SPR (Surface Plasmon Resonance) optical fiber probe and application thereof in detecting type II ribosome inactivation protein

Abstract

The invention relates to a surface plasmon resonance (SPR) optical fiber probe, further relates to application of the SPR optical fiber probe in detection of type II ribosome inactivating protein (such as ricin and abrus precatorius toxin), further relates to a kit and a detection system comprising the SPR optical fiber probe, and further relates to a method for detecting the type II ribosome inactivating protein by using the SPR optical fiber probe. The metal nanoparticle layer on the surface of the SPR optical fiber probe is not easy to fall off, has good stability, can be repeatedly used, and can improve the detection stability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of analytical chemistry and medical defense against chemical weapons, and particularly relates to a surface plasmon resonance (SPR) optical fiber probe and its application in detection of type II ribosome inactivating proteins (e.g., ricin and abrin). BACKGROUND

[0002] Ricin and abrin belong to type II ribosome inactivating proteins (RIP-II) and are composed of A and B chains, with the molecular weight of the A chain being about 30 kDa and the molecular weight of the B chain being about 35 kDa. The two chains are connected by a disulfide bond, and the B chain has lectin activity and can bind to glycosyl structures such as galactose and N-acetylgalactosamine on the cell surface, thereby guiding the RIP-II toxin protein into the cell. This process is a prerequisite for the A chain to exert enzymatic activity and inhibit protein synthesis, ultimately leading to cell death. Ricin and abrin are highly toxic, easy to prepare, and highly concealed (with a latent period in the body), and there is currently no specific antidote, only symptomatic treatment. Therefore, how to sensitively and accurately detect trace amounts of active ricin and abrin proteins has important practical significance and has become a problem that needs to be solved in public health, food safety, and poisoning diagnosis and treatment.

[0003] Surface plasmon resonance (SPR) is an excellent method for studying molecular interactions. Based on the study of the interaction of molecules with different characteristics and their combinations on the material surface with incident light, it can be used to monitor, label-free, and low sample requirement, and has the advantages of rapid detection process, high throughput, and high sensitivity, which has great potential in the field of toxin protein sensing detection. For example, in 2008, Hung et al. used antibodies as affinity elements to combine with commercial SPR instruments to detect ricin in environmental samples, with a LOD of 0.5 ng / mL and a linear range of 3 orders of magnitude (Toxicon, 2008, 52(4): 582-588). In 2022, the inventors' team developed a protein G-directed SPR resonance immunosensing method based on a commercial SPR instrument, which realized the label-free differentiation and quantification of RCA60, ABA60 and their lectins, and successfully applied it to steviol glycosides, protein powder and other matrices with a LOD as low as 0.6 ng / mL and a linear range of 2-3 orders of magnitude (Talanta, 2022, 238:122860). In 2023, the inventors' team screened different polypeptides as affinity elements and established a polypeptide-based SPR sensing method to detect ricin, which had a good linear relationship and a LOD of 0.5 nM (30 ng / mL) (Toxins, 2023, 15(8)). The above work laid a good foundation for the development of portable sensing analysis methods based on SPR technology for toxin proteins.

[0004] However, this technology is often limited by the instrument structure when applied. Most commercial SPR instruments adopt a prism structure, which is often bulky and complex to operate, and can only be operated in an off-site laboratory, making them unsuitable for use as field testing equipment. For example, the Biacore T200 uses a prism structure and employs complex microfluidic technology to analyze samples, requiring cumbersome and complex cleaning steps between the analysis of different samples. Fiber-coupled SPR is another SPR coupling method, which is suitable for the development of miniaturized SPR instruments. Suzuki et al. successfully fabricated a fiber optic SPR sensor by depositing a uniformly thick gold film layer on the surface of an optical fiber (Sensors and Actuators B: Chemical, 2008, 132(1): 26-33). This uniform coating method has been widely recognized and applied, becoming a classic approach for fabricating fiber optic SPR sensors. To further improve the sensitivity of sensors, researchers have explored various methods, one of which is to change the substrate structure of the optical fiber, including D-type, U-type, tapered, and multimode fiber-coreless fiber-multimode fiber (MMF-NCF-MMF) structures (Sensors, 2019, 19(19): 4345). This allows the light field in the fiber to leak from the fiber core, thereby exciting more evanescent waves, improving the sensor's sensitivity, and achieving better sensing performance. However, optical fibers are thin and fragile, and changing the substrate structure is not only complex but also reduces the mechanical strength of the sensor structure, making it difficult to ensure the sensor's stability. Another method is to optimize the film material by adding metal oxide films or two-dimensional materials, such as TiO2, Ta2O5, MoS2, and graphene, to the surface of the sensor's metal film layer. This aims to change the electric field distribution in the sensing film layer, enhance the electric field strength, and ultimately improve the performance of the fiber optic SPR sensor (Optik, 2021, 226 (P1): 165842). However, this type of method requires complex chemical processing, which is costly and increases the difficulty of fabricating fiber optic SPR sensors. Furthermore, it may lead to an increase in the full width at half maximum (FWHM) of the resonant wavelength, reducing the sensor's performance and stability. Patent document CN119738388A discloses a fiber optic SPR sensor, its fabrication method, and a detection system. The gold film particle size on the surface of this fiber optic SPR sensor exhibits a gradient change along the fiber axis, resulting in a wide detection range and high detection sensitivity. This to some extent meets the requirements for instrument miniaturization, simplified operation, and portability. However, the SPR fiber optic sensor fabricated using this method has poor gold film stability, which greatly restricts its widespread use in the field of rapid detection. Summary of the Invention

[0005] This application provides an SPR fiber probe, comprising: a fiber substrate, a first dielectric layer and a metal nanoparticle layer, wherein the fiber substrate is a quartz fiber substrate, and the first dielectric layer is formed by a mercaptosilane coupling agent or an aminosilane coupling agent and is located between the fiber substrate and the metal nanoparticle layer.

[0006] In some embodiments, the first dielectric layer is covalently connected to the optical fiber substrate and to the metal nanoparticle layer via metal-nitrogen coordination bonds or metal-sulfur covalent bonds.

[0007] In some embodiments, the mercaptosilane coupling agent or aminosilane coupling agent of the first dielectric layer undergoes hydrolytic condensation with the surface of the optical fiber substrate to form Si-O-Si covalent bonds, and forms metal-nitrogen coordination bonds or metal-sulfur covalent bonds with the metal nanoparticles of the metal nanoparticle layer.

[0008] The hydrolyzable siloxane groups in mercaptosilane coupling agents or aminosilane coupling agents can undergo hydrolysis and condensation reactions with the silanol groups on the surface of the optical fiber substrate to form Si-O-Si covalent bonds; the mercapto groups in mercaptosilane coupling agents can form metal-sulfur covalent bonds between the metal nanoparticles in the metal nanoparticle layer; the amino groups in aminosilane coupling agents can form metal-nitrogen coordination bonds between the metal nanoparticles in the metal nanoparticle layer, thereby making the metal nanoparticles stably bonded to the surface of the optical fiber substrate and not easily detached.

[0009] In some embodiments, the mercaptosilane coupling agent is 3-mercaptopropyltrimethoxysilane, 3-mercaptopropyltriethoxysilane, or mercaptomethyltrimethoxysilane. In some embodiments, the aminosilane coupling agent is 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-aminopropylmethyldimethoxysilane, N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane, or N-(β-aminoethyl)-γ-aminopropyltriethoxysilane.

[0010] In some embodiments, the first dielectric layer is formed of a mercaptosilane coupling agent, wherein the mercaptosilane coupling agent is 3-mercaptopropyltrimethoxysilane.

[0011] In some embodiments, the metal nanoparticle layer includes metal nanoparticles, such as gold, silver, copper, platinum, aluminum, and titanium nanoparticles.

[0012] In some embodiments, the metal nanoparticles are spherical, star-shaped, rod-shaped, spindle-shaped, or triangular nanoparticles.

[0013] In some embodiments, the metal nanoparticles are gold nanoparticles, and the first dielectric layer and the metal nanoparticle layer are connected by gold-sulfur covalent bonds.

[0014] In some embodiments, the SPR fiber probe further includes a metal oxide layer attached to the metal nanoparticle layer.

[0015] In some embodiments, the metal oxide layer comprises a metal oxide, such as titanium dioxide, tantalum pentoxide, or aluminum oxide. In some embodiments, the metal oxide is titanium dioxide.

[0016] In some embodiments, the SPR fiber probe further includes a second dielectric layer and a functional modification layer, wherein the second dielectric layer is formed of epichlorohydrin or an analogue thereof (e.g., methyl epichlorohydrin) and is connected to the metal oxide layer via ether bonds; the functional modification layer includes carboxylated dextran and is connected to the second dielectric layer via ether bonds.

[0017] After treatment with a strong alkaline aqueous solution, exposed hydroxyl groups are formed on the surface of the metal oxide layer. Chlorine atoms of epichlorohydrin or its analogues undergo Williamson synthesis with these hydroxyl groups to form ether bonds, thereby forming ethylene oxide-2-ylmethoxy groups on the surface of the metal oxide layer. Ethyl oxide-2-yl (i.e., epoxy groups) forms ether bonds with dextran through a ring-opening reaction, modifying the metal oxide surface with dextran. Further, the hydroxyl groups on the dextran surface are oxidized to carboxyl groups, thus completing the connection of the functional modification layer.

[0018] This application also provides a detection system, including a light source, a spectrometer, and the SPR fiber optic probe described in any embodiment of this application.

[0019] This application also provides a kit comprising the SPR fiber probe described in any embodiment of this application.

[0020] In some embodiments, the kit further includes one or more substances from group A consisting of desialylated fetoglobulin, gold nanoparticles, protein A, recombinant humanized anti-ricin monoclonal antibody, EDC (1-Ethyl-3-(3-dimethylaminopropyl)Carbodiimide), NHS (N-Hydroxy succinimide), acetate-sodium acetate buffer, PBS buffer, and NaOH.

[0021] In some embodiments, the kit further includes one or more substances from group B consisting of desialylated fetoglobulin, gold nanoparticles, biotin, concanavalin A, streptavidin, EDC, NHS, acetate-sodium acetate buffer, HBS-BT buffer, and HCl solution.

[0022] In some embodiments, the kit further includes one or more substances from group C, consisting of desialylated fetoglobulin, gold-coated magnetic nanoparticles, protein A, recombinant humanized anti-ricin monoclonal antibody, EDC, NHS, acetate-sodium acetate buffer, PBS buffer, and NaOH.

[0023] In some embodiments, the kit further includes one or more substances from group D consisting of desialylated fetoglobulin, gold-coated magnetic nanoparticles, biotin, concanavalin A, streptavidin, EDC, NHS, acetate-sodium acetate buffer, HBS-BT buffer, and HCl solution.

[0024] In some embodiments, the kit further includes one or more substances from group E consisting of gold nanoparticles surface-conjugated with desialylate-fetoprotein, protein A, recombinant humanized monoclonal antibody against ricin, EDC, NHS, acetate-sodium acetate buffer, PBS buffer, and NaOH.

[0025] In some embodiments, the kit further includes one or more substances from group F, consisting of gold nanoparticles surface-coupled with desialylated fetoglobulin, biotin-labeled concanavalin A, streptavidin, EDC, NHS, acetate-sodium acetate buffer, HBS-BT buffer, and HCl solution.

[0026] In some embodiments, the kit further includes one or more substances from group G consisting of gold-coated magnetic nanoparticles with desialylated fetoglobulin surface-coupled, protein A, recombinant humanized anti-ricin monoclonal antibody, EDC, NHS, acetate-sodium acetate buffer, PBS buffer, and NaOH.

[0027] In some embodiments, the kit further includes one or more substances from group H, consisting of gold-coated magnetic nanoparticles with desialylated fetoglobulin surface-coupled, biotin-labeled concanavalin A, streptavidin, EDC, NHS, acetate-sodium acetate buffer, HBS-BT buffer, and HCl solution.

[0028] This application also provides the use of the SPR fiber probe, detection system or kit described in any embodiment of this application in the detection of type II ribosome inactivating proteins.

[0029] This application also provides a method for detecting type II ribosome inactivating proteins in a sample, comprising: 1) bringing the SPR fiber optic probe described in any embodiment of this application into contact with the sample to be tested; and 2) detecting changes in the SPR signal.

[0030] In some embodiments, the method for detecting type II ribosome-inactivating proteins in a sample includes: Activate the SPR fiber probe and bring it into contact with protein A, then attach protein A to the surface of the SPR fiber probe. The SPR fiber probe is brought into contact with the recombinant anti-ricin humanized monoclonal antibody, and the recombinant anti-ricin humanized monoclonal antibody is attached to protein A on the surface of the SPR fiber probe. The SPR fiber probe is brought into contact with the sample to capture type II ribosome-inactivating proteins in the sample. The SPR fiber probe is brought into contact with gold nanoparticles whose surface is coupled with desialylated fetoglobulin. Detect changes in the SPR signal.

[0031] In some embodiments, the method for detecting type II ribosome-inactivating proteins in a sample includes: The SPR fiber probe is activated and brought into contact with streptavidin, thereby attaching streptavidin to the surface of the SPR fiber probe. The SPR fiber probe is brought into contact with biotin-labeled concanavalin A, and the biotin-labeled concanavalin A is attached to streptavidin on the surface of the SPR fiber probe. The SPR fiber probe is brought into contact with the sample to capture type II ribosome-inactivating proteins in the sample. The SPR fiber probe is brought into contact with gold nanoparticles whose surface is coupled with desialylated fetoglobulin. Detect changes in the SPR signal.

[0032] In some embodiments, the method for detecting type II ribosome-inactivating proteins in a sample includes: Activate the SPR fiber probe and bring it into contact with protein A, then attach protein A to the surface of the SPR fiber probe. The SPR fiber probe is brought into contact with the recombinant anti-ricin humanized monoclonal antibody, and the recombinant anti-ricin humanized monoclonal antibody is attached to protein A on the surface of the SPR fiber probe. The sample to be tested was brought into contact with gold-coated magnetic nanoparticles on which desialylated fetoglobulin was coupled to the surface, and the type II ribosome inactivating protein in the sample to be tested was captured by the gold-coated magnetic nanoparticles. The SPR fiber probe was brought into contact with gold-coated magnetic nanoparticles that had captured type II ribosome inactivating proteins. Detect changes in the SPR signal.

[0033] In some embodiments, the method for detecting type II ribosome-inactivating proteins in a sample includes: The SPR fiber probe is activated and brought into contact with streptavidin, thereby attaching streptavidin to the surface of the SPR fiber probe. The SPR fiber probe is brought into contact with biotin-labeled concanavalin A, and the biotin-labeled concanavalin A is attached to streptavidin on the surface of the SPR fiber probe. The sample to be tested was brought into contact with gold-coated magnetic nanoparticles on which desialylated fetoglobulin was coupled to the surface, and the type II ribosome inactivating protein in the sample to be tested was captured by the gold-coated magnetic nanoparticles. The SPR fiber probe was brought into contact with gold-coated magnetic nanoparticles that had captured type II ribosome inactivating proteins. Detect changes in the SPR signal.

[0034] During detection, qualitative analysis of the analyte is performed based on the SPR absorption wavelength shift, and quantitative analysis is performed based on the magnitude of the signal response value.

[0035] In some embodiments, the sample to be tested is a urine sample or a blood sample. When the sample to be tested is a blood sample, it can be tested by contacting the sample with gold-coated magnetic nanoparticles with desialylate-fetoprotein coupled to their surface.

[0036] In some embodiments, the type II ribosome inactivating protein is ricin or absinthecin.

[0037] This application also provides a method for preparing the SPR fiber probe described in any embodiment of this application, comprising: Hydroxylation treatment is performed on the optical fiber substrate; A mercaptosilane coupling agent or an aminosilane coupling agent is modified onto the surface of the optical fiber substrate to form a first dielectric layer; A layer of metal nanoparticles is deposited on the surface of an optical fiber substrate.

[0038] In some embodiments, the method for preparing the SPR fiber probe according to any embodiment of this application further includes one or more of the following operations: A metal oxide layer is coated on the surface of the optical fiber substrate; The metal oxide layer on the surface of the optical fiber substrate is subjected to hydroxylation treatment; Epichlorohydrin or methyl epichlorohydrin is modified onto the surface of the optical fiber substrate to form a second dielectric layer; Dextran was modified onto the surface of the optical fiber substrate; Carboxylation treatment was performed on the surface of the optical fiber substrate.

[0039] In this application, the silica optical fiber is an optical fiber made primarily of SiO2. During the manufacture of the silica optical fiber, the refractive index distribution of the fiber can be altered by controlling the doping amount as needed.

[0040] In this application, the aminosilane coupling agent is a class of organosilicon compounds containing both an amino group (-NH2 or -NHR) and a hydrolyzable siloxane group (such as -Si(OR)3, where R is an alkyl group), including 3-aminopropyltrimethoxysilane (H2N-(CH2)3-Si(OCH3)3), 3-aminopropyltriethoxysilane (H2N-(CH2)3-Si(OEt)3), 3-aminopropylmethyldimethoxysilane (H2N-(CH2)3-Si(OEt)3), and 3-aminopropylmethyldimethoxysilane (H2N-(CH2)3-Si(OEt)3). Monoamino silane coupling agents such as H2N-(CH2)3-Si(CH3)(OCH3)2, and diamino silane coupling agents such as N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane (H2N-(CH2)2-NH-(CH2)3-Si(OCH3)3) and N-(β-aminoethyl)-γ-aminopropyltriethoxysilane (H2N-(CH2)2-NH-(CH2)3-Si(OEt)3).

[0041] In this application, the mercaptosilane coupling agent is a class of organosilicon compounds that simultaneously contain a mercapto group (-SH) and a hydrolyzable siloxane group (such as -Si(OR)3), including mono-mercaptosilane coupling agents such as 3-mercaptopropyltrimethoxysilane (HS-(CH2)3-Si(OCH3)3), 3-mercaptopropyltriethoxysilane (HS-(CH2)3-Si(OEt)3), and mercaptomethyltrimethoxysilane (HS-CH2-Si(OCH3)3).

[0042] In this application, the carboxylated dextran refers to a functional polysaccharide derivative obtained by chemically modifying the hydroxyl groups of the glucose units of dextran to introduce carboxyl groups (-COOH). In this application, when modifying the SPR fiber probe with carboxylated dextran, the dextran can be first modified onto the surface of the SPR fiber probe, and then the hydroxyl groups on the surface of the dextran can be further oxidized to carboxyl groups, thereby completing the modification of the carboxylated dextran.

[0043] In this application, coupling desialylated fetoprotein to the surface of gold nanoparticles or gold-coated magnetic nanoparticles is a routine operation in the art. For example, the strong interaction of gold-sulfur bonds (Au-S) can be utilized, and the connection can be achieved through the thiol group (-SH) in the ASF molecule or by introducing thiol modification, while ensuring protein activity and nanoparticle stability.

[0044] In this application, the HBS-N buffer substance refers to a substance that can be formulated into an HBS-N buffer solution, which can exist in solution or dry powder form, including HEPES buffer substance and NaCl. It can be formulated as needed, for example, into a buffer solution containing 10 mM HEPES and 137 mM NaCl with a pH of 7.5.

[0045] In this application, the HBS-T buffer material refers to a substance that can be formulated into an HBS-T buffer solution, which can exist in solution or dry powder form, including HEPES buffer material, NaCl and Tween-20. It can be formulated as needed, for example, into a buffer solution containing 10 mM HEPES, 150 mM NaCl and 0.05% (v / v) Tween-20, with a pH of 7.2-7.4.

[0046] In this application, the HBS-BT buffer material refers to a substance that can be formulated into an HBS-BT buffer solution, which can exist in solution or dry powder form, including HEPES buffer material, NaCl, Tween-20 and bovine serum albumin (BSA). It can be formulated as needed, for example, into a buffer solution containing 10 mM HEPES, 150 mM NaCl, 0.05% (v / v) Tween-20 and 0.1% BSA, with a pH of 7.2-7.4.

[0047] In this application, the PBS buffer substance refers to a substance that can be formulated into a PBS buffer solution, which can exist in solution or dry powder form, including disodium hydrogen phosphate (Na2HPO4), potassium dihydrogen phosphate (KH2PO4), and sodium chloride (NaCl). It can be formulated as needed, for example, into a 10 mM, 0.8% NaCl buffer solution with a pH of 7.2 to 7.4.

[0048] In this application, the acetate-sodium acetate buffer substance can exist in solution or dry powder form, referring to a substance that can be formulated into an acetate-sodium acetate buffer solution. In use, the acetate-sodium acetate buffer substance can be formulated into buffer solutions of different pH values ​​as needed, for example, a 10 mM buffer solution with pH 4.0.

[0049] In this application, the HEPES buffer substance can exist in solution or dry powder form, and refers to a substance that can be formulated into HEPES buffer solution, including HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid).

[0050] Beneficial technical effects of this application 1. The metal nanoparticle layer on the surface of the SPR fiber probe provided in this application is not easily detached, has good stability, can be reused, and can improve the stability of detection; 2. Using the SPR fiber optic probe provided in this application to detect type II ribosome-inactivating proteins (such as ricin or abrinogen toxin) can improve the detection sensitivity; 3. The method provided in this application for detecting type II ribosome inactivating proteins (such as ricin or abrinogen) has high sensitivity, a detection limit as low as 1 ng / mL (LOD of 0.6 ng / mL), and is simple to operate. It can be used with the help of magnetic particles to perform rapid on-site detection of analytes in complex matrices such as plasma. 4. The SPR fiber optic probe and detection method provided in this application can be adapted to small portable instruments as well as desktop SPR detection instruments based on the same principle, and can be easily expanded from single-channel to multi-channel, thus having a wide range of applications. Attached Figure Description

[0051] Figure 1 Image (A) of the gold film in the sensing area of ​​the control sample under a 40x microscope and image (B) of the gold film in the sensing area of ​​the SPR fiber probe prepared in the embodiments of this application. Figure 2 The image shows the UV-Vis absorption spectrum of AuNPs. Figure 3 The BCA standard curve plotted for the preparation of ASF@AuNPs; Figure 4 The UV-Vis absorption spectra before and after GMNP modification are shown. Figure 5 The BCA standard curve plotted for the preparation of ASF@GMNPs; Figure 6 Linear curve of RCA60 detection using antibody-glycoprotein sandwich assay; Figure 7 The results of antibody-glycoprotein sandwich assay for detecting ricin and abrinogen toxin are shown in the figure. Figure 8 Standard curve for RCA60 detection using the disaccharide-protein sandwich method; Figure 9 A standard curve for the detection of ABA60 using the disaccharide sandwich method; Figure 10 The results of direct detection of RCA60 in urine samples using the antibody-glycoprotein sandwich method are shown in the figure. Figure 11 A standard curve for detecting RCA60 in plasma samples using the antibody-glycoprotein sandwich method with ASF@GMNPs; Figure 12 A standard curve for detecting ABA60 in plasma samples using the ASF@GMNPs disaccharide sandwich method. Detailed Implementation

[0052] The following specific embodiments further illustrate the substantive content of this application. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this application. In the following embodiments, unless specific conditions are specified, conventional conditions or manufacturer recommendations are followed. Raw materials whose manufacturers are not specified are all commercially available conventional products.

[0053] While many of the materials and operating methods used in the following embodiments are well known in the art, this application still describes them in as much detail as possible. It will be apparent to those skilled in the art that, unless otherwise stated, the materials and operating methods used in the following embodiments are well known in the art.

[0054] Example 1: Preparation and stability evaluation of gold film on the sensing area surface of SPR fiber optic probe 1. Fabrication of gold film on the surface of the sensing region of SPR fiber optic probe (1) SPR fiber probe pretreatment: Wipe the surface of the SPR fiber probe sensing area (i.e. fiber substrate) with lint-free paper dipped in alcohol (the fiber probe was purchased from Suzhou Feibosichuang Optoelectronic Technology Co., Ltd., specification model: TZ-HP400-L37.7-6). Then, immerse the SPR fiber probe sensing area in water, anhydrous ethanol and acetone in sequence and sonicate for 5 min each. After sonication, let it stand at room temperature to dry.

[0055] (2) Hydroxylation of SPR fiber probe: The sensing area of ​​the pretreated SPR fiber probe is immersed in 1 M sodium hydroxide aqueous solution for 1 hour. After immersion, it is cleaned with ultrapure water and then with anhydrous ethanol. After cleaning, it is placed in an 80℃ oven to dry for 1 hour. Then the SPR fiber probe is taken out and allowed to return to room temperature.

[0056] (3) Modify the surface of the sensing area of ​​the SPR fiber probe with 3-mercaptopropyltrimethoxysilane (MTS) to form the first dielectric layer: Place the pretreated and hydroxylated SPR fiber probe in a 2% (v / v) anhydrous ethanol solution of MTS and incubate it on a shaker at 37°C for 2 h. This allows the MTS to undergo a hydrolysis-condensation reaction with the silanol groups in the sensing area of ​​the fiber SPR fiber probe and covalently connect them, thereby generating a layer of exposed thiol groups on the surface of the sensing area of ​​the fiber probe. After the reaction is complete, clean the fiber surface with anhydrous ethanol and dry it with nitrogen.

[0057] (4) Gold plating to form a gold nanoparticle layer on the SPR fiber probe: The sensing area of ​​the SPR fiber probe was gold-plated using an SBC-12 miniature ion sputtering instrument (Beijing Zhongke Keyi Co., Ltd.). The gold plating parameters were: current 15 mA, time 20 s (Journal of Process Engineering, 1-10 [2025-10-19]). The exposed thiol groups in the sensing area of ​​the SPR fiber probe formed gold-sulfur bonds with strong interactions with the gold atoms of the gold film.

[0058] In addition, without hydroxylation and MTS modification, the pretreated SPR fiber probe was directly plated with gold according to the method in step (4) to obtain a control sample.

[0059] 2. The stability of the gold film on the sensing area surface of the prepared SPR fiber probe was evaluated. The SPR fiber probe and control sample prepared in this embodiment were processed in the following manner (Journal of Process Engineering, 1-10 [2025-10-19]). After processing, the gold film shedding in the sensing area was observed.

[0060] (1) Connection of mercaptoalcohol on the surface of the gold film of the sensing area: The sensing area is placed in 5mM (1.02185g / L) 11-mercaptoundecanool (solvent: 80% ethanol) and reacted at 40℃ for 30min. The optical fiber is then removed and repeatedly washed with 80% ethanol solution and deionized water to remove the residual solution on the surface, and then dried with nitrogen.

[0061] (2) Epoxy group bonding: The SPR fiber probe sensing area modified with mercaptoalkanol was placed in a 0.6 M epichlorohydrin solution (the solvent was a 1:1 volume ratio of 0.4 M sodium hydroxide solution and diethylene glycol dimethyl ether) and incubated on a shaker at room temperature for 4 h to form an epoxy group layer on the gold film surface of the SPR fiber probe sensing area. After incubation, the surface was repeatedly washed with ultrapure water and 80% ethanol aqueous solution alternately to remove residual solution, and then dried with nitrogen gas.

[0062] (3) Dextran bonding: The epoxy-modified SPR fiber probe sensing region was placed in a 0.3 g / mL dextran (molecular weight 500,000) solution in a 0.1 M sodium hydroxide aqueous solution and incubated on a shaker at room temperature for 20 h. A layer of dextran was formed on the gold film surface of the SPR fiber probe sensing region through covalent bonds. After incubation, the residual dextran on the surface of the SPR fiber probe was washed with ultrapure water at 50 °C, and then dried with nitrogen gas.

[0063] (4) Carboxylation of dextran: The dextran-modified SPR fiber probe sensing area was placed in a 1 M bromoacetic acid solution with a 2 M sodium hydroxide aqueous solution as the solvent and incubated on a shaker at room temperature for 16 h. The hydroxyl groups on the surface of the dextran were oxidized to carboxyl groups by the oxidizing property of bromoacetic acid. After incubation, the residual solution on the surface of the SPR fiber probe was washed with ultrapure water and then dried with nitrogen.

[0064] The gold film detachment in the sensing area of ​​the SPR fiber probe and the control sample prepared in this embodiment after the above steps was observed, and the gold film in the sensing area was observed under a 40x optical microscope. The results are as follows: Figure 1 As shown, there is no obvious difference between the two under a microscope. However, as shown in Table 1, the gold film shedding rate of the SPR fiber probe prepared in this embodiment was 2% after treatment, while the gold film shedding rate of the control sample was 20% after modification. This shows that the gold film of the SPR fiber probe prepared in this embodiment has higher stability.

[0065] Table 1 Comparison of gold film stability in the sensing region of SPR fiber optic probe

[0066] Example 2: Functional Modification of SPR Fiber Probes 1. Modification with titanium dioxide thin film layer (i.e., metal oxide layer) (1) Preparation of titanium dioxide sol Take 2 mL of 99.5% acetic acid and add it to 48 mL of ultrapure water. Mix well to obtain 50 mL of 4% acetic acid aqueous solution. Accurately weigh 250 mg of chitosan powder (degree of deacetylation ≥95%, viscosity 100-200 mPa·s). Add the chitosan powder to the 4% acetic acid aqueous solution while stirring. After the addition is complete, use a magnetic stirrer to continuously stir at 1150 rpm at 40℃ until the chitosan is completely dissolved. Then, accurately weigh 50 mg of small-diameter titanium dioxide nanoparticles (5~10 nm) and add them to the above chitosan solution while stirring. After the addition is complete, stir at room temperature for 5 min, then sonicate for 30 min to disperse the titanium dioxide particles. After sonication, stir overnight at 1150 rpm at room temperature to obtain titanium dioxide sol.

[0067] (2) Preparation of 4% polyacrylic acid aqueous solution Weigh 2 g of polyacrylic acid powder (average Mv~450000), add it to 50 mL of ultrapure water, and dissolve it to obtain a 4% polyacrylic acid aqueous solution.

[0068] (3) Deposition of titanium dioxide thin film layer ① The sensing area of ​​the SPR fiber probe prepared in Example 1 was immersed in titanium dioxide sol and pulled out at a speed of 1 mm / s after 5 min. ② Dry at room temperature for 10 minutes; ③ Rinse with deionized water for 1 minute, then dry at room temperature for 10 minutes; ④ Replace TiO2 sol with 4% polyacrylic acid solution to complete process (1)-(3); ⑤ Dry in a vacuum drying oven at 60℃ for 1 hour.

[0069] After the above operations, the titanium dioxide thin film layer is modified on the gold film surface of the sensing area of ​​the SPR fiber probe.

[0070] 2. Functional modification (1) Hydroxylation: The sensing area of ​​the SPR fiber probe with a titanium dioxide thin film layer on the surface is immersed in a 1 M sodium hydroxide aqueous solution for 1 hour. After immersion, it is cleaned with ultrapure water and then with anhydrous ethanol. After cleaning, it is placed in an 80℃ oven to dry for 1 hour. Then, the SPR fiber probe is taken out and allowed to return to room temperature, thus completing the hydroxylation of the sensing area of ​​the SPR fiber probe.

[0071] (2) Epoxy group bonding: The hydroxyl-modified SPR fiber probe sensing area was placed in a 0.6 M epichlorohydrin solution, with the solvent being a 1:1 volume ratio of 0.4 M sodium hydroxide solution and diethylene glycol dimethyl ether. The solution was incubated on a shaker at room temperature for 4 h to generate epoxy groups on the fiber surface. After incubation, the surface was repeatedly washed with ultrapure water and 80% ethanol aqueous solution alternately to remove residual solution, and then dried with nitrogen gas. This process forms an epoxy group layer on the gold film surface of the SPR fiber probe sensing area.

[0072] (3) Dextran bonding: The epoxy-modified SPR fiber probe sensing region was placed in a 0.3 g / mL dextran (molecular weight 500,000) solution in a 0.1 M sodium hydroxide aqueous solution and incubated on a shaker at room temperature for 20 h to form a layer of dextran on the surface of the SPR fiber probe sensing region through covalent bonds. After incubation, the residual dextran on the surface of the SPR fiber probe was washed with ultrapure water at 50 °C and then dried with nitrogen.

[0073] (4) Carboxylation of dextran: The dextran-modified SPR fiber probe sensing area was placed in a 1 M bromoacetic acid solution with a 2 M sodium hydroxide aqueous solution as the solvent and incubated on a shaker at room temperature for 16 h. The hydroxyl groups on the surface of the dextran were oxidized to carboxyl groups by the oxidizing property of bromoacetic acid. After incubation, the residual solution on the surface of the SPR fiber probe was washed with ultrapure water and then dried with nitrogen gas, thus completing the surface functionalization modification of the SPR fiber probe sensing area.

[0074] Example 3 Preparation and characterization of ASF@AuNPs Preparation of gold nanoparticles (AuNPs): AuNPs were prepared according to Nature-Physical Science, 1973, 241(105):20-22. All glassware used (including stir bar) was soaked overnight in aqua regia (concentrated HCl:concentrated HNO3, 3:1, v / v) to remove tiny seed crystals that might affect crystallization. After soaking, the glassware was rinsed with ultrapure water and dried in an oven. Note that the aqua regia should be kept away from light during overnight soaking, and the openings of the glassware should be sealed with sealing film to prevent the aqua regia from evaporating. 50 mL of 0.01% (w / v) chloroauric acid aqueous solution was added to a clean three-necked round-bottom flask, a stir bar was placed inside, and a spherical condenser was connected. The flask was heated and stirred at 1100 rpm in an oil bath at 130°C. After the chloroauric acid solution began to show obvious reflux and persisted for 30 minutes, 1.55 mL of 1% (w / v) sodium citrate aqueous solution was quickly added in one go, and heating and stirring were continued to maintain reflux for another 40 minutes. After 40 minutes, heating was stopped, the rotation speed was adjusted to 600 rpm, and the solution was allowed to cool to room temperature. The solution was then aliquoted into 50 mL centrifuge tubes to obtain AuNPs. Subsequently, a small amount of the AuNPs solution was used to characterize the plasma absorption peaks of the AuNPs using ultraviolet-visible spectrophotometry (UV-Vis), and the position of the maximum UV-Vis absorption peak was measured. Figure 2 As shown, the maximum absorption peak wavelength is 520 nm. Referring to Anal. Chem. 2007, 79, 4215-4221, the calculated AuNPs particle size is approximately 13 nm. The prepared AuNPs solution needs to be stored refrigerated at 4°C in the dark and should be used within one month of preparation.

[0075] Add 8 μL of desialized fetoglobulin (ASF) (5 mg / mL, dissolved in ultrapure water, purchased from Sigma-Aldrich, lot number: SLCK5437) and 10 μL of PEGNHS ester disulfide (n=7) (4,7,10,13,16,19,22,25,32,35,38,41,44,47,50,53-hexadecanooxa-28,29-dithiapentahexadecanoic acid di-N-succinimide ester, purchased from Sigma-Aldrich, 1 mg / mL, dissolved in DMSO) to 482 μL of HBS-N buffer (10 mM HEPES, 137 mM NaCl, pH 7.5), mix well, and incubate overnight at room temperature by rotation. This will successfully couple ASF with PEG NHS ester disulfide to form the ASF-PEG7-SS-PEG7-ASF complex. Take 10 mL of freshly prepared AuNPs, centrifuge at 12000 g for 20 min to remove the supernatant, and reconstitute the precipitate in 1.5 mL of 1.8 mM K2CO3 solution. Mix the above ASF-PEG7-SS-PEG7-ASF solution with the 1.8 mM K2CO3 solution of AuNPs thoroughly, incubate at room temperature in the dark for 4 h, then freeze at -20ºC for 2 h. After freezing, remove and reconstitute at room temperature, centrifuge at 12000 g for 20 min to remove the supernatant, wash twice with ultrapure water, and reconstitute in 100 μL HBS-T buffer (10 mM HEPES, 150 mM NaCl, 0.05% (v / v) Tween-20, pH 7.2-7.4) to prepare ASF@AuNPs. Store the prepared ASF@AuNPs at 4℃ in the dark.

[0076] The ASF content in the supernatant was determined by the Bicinchoninic Acid (BCA) method to indirectly characterize ASF ligation efficiency. A BCA standard curve was plotted, as shown below. Figure 3 As shown in Table 2, the coupling efficiency of ASF during the preparation of ASF@AuNPs was calculated, and the results are shown in Table 2. The coupling efficiency reached 80%, indicating that the ASF coupling was successful.

[0077] Table 2 Calculation results of ASF coupling efficiency

[0078] Example 4 Preparation and characterization of ASF@GMNPs 40 μg (8 μL) of ASF (5 mg / mL, dissolved in PBS) and 10 μg (10 μL) of PEG NHS ester disulfide (n=7) (1 mg / mL, dissolved in DMSO) were added to 482 μL of HBS-N solution (10 mM HEPES, 137 mM NaCl, pH 7.5). The mixture was then incubated overnight at room temperature in the dark by rotation, which successfully coupled ASF to the surface of PEG NHS ester disulfide to form an ASF-PEG7-SS-PEG7-ASF complex. Take 0.5 mL of gold-coated magnetic nanoparticles (GMNPs, purchased from Xi'an Ruixi Biotechnology Co., Ltd., 2 mg / mL, particle size 100 nm), remove the supernatant by magnetic separation, reconstitute to 0.5 mL of HBS-N buffer, add the above ASF-PEG7-SS-PEG7-ASF solution, incubate at room temperature in the dark for 8 h, wash twice with ultrapure water, and reconstitute to 0.5 mL of HBS-T buffer (10 mM HEPES, 150 mM NaCl, 0.05% (v / v) Tween-20, pH 7.2-7.4) for later use.

[0079] The plasma absorption peaks before and after GMNP modification were characterized using UV-Vis, and the results are as follows: Figure 4 As shown, the SPR absorption peak red-shifted after GMNP modification. The ASF content in the supernatant was detected by the BCA method to indirectly characterize ASF connectivity. A BCA standard curve was plotted, as shown below. Figure 5 As shown in Table 3, the coupling efficiency of ASF was calculated. The results show that the ASF coupling rate reached 60%, indicating that the ASF coupling was successful.

[0080] Table 3. Calculation results of ASF coupling efficiency for ASF@GMNPs.

[0081]

[0082] Example 5: Antibody-glycoprotein sandwich method for the detection of ricin (RCA60) This embodiment uses the SPR fiber probe prepared in Example 2 of this application to detect ricin (RCA60) by antibody-glycoprotein sandwich method.

[0083] The signal transmission end of the SPR fiber probe prepared in Example 2 of this application was connected to a light source and a spectrometer via an optical fiber to build a detection system. The light source used was a tungsten lamp (purchased from Hangzhou Jingfei Technology, FLE1003). The spectrometer used was also purchased from Hangzhou Jingfei Technology, FLA5300, a 2048-line CCD detector with a wavelength range of 350-1100 nm, a 70 μm slit, and a wavelength resolution of 0.1 nm. The optical fiber used was an HF400 / 430-37 type multimode fiber, purchased from Beijing Feibosida Technology, with a numerical aperture of 0.37 and a core diameter of 400 μm.

[0084] During detection, the SPR fiber optic probe is brought into contact with the sample to be tested. Light emitted from the light source is transmitted to the fiber optic probe through the fiber optic cable. Within the fiber optic probe, light is continuously reflected and reaches the sensing area. The signal generated by the interaction between the fiber optic probe and the analytes in the sample is received by the spectrometer.

[0085] The principle of the antibody-glycoprotein sandwich method for detecting RCA60 is as follows. After the dextran carboxyl groups in the sensing region of the SPR fiber optic probe prepared in Example 2 are activated, they are transmitted through amino-coupled protein A. Protein A recognizes and captures the Fc segment of the recombinant humanized anti-ricin monoclonal antibody (MIL50, a humanized antibody against the anti-ricin neutralizing monoclonal antibody 4C13, donated to the research group of Professor Feng Jiannan at the Academy of Military Medical Sciences of the Chinese People's Liberation Army), exposing its Fab segment. MIL50 recognizes and captures the A chain of RCA60 in the test sample through the Fab segment, while ASF specifically recognizes and binds to the B chain of RCA60. MIL50 and ASF form a sandwich structure by recognizing different fragments of RCA60.

[0086] The SPR fiber probe prepared in Example 2 of this application employs wavelength modulation, using white light from a tungsten lamp source as the incident light, irradiated at a fixed incident angle. The change in reflectance at different wavelengths is detected by a spectrometer, yielding a spectral curve showing the reflectance versus wavelength. The wavelength corresponding to the lowest reflectance is the SPR resonance wavelength at the corresponding incident angle. When the refractive index of the solution on the gold film surface of the sensing area increases, the resonance condition changes, resulting in a redshift (wavelength increase) of the corresponding resonance wavelength. In this example, antibody MIL50 is immobilized on the surface of the sensing area of ​​the SPR fiber probe. MIL50 captures the analyte (RCA60) in the sample, increasing the refractive index of the medium on the surface of the sensing area of ​​the SPR fiber probe, causing a redshift of the resonance wavelength. The amount of redshift is positively correlated with the amount of analyte captured. Similarly, the MIL50-RCA60 complex couples with ASF in ASF@AuNPs, forming a sandwich complex, which also generates a redshift signal of the resonance wavelength. Qualitative and quantitative detection of the analyte can be achieved by detecting the redshift signal.

[0087] The testing steps are as follows: (1) Activation of dextran carboxyl groups: The SPR fiber probe sensing area modified with carboxylated dextran prepared in Example 2 was immersed in EDC / NHS (EDC: 37.5 mg / mL, NHS: 5.75 mg / mL, dissolved in water) activating reagent and activated for 30 min.

[0088] (2) Protein A coupling: The activated SPR fiber probe sensing region was immersed in acetate-sodium acetate buffer (10 mM, pH 4.5) and equilibrated for 2 min. Then, the SPR fiber probe sensing region was immersed in protein A solution (200 μg / mL, dissolved in the above acetate-sodium acetate buffer, purchased from Shanghai Yuanye Biotechnology Co., Ltd.). Protein A is linked to the activated carboxyl group through its amino group and reacted for 15 min.

[0089] (3) Antibody (MIL50)-glycoprotein (ASF) sandwich method for RCA60 detection: The sensing area of ​​the SPR fiber optic probe is immersed in different reagents in the following order and time to achieve RCA60 detection: ① PBS buffer (10 mM, 0.8% NaCl, pH 7.2~7.4): 2 min ② MIL50 (5 μg / mL, dissolved in PBS buffer): 10 min ③ PBS buffer: 2 min ④ Test sample (RCA60, dissolved in PBS buffer): 10 min ⑤ PBS buffer: 2 min ⑥ ASF@AuNPs (ASF concentration 50 nM, dissolved in PBS buffer): 10 min ⑦ PBS buffer: 2 min ⑧ NaOH solution (20 mM): 30s ⑨ PBS buffer: 2 min The above procedure was followed to detect different concentrations of RCA60, with concentration gradients of 0.01 nM, 0.02 nM, 0.1 nM, 0.5 nM, 1 nM, and 2 nM. Each concentration was measured twice, and the average value was taken. Analysis showed a good linear relationship for the first five concentration points, and a standard curve was obtained by plotting the curve. Figure 6 (All data obtained were obtained after subtracting the blank control group.) As shown, the linear regression equation of the standard curve is y = 10.54x + 1.67 (R²). 2 =0.98), indicating that the detection method established in this embodiment has a good linear relationship in the range of 0.01~1 nM, and the LOD is 0.01 nM (0.6 ng / mL).

[0090] Example 6: Selective analysis of ricin and abrinogen (ABA60) using the method established in Example 5. Referring to the method in Example 5, RCA60 at a concentration of 2 nM and ABA60 at concentrations of 2 nM, 20 nM, 50 nM, and 100 nM were detected respectively, and the selectivity of the method for the two toxin proteins was analyzed.

[0091] like Figure 7 As shown, this method produced a significant signal response for 2 nM RCA60, but no significant signal response was observed for 1, 10, 25, and 50 times the concentration of ABA60, indicating that the MIL50-ASF sandwich detection system has at least 50 times selectivity for RCA60 and ABA60.

[0092] Example 7: Detection of RCA60 / ABA60 using the disaccharide-protein sandwich method In this embodiment, the SPR fiber probe prepared in Example 2 of this application is used to detect RCA60 / ABA60 by the disaccharide-protein sandwich method.

[0093] The principle of the disaccharide-protein sandwich method for detecting RCA60 / ABA60 is as follows. After the dextran carboxyl groups in the sensing region of the SPR fiber probe prepared in Example 2 are activated, they are coupled with streptavidin (SA) via amino-coupling. SA can recognize and capture biotinylated concanavalin A (ConA). ConA can recognize and capture one site of the RCA60 / ABA60 B chain in the sample, while ASF can specifically recognize and bind to another site of the RCA60 / ABA60 B chain. ConA and ASF form a sandwich structure by binding to different sites of the RCA60 / ABA60 B chain.

[0094] The detection system is the same as in Example 5, and the detection steps are as follows: (1) Biotinylation of Cona lectin A (ConA): ConA (purchased from Sigma-Aldrich) was biotinylated using the Biotin Labeling Kit-NH2 Biotinylation Kit (purchased from DOJINDO, batch number: AJ801).

[0095] (2) Activation of dextran carboxyl groups: The SPR fiber probe sensing area modified with carboxylated dextran prepared in Example 2 was immersed in EDC / NHS (EDC: 37.5 mg / mL, NHS: 5.75 mg / mL, dissolved in water) activating reagent and activated for 30 min.

[0096] (3) SA Coupling: The activated SPR fiber probe sensing region was immersed in acetate-sodium acetate buffer (10 mM, pH 4.0) for equilibration for 2 min. Then, the SPR fiber probe sensing region was immersed in SA solution (200 μg / mL, dissolved in the above acetate-sodium acetate buffer, purchased from Beijing Bio-Tech Technology Co., Ltd.). SA was coupled to the activated carboxyl group via amino groups for 20 min. After coupling, the SPR fiber probe sensing region was immersed in acetate-sodium acetate buffer, and the red shift of the SPR absorption peak in acetate-sodium acetate buffer was observed before and after the coupling to determine whether SA was successfully coupled to the fiber sensing region and the amount of coupling.

[0097] (4) ConA immobilization: The SPR fiber probe sensing region coupled with SA was immersed in HBS-BT buffer (10 mM HEPES, 150 mM NaCl, 0.05% (v / v) Tween-20, 0.1% BSA, pH 7.2-7.4) and equilibrated for 2 min. Then the SPR fiber probe sensing region was immersed in Bio-ConA solution (25 μg / mL, dissolved in HBS-BT buffer). ConA was immobilized on the surface of the fiber sensing region through the interaction between biotin and streptavidin and ligated for 10 min.

[0098] (5) ConA-ASF sandwich method for detecting RCA60 / ABA60: The sensing area of ​​the SPR fiber probe is immersed in different reagents in the following order and time to achieve the detection of RCA60 / ABA60: ① HBS-BT buffer: 2 min ② Test sample (RCA60 / ABA60 dissolved in HBS-BT buffer): 10 min ③ HBS-BT buffer: 2 min ④ ASF@AuNPs (ASF concentration 50 nM, dissolved in HBS-BT buffer): 10 min ⑤ HBS-BT buffer: 2 min ⑥ HCl solution (50 mM): 30 s ⑦ HBS-BT buffer: 2 min The above procedure was followed to detect different concentrations of RCA60 / ABA60, with concentration gradients of 0.0006, 0.003, 0.006, 0.03, 0.06, and 0.12 μg / mL. Each concentration was measured twice, and the average value was taken. A standard curve for RCA60 / ABA60 was plotted. Figure 8 , 9As shown (all data were obtained after subtracting the blank control group). The linear regression equation of the RCA60 standard curve is y = 17.658x + 0.3595 (R 2 = 0.99), the linear range was 5.66~120 ng / mL, and the LOD was 0.6 ng / mL; the linear regression equation of the ABA60 standard curve was y = 50.253x + 0.1929 (R² = 0.99). 2 = 0.97), the linear range was 1.99~120 ng / mL, and the LOD was 0.6 ng / mL; the selectivity of this method for the two toxin proteins was 1:3.

[0099] Example 8 Detection of RCA60 in urine or plasma RCA60 was added to urine to prepare urine test samples with concentrations of 0.02 nM and 0.5 nM. Following the method described in Example 5, the RCA60 in the test samples was directly detected. The results are as follows: Figure 10 As shown in the figure, this method can be used to detect RCA60 in urine.

[0100] Rat plasma was diluted 10-fold, and RCA60 was added to prepare plasma test samples of different concentrations. ASF@GMNPs were mixed with the plasma test samples to a final ASF concentration of 50 nM. The mixture was incubated at room temperature for 15 min by rotation. ASF@GMNPs were magnetically separated to obtain the RCA60–ASF@GMNPs complex. The complex was washed twice with PBS buffer and reconstituted in 200 μL PBS buffer. Following the method in Example 5, the dextran carboxyl groups on the surface of the SPR fiber probe sensing area were activated and coupled with protein A. Then, the fiber optic SPR fiber probe sensing area was immersed in different reagents in the following order and at the following times to achieve the detection of RCA60 in the test samples: ① PBS buffer (10 mM, 0.8% NaCl, pH 7.2-7.4): 2 min ② MIL50 (5 μg / mL, dissolved in PBS buffer): 10 min ③ PBS buffer: 2 min ④ Test sample (RCA60–ASF@GMNPs complex, dissolved in PBS buffer): 10 min ⑤ PBS buffer: 2 min ⑥ NaOH solution (20 mM): 30s ⑦ PBS buffer: 2 min The above procedure was followed to test plasma samples with different concentrations of RCA60. The concentration gradients were 0.01, 0.05, 0.1, and 1 nM. Each concentration was tested twice, and the average value was taken. A standard curve was obtained by plotting the values. Figure 11 As shown (all data were obtained after subtracting the blank control group), the linear regression equation of the standard curve is y = 0.275x + 0.723 (R²). 2 =0.9995), and LOD is 0.01 nM (0.6 ng / mL), indicating that this method can detect RCA60 in blood samples.

[0101] Example 9 Detection of ABA60 in plasma Rat plasma was diluted 10-fold, and ABA60 was added to prepare plasma test samples of different concentrations. ASF@GMNPs were mixed with the plasma test samples to a final ASF concentration of 50 nM. The mixture was incubated at room temperature for 10 min by rotation. ASF@GMNPs were then magnetically separated to obtain the ABA60-ASF@GMNPs complex. The complex was washed twice with HBS-T buffer (10 mM HEPES, 150 mM NaCl, 0.05% (v / v) Tween-20, pH 7.2-7.4) and reconstituted in 200 μL of HBS-T buffer. Following the method in Example 7, the dextran carboxyl groups on the surface of the SPR fiber probe sensing area were activated, and SA and ConA were sequentially linked. The SPR fiber probe sensing area was then immersed in different reagents in the following order and for the following time periods to detect ABA60 in the test samples: ① HBS-T buffer: 2 min ② Test sample (ABA60-ASF@GMNPs complex, dissolved in HBS-T buffer): 10 min ③ HBS-T buffer: 2 min ④ HCl solution (50 mM): 30s ⑤ HBS-T buffer: 2 min The above procedure was followed to test plasma samples with different concentrations of ABA60. The concentration gradient was 0.01, 0.05, 0.1, 0.5, 1, and 2 nM. Each concentration was measured twice, and the average value was taken. The last five points were plotted to obtain a standard curve. Figure 12 As shown (all data were obtained after subtracting the blank control group), the linear regression equation of the standard curve is y = 0.1534x + 0.348 (R²). 2 =0.9859), and LOD was 0.01 nM (0.6 ng / mL), indicating that this method can detect ABA60 in plasma.

[0102] Although specific embodiments of this application have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and such changes are all within the scope of protection of this application. The full scope of this application is given by the appended claims and any equivalents.

Claims

1. An SPR fiber optic probe, comprising: An optical fiber substrate, a first dielectric layer, and a metal nanoparticle layer, wherein the optical fiber substrate is a quartz optical fiber substrate, and the first dielectric layer is formed by a mercaptosilane coupling agent or an aminosilane coupling agent and is located between the optical fiber substrate and the metal nanoparticle layer.

2. The SPR fiber probe of claim 1, wherein the first dielectric layer is covalently connected to the fiber substrate and to the metal nanoparticle layer via metal-nitrogen coordination bonds or metal-sulfur covalent bonds.

3. The SPR fiber probe of claim 1, wherein the mercaptosilane coupling agent is 3-mercaptopropyltrimethoxysilane, 3-mercaptopropyltriethoxysilane, or mercaptomethyltrimethoxysilane; and the aminosilane coupling agent is 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-aminopropylmethyldimethoxysilane, N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane, or N-(β-aminoethyl)-γ-aminopropyltriethoxysilane.

4. The SPR fiber probe of claim 1, wherein the metal nanoparticle layer comprises metal nanoparticles, wherein the metal nanoparticles are gold, silver, copper, platinum, aluminum or titanium nanoparticles.

5. The SPR fiber probe of claim 4, wherein the metal nanoparticles are spherical, star-shaped, rod-shaped, spindle-shaped, or triangular nanoparticles.

6. The SPR fiber probe of claim 4, wherein the metal nanoparticles are gold nanoparticles, and the first dielectric layer and the metal nanoparticle layer are connected by gold-sulfur covalent bonds.

7. The SPR fiber probe according to any one of claims 1-6, further comprising a metal oxide layer attached to the metal nanoparticle layer.

8. The SPR fiber probe of claim 7, wherein the metal oxide layer comprises a metal oxide, wherein the metal oxide is titanium dioxide, tantalum pentoxide or aluminum oxide.

9. The SPR fiber probe of claim 7 further comprises a second dielectric layer and a functional modification layer, wherein the second dielectric layer is formed of epichlorohydrin or methyl epichlorohydrin and is connected to the metal oxide layer via ether bonds; the functional modification layer comprises carboxylated dextran and is connected to the second dielectric layer via ether bonds.

10. A detection system comprising a light source, a spectrometer, and the SPR fiber optic probe as described in any one of claims 1-9.

11. A kit comprising the SPR fiber probe according to any one of claims 1-9.

12. The kit of claim 11, further comprising: a) One or more substances from group A, consisting of desialylated fetoglobulin, gold nanoparticles, protein A, recombinant humanized anti-ricin monoclonal antibody, EDC, NHS, acetate-sodium acetate buffer, PBS buffer, and NaOH; or b) One or more substances from group B consisting of desialized fetoglobulin, gold nanoparticles, biotin, concanavalin A, streptavidin, EDC, NHS, acetate-sodium acetate buffer, HBS-BT buffer, and HCl solution; or c) One or more substances from group C, consisting of desialized fetoglobulin, gold-coated magnetic nanoparticles, protein A, recombinant humanized anti-ricin monoclonal antibody, EDC, NHS, acetate-sodium acetate buffer, PBS buffer, and NaOH; or d) One or more substances from group D, consisting of desialylated fetoglobulin, gold-coated magnetic nanoparticles, biotin, concanavalin A, streptavidin, EDC, NHS, acetate-sodium acetate buffer, HBS-BT buffer, and HCl solution, or e) One or more substances from group E consisting of gold nanoparticles surface-coupled with desialylate-fetoprotein, protein A, recombinant humanized anti-ricin monoclonal antibody, EDC, NHS, acetate-sodium acetate buffer, PBS buffer, and NaOH; or f) One or more substances from group F, consisting of gold nanoparticles surface-coupled with desialylated fetoglobulin, biotin-labeled concanavalin A, streptavidin, EDC, NHS, acetate-sodium acetate buffer, HBS-BT buffer, and HCl solution; or g) One or more substances from group G consisting of gold-coated magnetic nanoparticles with desialylate-fetoprotein surface-coupled, protein A, recombinant humanized anti-ricin monoclonal antibody, EDC, NHS, acetate-sodium acetate buffer, PBS buffer, and NaOH; or h) One or more substances from group H consisting of gold-coated magnetic nanoparticles with desialylated fetoglobulin on their surface, biotin-labeled concanavalin A, streptavidin, EDC, NHS, acetate-sodium acetate buffer, HBS-BT buffer, and HCl solution.

13. The use of the SPR fiber probe according to any one of claims 1-9, the detection system according to claim 10, or the kit according to claim 11 or 12 in the detection of type II ribosome inactivating proteins.

14. A method for detecting type II ribosome-inactivating proteins in a sample, comprising: 1) contacting the SPR fiber optic probe according to any one of claims 1-9 with the sample to be tested; and 2) detecting signal changes.

15. The method of claim 14, wherein a) The method includes: Activate the SPR fiber probe and bring it into contact with protein A, then attach protein A to the surface of the SPR fiber probe. The SPR fiber probe is brought into contact with the recombinant anti-ricin humanized monoclonal antibody, and the recombinant anti-ricin humanized monoclonal antibody is attached to protein A on the surface of the SPR fiber probe. The SPR fiber probe is brought into contact with the sample to capture type II ribosome-inactivating proteins in the sample. The SPR fiber probe is brought into contact with gold nanoparticles whose surface is coupled with desialylated fetoglobulin. Detect signal changes; or b) The method includes: The SPR fiber probe is activated and brought into contact with streptavidin, thereby attaching streptavidin to the surface of the SPR fiber probe. The SPR fiber probe is brought into contact with biotin-labeled concanavalin A, and the biotin-labeled concanavalin A is attached to streptavidin on the surface of the SPR fiber probe. The SPR fiber probe is brought into contact with the sample to capture type II ribosome-inactivating proteins in the sample. The SPR fiber probe is brought into contact with gold nanoparticles whose surface is coupled with desialylated fetoglobulin. Detect signal changes; or c) The method includes: Activate the SPR fiber probe and bring it into contact with protein A, then attach protein A to the surface of the SPR fiber probe. The SPR fiber probe is brought into contact with the recombinant anti-ricin humanized monoclonal antibody, and the recombinant anti-ricin humanized monoclonal antibody is attached to protein A on the surface of the SPR fiber probe. The sample to be tested was brought into contact with gold-coated magnetic nanoparticles on which desialylated fetoglobulin was coupled to the surface, and the type II ribosome inactivating protein in the sample to be tested was captured by the gold-coated magnetic nanoparticles. The SPR fiber probe was brought into contact with gold-coated magnetic nanoparticles that had captured type II ribosome inactivating proteins. Detect signal changes; or d) The method includes: The SPR fiber probe is activated and brought into contact with streptavidin, thereby attaching streptavidin to the surface of the SPR fiber probe. The SPR fiber probe is brought into contact with biotin-labeled concanavalin A, and the biotin-labeled concanavalin A is attached to streptavidin on the surface of the SPR fiber probe. The sample to be tested was brought into contact with gold-coated magnetic nanoparticles on which desialylated fetoglobulin was coupled to the surface, and the type II ribosome inactivating protein in the sample to be tested was captured by the gold-coated magnetic nanoparticles. The SPR fiber probe was brought into contact with gold-coated magnetic nanoparticles that had captured type II ribosome inactivating proteins. Detect signal changes.

16. The application of claim 13 or the method of claim 14, wherein the type II ribosome inactivating protein is ricin or absinthecin.

17. A method for preparing the SPR fiber probe according to any one of claims 1-9, comprising: Hydroxylation treatment is performed on the optical fiber substrate; A mercaptosilane coupling agent or an aminosilane coupling agent is modified onto the surface of the optical fiber substrate to form a first dielectric layer; A layer of metal nanoparticles is deposited on the surface of an optical fiber substrate.

18. The method of claim 17, further comprising one or more of the following operations: A metal oxide layer is coated on the surface of the optical fiber substrate; The metal oxide layer on the surface of the optical fiber substrate is subjected to hydroxylation treatment; Epichlorohydrin or methyl epichlorohydrin is modified onto the surface of the optical fiber substrate to form a second dielectric layer; Dextran was modified onto the surface of the optical fiber substrate; Carboxylation treatment was performed on the surface of the optical fiber substrate.

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