Optical fiber SPR (Surface Plasmon Resonance) probe based on nano antibody, preparation method and detection system

Abstract

The invention discloses an optical fiber SPR (Surface Plasmon Resonance) probe based on a nano antibody, a preparation method and a detection system. The probe comprises a multimode optical fiber, the tail end of a fiber core is cut flat through an optical fiber cutting knife, a cladding near the end face is removed, the fiber core of the optical fiber is exposed, and the surface of the fiber core sequentially comprises a precious metal layer, a near guided wave dielectric layer and a reaction layer from bottom to top. A two-dimensional material is uniformly doped in the near-guided wave dielectric layer; the reaction layer comprises a biochemical detection reagent and serum albumin; the biochemical detection reagent comprises an antibody connection reagent, a nano antibody and metal ions; the nano antibody is provided with a His label and is a single-domain antibody only containing a heavy chain variable region. The prepared probe is suitable for high-sensitivity and low-concentration biological small molecule detection.

Description

Technical Field

[0001] The invention belongs to the field of biomedical optical detection, and in particular relates to an optical fiber SPR probe based on nano-antibodies, a preparation method and a sensing detection system. Background Art

[0002] The surface plasmon resonance (SPR) phenomenon refers to a collective resonance phenomenon generated by electromagnetic waves exciting free electrons in materials. SPR is very sensitive to changes in the dielectric constant of the local environment, so it is often used to detect changes in liquid concentration and real-time monitoring of biomolecule adsorption and binding. The binding of antibodies coupled to the metal surface with analytes can cause changes in the environment around the SPR sensor. By monitoring the changes in the position of the resonance valley, the affinity, affinity constant, binding kinetics and other information of the biomolecular interaction can be inferred. Therefore, SPR technology has wide applications and important value in the fields of drug screening, protein-protein interaction research, antibody-antigen binding, etc. Fiber-optic SPR sensors have the advantages of anti-electromagnetic interference, corrosion resistance, small size, and strong flexibility, and can achieve 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 SPR biochemical sensor based on a metal micro-nano array structure and a preparation method thereof, wherein the sensitivity of the sensor is improved by constructing a metal micro-nano array structure on the arc surface of the sensing optical fiber; the biorecognition molecules with carboxyl groups on the surface are fixed on the surface of the nanomaterial through coordination bond coupling to achieve biochemical detection. CN112461794A discloses a long-range SPR sensor and a preparation method thereof, wherein the sensitivity of the sensor is improved through the plasma coupling effect between the nanometal material and the metal layer in the SPR sensor, the antibody is fixed on the surface of the long-range SPR sensor through a coupling agent, and the double antibody sandwich immunoassay is used to achieve biological detection.

[0004] Traditional fiber-optic SPR sensors can only bind to common IgG antibodies, which have the disadvantages of large molecular weight (about 150kDa) and poor stability, making it difficult to detect small molecules. Compared with traditional IgG antibodies, nanoantibodies have the advantages of small molecular weight (about 15kDa), high affinity, strong stability, the ability to identify hidden antigen epitopes, and the ability to specifically bind to small biological molecules. At the same time, nanoantibodies can improve sensor sensitivity and reduce sensor detection limits.

[0005] However, nanobodies have a relatively simple structure, with only one specific binding site and no other amino groups. The traditional method of combining the amino group at the tail of IgG antibodies with optical fiber SPR sensor coupling agents (such as cysteamine, polydopamine, gold-sulfur bonds, etc.) is not suitable for nanobodies, and a connection method for directional fixation of nanobodies needs to be invented. Summary of the invention

[0006] The purpose of the present invention is to overcome the problem that the existing fiber SPR sensor is difficult to detect low-concentration biological small molecules, and proposes a fiber SPR probe based on nano-antibodies, a preparation method and a detection system for biological small molecules detection. The present invention introduces a polydopamine film layer uniformly doped with two-dimensional materials as a near-waveguide dielectric layer, which is solidified on the surface of the noble metal layer to form a near-waveguide fiber SPR probe. The nano-antibody is fixed on the surface of the fiber SPR probe by the coupling reaction between polydopamine material, trinitrotriacetic acid (Tris-NTA), nickel ions and nano-antibodies. The advantage of the small molecular weight of the nano-antibody that can specifically bind to biological small molecules is used to achieve biological small molecule measurement. The polydopamine material film layer has the advantages of being dense and having strong adhesion, which can prevent each film layer from falling off and improve the stability of the sensor. Two-dimensional material nanosheets with high / complex dielectric constants are incorporated into the polydopamine material film layer to form a near-waveguide dielectric layer. The two-dimensional material has photoelectric properties such as high refractive index and high carrier mobility, which can enhance the surface electric field strength of the fiber SPR probe and improve the sensor sensitivity.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] The first aspect of the present invention is to disclose a nano-antibody-based optical fiber SPR probe, comprising a multimode optical fiber, one end of which has a core with a cladding and a coating removed, the core having a flat end face as an end face region, and the outer surface of the core being a sensing region;

[0009] The fiber core includes a noble metal layer, a near-waveguide dielectric layer and a reaction layer in order from the fiber core to the outside;

[0010] The noble metal layer is solidified in the sensing area and the end surface area, wherein the noble metal layer in the sensing area is used to stimulate the SPR effect, the noble metal layer in the end surface area is used to reflect light, and the thickness of the noble metal layer in the end surface 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 area, and includes biochemical detection reagents and serum albumin; the biochemical detection reagents include antibody linking reagents, nano antibodies and metal ions; the nano antibodies carry a polyhistidine (His) tag and are single-domain antibodies containing only one heavy chain variable region; the metal ions cooperate with the antibody linking reagent to solidify the nano antibodies on the upper surface of the near-waveguide dielectric layer; the serum albumin is used to block the area on the upper surface of the near-waveguide dielectric layer that has not reacted with the antibody linking reagent.

[0013] In some specific embodiments, the fiber core length is in 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 cut flat by using a cutting knife.

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

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

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

[0018] In some specific embodiments, the near-waveguide dielectric layer is nanometer-sized and has a thickness of 10-15 nm.

[0019] In some specific embodiments, the nanobody is the smallest complete antigen binding fragment, and the metal ion can undergo a chelation reaction with the Tris-NTA and the nanobody, and the chelation force can solidify the nanobody on the outer surface of the near-waveguide dielectric layer to form a biorecognition interface. When the substance to be tested is close to the surface of the optical fiber SPR probe, the nanobody can specifically bind to the substance to be tested to form an antibody-antigen complex. This specific binding can change the equivalent refractive index of the sensor surface, and the SPR resonance valley will move. By judging the movement of the SPR resonance valley, the concentration detection of small molecule substances is achieved.

[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] The second aspect of the present invention is to disclose a detection system of a nano-antibody-based optical fiber SPR probe, which includes a spectrum analyzer, a broadband light source, and an optical fiber SPR probe with a multimode optical fiber as an optical path; the input end of the optical fiber SPR probe transmits light emitted by the broadband light source to the optical fiber SPR probe through an optical fiber jumper, the output end of the optical fiber SPR probe is connected to the spectrum analyzer through an optical fiber jumper, and the spectrum analyzer is connected to a computer through a data interface; the nano-antibody on the surface of the optical fiber SPR probe specifically binds to a substance to be tested, so that the equivalent refractive index of the surface of the optical fiber SPR probe changes, and the concentration detection of small molecule substances is achieved by judging the movement of the SPR resonance valley.

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

[0024] The third aspect of the present invention is to disclose a method for preparing the nano-antibody-based optical fiber SPR probe, comprising:

[0025] Pre-processed multimode fiber:

[0026] Using an optical fiber cutter to cut the end of the multimode optical fiber flat, using a flat end face as the end face area, removing the coating layer and cladding near the end face to expose the centimeter-level fiber core, and the outer surface of the fiber core is used as the sensing area;

[0027] Preparation of precious metal layer:

[0028] A magnetron sputtering device is used to sputter noble metals at the end face region and the sensing region of the fiber core to form a noble metal layer, wherein the noble metal layer at the end face region forms a reflector, and the noble metal layer at the sensing region excites the SPR effect;

[0029] Plating of near-waveguide dielectric layer:

[0030] A tris-HCl buffer solution is mixed with dopamine hydrochloride powder to prepare a dopamine solution with a concentration of 1.9-2.1 mg / ml and a pH value of 8-9; a single layer of two-dimensional material nanosheets and the dopamine solution are mixed and shaken evenly; and a fiber core prepared with a noble metal layer is immersed in the mixed solution and oscillated at a constant speed to obtain a uniform polydopamine near-waveguide dielectric layer doped with the two-dimensional material nanosheets;

[0031] Preparation of reaction layer:

[0032] The fiber core coated with the near-waveguide dielectric layer is immersed in an antibody connection reagent solution and incubated overnight; the quinone group on the surface of the polydopamine near-waveguide dielectric layer is coupled with the amino group of Tris-NTA, and the Tris-NTA can be solidified on the upper surface of the near-waveguide dielectric layer;

[0033] Then, immersing the fiber core in a serum albumin solution to seal the unreacted area on the surface of the near-waveguide dielectric layer;

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

[0035] The prepared fiber core is immersed in the nanoantibody solution and incubated overnight; through chelation, the chelating force between the His tag of the nanoantibody and the metal ions is utilized to fix the nanoantibody on the Tris-NTA surface of the sensing area, thereby completing the preparation of the nanoantibody-based optical fiber SPR probe.

[0036] The beneficial effects of the present invention are:

[0037] 1. Compared with the conventional fiber SPR sensor coupled with common antibodies, the present invention utilizes the coupling effect between polydopamine material, Tris-NTA, metal ions and nano antibodies to fix the nano antibodies on the surface of the fiber SPR probe to form the fiber SPR probe based on nano antibodies, which can be used for biological small molecule detection. Taking advantage of the small molecular weight and strong specificity of nano antibodies, which can identify relatively hidden antigen epitopes that are difficult to identify with traditional antibodies, the fiber SPR probe based on nano antibodies can realize high sensitivity and rapid detection of biological small molecules.

[0038] 2. The polydopamine material has the advantage of a high negative refractive index. Evenly coating it on the surface of the precious metal layer can stimulate the near-waveguide effect. The two-dimensional material nanosheets evenly doped in the polydopamine near-waveguide dielectric layer have the advantages of a high negative refractive index and high carrier mobility. The synergistic sensitization between the two can effectively improve the sensitivity of the optical fiber SPR probe.

[0039] 3. Polydopamine material has an adhesion mechanism similar to that of mussels, which can adhere the two-dimensional material nanosheets to the surface of the optical fiber SPR probe and form a uniform and dense film, avoiding chemical deformation and shedding of the sensor membrane layer, which will significantly improve the stability of the sensor. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a schematic structural diagram of the nano-antibody-based optical fiber SPR probe of the present invention;

[0041] Figure 2 It is a schematic diagram of the structure of the detection system of the present invention;

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

[0043] Figure 4 A fluorescence microscopy image of the nanobody-based fiber-optic SPR probe in the example is shown, which is bound to red fluorescent protein.

[0044] In the figure:

[0045] 1: nanobody; 2: metal ion; 3: Tris-NTA; 4: fiber core; 5: noble metal layer;

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

[0047] 1': computer; 2': spectrum analyzer; 3': broadband light source; 4': z-axis vertical displacement platform;

[0048] 5': Fiber-optic SPR probe based on nanoantibodies; 6': analyte solution sample. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solution, beneficial effects and significant progress of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below in conjunction with the drawings provided in the examples of the present invention. Obviously, all the described embodiments are only partial embodiments of the present invention, rather than all embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0050] In the description of this application, unless otherwise clearly specified and limited, the terms "first", "second", and "third" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance; the term "plurality" refers to two or more; unless otherwise specified or explained, the terms "connected", "fixed", etc. should be understood in a broad sense, 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. For ordinary technicians in this field, the specific meanings of the above terms in this application can be understood according to specific circumstances.

[0051] like Figure 2As shown, the detection system of the fiber SPR probe 5' based on nano-antibodies includes a spectrum analyzer 2', a broadband light source 3' and a fiber SPR probe 5' with a multimode optical fiber as an optical path. The input end of the fiber SPR probe 5' transmits the light emitted by the broadband light source 3' to the fiber SPR probe 5' through a Y-type optical fiber jumper, and the output end of the fiber SPR probe 5' is connected to the spectrum analyzer 2' through a Y-type optical fiber jumper, and the spectrum analyzer 2' is connected to a computer 1' through a data interface. Moreover, 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, and the fiber end face is vertically facing downward, and the fiber end face faces the solution sample 6' of the object to be detected; the fiber optic SPR probe 5' is controlled by the z-axis vertical displacement platform to vertically lift and move along the z-axis vertical displacement platform 4', and the probe immersion depth in the liquid is controlled by the platform lifting and lowering, so that the fiber optic sensing area is completely immersed in the solution of the solution sample 6' of the object to be detected, so as to realize detection.

[0052] like Figure 1 As shown, the nano-antibody-based optical fiber SPR probe 5' comprises a multimode optical fiber 8, and the multimode optical fiber 8 is any multimode optical fiber that can be used as an SPR probe substrate, and one end of the multimode optical 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 area, and the cladding and coating 2 cm away from the end face expose the multimode optical fiber core 4 as a sensing area, and the core length of the sensing area is at the centimeter level; and the core 4 has a diameter of 600 μm.

[0053] From the outer surface of the core 4 to the outside, it includes a noble metal layer 5, a near-waveguide dielectric layer 6 and a reaction layer in sequence; the noble metal layer 5 is a dense gold film solidified in the sensing area (i.e., the outer surface of the core) and the end face area of ​​the core 4, and the end face and the sensing area have different thicknesses, wherein the thickness of the end face area is 295-305nm, and the thickness of the sensing area is 45-55nm, that is, the noble metal layer in the sensing area is used to generate the SPR effect, wherein the noble metal layer in the end face area only plays a reflective role and does not stimulate the SPR effect.

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

[0055] A reaction layer is provided on the upper surface of the near-waveguide dielectric layer 6 in the sensing area, and the reaction layer includes biochemical detection reagents and serum albumin 7. In this embodiment, the serum albumin 7 is selected from bovine serum albumin; the biochemical detection reagent includes commercially available Tris-NTA 3, metal ions 2 and nano antibodies 1. Tris-NTA is an antibody linking reagent that can be used to bind nano antibodies with His tags. And the metal ions 2 and nano antibodies 1 are solidified on the upper surface of the Tris-NTA 3, wherein the metal ions 2 are nickel ions, which can undergo a chelating reaction with the Tris-NTA, and the nano antibodies 1 are single-domain antibodies containing only one heavy chain variable region, which are the smallest complete antigen binding fragments and are nano antibodies with His tags. The Tris-NTA and the nickel ions play a role in solidifying the nano antibodies 1. The bovine serum albumin plays a role in sealing the unreacted areas on the surface of the near-waveguide dielectric layer.

[0056] By fixing the nano-antibody on the surface of the optical fiber SPR probe, a biorecognition interface can be formed. When the substance to be tested is close to the surface of the optical fiber SPR probe, the nano-antibody can specifically bind to the substance to be tested to form an antibody-antigen complex. This specific binding can change the equivalent refractive index of the sensor surface, and the SPR resonance valley will move. Since the nano-antibody has a small molecular weight, it can stably bind to small molecules. By judging the movement of the SPR resonance valley, the concentration of small molecules can be detected.

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

[0058] Step 1: Pre-treating the multimode fiber

[0059] A multimode optical fiber 8 with a cladding diameter of 630 μm and a core diameter of 600 μm was selected, and the end face of the optical fiber was cut flat with an optical fiber cutter as the end face area, and the cladding and coating layer of 2 cm near the end face were removed to expose the multimode optical fiber core 4 as the sensing area;

[0060] Step 2: Sputtering of precious metal layer on the core end face

[0061] The outer surface of the multimode optical fiber core 4 prepared in step 1 is covered to expose the optical fiber end face, and gold is sputtered on the end face of the fiber core using a magnetron sputtering apparatus to form a precious metal layer 5 with a thickness of 295 nm in the end face area to form a reflector; the sputtering power is set to 30w and the vacuum degree is 5×10 -4 Pa, sputtering time 30 min;

[0062] Step 3: Sputtering precious metal layer on the outer surface of the core

[0063] Remove the shielding on the outer surface of the multimode optical fiber core, and use a magnetron sputtering instrument to sputter gold on the outer surface of the core 4 of the multimode optical fiber obtained in step 2 to form a noble metal layer 5 with a thickness of 45nm in the sensing area; set the sputtering power to 30w and the vacuum degree to 5×10 -4 Pa, sputtering time 5 min;

[0064] Step 4: Plating the near-waveguide dielectric layer

[0065] Mix 10 mL of Tris-HCl buffer with 0.02 g of dopamine hydrochloride powder to prepare a dopamine solution with a concentration of 2 mg / ml and a pH value of 8-9; mix the monolayer two-dimensional material nanosheet with the dopamine solution and shake well;

[0066] Immerse the sensing area of ​​the multimode optical fiber 8 prepared in step 3 in the mixed solution and shake at a constant speed for 2-4 hours;

[0067] Dopamine monomers can self-polymerize into polydopamine layers, and their mussel-like adhesion mechanism can solidify the two-dimensional material nanosheets on the surface of the noble metal layer to form a uniform polydopamine near-waveguide dielectric layer doped with two-dimensional material nanosheets, wherein the near-waveguide dielectric layer 6 has a thickness of 10-15 nm; the thickness of the near-waveguide dielectric layer can be controlled by controlling the oscillation duration;

[0068] Step 5: Fixation of Tris-NTA

[0069] The sensing area prepared in step 4 is immersed in a Tris-NTA solution with a concentration of 1 mM and incubated overnight; the quinone group on the surface of the dielectric layer near the waveguide of polydopamine reacts with the amino group of the Tris-NTA3 to solidify the Tris-NTA3 on the upper surface of the dielectric layer near the waveguide 6;

[0070] After taking it out, rinse the optical fiber SPR probe with deionized water and blow it dry with nitrogen. Then immerse the sensing area in a bovine serum albumin solution with a concentration of 10 mg / ml and incubate for 1 hour to block the unreacted area on the surface of the near-waveguide dielectric layer 6;

[0071] Step 6: Pre-loading Nickel Ions

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

[0073] Step 7: Immobilization of Nanobodies

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

[0075] When used, the detection system of the nanobody-based fiber optic SPR probe 5' works as follows:

[0076] The broadband light source 3' generates incident light, which is transmitted to the nano-antibody-based optical fiber SPR probe 5' through a Y-type jumper, and the reflected outgoing light is transmitted to the spectrum analyzer 2' by the jumper for real-time monitoring of the optical power loss in the output spectrum. The optical fiber SPR probe 5' controls the sensing area of ​​the probe 5' to be completely immersed in the sample solution 6' of the object to be tested through the z-axis vertical displacement platform 4', stimulates the SPR effect, and the electric field penetrates the near-waveguide dielectric layer 6 to reach the sample solution 6' of the object to be tested, at which time the SPR resonance valley appears. As the refractive index of the sample solution 6' of the object to be tested increases, the SPR resonance valley redshifts; the spectrum data is processed by the computer 1' to explore the movement law of the SPR resonance valley and determine the refractive index of the sample to be tested.

[0077] In order to verify the sensitivity of the prepared nanobody-based optical fiber SPR probe, the optical fiber SPR probe was used to measure glycerol aqueous solutions with different refractive indices:

[0078] The optical fiber SPR probe based on nanobody 1 was used to detect a glycerol aqueous solution with a refractive index range of 1.333-1.356, and the resonance wavelength value obtained was as follows: Figure 3 The wavelength value is fitted quadratically, and the slope of the tangent at each point is the corresponding sensitivity. The average sensitivity of the fiber optic SPR probe based on nanoantibodies is 3191.61 nm / RIU.

[0079] In order to verify the effectiveness of the surface-immobilized nanoantibodies of the prepared nanoantibody-based optical fiber SPR probe, red fluorescent protein with a His tag was selected to replace the nanoantibody, and the red fluorescent protein with a His tag was connected to the surface of the near-waveguide optical fiber SPR sensing probe through Tris-NTA3 and nickel ions. The probe observed under a fluorescence microscope is as follows Figure 4 As shown, the red fluorescent protein with His tag was successfully immobilized on the surface of the near-guide fiber SPR sensor probe. This proves that the nanobody 1 with the same His tag can also be immobilized on the surface of the near-guide fiber SPR sensor probe.

[0080] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode includes only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in the embodiments may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A nano-antibody-based optical fiber SPR probe, comprising a multimode optical fiber (8), wherein one end of the multimode optical fiber (8) has a core (4) with a cladding and a coating removed, the core (4) having a flat end face as an end face region, and the outer surface of the core (4) is a sensing region; characterized in that: The fiber core (4) includes a noble metal layer (5), a near-waveguide dielectric layer (6) and a reaction layer in order from the fiber core (4) outward; The noble metal layer (5) is solidified in the sensing region and the end face region, wherein the noble metal layer (5) located in the sensing region is used to stimulate the SPR effect, and the noble metal layer (5) located in the end face region is used to reflect light, and the noble metal layer (5) in the end face region has a thickness greater than that of the noble metal layer (5) in the sensing region; The near-waveguide dielectric layer (6) is solidified on the outer surface of the noble metal layer (5), and the near-waveguide dielectric layer (6) is uniformly doped with two-dimensional material nanosheets; The reaction layer is solidified on the near-waveguide dielectric layer (6) located in the sensing area, and comprises a biochemical detection reagent and serum albumin (7); the biochemical detection reagent comprises an antibody linking reagent, a nano antibody (1) and a metal ion (2); the nano antibody (1) carries a His tag and is a single domain antibody containing only one heavy chain variable region; the metal ion (2) cooperates with the antibody linking reagent to solidify the nano antibody (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 reagent.

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

3. The optical fiber SPR probe based on nanoantibodies 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 surface area is 295-305 nm, and the thickness of the noble metal layer (5) in the sensing area is 45-55 nm.

4. The optical fiber SPR probe based on nanoantibodies according to claim 2, characterized in that: The fiber core length is 1-1.5 cm, and the near-waveguide dielectric layer (6) thickness is 10-15 nm.

5. The optical fiber SPR probe based on nanoantibodies according to claim 1, characterized in that: The antibody linking reagent is Tris-NTA (3), and the near-waveguide dielectric layer (6) is a polydopamine film layer uniformly doped with a two-dimensional material, with a quinone group on the surface that can react with the amino group of Tris-NTA (3) to form a stable covalent bond.

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

7. The optical fiber SPR probe based on nanoantibodies 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 the optical fiber SPR probe according to any one of claim 5, comprising: Pre-processed multimode fiber: Using an optical fiber cutter to cut the end of a multimode optical fiber (8) flat, leaving a flat end face as the end face region, removing the coating layer and cladding layer near the end face to expose a centimeter-level fiber core (4), wherein the outer surface of the fiber core serves as a sensing region; Preparation of precious metal layer: Using a magnetron sputtering device to sputter noble metals on the end face region and the sensing region of the fiber core (4) to form noble metal layers, wherein the noble metal layer (5) in the end face region forms a reflector, and the noble metal layer (5) in the sensing region excites the SPR effect; Plating of near-waveguide dielectric layer: The tris(hydroxymethyl)aminomethane hydrochloride buffer solution is mixed with dopamine hydrochloride powder to prepare a dopamine solution with a concentration of 1.9-2.1 mg / ml and a pH value of 8-9; the single-layer two-dimensional material nanosheet and the dopamine solution are mixed and shaken evenly; the fiber core prepared with the noble metal layer (5) is immersed in the mixed solution and shaken at a constant speed, thereby obtaining a uniform polydopamine near-waveguide dielectric layer doped with the two-dimensional material nanosheet; Preparation of reaction layer: The fiber core coated with the near-waveguide dielectric layer is immersed in an antibody linking reagent 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), and the antibody linking reagent can be solidified on the upper surface of the near-waveguide dielectric layer (6); Then, the sensing area is immersed in a serum albumin solution to seal the unreacted area on the surface of the near-waveguide dielectric layer (6); Immerse the sensing area in a metal ion solution and incubate for 1-2 hours; preload the metal ions (2) on the surface of Tris-NTA (3) through chelation; The prepared sensing area is immersed in a nano-antibody solution and incubated overnight; the nano-antibody is fixed on the Tris-NTA surface of the sensing area by chelation using the chelating force between the His tag of the nano-antibody (1) and the metal ion, thereby completing the preparation of the nano-antibody-based optical fiber SPR probe.

9. A sensing detection system, comprising a spectrum analyzer (2'), a broadband light source (3') and a fiber SPR probe (5') according to any one of claims 1 to 6; the input end of the fiber SPR probe (5') transmits light emitted by the broadband light source (3') to the fiber SPR probe (5') via an optical fiber jumper, the output end of the fiber SPR probe (5') is connected to the spectrum analyzer (2') via an optical fiber jumper, and the spectrum analyzer (2') is connected to a computer (1') via a data interface; the nano antibody (1) on the surface of the fiber SPR probe (5') specifically binds to a substance to be detected, so that the equivalent refractive index of the surface of the fiber SPR probe (5') changes, and the concentration detection of small molecule substances is achieved by judging the movement of the SPR resonance valley.

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