A method and kit for detecting ribosome inactivation proteins of type ii

Abstract

The application relates to a surface plasmon resonance (SPR) optical fiber probe, application of the SPR optical fiber probe in detection of type II ribosome inactivating proteins (such as ricin and abrin), a kit and a detection system comprising the SPR optical fiber probe, and a method for detecting type II ribosome inactivating proteins 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 stability of detection.

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 that 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, with low sample requirement, rapid detection process, high throughput, high sensitivity, and other advantages, and 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, with 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] The application provides an SPR optical fiber probe, comprising: an optical fiber substrate, a first medium layer and a metal nanoparticle layer, wherein the optical fiber substrate is a quartz optical fiber substrate, the first medium layer is formed by a mercapto silane coupling agent or an amino silane coupling agent, and is located between the optical fiber substrate and the metal nanoparticle layer.

[0006] In some embodiments, the first medium layer is connected to the optical fiber substrate by a covalent bond, and is connected to the metal nanoparticle layer by a metal-nitrogen coordination bond or a metal-sulfur covalent bond.

[0007] In some embodiments, the mercapto silane coupling agent or the amino silane coupling agent of the first medium layer hydrolyzes and condenses with the surface of the optical fiber substrate to form a Si-O-Si covalent bond, and forms a metal-nitrogen coordination bond or a metal-sulfur covalent bond with the metal nanoparticles of the metal nanoparticle layer.

[0008] The hydrolysable siloxane group in the mercapto silane coupling agent or the amino silane coupling agent can hydrolyze and condense with the silicon hydroxyl group on the surface of the optical fiber substrate to form a Si-O-Si covalent bond; the mercapto group in the mercapto silane coupling agent can form a metal-sulfur covalent bond with the metal nanoparticles of the metal nanoparticle layer; and the amino group in the amino silane coupling agent can form a metal-nitrogen coordination bond with the metal nanoparticles of the metal nanoparticle layer, so that the metal nanoparticles are stably combined on the surface of the optical fiber substrate and are not easy to fall off.

[0009] In some embodiments, the mercapto silane coupling agent is 3-mercaptopropyl trimethoxysilane, 3-mercaptopropyl triethoxysilane or mercaptomethyl trimethoxysilane. In some embodiments, the amino silane coupling agent is 3-aminopropyl trimethoxysilane, 3-aminopropyl triethoxysilane, 3-aminopropyl methyl dimethoxysilane, N-(β-aminoethyl)-γ-aminopropyl trimethoxysilane or N-(β-aminoethyl)-γ-aminopropyl triethoxysilane.

[0010] In some embodiments, the first medium layer is formed by a mercapto silane coupling agent, and the mercapto silane coupling agent is 3-mercaptopropyl trimethoxysilane.

[0011] In some embodiments, the metal nanoparticle layer comprises metal nanoparticles, and the metal nanoparticles are gold, silver, copper, platinum, aluminum or 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 medium layer is connected to the metal nanoparticle layer by a gold-sulfur covalent bond.

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

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

[0016] In some embodiments, the SPR fiber-optic probe further comprises a second medium layer formed by epichlorohydrin or its analogs (e.g. methyl epichlorohydrin) and connected to the metal oxide layer via ether bond, and a functional modification layer comprising carboxylated dextran connected to the second medium layer via ether bond.

[0017] The metal oxide layer is treated with strong alkaline aqueous solution to form exposed hydroxyl groups on the surface. The chlorine atoms of epichlorohydrin or its analogs react with these hydroxyl groups via Williamson synthesis to form ether bond, thus forming oxirane-2-ylmethoxy groups on the surface of the metal oxide layer. The ether bond between oxirane-2-yl (i.e. epoxy group) and dextran is formed via ring-opening reaction, thus modifying dextran on the surface of the metal oxide. Further, the surface hydroxyl groups of dextran are oxidized to carboxyl groups, thus completing the connection of the functional modification layer.

[0018] The present application also provides a detection system comprising a light source, a spectrum analyzer and the SPR fiber-optic probe of any of the embodiments of the present application.

[0019] The present application also provides a kit comprising the SPR fiber-optic probe of any of the embodiments of the present application.

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

[0021] In some embodiments, the kit further comprises one or more substances in Group B consisting of desialylated fetuin, gold nanoparticles, biotin, concanavalin A, streptavidin, EDC, NHS, acetic acid-sodium acetate buffer, HBS-BT buffer and HCl solution.

[0022] In certain embodiments, the kit further comprises one or more of the following in Group C: desialylated Fetuin, gold-coated magnetic nanoparticles, Protein A, recombinant anti-ricin humanized monoclonal antibody, EDC, NHS, acetic acid-sodium acetate buffer, PBS buffer, and NaOH.

[0023] In certain embodiments, the kit further comprises one or more of the following in Group D: desialylated Fetuin, gold-coated magnetic nanoparticles, biotin, Concanavalin A, Streptavidin, EDC, NHS, acetic acid-sodium acetate buffer, HBS-BT buffer, and HCl solution.

[0024] In certain embodiments, the kit further comprises one or more of the following in Group E: gold nanoparticles coupled with desialylated Fetuin, Protein A, recombinant anti-ricin humanized monoclonal antibody, EDC, NHS, acetic acid-sodium acetate buffer, PBS buffer, and NaOH.

[0025] In certain embodiments, the kit further comprises one or more of the following in Group F: gold nanoparticles coupled with desialylated Fetuin, biotin-labeled Concanavalin A, Streptavidin, EDC, NHS, acetic acid-sodium acetate buffer, HBS-BT buffer, and HCl solution.

[0026] In certain embodiments, the kit further comprises one or more of the following in Group G: gold-coated magnetic nanoparticles coupled with desialylated Fetuin, Protein A, recombinant anti-ricin humanized monoclonal antibody, EDC, NHS, acetic acid-sodium acetate buffer, PBS buffer, and NaOH.

[0027] In certain embodiments, the kit further comprises one or more of the following in Group H: gold-coated magnetic nanoparticles coupled with desialylated Fetuin, biotin-labeled Concanavalin A, Streptavidin, EDC, NHS, acetic acid-sodium acetate buffer, HBS-BT buffer, and HCl solution.

[0028] The present application also provides the use of the SPR optical fiber probe, the detection system or the kit of any of the embodiments of the present application in detecting the type II ribosome inactivating proteins.

[0029] The present application also provides a method for detecting the type II ribosome inactivating proteins in a sample, comprising 1) contacting the SPR optical fiber probe of any of the embodiments of the present application with the sample to be detected; and 2) detecting the change of the SPR signal.

[0030] In certain embodiments, the method of detecting a ribosome inactivating protein of type II in a sample comprises:

[0031] activating the SPR fiber optic probe and contacting it with protein A, wherein the protein A is attached to the surface of the SPR fiber optic probe;

[0032] contacting the SPR fiber optic probe with a recombinant anti-ricin humanized monoclonal antibody, wherein the recombinant anti-ricin humanized monoclonal antibody is attached to the protein A on the surface of the SPR fiber optic probe;

[0033] contacting the SPR fiber optic probe with a sample to be tested, wherein the ribosome inactivating protein of type II in the sample to be tested is captured;

[0034] contacting the SPR fiber optic probe with gold nanoparticles having desialidated fetuin coupled to the surface of the gold nanoparticles;

[0035] detecting a change in the SPR signal.

[0036] In certain embodiments, the method of detecting a ribosome inactivating protein of type II in a sample comprises:

[0037] activating the SPR fiber optic probe and contacting it with streptavidin, wherein the streptavidin is attached to the surface of the SPR fiber optic probe;

[0038] contacting the SPR fiber optic probe with biotin-labeled lectin A, wherein the biotin-labeled lectin A is attached to the streptavidin on the surface of the SPR fiber optic probe;

[0039] contacting the SPR fiber optic probe with a sample to be tested, wherein the ribosome inactivating protein of type II in the sample to be tested is captured;

[0040] contacting the SPR fiber optic probe with gold nanoparticles having desialidated fetuin coupled to the surface of the gold nanoparticles;

[0041] detecting a change in the SPR signal.

[0042] In certain embodiments, the method of detecting a ribosome inactivating protein of type II in a sample comprises:

[0043] activating the SPR fiber optic probe and contacting it with protein A, wherein the protein A is attached to the surface of the SPR fiber optic probe;

[0044] contacting the SPR fiber optic probe with a recombinant anti-ricin humanized monoclonal antibody, wherein the recombinant anti-ricin humanized monoclonal antibody is attached to the protein A on the surface of the SPR fiber optic probe;

[0045] contacting the sample to be tested with gold-coated magnetic nanoparticles coupled with asialofetuin, and capturing the ribosome inactivating protein of type II in the sample to be tested by the gold-coated magnetic nanoparticles;

[0046] contacting the SPR fiber probe with the gold-coated magnetic nanoparticles capturing the ribosome inactivating protein of type II;

[0047] detecting the change of SPR signal.

[0048] In some embodiments, the method for detecting the ribosome inactivating protein of type II in the sample comprises:

[0049] activating the SPR fiber probe and contacting it with streptavidin, and connecting the streptavidin to the surface of the SPR fiber probe;

[0050] contacting the SPR fiber probe with biotin-labeled concanavalin A, and connecting the biotin-labeled concanavalin A to the streptavidin on the surface of the SPR fiber probe;

[0051] contacting the sample to be tested with gold-coated magnetic nanoparticles coupled with asialofetuin, and capturing the ribosome inactivating protein of type II in the sample to be tested by the gold-coated magnetic nanoparticles;

[0052] contacting the SPR fiber probe with the gold-coated magnetic nanoparticles capturing the ribosome inactivating protein of type II;

[0053] detecting the change of SPR signal.

[0054] In the detection, the analyte is qualitatively analyzed according to the SPR absorption wavelength shift, and quantitatively analyzed according to the signal response value.

[0055] 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, the sample to be tested can be contacted with gold-coated magnetic nanoparticles coupled with asialofetuin before detection.

[0056] In some embodiments, the ribosome inactivating protein of type II is ricin or abrin.

[0057] The application also provides a method for preparing the SPR fiber probe according to any of the embodiments of the application, comprising:

[0058] hydroxylating the fiber substrate;

[0059] modifying a mercapto silane coupling agent or an amino silane coupling agent to the surface of the fiber substrate to form a first medium layer;

[0060] arranging a metal nanoparticle layer on the surface of the fiber substrate.

[0061] In certain embodiments, the method of making the SPR fiber optic probe of any of the embodiments described herein further comprises one or more of the following operations:

[0062] coating a metal oxide layer on the surface of the fiber optic substrate;

[0063] hydroxylating the metal oxide layer on the surface of the fiber optic substrate;

[0064] modifying an epoxy chloropropane or a methyl epoxy chloropropane to the surface of the fiber optic substrate to form a second dielectric layer;

[0065] modifying a dextran to the surface of the fiber optic substrate;

[0066] carboxylating the dextran on the surface of the fiber optic substrate.

[0067] In the present application, the quartz fiber is a fiber made of SiO2 as the main material. When manufacturing the quartz fiber, the refractive index distribution of the fiber can be changed by controlling the doping amount as needed.

[0068] In the present application, the amino silane coupling agent is a kind of organosilicon compound containing both amino group (-NH2 or -NHR) and hydrolysable siloxane group (such as -Si(OR)3, R is alkyl), including 3-aminopropyltrimethoxysilane (H2N-(CH2)3-Si(OCH3)3), 3-aminopropyltriethoxysilane (H2N-(CH2)3-Si(OEt)3), 3-aminopropylmethyldimethoxysilane (H2N-(CH2)3-Si(CH3)(OCH3)2) and other mono-amino silane coupling agents, and N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane (H2N-(CH2)2-NH-(CH2)3-Si(OCH3)3), N-(β-aminoethyl)-γ-aminopropyltriethoxysilane (H2N-(CH2)2-NH-(CH2)3-Si(OEt)3) and other bis-amino silane coupling agents.

[0069] In the present application, the mercapto silane coupling agent is a kind of organosilicon compound containing both mercapto group (-SH) and hydrolysable siloxane group (such as -Si(OR)3), including 3-mercaptopropyltrimethoxysilane (HS-(CH2)3-Si(OCH3)3), 3-mercaptopropyltriethoxysilane (HS-(CH2)3-Si(OEt)3), mercaptomethyltrimethoxysilane (HS-CH2-Si(OCH3)3) and other mono-mercapto silane coupling agents.

[0070] In the present application, the carboxylated dextran refers to a functional polysaccharide derivative obtained by introducing carboxyl (-COOH) on the hydroxyl of the glucose unit of dextran through chemical modification. In the present application, when the carboxylated dextran is modified to the SPR optical fiber probe, the dextran can be first modified to the surface of the SPR optical fiber probe, and then the hydroxyl on the surface of the dextran is further oxidized to carboxyl, thereby completing the modification of the carboxylated dextran.

[0071] In the present application, coupling of asialofetuin 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 bond (Au-S) can be used to realize the connection through the thiol group (-SH) in the ASF molecule or by introducing thiol modification, while ensuring the activity of the protein and the stability of the nanoparticles.

[0072] In the present application, the HBS-N buffer substance refers to a substance capable of being formulated into an HBS-N buffer, which can exist in the form of a solution or a dry powder, including HEPES buffer substance and NaCl, which can be formulated as needed during use, for example, into a buffer containing 10 mM HEPES and 137 mM NaCl, pH 7.5.

[0073] In the present application, the HBS-T buffer substance refers to a substance capable of being formulated into an HBS-T buffer, which can exist in the form of a solution or a dry powder, including HEPES buffer substance, NaCl and Tween-20, which can be formulated as needed during use, for example, into a buffer containing 10 mM HEPES, 150 mM NaCl and 0.05% (v / v) Tween-20, pH 7.2-7.4.

[0074] In the present application, the HBS-BT buffer substance refers to a substance capable of being formulated into an HBS-BT buffer, which can exist in the form of a solution or a dry powder, including HEPES buffer substance, NaCl, Tween-20 and bovine serum albumin (BSA), which can be formulated as needed during use, for example, into a buffer containing 10 mM HEPES, 150 mM NaCl, 0.05% (v / v) Tween-20 and 0.1% BSA, pH 7.2-7.4.

[0075] In the present application, the PBS buffer substance refers to a substance capable of being formulated into a PBS buffer, which can exist in the form of a solution or a dry powder, including disodium hydrogen phosphate (Na2HPO4), potassium dihydrogen phosphate (KH2PO4) and sodium chloride (NaCl), which can be formulated as needed during use, for example, into a buffer containing 10 mM, 0.8% NaCl, pH 7.2-7.4.

[0076] In the present application, the acetic acid-sodium acetate buffer substance can exist in the form of a solution or a dry powder, which refers to a substance capable of being prepared into an acetic acid-sodium acetate buffer. When used, the acetic acid-sodium acetate buffer substance can be prepared into a buffer of different pH values according to the needs, for example, a 10 mM, pH 4.0 buffer.

[0077] In the present application, the HEPES buffer substance can exist in the form of a solution or a dry powder, which refers to a substance capable of being prepared into a HEPES buffer, including HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid).

[0078] Beneficial technical effects of the present application

[0079] 1. The metal nanoparticle layer on the surface of the SPR optical fiber probe provided by the present application is not easy to fall off, has good stability, can be reused, and can improve the stability of detection;

[0080] 2. Using the SPR optical fiber probe provided by the present application to detect type II ribosome inactivating proteins (such as ricin or abrin) can improve the sensitivity of detection;

[0081] 3. The method for detecting type II ribosome inactivating proteins (such as ricin or abrin) provided by the present application has high sensitivity, with a detection limit as low as 1 ng / mL (LOD is 0.6 ng / mL), and is simple to operate, and with the aid of magnetic particles, the analyte in a complex matrix such as plasma can be detected on site and quickly;

[0082] 4. The SPR optical fiber probe and detection method provided by the present application can be adapted to small portable instruments, and can also be adapted to desktop SPR detection instruments of the same principle, and can be conveniently expanded from a single channel to multiple channels, and has a wide range of applications. BRIEF DESCRIPTION OF DRAWINGS

[0083] Figure 1 It is the image (A) of the gold film in the sensing area of the control sample under 40 times microscope and the image (B) of the gold film in the sensing area of the SPR optical fiber probe prepared in the present application;

[0084] Figure 2 It is the ultraviolet-visible absorption spectrum of AuNPs;

[0085] Figure 3 It is the standard curve graph of BCA method drawn when preparing ASF@AuNPs;

[0086] Figure 4 It is the ultraviolet-visible absorption spectrum of GMNPs before and after modification;

[0087] Figure 5BCA standard curve graph plotted for preparation of ASF@GMNPs;

[0088] Figure 6 RCA60 linear graph for antibody-glycoprotein sandwich method;

[0089] Figure 7 Ricin and abrin results graph for antibody-glycoprotein sandwich method;

[0090] Figure 8 RCA60 standard curve graph for double glycoprotein sandwich method;

[0091] Figure 9 ABA60 standard curve graph for double glycoprotein sandwich method;

[0092] Figure 10 RCA60 direct detection graph for antibody-glycoprotein sandwich method in urine sample;

[0093] Figure 11 RCA60 standard curve graph for antibody-glycoprotein sandwich method in plasma sample using ASF@GMNPs;

[0094] Figure 12 ABA60 standard curve graph for double glycoprotein sandwich method in plasma sample using ASF@GMNPs. DETAILED DESCRIPTION

[0095] The essential content of the present application will be further described in combination with the specific embodiments of the present application. It should be understood that the following embodiments are only used to illustrate the present application, but not to limit the scope of protection of the present application. The specific conditions are not specified in the following examples, which are carried out according to the conventional conditions or the manufacturer's recommendations. The raw materials used are not specified by the manufacturer, which are conventional products that can be obtained by purchase.

[0096] Although many of the materials and methods used in the following examples are well known in the art, the present application still describes them as much as possible. It is clear to those skilled in the art that the materials and methods used in the following examples are well known in the art if not specifically mentioned. The following examples are used to illustrate the present application.

[0097] Example 1 Preparation and stability evaluation of gold film on the sensing area surface of SPR fiber probe

[0098] 1. Preparation of gold film on the sensing area surface of SPR fiber probe

[0099] (1) SPR fiber probe pretreatment: Dabbing the surface of the SPR fiber probe sensing area (i.e. fiber base) with alcohol with a dust-free paper (the SPR fiber probe is purchased from Suzhou Feibo Sichuang Optoelectronic Technology Co., Ltd., specification: TZ-HP400-L37.7-6), then immerse the SPR fiber probe sensing area into water, anhydrous ethanol and acetone in turn for 5 min of ultrasonic, and then air dry at room temperature after ultrasonic.

[0100] (2) Hydroxylation of the SPR fiber probe: immerse the pretreated SPR fiber probe sensing area into 1 M sodium hydroxide aqueous solution for 1 hour, then wash with ultrapure water, and then wash with anhydrous ethanol, and then dry in an oven at 80℃ for 1 hour, and then take out the SPR fiber probe to restore to room temperature.

[0101] (3) Modification of the surface of the SPR fiber probe sensing area with 3-mercaptopropyltrimethoxysilane (MTS) to form a first medium layer: place the pretreated and hydroxylated SPR fiber probe into a 2% volume fraction MTS anhydrous ethanol solution, and then incubate on a shaking table at 37℃ for 2 h, so that the MTS and the silicon hydroxyl groups on the sensing area of the fiber SPR fiber probe undergo hydrolysis and condensation reaction to covalently connect, and then a layer of exposed mercapto groups is generated on the surface of the sensing area of the fiber probe, and then clean the surface of the fiber with anhydrous ethanol, and then dry with nitrogen.

[0102] (4) Gold plating of the SPR fiber probe to form a gold nanoparticle layer: use an SBC-12 small ion sputtering instrument (Beijing Zhongke Keyi Co., Ltd.) to perform gold plating on the sensing area of the SPR fiber probe, and the gold plating parameters are: current 15 mA, time 20 s (Process Engineering, 1-10 [2025-10-19]). The exposed mercapto groups on the sensing area of the SPR fiber probe form a gold-sulfur bond with the gold atoms of the gold film, which has a strong interaction.

[0103] In addition, without hydroxylation and MTS modification, the pretreated SPR fiber probe is directly gold plated according to the method of step (4) to obtain a control sample.

[0104] 2. Stability evaluation of the gold film on the sensing area surface of the prepared SPR fiber probe

[0105] The SPR fiber probe prepared in this example and the control sample are treated in the following manner (Process Engineering, 1-10 [2025-10-19]), and then the shedding of the gold film on the sensing area is observed.

[0106] (1) Thiol alkyl alcohol connection on the surface of the gold film of the sensing area: the sensing area was placed in 5 mM (1.02185 g / L) 11-thiol undecanol (solvent: 80% ethanol) and reacted at 40°C for 30 min. The optical fiber was taken out and washed repeatedly with 80% ethanol solution and deionized water to remove the residual solution on the surface and then dried with nitrogen.

[0107] (2) Epoxy group connection: the sensing area of the SPR optical fiber probe modified with thiol alkyl alcohol was placed in 0.6 M epichlorohydrin solution (solvent: 0.4 M sodium hydroxide solution and diethylene glycol dimethyl ether in a volume ratio of 1:1) and incubated at room temperature on a shaker for 4 h to form an epoxy group layer on the surface of the gold film of the sensing area of the SPR optical fiber probe. After incubation, the residual solution on the surface was removed by washing repeatedly with ultrapure water and 80% ethanol solution, and then dried with nitrogen.

[0108] (3) Dextran connection: the sensing area of the SPR optical fiber probe modified with the epoxy group was placed in 0.3 g / mL dextran (molecular weight 500000) solution (solvent: 0.1 M sodium hydroxide aqueous solution) and incubated at room temperature on a shaker for 20 h to form a dextran layer on the surface of the gold film of the sensing area of the SPR optical fiber probe by covalent bond. After incubation, the residual dextran on the surface of the SPR optical fiber probe was removed by washing with 50°C ultrapure water, and then dried with nitrogen.

[0109] (4) Carboxylation of dextran: the sensing area of the SPR optical fiber probe modified with dextran was placed in 1 M bromoacetic acid solution (solvent: 2 M sodium hydroxide aqueous solution) and incubated at room temperature on a shaker for 16 h to oxidize the hydroxyl group on the surface of the dextran to carboxyl group by the oxidizing property of bromoacetic acid. After incubation, the residual solution on the surface of the SPR optical fiber probe was removed by washing with ultrapure water, and then dried with nitrogen.

[0110] The SPR optical fiber probe prepared in the above steps and the control sample were observed for the shedding of the gold film on the sensing area, and the gold film on the sensing area was observed under a 40-fold optical microscope. As shown in Table 1, the SPR optical fiber probe prepared in the present embodiment had a shedding rate of 2% of the gold film on the sensing area after treatment, while the control sample had a shedding rate of 20% of the gold film on the sensing area after modification, indicating that the SPR optical fiber probe prepared in the present embodiment had higher stability. Figure 1

[0111] Table 1 Comparison of the stability of the gold film on the sensing area of the SPR optical fiber probe

[0112]

[0113] Example 2 Functional modification of the SPR optical fiber probe ​

[0114] 1. Titanium dioxide film layer (i.e. metal oxide layer) modification

[0115] (1) Preparation of titanium dioxide sol

[0116] Take 2 mL of 99.5% acetic acid, add 48 mL of ultrapure water, mix well to obtain 50 mL of 4% acetic acid aqueous solution. Precisely weigh 250 mg of chitosan powder (degree of deacetylation ≥95%, viscosity 100-200 mpa·s), add the chitosan powder into the 4% acetic acid aqueous solution while stirring, after adding, use a magnetic stirrer to stir at 40°C and a speed of 1150 rpm continuously until the chitosan is completely dissolved, then precisely weigh 50 mg of small particle size titanium dioxide nanoparticles (5-10 nm), add the titanium dioxide nanoparticles into the above chitosan solution while stirring. After adding, first stir at room temperature for 5 min, then ultrasonic for 30 min to disperse the titanium dioxide particles, after ultrasonic, stir at room temperature and a speed of 1150 rpm overnight, to obtain the titanium dioxide sol.

[0117] (2) Preparation of 4% polyacrylic acid aqueous solution

[0118] Weigh 2 g of polyacrylic acid powder (average Mv~450000), add 50 mL of ultrapure water, dissolve to obtain 4% polyacrylic acid aqueous solution.

[0119] (3) Deposition of titanium dioxide film layer

[0120] ① Immerse the sensing area of the SPR fiber probe prepared in Example 1 into the titanium dioxide sol, and after 5 min, pull it out at a speed of 1 mm / s;

[0121] ② Dry at room temperature for 10 min;

[0122] ③ Rinse with deionized water for 1 min, then dry at room temperature for 10 min;

[0123] ④ Replace the TiO2 sol with 4% polyacrylic acid solution, and complete the process of (1)-(3);

[0124] ⑤ Dry in a vacuum drying oven at 60°C for 1 h.

[0125] After the above operations, the modification of the titanium dioxide film layer on the surface of the gold film of the sensing area of the SPR fiber probe is completed.

[0126] 2. Functional modification

[0127] (1) Hydroxylation: The SPR fiber probe sensing area with a titanium dioxide film layer was immersed in 1 M sodium hydroxide solution for 1 hour, and then washed with ultrapure water and anhydrous ethanol. After washing, the SPR fiber probe was placed in an 80°C oven for 1 hour to dry, and then taken out to restore to room temperature, thereby completing the hydroxylation of the SPR fiber probe sensing area.

[0128] (2) Epoxy group connection: The SPR fiber probe sensing area with a hydroxyl group was placed in a 0.6 M epichlorohydrin solution, and the solvent was a 0.4 M sodium hydroxide solution and diethylene glycol dimethyl ether with a volume ratio of 1:1. The fiber surface was incubated at room temperature for 4 hours to generate an epoxy group. After incubation, the surface residual solution was washed with ultrapure water and 80% ethanol solution to remove the surface residual solution, and then dried with nitrogen, thereby forming an epoxy group on the surface of the SPR fiber probe sensing area.

[0129] (3) Dextran connection: The SPR fiber probe sensing area with an epoxy group was placed in a 0.3 g / mL dextran (molecular weight 500000) solution, and the solvent was a 0.1 M sodium hydroxide solution. The SPR fiber probe sensing area was incubated at room temperature for 20 hours to form a layer of dextran on the surface of the SPR fiber probe sensing area through covalent bonds. After incubation, the surface residual dextran of the SPR fiber probe was washed with 50°C ultrapure water, and then dried with nitrogen.

[0130] (4) Carboxylation of dextran: The SPR fiber probe sensing area with dextran was placed in a 1 M bromoacetic acid solution, and the solvent was a 2 M sodium hydroxide solution. The SPR fiber probe sensing area was incubated at room temperature for 16 hours to oxidize the hydroxyl group on the surface of the dextran to a carboxyl group by the oxidation of bromoacetic acid. After incubation, the surface residual solution of the SPR fiber probe was washed with ultrapure water, and then dried with nitrogen, thereby completing the functional modification of the SPR fiber probe sensing area.

[0131] Example 3 Preparation and characterization of ASF@AuNPs

[0132] 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 HC1: concentrated HNO3, 3: 1, v / v) to remove small seeds that could affect crystallization, and then rinsed with ultrapure water and dried in an oven for future use. Note that the aqua regia needs to be kept away from light during the overnight soaking, and the opening of the glassware needs to be tightly sealed with parafilm to prevent the evaporation of the aqua regia. 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 inserted, and a spherical condenser was connected. The solution was heated and stirred at 130 °C under oil bath with a rotation speed of 1100 rpm. After 30 min of reflux, 1.55 mL of 1% (w / v) sodium citrate aqueous solution was added quickly, and the solution was continuously refluxed for another 40 min. After 40 min, the 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 AuNPs solution was used to characterize the plasmonic absorption peak of AuNPs by UV-Vis spectrophotometry. As shown in FIG. 1, the maximum UV-Vis absorption peak was at 520 nm. According to Anal. Chem. 2007, 79, 4215-4221, the size of AuNPs was calculated to be about 13 nm. The prepared AuNPs solution needed to be stored at 4 °C in the dark and used up within one month after preparation. Figure 2

[0133] ​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.

[0134] 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.

[0135] Table 2 Calculation results of ASF coupling efficiency

[0136]

[0137] Example 4: Preparation and Characterization of ASF@GMNPs

[0138] 482 μL HBS-N solution (10 mM HEPES, 137 mM NaCl, pH 7.5) was added with 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), and incubated overnight at room temperature in the dark with rotation, so as to successfully couple the ASF to the surface of the PEG NHS ester disulfide to form an ASF-PEG7-S-S-PEG7-ASF complex. 0.5 mL of gold-coated magnetic nanoparticles (Gold coated magnetic nanoparticles, GMNPs, purchased from Xi'an Ruishi Biological Technology Co., Ltd., 2 mg / mL, particle size 100 nm) was taken, the supernatant was removed by magnetic separation, and then resuspended in 0.5 mL of HBS-N buffer. Then, the above-mentioned ASF-PEG7-S-S-PEG7-ASF solution was added, and incubated at room temperature in the dark for 8 h with rotation. After washing twice with ultrapure water, the GMNPs were resuspended in 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 standby.

[0139] The SPR absorption peaks of the GMNPs before and after modification were characterized by UV-Vis, and the results are shown in Figure 4 , indicating that the SPR absorption peak of the GMNPs was red-shifted after modification. The content of ASF in the supernatant was detected by BCA method, and the ASF connection was indirectly characterized. The standard curve of BCA method was drawn, as shown in Figure 5 , and the coupling efficiency of ASF was calculated, as shown in Table 3. The results showed that the coupling rate of ASF reached 60%, indicating that the ASF coupling was successful.

[0140] Table 3 Calculation results of ASF coupling efficiency of ASF@GMNPs.

[0141]

[0142] Example 5 Detection of ricin (RCA60) by antibody-glycoprotein sandwich method

[0143] In this example, the SPR fiber probe prepared in Example 2 of the present application was used to detect ricin (RCA60) by the antibody-glycoprotein sandwich method.

[0144] The signal transmission end of the SPR optical fiber probe prepared in Example 2 of the present application is connected with a light source and a spectrometer through an optical fiber to build a detection system. The light source used is a tungsten lamp light source (purchased from Hangzhou Jinhui Technology, FLE1003). The spectrometer used is purchased from Hangzhou Jinhui Technology, FLA5300, 2048 line array CCD detection, wavelength range 350-1100 nm, 70 μm slit, wavelength resolution 0.1 nm. The optical fiber used is HF400 / 430-37 type multimode optical fiber, purchased from Beijing Feibosida Technology, numerical aperture 0.37, core diameter 400 μm.

[0145] During detection, the SPR optical fiber probe is brought into contact with the sample to be detected, and the light emitted from the light source is transmitted to the optical fiber probe through the optical fiber, reaches the sensing area through continuous reflection in the optical fiber probe, and the signal generated by the interaction between the optical fiber probe and the analyte in the sample to be detected is received by the spectrometer.

[0146] The principle of detecting RCA60 by the antibody-glycoprotein sandwich method is described as follows. After the carboxyl groups of dextran on the sensing area of the SPR optical fiber probe prepared in Example 2 are activated, protein A is coupled through the amino group, and protein A can recognize and capture the Fc segment of recombinant anti-ricin humanized monoclonal antibody (MIL50, anti-ricin neutralizing monoclonal antibody 4C13 humanized antibody, donated by the research group of Professor Feng Jiannan, Academy of Military Medical Sciences, Chinese People's Liberation Army), so that the Fab segment is exposed. MIL50 recognizes and captures the A chain of RCA60 in the sample to be detected through the Fab segment, ASF can specifically recognize and bind the B chain of RCA60, and MIL50 and ASF form a sandwich structure by recognizing different fragments of RCA60.

[0147] The SPR optical fiber probe prepared in Example 2 of the present application uses wavelength modulation, white light of a tungsten lamp light source is used as incident light, and irradiation is carried out at a fixed incident angle, the change in reflectivity at different wavelengths is detected by a spectrometer, and a spectral curve of reflectivity changing with wavelength can be obtained. The wavelength corresponding to the minimum reflectivity is the SPR resonance wavelength at the corresponding incident angle. When the refractive index of the solution on the surface of the sensing area of the gold film changes, the resonance condition changes, and the corresponding resonance wavelength red shifts (the wavelength increases). In this example, the antibody MIL50 is fixed on the surface of the sensing area of the SPR optical fiber probe, and MIL50 captures the analyte (RCA60) in the sample to be detected, so that the medium refractive index on the surface of the sensing area of the SPR optical fiber probe changes, the resonance wavelength red shifts, and the red shift amount is positively correlated with the capture amount of the analyte. Similarly, the MIL50-RCA60 complex is coupled with ASF in the ASF@AuNPs to form a sandwich complex, which also produces a resonance wavelength red shift signal. By detecting the red shift signal, qualitative and quantitative detection of the analyte can be achieved.

[0148] The detection steps are as follows:

[0149] (1) Activation of carboxyl groups of dextran: The SPR fiber probe sensing region 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) activation reagent for 30 min.

[0150] (2) Protein A coupling: The activated SPR fiber probe sensing region was immersed in acetic acid-sodium acetate buffer (10 mM, pH 4.5) for 2 min. Then the SPR fiber probe sensing region was immersed in protein A solution (200 μg / mL, dissolved in the above-mentioned acetic acid-sodium acetate buffer, purchased from Shanghai Yuan Ye Biological Technology Co., Ltd.) for 15 min. Protein A was connected to the activated carboxyl group through its amino group.

[0151] (3) Detection of RCA60 by antibody (MIL50)-glycoprotein (ASF) sandwich method: The SPR fiber probe sensing region was immersed in different reagents in the following order and time to realize the detection of RCA60:

[0152] ① PBS buffer (10 mM, 0.8% NaCl, pH 7.2~7.4): 2 min

[0153] ② MIL50 (5 μg / mL, dissolved in PBS buffer): 10 min

[0154] ③ PBS buffer: 2 min

[0155] ④ Sample to be tested (RCA60, dissolved in PBS buffer): 10 min

[0156] ⑤ PBS buffer: 2 min

[0157] ⑥ ASF@AuNPs (ASF concentration is 50 nM, dissolved in PBS buffer): 10 min

[0158] ⑦ PBS buffer: 2 min

[0159] ⑧ NaOH solution (20 mM): 30 s

[0160] ⑨ PBS buffer: 2 min

[0161] The above operation was performed on different concentrations of RCA60, and the concentration gradient was: 0.01 nM, 0.02 nM, 0.1 nM, 0.5 nM, 1 nM, 2 nM, each concentration was detected twice to take the average value, and the linear relationship of the first five concentration points was good, and the standard curve was plotted as Figure 6The linear regression equation of the standard curve is y = 10.54x + 1.67 (R = 0.98), which shows 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). 2 =0.98), which shows 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).

[0162] Example 6 Selectivity analysis of the method established in Example 5 for ricin and abrin (ABA60)

[0163] Referring to the method of Example 5, RCA60 with a concentration of 2 nM, and ABA60 with concentrations of 2 nM, 20 nM, 50 nM and 100 nM were detected respectively to analyze the selectivity of the method to the two toxin proteins.

[0164] As shown in Figure 7 , for 2 nM of RCA60, the method produced a significant signal response, but for ABA60 with a concentration of 1 times, 10 times, 25 times and 50 times, there was no significant signal response, indicating that the MIL50-ASF sandwich detection system has at least 50 times selectivity for RCA60 and ABA60.

[0165] Example 7 Detection of RCA60 / ABA60 by double glycoprotein sandwich method

[0166] In this embodiment, the SPR optical fiber probe prepared in Example 2 is used to detect RCA60 / ABA60 by a double glycoprotein sandwich method.

[0167] The principle of detecting RCA60 / ABA60 by a double glycoprotein sandwich method is described as follows. After the carboxyl groups of the dextran in the sensing region of the SPR optical fiber probe prepared in Example 2 are activated, streptavidin (SA) is coupled through the amino group. The SA can recognize and capture biotinylated concanavalin A (ConA), the ConA can recognize and capture a site of the B chain of RCA60 / ABA60 in the sample to be detected, and the ASF can specifically recognize and bind another site of the B chain of RCA60 / ABA60, so that the ConA and the ASF form a sandwich structure by binding to different sites of the B chain of RCA60 / ABA60.

[0168] The detection system is the same as that in Example 5, and the detection steps are as follows:

[0169] (1) Biotinylated concanavalin A (ConA): Biotin Labeling Kit-NH2 biotinylation kit (purchased from DOJINDO, batch number: AJ801) was used to biotinylate and modify the ConA (purchased from Sigma-Aldrich).

[0170] (2) Activation of carboxyl groups of dextran: The SPR fiber probe sensing region 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) activation reagent for 30 min.

[0171] (3) SA coupling: The activated SPR fiber probe sensing region was immersed in acetic acid-sodium acetate buffer (10 mM, pH 4.0) for 2 min. Then the SPR fiber probe sensing region was immersed in a SA solution (200 μg / mL, dissolved in the above-mentioned acetic acid-sodium acetate buffer, purchased from Beijing Biotopped Technology Co., Ltd.) for 20 min. The SA was coupled to the activated carboxyl groups through amino coupling. After the coupling was completed, the SPR fiber probe sensing region was immersed in acetic acid-sodium acetate buffer, and the red shift amount of the SPR absorption peak in the acetic acid-sodium acetate buffer before and after the two immersions was observed to determine whether the SA was successfully coupled to the fiber sensing region and the coupling amount.

[0172] (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) for 2 min. Then the SPR fiber probe sensing region was immersed in a Bio-ConA solution (25 μg / mL, dissolved in HBS-BT buffer) for 10 min. The ConA was immobilized on the surface of the fiber sensing region through the interaction between biotin and streptavidin.

[0173] (5) Detection of RCA60 / ABA60 by ConA-ASF sandwich method: The SPR fiber probe sensing region was immersed in different reagents in the following order and time to realize the detection of RCA60 / ABA60:

[0174] ① HBS-BT buffer: 2 min

[0175] ② Test sample (RCA60 / ABA60 dissolved in HBS-BT buffer): 10 min

[0176] ③ HBS-BT buffer: 2 min

[0177] ④ ASF@AuNPs (ASF concentration of 50 nM, dissolved in HBS-BT buffer): 10 min

[0178] ⑤ HBS-BT buffer: 2 min

[0179] ⑥ HCl solution (50 mM): 30 s

[0180] ⑦ HBS-BT buffer: 2 min

[0181] The different concentrations of RCA60 / ABA60 were detected according to the above operation, and the concentration gradient was: 0.0006, 0.003, 0.006, 0.03, 0.06, 0.12 μg / mL, each concentration was detected twice to take the average value, and the standard curve of RCA60 / ABA60 was drawn as shown in Figure 8 、 9 The linear regression equation of the RCA60 standard curve was 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 (R 2 = 0.97), the linear range was 1.99~120 ng / mL, and the LOD was 0.6 ng / mL; the selectivity of the method for the two toxin proteins was 1:3.

[0182] Example 8: Detection of RCA60 in urine or plasma

[0183] RCA60 was added to the urine, and urine samples with concentrations of 0.02 nM and 0.5 nM were prepared. According to the method in Example 5, RCA60 in the test sample was directly detected, and the results are shown in Figure 10 It can be seen that the method can realize the detection of RCA60 in urine.

[0184] After diluting the rat plasma 10 times, RCA60 was added to prepare plasma test samples with different concentrations. ASF@GMNPs were mixed with the plasma test samples to make the final concentration of ASF 50 nM, and incubated at room temperature for 15 min. ASF@GMNPs were magnetically separated to obtain RCA60-ASF@GMNPs complexes, which were washed twice with PBS buffer and resuspended in 200 μL PBS buffer. According to the method in Example 5, the dextran carboxyl groups on the surface of the SPR optical fiber probe sensing area were activated and coupled with protein A. Then the optical fiber SPR optical fiber probe sensing area was immersed in different reagents in the following order and time to realize the detection of RCA60 in the test sample:

[0185] ① PBS buffer (10 mM, 0.8% NaCl, pH 7.2-7.4): 2 min

[0186] ② MIL50 (5 μg / mL, dissolved in PBS buffer): 10 min

[0187] ③ PBS buffer: 2 min

[0188] ④ Test sample (RCA60-ASF@GMNPs complex, dissolved in PBS buffer): 10 min

[0189] ⑤ PBS buffer: 2 min

[0190] ⑥ NaOH solution (20 mM): 30 s

[0191] ⑦ PBS buffer: 2 min

[0192] The plasma test sample of different concentrations of RCA60 was detected according to the above operation, and the concentration gradient was: 0.01, 0.05, 0.1, 1 nM, each concentration was detected twice to take the average value, and the standard curve was plotted as shown in Figure 11 The linear regression equation of the standard curve was y=0.275x+0.723 (R 2 =0.9995), and the LOD was 0.01 nM (0.6 ng / mL). It can be seen that the method can realize the detection of RCA60 in blood samples.

[0193] Example 9: Detection of ABA60 in plasma

[0194] After diluting the rat plasma 10 times, ABA60 was added to prepare plasma test samples of different concentrations. ASF@GMNPs was mixed with the plasma test sample to make the final concentration of ASF 50 nM, and incubated at room temperature for 10 min. The ASF@GMNPs was magnetically separated to obtain ABA60-ASF@GMNPs complex, which was washed twice with HBS-T (10 mM HEPES, 150 mM NaCl, 0.05% (v / v) Tween-20, pH 7.2-7.4) buffer and resuspended in 200 μL HBS-T buffer. Referring to the method of Example 7, the dextran carboxyl groups on the surface of the SPR optical fiber probe sensing area were activated and SA and ConA were connected in turn, and then the SPR optical fiber probe sensing area was immersed in different reagents in the following order and time to realize the detection of ABA60 in the test sample:

[0195] ① HBS-T buffer: 2 min

[0196] ② Test sample (ABA60-ASF@GMNPs complex, dissolved in HBS-T buffer): 10 min

[0197] ③ HBS-T buffer: 2 min

[0198] (4) HCl solution (50 mM): 30 s

[0199] (5) HBS-T buffer: 2 min

[0200] The plasma samples to be tested of different concentrations of ABA60 were detected according to the above operation, and the concentration gradient was: 0.01, 0.05, 0.1, 0.5, 1, 2 nM, each concentration was detected twice to take the average value, and the last 5 points were plotted to obtain the standard curve, as shown in Figure 12 The linear regression equation of the standard curve was y = 0.1534x + 0.348 (R 2 = 0.9859), and the LOD was 0.01 nM (0.6 ng / mL). It can be seen that the method can realize the detection of ABA60 in plasma.

[0201] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details according to all the teachings disclosed herein, and these changes are within the scope of protection of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. A method for detecting a type II ribosome inactivating protein in a sample, wherein: a) the method comprises: activating an SPR fiber-optic probe and contacting the SPR fiber-optic probe with protein A, linking the protein A to the surface of the SPR fiber-optic probe; contacting the SPR fiber-optic probe with a recombinant anti-ricin humanized monoclonal antibody, linking the recombinant anti-ricin humanized monoclonal antibody to the protein A on the surface of the SPR fiber-optic probe; contacting the SPR fiber-optic probe with the sample to be tested, capturing the type II ribosome inactivating protein in the sample to be tested; contacting the SPR fiber-optic probe with gold nanoparticles having desialidated fetuin coupled to the surface; detecting a change in signal; or b) the method comprises: activating an SPR fiber-optic probe and contacting the SPR fiber-optic probe with streptavidin, linking the streptavidin to the surface of the SPR fiber-optic probe; contacting the SPR fiber-optic probe with biotin-labeled concanavalin A, linking the biotin-labeled concanavalin A to the streptavidin on the surface of the SPR fiber-optic probe; contacting the SPR fiber-optic probe with the sample to be tested, capturing the type II ribosome inactivating protein in the sample to be tested; contacting the SPR fiber-optic probe with gold nanoparticles having desialidated fetuin coupled to the surface; detecting a change in signal; or c) the method comprises: activating an SPR fiber-optic probe and contacting the SPR fiber-optic probe with protein A, linking the protein A to the surface of the SPR fiber-optic probe; contacting the SPR fiber-optic probe with a recombinant anti-ricin humanized monoclonal antibody, linking the recombinant anti-ricin humanized monoclonal antibody to the protein A on the surface of the SPR fiber-optic probe; contacting the sample to be tested with gold-coated magnetic nanoparticles having desialidated fetuin coupled to the surface, the type II ribosome inactivating protein in the sample to be tested being captured by the gold-coated magnetic nanoparticles; contacting the SPR fiber-optic probe with the gold-coated magnetic nanoparticles having captured the type II ribosome inactivating protein; detecting a change in signal; or d) the method comprises: activating an SPR fiber-optic probe and contacting the SPR fiber-optic probe with streptavidin, linking the streptavidin to the surface of the SPR fiber-optic probe; contacting the SPR fiber-optic probe with biotin-labeled concanavalin A, linking the biotin-labeled concanavalin A to the streptavidin on the surface of the SPR fiber-optic probe; contacting the sample to be tested with gold-coated magnetic nanoparticles having desialidated fetuin coupled to the surface, the type II ribosome inactivating protein in the sample to be tested being captured by the gold-coated magnetic nanoparticles; contacting the SPR fiber-optic probe with the gold-coated magnetic nanoparticles having captured the type II ribosome inactivating protein; detecting a change in signal, wherein the SPR fiber-optic probe comprises: a fiber-optic base, a first medium layer and a metal nanoparticle layer, wherein the fiber-optic base is a quartz fiber-optic base, the first medium layer is formed by a mercapto-silane coupling agent or an amino-silane coupling agent, and is located between the fiber-optic base and the metal nanoparticle layer. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 2. The method of claim 1, wherein the first dielectric layer is connected to the optical fiber substrate by covalent bond, and connected to the metal nanoparticle layer by metal-nitrogen coordination bond or metal-sulfur covalent bond.

3. The method of claim 1, wherein the mercapto silane coupling agent is 3-mercaptopropyl trimethoxysilane, 3-mercaptopropyl triethoxysilane or mercaptomethyl trimethoxysilane; the amino silane coupling agent is 3-aminopropyl trimethoxysilane, 3-aminopropyl triethoxysilane, 3-aminopropyl methyl dimethoxysilane, N-(β-aminoethyl)-γ-aminopropyl trimethoxysilane or N-(β-aminoethyl)-γ-aminopropyl triethoxysilane.

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

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

6. The method of claim 4, wherein the metal nanoparticles are gold nanoparticles, the first dielectric layer is connected to the metal nanoparticle layer by gold-sulfur covalent bond.

7. The method of any one of claims 1-6, wherein the SPR optical fiber probe further comprises a metal oxide layer, attached to the metal nanoparticle layer.

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

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

10. The method of any one of claims 1-6, wherein the type II ribosome inactivating protein is ricin or abrin.

11. A kit for detecting type II ribosome inactivating protein, comprising an SPR optical fiber probe, and a) asialofetuin, gold nanoparticles, protein A, recombinant anti-ricin humanized monoclonal antibody, and one or more substances in group A consisting of EDC, NHS, acetic acid-sodium acetate buffer, PBS buffer and NaOH; or b) asialofetuin, gold nanoparticles, biotin, concanavalin A, streptavidin, and one or more substances in group B consisting of EDC, NHS, acetic acid-sodium acetate buffer, HBS-BT buffer and HCl solution; or c) asialofetuin, gold-coated magnetic nanoparticles, protein A, recombinant anti-ricin humanized monoclonal antibody, and one or more substances in group C consisting of EDC, NHS, acetic acid-sodium acetate buffer, PBS buffer and NaOH. ​ ​ ​ d) desialylated fetuin, gold-coated magnetic nanoparticles, biotin, concanavalin A, streptavidin, and one or more of the following: EDC, NHS, acetic acid-sodium acetate buffer, HBS-BT buffer, and HCl solution, or e) gold nanoparticles with desialylated fetuin coupled to the surface, protein A, recombinant anti-ricin humanized monoclonal antibody, and one or more of the following: EDC, NHS, acetic acid-sodium acetate buffer, PBS buffer, and NaOH; or f) gold nanoparticles with desialylated fetuin coupled to the surface, biotin-labeled concanavalin A, streptavidin, and one or more of the following: EDC, NHS, acetic acid-sodium acetate buffer, HBS-BT buffer, and HCl solution; or g) gold-coated magnetic nanoparticles with desialylated fetuin coupled to the surface, protein A, recombinant anti-ricin humanized monoclonal antibody, and one or more of the following: EDC, NHS, acetic acid-sodium acetate buffer, PBS buffer, and NaOH; or h) gold-coated magnetic nanoparticles with desialylated fetuin coupled to the surface, biotin-labeled concanavalin A, streptavidin, and one or more of the following: EDC, NHS, acetic acid-sodium acetate buffer, HBS-BT buffer, and HCl solution, wherein the SPR fiber probe comprises: an optical fiber substrate, a first dielectric layer, and a metal nanoparticle layer, wherein the optical fiber substrate is a quartz optical fiber substrate, the first dielectric layer is formed of a mercapto-silane coupling agent or an amino-silane coupling agent, and is located between the optical fiber substrate and the metal nanoparticle layer.

12. The kit of claim 11, wherein the first dielectric layer is connected to the optical fiber substrate by a covalent bond, and is connected to the metal nanoparticle layer by a metal-nitrogen coordination bond or a metal-sulfur covalent bond.

13. The kit of claim 11, wherein the mercapto-silane coupling agent is 3-mercaptopropyltrimethoxysilane, 3-mercaptopropyltriethoxysilane, or mercaptomethyltrimethoxysilane; and the amino-silane coupling agent is 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-aminopropylmethyldimethoxysilane, N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane, or N-(β-aminoethyl)-γ-aminopropyltriethoxysilane.

14. The kit of claim 11, wherein the metal nanoparticle layer comprises metal nanoparticles, and the metal nanoparticles are gold, silver, copper, platinum, aluminum, or titanium nanoparticles.

15. The kit of claim 14, wherein the metal nanoparticles are spherical, star-shaped, rod-shaped, spindle-shaped, or triangular nanoparticles.

16. The kit of claim 14, wherein the metal nanoparticles are gold nanoparticles, and the first dielectric layer is connected to the metal nanoparticle layer by a gold-sulfur covalent bond.

17. The kit of any one of claims 11-16, wherein the SPR optical fiber probe further comprises a metal oxide layer attached to the metal nanoparticle layer.

18. The kit of claim 17, wherein the metal oxide layer comprises a metal oxide that is titanium dioxide, tantalum pentoxide, or aluminum oxide.

19. The kit of claim 17, wherein the SPR optical fiber probe further comprises a second dielectric layer formed from epichlorohydrin or methyl epichlorohydrin and connected to the metal oxide layer by ether bonds, and a functional modification layer comprising carboxylated dextran connected to the second dielectric layer by ether bonds.

20. The kit of any one of claims 11-16, wherein the type II ribosome inactivating protein is ricin or abrin.

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