A nanobody-based fiber-optic SPR probe, a preparation method and a detection system

Abstract

The application discloses a kind of nanobody-based optical fiber SPR probe, preparation method and detection system;The probe includes multimode optical fiber, and the end of fiber core is cut flat using optical fiber cutting knife, and the cladding near end face is removed, and the fiber core is exposed, and the surface of fiber core includes from bottom to top in sequence: noble metal layer, near waveguide dielectric layer and reaction layer.Near waveguide dielectric layer is uniformly doped with two-dimensional material;Reaction layer includes biochemical detection reagent and serum albumin;The biochemical detection reagent includes antibody connecting reagent, nanobody and metal ion;The nanobody is with His label, and is single-domain antibody including only one heavy chain variable region.The prepared probe is suitable for high-sensitivity, low-concentration biological small molecule detection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical optical detection, specifically relating to an optical fiber SPR probe based on nanobodies, its preparation method, and a sensing and detection system. Background Technology

[0002] Surface plasmon resonance (SPR) is a collective resonance phenomenon generated by electromagnetic waves exciting free electrons in a material. SPR is highly sensitive to changes in the dielectric constant of the local environment, and therefore is commonly used for detecting changes in liquid concentration and real-time monitoring of biomolecule adsorption and binding. The binding of antibodies coupled to analytes on metal surfaces can alter the environment surrounding the SPR sensor. By monitoring changes in the location of resonance valleys, information such as the affinity, affinity constant, and binding kinetics of biomolecule interactions can be inferred. Therefore, SPR technology has wide applications and significant value in drug screening, protein-protein interaction studies, and antibody-antigen binding. Fiber optic SPR sensors offer advantages such as resistance to electromagnetic interference, corrosion resistance, small size, and high flexibility, enabling real-time, convenient, and multi-parameter detection of target analytes. Therefore, fiber optic SPR sensors are highly favored in the field of biochemical sensing.

[0003] CN116106270A discloses a fiber optic SPR biochemical sensor based on a metal micro / nano array structure and its fabrication method. By constructing a metal micro / nano array structure on the curved surface of the sensing fiber, the sensor sensitivity is improved. Biorecognition molecules with carboxyl groups on their surface are immobilized on the nanomaterial surface through coordination bond coupling, achieving biochemical detection. CN112461794A discloses a long-range SPR sensor and its fabrication method. The sensor sensitivity is improved through the plasmonic coupling effect between the nanomaterial and the metal layer in the SPR sensor. Antibodies are immobilized on the surface of the long-range SPR sensor using a coupling agent, and biochemical detection is achieved using a double-antibody sandwich immunoassay.

[0004] Traditional fiber optic SPR sensors can only bind to ordinary IgG antibodies, which have drawbacks such as large molecular weight (approximately 150 kDa) and poor stability, making them unsuitable for detecting small molecules. Nanobodies, compared to traditional IgG antibodies, offer advantages such as small molecular weight (approximately 15 kDa), high affinity, strong stability, the ability to recognize hidden antigenic epitopes, and the ability to specifically bind to small biological molecules. Furthermore, nanobodies can improve sensor sensitivity and lower the sensor's detection limit.

[0005] However, nanobodies have a relatively simple structure with only one specific binding site and no other amino groups. The traditional method of binding the amino group at the tail of IgG antibodies to coupling agents for fiber optic SPR sensors (such as cysteine, polydopamine, and gold-sulfur bonds) is not suitable for nanobodies. Therefore, a new method for directionally immobilizing nanobodies needs to be developed. Summary of the Invention

[0006] The purpose of this invention is to overcome the problem of existing fiber optic SPR sensors' inability to detect low concentrations of small biomolecules. It proposes a fiber optic SPR probe based on nanobodies, its preparation method, and a detection system for the detection of small biomolecules. This invention introduces a uniformly doped two-dimensional polydopamine film as a near-waveguide dielectric layer, which is solidified on the surface of a noble metal layer to form a near-waveguide fiber optic SPR probe. The nanobodies are immobilized on the surface of the fiber optic SPR probe by utilizing the coupling reaction between polydopamine, trinitrotriacetic acid (Tris-NTA), nickel ions, and the nanobodies. The small molecular weight of the nanobodies allows for specific binding to small biomolecules, enabling the measurement of small biomolecules. The polydopamine film has advantages such as density and strong adhesion, preventing film detachment and improving sensor stability. Two-dimensional nanosheets with high / complex dielectric constants are incorporated into the polydopamine film to form the near-waveguide dielectric layer. The high refractive index and high carrier mobility of the two-dimensional material enhance the surface electric field strength of the fiber optic SPR probe, improving sensor sensitivity.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] The first aspect of this invention discloses a fiber optic SPR probe based on nanobody, comprising a multimode fiber, wherein one end of the multimode fiber has a core with the cladding and coating removed, the core has a flat end face as an end face region, and the outer surface of the core is a sensing region.

[0009] From the fiber core outwards, it comprises a noble metal layer, a near-waveguide dielectric layer, and a reactive layer;

[0010] The noble metal layer is solidified in the sensing area and the end face area, wherein the noble metal layer located in the sensing area is used to excite the SPR effect, the noble metal layer located in the end face area is used for reflection, and the thickness of the noble metal layer in the end face area is greater than that of the noble metal layer in the sensing area.

[0011] The near-waveguide dielectric layer is solidified on the outer surface of the noble metal layer, and the near-waveguide dielectric layer is uniformly doped with two-dimensional material nanosheets.

[0012] The reaction layer is solidified on the near-waveguide dielectric layer located in the sensing region and includes biochemical detection reagents and serum albumin. The biochemical detection reagents include antibody-binding reagents, nanobodies, and metal ions. The nanobodies are labeled with polyhistidine (His) and are single-domain antibodies containing only one heavy chain variable region. The metal ions cooperate with the antibody-binding reagents to solidify the nanobodies on the upper surface of the near-waveguide dielectric layer. The serum albumin is used to seal the areas on the upper surface of the near-waveguide dielectric layer that have not reacted with the antibody-binding reagents.

[0013] In some specific embodiments, the fiber core length is on the order of centimeters. Preferably, the fiber core length is 1-2 cm, and particularly preferably, the fiber core length is 1-1.5 cm.

[0014] In some specific embodiments, the fiber core end face is flattened by using a cutting blade.

[0015] In some specific embodiments, the noble metal layer is on the nanometer scale, and the thickness of the noble metal layer in the end face region is 295-305 nm, while the thickness of the noble metal layer in the sensing region is 45-55 nm.

[0016] Furthermore, the precious metal layer is a dense and flat layer, and the preferred material is gold.

[0017] In some specific embodiments, the two-dimensional material contained in the near-waveguide dielectric layer can react with Tris-NTA to form stable covalent bonds. Specifically, the near-waveguide dielectric layer is a polydopamine film uniformly doped with two-dimensional material, with quinone groups on its surface, which can undergo Schiff base reactions and Michelson reactions with the amino groups of Tris-NTA to form stable covalent bonds.

[0018] In some specific embodiments, the near-guided dielectric layer is on the nanometer scale with a thickness of 10-15 nm.

[0019] In some specific embodiments, the nanobody is the smallest complete antigen-binding fragment. The metal ions can chelate with the Tris-NTA and the nanobody. The chelating force can solidify the nanobody on the outer surface of the near-guided dielectric layer, forming a biorecognition interface. When the analyte approaches the surface of the fiber optic SPR probe, the nanobody can specifically bind to the analyte, forming an antibody-antigen complex. This specific binding can change the equivalent refractive index of the sensor surface, causing the SPR resonance valley to shift. By determining the amount of SPR resonance valley shift, the concentration of small molecules can be detected.

[0020] Furthermore, the metal ion is selected from one of the following: copper ion, nickel ion, and calcium ion.

[0021] Furthermore, the two-dimensional material is a transition metal sulfide, a transition metal selenide, or a transition metal telluride.

[0022] A second aspect of this invention discloses a detection system for a nanobody-based fiber optic SPR probe, comprising a spectrometer, a broadband light source, and a fiber optic SPR probe with a multimode fiber as the optical path. The input end of the fiber optic SPR probe transmits light emitted from the broadband light source to the fiber optic SPR probe via a fiber optic patch cord. The output end of the fiber optic SPR probe is connected to the spectrometer via a fiber optic patch cord, and the spectrometer is connected to a computer via a data interface. The nanobody on the surface of the fiber optic SPR probe specifically binds to the analyte, causing a change in the equivalent refractive index of the fiber optic SPR probe surface. By determining the amount of SPR resonance valley shift, the concentration of small molecules is detected.

[0023] Furthermore, the fiber optic SPR probe is fixed on a z-axis vertical displacement platform with its end face facing the analyte solution sample. The fiber optic SPR probe is raised and lowered by the z-axis vertical displacement platform so that its sensing area is completely immersed in the solution of the analyte solution sample to achieve detection.

[0024] A third aspect of this invention discloses a method for preparing the aforementioned nanobody-based fiber optic SPR probe, comprising:

[0025] Pre-processed multimode fiber:

[0026] The end of the multimode fiber is cut flat using a fiber optic cleaver to create a flat end face. The coating and cladding near the end face are removed to expose a fiber core on the order of centimeters. The outer surface of the fiber core is used as the sensing area.

[0027] Preparation of noble metal layer:

[0028] Noble metal layers are formed by sputtering noble metals on the end face region and the sensing region of the fiber core using a magnetron sputtering instrument. The noble metal layer in the end face region forms a mirror, and the noble metal layer in the sensing region excites the SPR effect.

[0029] Preparing a near-waveguide dielectric layer:

[0030] Tris-HCl buffer solution was mixed with dopamine hydrochloride powder to prepare a dopamine solution with a concentration of 1.9-2.1 mg / ml and a pH of 8-9. The monolayer two-dimensional material nanosheets were mixed with the dopamine solution and shaken well. The fiber core with the noble metal layer was then immersed in the mixed solution and oscillated at a constant speed to obtain a uniform polydopamine near-waveguide dielectric layer doped with two-dimensional material nanosheets.

[0031] Preparation of the reaction layer:

[0032] The fiber core with the near-waveguide dielectric layer is immersed in the antibody linkage reagent solution and incubated overnight; the quinone group on the surface of the polydopamine near-waveguide dielectric layer undergoes a coupling reaction with the amino group of Tris-NTA, thereby solidifying the Tris-NTA on the upper surface of the near-waveguide dielectric layer.

[0033] The fiber core is then immersed in a serum albumin solution to seal off the unreacted area on the surface of the near-guided dielectric layer;

[0034] The fiber optic SPR sensing probe was immersed in a metal ion solution and incubated for 1-2 hours; nickel ions were preloaded onto the Tris-NTA surface through chelation.

[0035] The prepared fiber core was immersed in a nanobody solution and incubated overnight. Through chelation, the nanobody was immobilized on the Tris-NTA surface of the sensing region by utilizing the chelating force between the His tag of the nanobody and the metal ions, thereby completing the preparation of the fiber optic SPR probe based on nanobody.

[0036] The beneficial effects of this invention are as follows:

[0037] 1. Compared to traditional fiber optic SPR sensors that conjugate ordinary antibodies, this invention utilizes the coupling effect between polydopamine material, Tris-NTA, metal ions, and nanobodies to immobilize nanobodies on the surface of a fiber optic SPR probe, forming the aforementioned nanobodies-based fiber optic SPR probe, which can be used for the detection of small biological molecules. Taking advantage of the small molecular weight and high specificity of nanobodies, which can recognize relatively elusive antigenic epitopes that are difficult for traditional antibodies to identify, the aforementioned nanobodies-based fiber optic SPR probe can achieve high-sensitivity and rapid detection of small biological molecules.

[0038] 2. Polydopamine materials have the advantage of high negative refractive index. When uniformly coated on the surface of a noble metal layer, they can excite near-guided wave effect. Two-dimensional material nanosheets uniformly doped in the polydopamine near-guided wave dielectric layer have the advantages of high negative refractive index and high carrier mobility. The two materials work together to enhance sensitivity and can effectively improve the sensitivity of fiber optic SPR probes.

[0039] 3. Polydopamine materials have a mussel-like adhesion mechanism, which can adhere two-dimensional material nanosheets to the surface of fiber optic SPR probes and form a uniform and dense film, avoiding chemical deformation and detachment of the sensor film layer, thus significantly improving the stability of the sensor. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the structure of the fiber optic SPR probe based on nanobody described in this invention.

[0041] Figure 2 This is a schematic diagram of the detection system structure described in this invention;

[0042] Figure 3 The refractive index response resonance wavelength and sensitivity fitting curve of the fiber optic SPR probe based on nanobody in the embodiment are shown.

[0043] Figure 4 The image shows a fluorescence microscope image of the nanobody-based fiber optic SPR probe bound to a red fluorescent protein in the embodiment.

[0044] In the picture:

[0045] 1: Nanobody; 2: Metal ions; 3: Tris-NTA; 4: Core; 5: Noble metal layer;

[0046] 6: Near-guided dielectric layer; 7: Serum albumin; 8: Multimode optical fiber;

[0047] 1': Computer; 2': Spectrometer; 3': Broadband light source; 4': Z-axis vertical displacement platform;

[0048] 5': Fiber optic SPR probe based on nanobody; 6': Analyte solution sample. Detailed Implementation

[0049] To make the objectives, technical solutions, beneficial effects, and significant advancements of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings provided in the examples of the present invention. Obviously, all the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0050] In the description of this application, unless otherwise expressly specified and limited, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance; the term "multiple" refers to two or more; unless otherwise specified or explained, the terms "connected," "fixed," etc., should be interpreted broadly. For example, "connected" can be a fixed connection, a detachable connection, an integral connection, or an electrical connection; "connected" can be a direct connection or an indirect connection through an intermediate medium. Those skilled in the art can understand the specific meaning of the above terms in this application according to the specific circumstances.

[0051] like Figure 2As shown, the detection system of the fiber optic SPR probe 5' based on nanobody includes a spectrometer 2', a broadband light source 3', and a fiber optic SPR probe 5' with a multimode fiber as the optical path. The input end of the fiber optic SPR probe 5' transmits the light emitted by the broadband light source 3' to the fiber optic SPR probe 5' through a Y-type fiber optic patch cord, and the output end of the fiber optic SPR probe 5' is connected to the spectrometer 2' through a Y-type fiber optic patch cord. The spectrometer 2' is connected to the computer 1' through a data interface. Furthermore, the fiber optic SPR probe 5' is fixed on the z-axis vertical displacement platform 4'; the z-axis vertical displacement platform 4' is a commercially available vertical lifting displacement platform, and the fiber optic SPR probe 5' is fixed on the z-axis vertical displacement platform 4' by a clamp, with the fiber end face vertically downward and facing the test solution sample 6'; the fiber optic SPR probe 5' is controlled by the z-axis vertical displacement platform to move vertically up and down along the z-axis vertical displacement platform 4', and the immersion depth of the probe in the liquid is controlled by the platform's lifting and lowering, so that the fiber optic sensing area is completely immersed in the solution of the test solution sample 6', thereby realizing detection.

[0052] like Figure 1 As shown, the nanobody-based fiber SPR probe 5' includes a multimode fiber 8, which can be any multimode fiber suitable for use as an SPR probe substrate. One end of the multimode fiber 8 has a core with the cladding and coating removed at the centimeter level. The core has a flat end face as an end face region, and the cladding and coating are exposed 2 cm away from the end face, forming a sensing region. The core length of the sensing region is at the centimeter level, and the core diameter is 600 μm.

[0053] The fiber core 4 comprises, sequentially from its outer surface outwards, a noble metal layer 5, a near-waveguide dielectric layer 6, and a reactive layer. The noble metal layer 5 is a dense gold film solidified in the sensing region (i.e., the outer surface of the fiber core) and the end face region of the fiber core 4. The end face and the sensing region have different thicknesses, with the end face region having a thickness of 295-305 nm and the sensing region having a thickness of 45-55 nm. That is, the noble metal layer in the sensing region is used to generate the SPR effect, while the noble metal layer in the end face region only serves to reflect light and does not excite the SPR effect.

[0054] The near-waveguide dielectric layer 6 is a 10-15 nm thick polydopamine film layer solidified on the end face and outer surface of the noble metal layer 5. Two-dimensional nanomaterials are uniformly doped within the near-waveguide dielectric layer 6 to enhance sensitivity. In this embodiment, the two-dimensional material is a transition metal sulfide, transition metal selenide, or transition metal telluride.

[0055] A reaction layer is disposed on the upper surface of the sensing area of ​​the near-waveguide dielectric layer 6. The reaction layer includes biochemical detection reagents and serum albumin 7. In this embodiment, bovine serum albumin is selected as the serum albumin 7. The biochemical detection reagents include commercially available Tris-NTA 3, metal ions 2, and nanobody 1. Tris-NTA is an antibody-binding reagent that can be used to bind His-tagged nanobodies. Metal ions 2 and nanobody 1 are immobilized on the upper surface of Tris-NTA 3. The metal ions 2 are nickel ions, which can chelate with Tris-NTA. Nanobody 1 is a single-domain antibody containing only one heavy chain variable region, which is the smallest complete antigen-binding fragment and is a His-tagged nanobody. Tris-NTA and nickel ions play the role of immobilizing nanobody 1. Bovine serum albumin plays the role of sealing the unreacted area on the surface of the near-waveguide dielectric layer.

[0056] Immobilizing nanobodies on the surface of an optical fiber SPR probe creates a biorecognition interface. When an analyte approaches the probe surface, the nanobodies specifically bind to it, forming an antibody-antigen complex. This specific binding alters the equivalent refractive index of the sensor surface, causing the SPR resonance valley to shift. Due to their small molecular weight, nanobodies can stably bind to small molecules, allowing for the detection of small molecule concentrations by assessing the extent of SPR resonance valley shift.

[0057] The fabrication method of the optical fiber SPR probe 5' includes the following steps:

[0058] Step 1: Pre-processing multimode fiber

[0059] A multimode fiber 8 with a cladding diameter of 630μm and a core diameter of 600μm was selected. The fiber end face was cut flat using a fiber cleaver to form the end face area. The cladding and coating layer in the vicinity of the end face were removed to expose the multimode fiber core 4 as the sensing area.

[0060] Step 2: Sputtering a noble metal layer onto the fiber core end face

[0061] The outer surface of the multimode fiber core 4 prepared in step 1 is covered to expose the fiber end face. Gold is sputtered onto the fiber end face using a magnetron sputtering instrument to form a noble metal layer 5 with a thickness of 295 nm in the end face region, which forms a reflector. The sputtering power is set to 30 W and the vacuum degree is 5 × 10⁻⁶. -4 Pa, sputtering time 30 minutes;

[0062] Step 3: Sputtering a noble metal layer onto the outer surface of the fiber core

[0063] After removing the shielding on the outer surface of the multimode fiber core, gold was sputtered onto the outer surface of the multimode fiber core 4 obtained in step 2 using a magnetron sputtering apparatus to form a noble metal layer 5 with a sensing region thickness of 45 nm; the sputtering power was set to 30 W and the vacuum degree to 5 × 10⁻⁶. -4 Pa, sputtering time 5 minutes;

[0064] Step 4: Deposit the near-guided dielectric layer

[0065] Prepare a dopamine solution with a concentration of 2 mg / mL by mixing 10 mL of Tris-HCl buffer with 0.02 g of dopamine hydrochloride powder and a pH of 8-9; mix the monolayer two-dimensional material nanosheets with the dopamine solution and shake well.

[0066] Immerse the sensing region of the multimode fiber 8 prepared in step 3 into the mixed solution and oscillate at a constant speed for 2-4 hours.

[0067] Dopamine monomers can self-polymerize into a polydopamine layer. Its mussel-like adhesion mechanism can solidify two-dimensional material nanosheets onto the surface of a noble metal layer, forming a uniform polydopamine near-waveguide dielectric layer doped with two-dimensional material nanosheets. The thickness of the near-waveguide dielectric layer is 10-15 nm. The thickness of the near-waveguide dielectric layer can be controlled by controlling the oscillation duration.

[0068] Step 5: Fix Tris-NTA

[0069] The sensing region prepared in step 4 is immersed in a 1 mM Tris-NTA solution and incubated overnight; the quinone group on the surface of the polydopamine near-guided wave dielectric layer undergoes a coupling reaction with the amino group of the Tris-NTA3, which can solidify the Tris-NTA3 on the upper surface of the near-guided wave dielectric layer 6.

[0070] After removal, the fiber optic SPR probe was rinsed with deionized water and dried with nitrogen. The sensing area was then immersed in a 10 mg / ml bovine serum albumin solution and incubated for 1 hour to seal the unreacted area on the surface of the near-guide dielectric layer 6.

[0071] Step 6: Preloading nickel ions

[0072] The sensing region prepared in step 5 was immersed in a nickel chloride solution with a concentration of 5 mM and incubated for 1 hour; nickel ions were preloaded on the Tris-NTA surface of the sensing region through chelation.

[0073] Step 7: Immobilize nanobodies

[0074] The sensing region prepared in step 6 was immersed in a 0.6 mg / ml solution of anti-breast cancer nanobody and incubated overnight. Through chelation, the nanobody was immobilized on the Tris-NTA surface of the sensing region using the chelating force between the His tag of nanobody 1 and nickel ions, thus completing the preparation of the nanobody-based fiber optic SPR probe.

[0075] In use, the detection system based on nanobody-based fiber optic SPR probe 5' operates on the following principle:

[0076] A broadband light source 3' generates incident light, which is transmitted to a fiber optic SPR probe 5' based on nanobody via a Y-type jumper. The reflected outgoing light is transmitted to a spectrometer 2' via the jumper for real-time monitoring of optical power loss in the output spectrum. The fiber optic SPR probe 5' is controlled by a z-axis vertical displacement platform 4' to completely immerse its sensing area in the analyte solution sample 6', exciting the SPR effect. The electric field penetrates the near-guide dielectric layer 6 and reaches the analyte solution sample 6', at which point an SPR resonance valley appears. As the refractive index of the analyte solution sample 6' increases, the SPR resonance valley undergoes a redshift. The spectral data is processed by a computer 1' to investigate the movement pattern of the SPR resonance valley and determine the refractive index of the sample.

[0077] To verify the sensitivity of the prepared nanobody-based fiber optic SPR probe, experiments were conducted using the fiber optic SPR probe to measure glycerol aqueous solutions with different refractive indices:

[0078] The fiber optic SPR probe based on nanobody 1 was used to detect the resonant wavelength values ​​in an aqueous solution of glycerol with a refractive index range of 1.333-1.356. Figure 3 As shown. A quadratic fit is performed on the wavelength values, and the slope of the tangent at each point represents the corresponding sensitivity. The average sensitivity of the nanobody-based fiber optic SPR probe is 3191.61 nm / RIU.

[0079] To verify the effectiveness of the surface-immobilized nanobody method for the prepared nanobody-based fiber optic SPR probe, a His-tagged red fluorescent protein was selected to replace the nanobody. The His-tagged red fluorescent protein was then linked to the surface of the near-guided fiber optic SPR sensing probe via Tris-NTA3 and nickel ions. The probe was observed under a fluorescence microscope as follows: Figure 4 As shown, a His-tagged red fluorescent protein was successfully immobilized on the surface of a near-waveguide fiber SPR sensing probe. This demonstrates that the His-tagged nanobody 1 can also be immobilized on the surface of a near-waveguide fiber SPR sensing probe.

[0080] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style of the specification is merely for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in the embodiments can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A fiber optic SPR probe based on nanobody, comprising a multimode fiber (8), one end of which has a core (4) with the cladding and coating removed, the core (4) having a flat end face as an end face region, and the outer surface of the core (4) being a sensing region; characterized in that, From the fiber core (4) outwards, it includes a noble metal layer (5), a near-waveguide dielectric layer (6), and a reactive layer; The noble metal layer (5) is solidified in the sensing area and the end face area. The noble metal layer (5) located in the sensing area is used to excite the SPR effect, and the noble metal layer (5) located in the end face area is used to reflect light. The thickness of the noble metal layer (5) in the end face area is greater than that of the noble metal layer (5) in the sensing area. The near-waveguide dielectric layer (6) is solidified on the outer surface of the noble metal layer (5), and two-dimensional material nanosheets are uniformly doped in the near-waveguide dielectric layer (6); The reaction layer is solidified on the near-waveguide dielectric layer (6) located in the sensing region, and includes biochemical detection reagents and serum albumin (7); the biochemical detection reagents include antibody-linking reagents, nanobodies (1) and metal ions (2); the nanobodies (1) are labeled with His and are single-domain antibodies containing only one heavy chain variable region; the metal ions (2) cooperate with the antibody-linking reagents to solidify the nanobodies (1) on the upper surface of the near-waveguide dielectric layer (6); the serum albumin (7) is used to seal the area on the upper surface of the near-waveguide dielectric layer (6) that has not reacted with the antibody-linking reagents.

2. The fiber optic SPR probe based on nanobody according to claim 1, characterized in that, The fiber core (4) is on the order of centimeters in length, the noble metal layer (5) is on the order of nanometers in length, and the near-guided dielectric layer (6) is on the order of nanometers in length.

3. The fiber optic SPR probe based on nanobody according to claim 2, characterized in that, The noble metal layer (5) is a dense and flat layer. The thickness of the noble metal layer (5) in the end face region is 295-305nm, and the thickness of the noble metal layer (5) in the sensing region is 45-55nm.

4. The fiber optic SPR probe based on nanobody according to claim 2, characterized in that, The fiber core has a length of 1-1.5 cm and the near-guided dielectric layer (6) has a thickness of 10-15 nm.

5. The fiber optic SPR probe based on nanobody according to claim 1, characterized in that, The antibody linker is Tris-NTA (3), and the near-waveguide dielectric layer (6) is a polydopamine film layer uniformly doped with two-dimensional material. The surface has quinone groups, which can react with the amino groups of Tris-NTA (3) to form stable covalent bonds.

6. The fiber optic SPR probe based on nanobody according to claim 1, characterized in that, The nanobody (1) is the smallest complete antigen-binding fragment.

7. The fiber optic SPR probe based on nanobody according to claim 1, characterized in that, The metal ion is selected from one of the following: copper ion, nickel ion, and calcium ion.

8. The method for preparing an optical fiber SPR probe according to any one of claims 5, comprising: Pre-processed multimode fiber: The end of the multimode fiber (8) is cut flat using a fiber optic cleaver to create a flat end face. The coating and cladding near the end face are removed to expose a centimeter-sized fiber core (4). The outer surface of the fiber core is used as the sensing area. Preparation of noble metal layer: Noble metal layers are formed by sputtering noble metals on the end face region and the sensing region of the fiber core (4) using a magnetron sputtering instrument. The noble metal layer (5) in the end face region forms a mirror, and the noble metal layer (5) in the sensing region excites the SPR effect. Preparing a near-waveguide dielectric layer: Tris(hydroxymethyl)aminomethane hydrochloride buffer solution was mixed with dopamine hydrochloride powder to prepare a dopamine solution with a concentration of 1.9-2.1 mg / ml and a pH of 8-9; a single-layer two-dimensional material nanosheet was mixed with the dopamine solution and shaken well; then the fiber core with the noble metal layer (5) was immersed in the mixed solution and oscillated at a uniform speed to obtain a uniform polydopamine near-guided wave dielectric layer doped with two-dimensional material nanosheets. Preparation of the reaction layer: The fiber core with the near-waveguide dielectric layer is immersed in the antibody linker solution and incubated overnight; the quinone group on the surface of the polydopamine near-waveguide dielectric layer (6) is coupled with the amino group of Tris-NTA (3) to solidify the antibody linker on the surface of the near-waveguide dielectric layer (6); The sensing area is then immersed in serum albumin solution to seal the unreacted area on the surface of the near-guided dielectric layer (6); The sensing area was immersed in a metal ion solution and incubated for 1-2 hours; metal ions (2) were preloaded on the surface of Tris-NTA (3) through chelation. The prepared sensing region was immersed in a nanobody solution and incubated overnight. Through chelation, the nanobody was immobilized on the Tris-NTA surface of the sensing region by utilizing the chelation force between the His tag of the nanobody (1) and the metal ions, thereby completing the preparation of the fiber optic SPR probe based on the nanobody.

9. A sensing and detection system, comprising a spectrometer (2'), a broadband light source (3'), and an optical fiber SPR probe (5') according to any one of claims 1-6; the input end of the optical fiber SPR probe (5') transmits light emitted from the broadband light source (3') to the optical fiber SPR probe (5') via an optical fiber jumper, the output end of the optical fiber SPR probe (5') is connected to the spectrometer (2') via an optical fiber jumper, and the spectrometer (2') is connected to a computer (1') via a data interface; the nanoantibody (1) on the surface of the optical fiber SPR probe (5') specifically binds to the analyte, causing a change in the equivalent refractive index of the surface of the optical fiber SPR probe (5'), and the concentration of small molecule substances is detected by judging the amount of SPR resonance valley movement.

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