, technical scheme and beneficial effects of the application more clear, the application provides the following drawings for description:

[0023] Figure 1 For micro-nano optical fiber refractive index sensitivity calibration (A: spectral response of spectrum with environmental refractive index; B: wavelength shift and environmental refractive index relationship).

[0024] Figure 2 For taper thin-core micro-nano optical fiber surface modification and biological functionalization process diagram;

[0025] Figure 3 For taper thin-core micro-nano optical fiber surface modification and biological functionalization process spectrum detection result (A: spectral change; B: wavelength shift change);

[0026] Figure 4 For taper thin-core micro-nano optical fiber biosensor structure diagram;

[0027] Figure 5 For micro-nano optical fiber biosensor specificity and clinical detection (A: spectral change curve diagram; B: wavelength shift amount); DETAILED DESCRIPTION

[0028] The application will be further described below in combination with the drawings and specific embodiments, so that those skilled in the art can better understand the application and implement the application, but the embodiments are not as a limitation on the application.

[0029] The application is supported by the Postgraduate Innovation Fund Project of Chongqing University of Technology (No. gzlcx20223354).

[0030] Before the cone-shaped thin-core micro-nano fiber is used in the application, a refractive index calibration experiment needs to be performed, and simulation analysis needs to be performed under different external environment refractive indexes. The RI sensitivity value of the micro-nano fiber is obtained by calculation. The specific method is to drop the same volume of deionized water and 1%-5% NaCl solution on the lumbar region of the cone-shaped thin-core micro-nano fiber to simulate different external environment refractive indexes for experiments, and the results are shown in Figure 1 The results show that the deionized water RI is 1.3313, the 1% NaCl solution RI is 1.3327, the 2% NaCl solution RI is 1.3347, the 3% NaCl solution RI is 1.3366, the 4% NaCl solution RI is 1.3384, and the 5% NaCl solution RI is 1.3392. From the results, it can be seen that the transmission spectrum diagram of the wavelength of the cone-shaped thin-core micro-nano fiber changes with the external environment refractive index, and the corresponding RI value increases from 1.3327 to 1.3392 as the concentration of the NaCl solution increases. In this process, the transmission spectrum of the cone-shaped thin-core micro-nano fiber is red-shifted. Through data fitting, the refractive index sensitivity of the cone-shaped thin-core micro-nano fiber is calculated to be 2360.468 nm / RIU.

[0031] Example 1, surface modification and biological functionalization of cone-shaped thin-core micro-nano fiber

[0032] The surface modification and biological functionalization of the cone-shaped thin-core micro-nano fiber includes the following steps, as shown in Figure 2

[0033] (1) Surface pretreatment of cone-shaped thin-core micro-nano fiber: the lumbar region of the cone-shaped thin-core micro-nano fiber is immersed in a 5% HNO3 solution at room temperature for 30 min to remove impurities on the surface of the fiber. The spectrum data is recorded in real time, and then the lumbar region of the cone-shaped thin-core micro-nano fiber is washed with pure water;

[0034] (2) Hydroxylation (-OH) of the surface of the cone-shaped thin-core micro-nano fiber: the lumbar region of the cone-shaped thin-core micro-nano fiber after pretreatment is activated with a 2 mol / L NaOH solution at room temperature for 60 min to activate the hydroxyl group on the surface of the fiber. The lumbar region of the cone-shaped thin-core micro-nano fiber is washed with deionized water;

[0035] ​(3) Silanization of the surface of the tapered thin-core micro-nano fiber (-NH2): The lumbar region of the tapered thin-core micro-nano fiber after hydroxylation was immersed in 600 μL of 5% methoxypropyltrimethoxysilane ethanol solution at room temperature for 10 min to make the surface of the fiber have a mercapto group (-SH), and then the lumbar region of the tapered thin-core micro-nano fiber was washed with deionized water.

[0036] (4) Coating of MoS2 on the surface of the tapered thin-core micro-nano fiber:

[0037] The lumbar region of the tapered thin-core micro-nano fiber after silanization was immersed in 500 μL of 2 mg / mL MoS2 solution at room temperature for 40 s, repeated 6 times, and in this process, the defect sites formed due to the loss of sulfur atoms on the surface of MoS2 can be combined with the mercapto group on the surface of the fiber, which can make MoS2 modified to the surface of the tapered thin-core micro-nano fiber, and then the lumbar region of the tapered thin-core micro-nano fiber was washed with PBS to obtain a MoS2 modified tapered thin-core micro-nano fiber sensor.

[0038] (5) Modification of Toxoplasma SAG1 antigen on the surface of MoS2: The lumbar region of the tapered thin-core micro-nano fiber after MoS2 surface modification was immersed in 200 μL of 50 μg / mL Toxoplasma SAG1 antigen solution at room temperature for 1 h, and the spectral data was recorded in real time, and every 20 min the slide was moved gently to make the Toxoplasma SAG1 antigen fully combined with the lumbar region of the tapered thin-core micro-nano fiber, and then the lumbar region of the tapered thin-core micro-nano fiber was washed with PBS to remove unbound TG SAG1 antigen molecules.

[0039] (6) Blocking of blank binding sites: The lumbar region of the tapered thin-core micro-nano fiber after antigen modification was immersed in 400 μL of 5% skim milk blocking solution at room temperature for 1 h, and the spectral data was recorded in real time, and the purpose was to block the unbound blank sites, and then the lumbar region of the tapered thin-core micro-nano fiber was washed with PBS to obtain a tapered thin-core micro-nano fiber with surface modification and biological functionalization.

[0040] The spectral data was recorded in real time during the modification process, and the results are shown in Figure 3 The results show that the wavelength red shift of the biosensor after surface modification of MoS2, modification of Toxoplasma SAG1 antigen, and blocking of blank sites is 2.34 nm, 4.68 nm, and 6.48 nm, respectively, compared with the bare fiber, indicating that the surface modification of the tapered thin-core micro-nano fiber is good. Therefore, compared with the bare fiber, the wavelength shift of the tapered thin-core micro-nano spectrum after coating MoS2 is 2.34 nm; during the process of coating MoS2 on the tapered thin-core micro-nano fiber and modifying Toxoplasma SAG1 antigen, the wavelength red shift is 2.34 nm, indicating that the Toxoplasma SAG1 antigen is successfully modified on the surface of the sensor.

[0041] Example 2, construction of a tapered thin-core micro-nano fiber biosensor

[0042] The construction of a tapered thin-core micro-nano fiber biosensor includes a broadband light source (BBS), an optical spectrum analyzer (OSA), and the surface-modified and bio-functionalized tapered thin-core micro-nano fiber prepared in Example 1. The broadband light emitted by the broadband light source is transmitted through the tapered thin-core micro-nano fiber (TTCMF) to the MoS2-modified tapered thin-core micro-nano fiber sensing region, and the resulting spectral changes are monitored in real time by the optical spectrum analyzer. In order to prevent the tapered thin-core micro-nano fiber sensing region from being disturbed by external air flow and other factors, it is placed in a dustproof operation table, as shown in Figure 4 .

[0043] Example 3, detection of a tapered thin-core micro-nano fiber biosensor

[0044] When the sensor constructed in Example 2 was used to detect five groups of positive serum samples and three groups of negative serum samples of Toxoplasma gondii, the experimental results were as shown in Figure 5 . The results showed that the spectra of the five groups of positive serum samples of Toxoplasma gondii had a significant red shift compared to the spectra after sealing, with red shift amounts of 4.6676 nm, 4.8763 nm, 4.9117 nm, 5.9236 nm, and 6.3248 nm, respectively, which were significantly higher than the wavelength red shift amounts of the three groups of negative serum samples, indicating that the micro-nano fiber biosensor had good clinicality.

[0045] In order to verify the specificity of the prepared sensor, the sensor was used to detect pseudorabies (PRV) antibodies and rabies (RV) antibodies for specificity experiments, and the detection results were as shown in Figure 5 . The results showed that when detecting PRV antibody solution and RV antibody solution, the spectrum of the sensor only had a slight red shift compared to the spectrum after sealing, with red shift amounts of about 0.2518 nm and 0.3015 nm, respectively. The wavelength red shift may be due to the fact that there are still blank sites on the surface of the sensor, which can bind to non-specific antibodies and cause spectral drift. When detecting Toxoplasma gondii positive serum solution, the wavelength red shift amount was more than 4.6676 nm. The wavelength change produced by the biosensor when detecting Toxoplasma gondii antibodies was significantly higher than that when detecting PRV Ab and RV Ab, indicating that the constructed micro-nano fiber biosensor had good specificity.

[0046] Sensitivity detection: In order to evaluate the detection sensitivity of the biosensor, the prepared Toxoplasma SAG1 monoclonal antibody was diluted with PBS (0.01M, pH 7.4) into 1 pg / mL, 10 pg / mL, 50 pg / mL, 100 pg / mL, 500 pg / mL, 1 ng / mL, 100 ng / mL, 500 ng / mL, respectively, and then 400 μL of the Toxoplasma SAG1 antibody solution from low concentration to high concentration was added to soak the lumbar region of the tapered thin-core micro-nano optical fiber, each concentration of the antibody was combined with the antigen for 15 min, then the optical fiber surface was washed with PBS to remove the unbound Toxoplasma SAG1 antibody, and the spectral data was recorded in real time. The results show that the spectrum is red-shifted with the increase of the concentration of the Toxoplasma antibody solution, and the wavelength shift is more obvious in the concentration range of 1 pg / mL to 100 ng / mL, indicating that the binding sites of the optical fiber surface and the Toxoplasma antigen gradually decrease with the increase of the concentration of the antibody solution, and when the concentration of the antibody is from 100 ng / mL to 500 ng / mL, the wavelength has only a slight red shift, indicating that the detection upper limit of the sensor is 100 ng / mL, and the detection range of the biosensor is about 1 pg / mL to 100 ng / mL.

[0047] The above-described embodiments are only preferred embodiments of the present application for fully illustrating the present application, and the protection scope of the present application is not limited thereto. Any equivalent replacement or transformation of the present application based on the present application is within the protection scope of the present application. The protection scope of the present application is subject to the claims.

Claims

1. A method for fabricating functionalized tapered fine-core micro / nano optical fibers, characterized in that: Includes the following steps: (1) The waist cone region of the cleaned tapered micro / nano fiber is treated with NaOH to activate the hydroxyl groups on the surface of the waist cone region of the tapered micro / nano fiber; the refractive index sensitivity of the tapered micro / nano fiber is 2360.468 nm / RIU. (2) The waist cone region of the tapered fine core micro-nano fiber activated in step (1) is soaked in a 5% methoxypropyltrimethoxysilane ethanol solution for 10 min to make the surface of the tapered fine core micro-nano fiber have mercapto groups. The fiber is then rinsed with water to obtain silanized tapered fine core micro-nano fiber. (3) The waist cone region of the tapered micro / nano fiber treated in step (2) was soaked in a MoS2 solution with a concentration of 2 mg / mL at room temperature for 40 s, repeated 6 times, and then the waist cone region of the tapered micro / nano fiber was rinsed with PBS solution to obtain MoS2 modified tapered micro / nano fiber. (4) Then, the detection antigen is modified on the surface of the waist cone region of the MoS2-modified tapered micro / nano fiber; (5) Close blank binding sites.

2. The method for fabricating functionalized tapered fine-core micro / nano optical fibers according to claim 1, characterized in that: Step (1) involves immersing the waist cone region of the cleaned tapered micro / nano fiber in a 2 mol / L NaOH solution for at least 30 min, and then rinsing it with water to obtain the activated tapered micro / nano fiber.

3. The method for fabricating functionalized tapered fine-core micro / nano optical fibers according to claim 1, characterized in that: Step (4) involves soaking the waist cone region of the MoS2-modified tapered micro / nano fiber in an antigen solution with a concentration of 50 μg / mL at room temperature for 1 h, and then rinsing the waist cone region of the tapered micro / nano fiber with PBS to remove unbound antigen molecules, thereby obtaining the antigen-modified tapered micro / nano fiber.

4. The method for fabricating functionalized tapered fine-core micro / nano optical fibers according to claim 1, characterized in that: Step (5) involves immersing the waist cone region of the antigen-modified tapered micro / nano fiber in skim milk powder blocking solution at room temperature for 1 hour to block unbound blank sites, and then rinsing the waist cone region of the tapered micro / nano fiber with PBS to obtain a functionalized tapered micro / nano fiber.

5. The method for fabricating functionalized tapered fine-core micro / nano optical fibers according to any one of claims 1 to 4, characterized in that: The antigen being detected is the Toxoplasma gondii SAG1 antigen.

6. Functionalized tapered fine-core micro / nano optical fibers prepared by the preparation method according to any one of claims 1 to 5.

7. A sensor comprising the functionalized tapered fine-core micro / nano optical fiber as described in claim 6, characterized in that: It includes a broadband light source, a spectrometer, and the functionalized tapered fine-core micro / nano optical fiber as described in any one of claims 1 to 5.

8. A method for detecting Toxoplasma gondii antibodies using a sensor derived from the functionalized tapered micro / nano optical fiber of claim 7, characterized in that: The lumbar region of a tapered, fine-core micro / nano optical fiber was soaked with the antibody to be tested for Toxoplasma gondii to allow the antibody to bind to the antigen and react. The surface of the optical fiber was then rinsed with PBS, and the spectral data was recorded in real time.

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