Synergistic modified optical fiber SPR (Surface Plasmon Resonance) sensor, preparation method, SPR sensing system and application

Abstract

The invention discloses a synergistically modified optical fiber SPR (Surface Plasmon Resonance) sensor, a preparation method, an SPR sensing system and application, and belongs to the technical field of optical fiber biosensing. The synergistically modified optical fiber SPR sensor is of a multimode-single mode optical fiber cascade structure; a single-mode optical fiber serves as a sensing area, the surface of the single-mode optical fiber is sequentially modified with a gold film, AuNPs, CNTs and DNA aptamers, and the optical fiber SPR sensor based on AuNPs / CNTs / DNA aptamer synergistic modification is formed. The SPR sensing system comprises the SPR sensor, a Y-shaped optical fiber patch cord, a light source, a spectrograph and a computer, and is suitable for rapid and high-sensitivity detection of the concentration of vancomycin in a low concentration range in a complex sample. According to the present invention, the SPR sensing interface having the multiple signal amplification function and the specific vancomycin recognition function is constructed, such that the sensitivity and the specificity of the vancomycin detection are significantly improved, and the blank of the vancomycin concentration detection of the optical fiber SPR sensor is filled.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of optical fiber biosensing, and particularly relates to a synergistically modified optical fiber SPR sensor, a preparation method, an SPR sensing system and application. BACKGROUND

[0002] Vancomycin (VCM) is a glycopeptide antibiotic produced by Actinomyces orientalis, and is also the main method for treating methicillin-resistant Staphylococcus aureus infection and Gram-positive bacterial infection. However, the therapeutic window (the minimum effective drug concentration of toxicity) of vancomycin is very narrow, and a concentration below the lower limit may lead to ineffective treatment or drug resistance, and a concentration above the upper limit may cause serious toxic side effects, including ototoxicity and nephrotoxicity. Therefore, regular and accurate monitoring of vancomycin concentration in patients is crucial for doctors to guide patients to take medicine, optimize drug dosage, and ensure the effectiveness and safety of the drug.

[0003] At present, the methods commonly used for vancomycin therapeutic drug monitoring include high performance liquid chromatography, enzyme immunoassay, chemiluminescence, electrochemistry and fluorescence. Among them, high performance liquid chromatography and enzyme immunoassay have the advantages of high sensitivity and high detection accuracy. However, they often require expensive instruments and equipment, complex operation procedures and long turnaround time (about 6h), which cannot meet the needs of rapid and immediate detection of blood vancomycin concentration in critically ill patients in hospitals. Electrochemistry, chemiluminescence and fluorescence have certain deficiencies in sensitivity and specificity. Therefore, it is particularly important to develop a vancomycin sensor with high sensitivity, good specificity, low cost and easy use.

[0004] Optical fiber surface plasmon resonance (SPR) technology is a core component of the field of optical fiber sensing, and has gained extensive research interest in the field of optical fiber biosensing due to its advantages of label-free, real-time response, miniaturization and flexible structure. However, there are still some deficiencies in the current optical fiber SPR sensor, including limited sensitivity, low efficiency of biomolecule immobilization and poor specificity of target molecule recognition, which makes it difficult to achieve high precision and specific continuous monitoring in the detection of trace drug molecules. There is almost no related report on vancomycin detection in the field of optical fiber SPR and even the entire field of optical fiber sensing. SUMMARY

[0005] In view of the problems in the prior art, the application aims to provide a synergistically modified optical fiber SPR sensor, a preparation method, an SPR sensing system and an application. Based on the significant localized surface plasmon resonance effect of gold nanoparticles (AuNPs) and the easy-to-functionalize modification feature, the electromagnetic field strength and signal response can be effectively enhanced. Based on the high specific surface area, excellent electrical conductivity and rich surface functional groups of carbon nanotubes (CNTs), the probe molecule loading capacity and interface charge transfer efficiency can be significantly improved. The DNA aptamer capable of specifically recognizing vancomycin is covalently modified on the surface of the probe, so as to further improve the detection specificity of vancomycin. The AuNPs / CNTs / DNA aptamer synergistically modified optical fiber SPR sensor can be applied to the detection of trace vancomycin in complex biological samples, and high-sensitivity and high-selectivity detection can be realized.

[0006] The application aims to achieve the following technical solutions:

[0007] In a first aspect, the application provides a synergistically modified optical fiber SPR sensor, which is a multi-mode-single-mode optical fiber cascade structure. The single-mode optical fiber serves as a sensing area, and the surface is sequentially modified with a gold film, AuNPs, CNTs and DNA aptamer, thereby forming an AuNPs / CNTs / DNA aptamer synergistically modified optical fiber SPR sensor.

[0008] Further, the thickness of the gold film on the surface of the single-mode optical fiber is 45nm-55nm, and the length of the single-mode optical fiber is 2mm-4mm.

[0009] In a second aspect, the application provides a preparation method of a synergistically modified optical fiber SPR sensor, which comprises the following steps:

[0010] Step 1: A portion of the coating layer is removed along the axial end face area of the multi-mode optical fiber and the single-mode optical fiber, and then wiped clean. Then, a flat end face is cut out, and a fusion splicer is used for fusion splicing to control the length of the single-mode optical fiber.

[0011] Step 2: The end of the fused optical fiber is cleaned and dried, and then vertically placed in an ion sputtering instrument. A layer of gold film is coated on the sensing area of the single-mode optical fiber to obtain a probe.

[0012] Step 3: In a dark environment, the sensing area of the single-mode optical fiber of the probe is immersed in a dithiothreitol (DTT) solution, so that the thiol group at one end of the dithiothreitol molecule is combined with the gold film. AuNPs solution is prepared by sodium citrate reduction method. Then, the sensing area of the single-mode optical fiber of the probe after the thiol group is combined with the gold film is immersed in the AuNPs solution, and the thiol group at the other end of the dithiothreitol molecule is combined with the AuNPs. A stable covalent structure is formed between the AuNPs and the gold film through self-assembly.

[0013] Step 4: After the probe is treated in step 3, the single-mode optical fiber sensing area is immersed in an 11-mercapto-undecanoic acid (MUA) solution to bind the carboxyl group to the surface of the gold nanoparticles, and then immersed in a mixed solution of 1-ethyl-(3-dimethoxy aminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to activate the carboxyl group on the surface of the gold nanoparticles;

[0014] Step 5: After the activation in step 4 is completed, the probe is cleaned, and then the single-mode optical fiber sensing area is immersed in an amino-functionalized CNTs solution to covalently bind the amino group of the CNTs to the carboxyl group on the surface of the gold nanoparticles;

[0015] Step 6: The single-mode optical fiber sensing area of the probe treated in step 5 is immersed in a DNA aptamer solution to covalently bind the carboxyl group of the DNA aptamer to the amino group of the CNTs, and the DNA aptamer is uniformly and firmly modified on the surface of the probe to obtain a synergistically modified optical fiber SPR sensor.

[0016] Further, in step 1, the lengths of the stripped coating layers of the multi-mode optical fiber and the single-mode optical fiber are 15 mm-21 mm, and the flat end faces are cut to have a distance of 4 mm-6 mm from the coating layer, and the length of the single-mode optical fiber is controlled to be 2 mm-4 mm;

[0017] In step 2, the thickness of the gold film is 45 nm-55 nm, the magnetron sputtering current is 15 mA-20 mA, and the time is 100 s-140 s.

[0018] Further, in step 3, the concentration of dithiothreitol is 10 mM, and the probe is soaked in the dithiothreitol solution for 20 h-26 h;

[0019] The AuNPs solution prepared by the sodium citrate reduction method is prepared by diluting a 1wt% chloroauric acid (HAuCl4) solution with deionized water, the volume ratio of chloroauric acid to deionized water is 1:100, after water bath heating to boiling, 1wt% sodium citrate (Na3C6H5O7) solution is quickly added, the volume ratio of chloroauric acid to sodium citrate is 1:7, stirring for 15 min-20 min, until the solution changes from purple black to wine red, stop heating and natural cooling, get AuNPs solution;

[0020] The concentration of the AuNPs solution is 5 mg / mL, and the probe is soaked in the AuNPs solution for 10 h-14 h.

[0021] Further, the concentration of 11-mercaptoundecanoic acid in step 4 is 50 mM, the probe is soaked in 11-mercaptoundecanoic acid for 10-14 hours; the concentration of ethyl-(3-dimethoxyaminopropyl) carbodiimide hydrochloride is 20 mM, the concentration of N-hydroxysuccinimide is 50 mM, and the two are mixed into a mixed solution in equal volume, and the probe is soaked in the mixed solution for 30-50 minutes;

[0022] In step 5, the concentration of the CNTs solution is 4 mg / mL, and the probe is soaked in the CNTs solution for 1-7 hours.

[0023] Further, in step 6, the concentration of the DNA aptamer solution is 1 μM, and the probe is soaked in the DNA aptamer solution for 5-7 hours.

[0024] The sequence of the DNA aptamer is 5'-COOH-CGA GGG TAC CGC AAT AGT ACT TAT TGT TCG CCTATT GTG GGT CGG-3', and the nucleotide sequence involved is shown as SEQ ID NO. 1.

[0025] In a third aspect, the present application provides an SPR sensing system, comprising: the above-mentioned synergistically modified optical fiber SPR sensor, a Y-type optical fiber jumper, a light source, a spectrometer and a computer, the tail end of the synergistically modified optical fiber SPR sensor is connected to one end of the Y-type optical fiber jumper through an SMA905 optical fiber adapter, the other two ends of the Y-type optical fiber jumper are connected to the light source and the spectrometer respectively, and the spectrometer is connected to the computer.

[0026] Further, the Y-type optical fiber jumper is 1x2 Y-type, the light source is a broadband light source, and the spectrometer is an Ocean Optics fiber spectrometer.

[0027] In a fourth aspect, the present application provides an application of the SPR sensing system, which is used for rapidly and highly sensitively detecting the concentration of vancomycin in a low concentration range. The vancomycin molecules in a biological sample are selectively captured by the synergistically modified optical fiber SPR sensor. After the DNA aptamer on the surface of the sensing area is combined with the vancomycin molecules, the conformation of the aptamer changes, thereby causing a change in the refractive index of the sensing interface of the single-mode optical fiber. After being transmitted to the spectrometer, the change in the wavelength on the reflection spectrum is displayed on the computer terminal. The corresponding wavelength is detected by using a standard concentration of vancomycin, and the functional relationship between the concentration of vancomycin and the wavelength is determined by fitting. In the actual detection of the concentration of the vancomycin sample, the concentration of vancomycin in the sample is calculated by the shift of the resonance valley on the reflection spectrum.

[0028] Advantages and effects of the present application:

[0029] 1、The present application constructs an SPR sensing interface with multiple signal amplification function and specific recognition vancomycin function by covalently modifying gold film, AuNPs, CNTs and DNA aptamer on the surface of single-mode optical fiber sensing area, which significantly improves the sensitivity and specificity of vancomycin detection, and fills the blank of optical fiber SPR sensor for vancomycin concentration detection. In addition, compared with electrochemical, fluorescent, chemiluminescence and other detection methods, the vancomycin sensor of the present application has the advantages of label-free, miniaturization, low cost, easy to use and the like.

[0030] 2、The present application cooperatively modifies the high refractive index sensitivity of the optical fiber SPR sensor, successfully applies the constructed SPR sensing system to the high-sensitivity detection of low-concentration vancomycin molecules, and the synergistic sensitization effect of AuNPs and CNTs shows good linear response in the range of 0 μM-400 μM. Combined with the selection of DNA aptamer, it shows high specificity and excellent stability, which provides a reliable way for accurate detection of vancomycin in complex samples. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 It is the modification process diagram of AuNPs / CNTs / DNA aptamer on the surface of single-mode optical fiber sensing area of the present application;

[0032] Figure 2 It is the schematic diagram of the SPR sensing system of the present application;

[0033] 1, multimode optical fiber, 2, single-mode optical fiber, 3, DNA aptamer, 4, AuNPs / CNTs, 5, gold film, 6, 1x2 Y-type optical fiber jumper;

[0034] Figure 3 It is the SPR response reflection spectrum diagram of the application of the SPR sensing system of Example 1 under different vancomycin concentrations;

[0035] Figure 4 It is the fitting straight line diagram drawn according to the SPR response result in the application of the SPR sensing system of Example 1;

[0036] Figure 5 It is the wavelength shift and concentration change trend comparison diagram of the interference substances and vancomycin detected by the cooperatively modified optical fiber sensor of Example 1;

[0037] Figure 6 It is the wavelength shift column comparison diagram of the interference substances and vancomycin detected by the cooperatively modified optical fiber sensor of Example 1;

[0038] Figure 7 It is the stability diagram of the cooperatively modified optical fiber sensor of Example 1 immersed in buffer solution;

[0039] Figure 8 SPR response reflectance spectrum of different concentrations of vancomycin detected by the fiber SPR sensor based on DNA aptamer of Comparative Example 1;

[0040] Figure 9 Fitting straight line graph plotted according to the SPR response results of Comparative Example 1;

[0041] Figure 10 SPR response reflectance spectrum of different concentrations of vancomycin detected by the fiber SPR sensor based on CNTs / DNA aptamer of Comparative Example 2;

[0042] Figure 11 Fitting straight line graph plotted according to the SPR response results of Comparative Example 2. DETAILED DESCRIPTION

[0043] The specific embodiments of the present application will be further described in detail below with reference to the accompanying drawings and examples.

[0044] A synergistically modified fiber SPR sensor, the fiber SPR sensor is a multi-mode-single mode fiber cascade structure; the single mode fiber serves as a sensing area, and the surface is sequentially modified with a gold film, AuNPs, CNTs, and DNA aptamer; the thickness of the gold film on the surface of the single mode fiber is 50±5 nm, and the length of the single mode fiber is 3±1 mm, thereby forming a fiber SPR sensor based on AuNPs / CNTs / DNA aptamer synergistic modification.

[0045] A preparation method of a synergistically modified fiber SPR sensor, comprising the following steps:

[0046] Step 1: The coating layer of 15 mm-21 mm in the axial end face region of the multi-mode fiber and the single mode fiber is removed, and then the end face is wiped clean with a mirror paper, and then a cutting knife is used to cut a flat end face, so that the distance from the coating layer to the end face is 4 mm-6 mm, and then a fusion splicer is used to observe whether the end faces of the two fibers are flat, and if not, the cutting is re-performed, and after being flat, the fusion is performed, and after the fusion is completed, the single mode fiber of a certain length is cut off again, and the length of the single mode fiber is controlled to be 2 mm-4 mm;

[0047] Step 2: The end of the fused fiber is cleaned with ultrapure water, and after being dried, the fiber is vertically placed in an ion sputtering instrument to plate a gold film, the magnetron sputtering current is controlled to be 15 mA-20 mA, the single mode fiber sensing area is plated for 100 s-140 s, a gold film with a thickness of 45 nm-55 nm is formed, and a probe is obtained;

[0048] Step 3: The single-mode optical fiber sensing area of the probe is immersed in a 10 mM DTT solution for 20-26 h in a dark environment, so that the thiol group at one end of the DTT molecule is combined with the gold film (see reaction formula (1)), and the gold film has high affinity and can form a covalent bond with the sulfur atom on the thiol group of the DTT structure; then the single-mode optical fiber sensing area of the probe is immersed in a 5 mg / mL AuNPs solution after the thiol group is combined with the gold film, for 10-14 h, so that the thiol group at the other end of the DTT molecule is combined with the AuNPs (see reaction formula (2)), and a stable covalent structure is formed between the AuNPs and the gold film by self-assembly; reaction formulas (1) and (2) are as follows:

[0049]

[0050] Step 4: After the treatment in step 3, the probe is washed with ultrapure water, and then the single-mode optical fiber sensing area is immersed in a 50 mM MUA solution for 10-14 h, so that the sulfur atom of the thiol group in the MUA structure forms a covalent bond with the AuNPs, and the carboxyl group is combined on the surface of the gold nanoparticles (see reaction formula (3)), and then the single-mode optical fiber sensing area is immersed in a mixed solution formed by mixing equal volumes of 20 mM EDC and 50 mM NHS for 30-50 min, so as to activate the carboxyl group on the surface of the gold nanoparticles; reaction formula (3) is as follows:

[0051]

[0052] Step 5: After the activation in step 4 is completed, the probe is washed with ultrapure water to remove physically adsorbed molecules, and then the single-mode optical fiber sensing area is immersed in a 4 mg / mL CNTs solution for 1-7 h, so that the amino group of the CNTs is covalently combined with the carboxyl group on the surface of the gold nanoparticles (see reaction formula (4)), so as to be fixed on the probe; reaction formula (4) is as follows:

[0053]

[0054] Step 6: The single-mode optical fiber sensing area of the probe after the treatment in step 5 is immersed in a 1 μM DNA aptamer solution for 5-7 h, so that the carboxyl group of the DNA aptamer is covalently combined with the amino group of the CNTs (see reaction formula (5)), and the DNA aptamer is uniformly and firmly modified on the surface of the probe, to obtain a synergistically modified fiber SPR sensor. Reaction formula (5) is as follows:

[0055]

[0056] The modification process of the AuNPs / CNTs / DNA aptamer on the surface of the single-mode optical fiber sensing area in steps 2 to 6 is shown in Figure 1

[0057] An SPR sensing system is provided, as shown in​Figure 2 The SPR sensing system shown, comprising: the above-mentioned synergistically modified optical fiber SPR sensor, 1x2Y optical fiber jumper 6, broadband light source, marine optical fiber spectrometer and computer, 1x2Y optical fiber jumper is composed of three sections of optical fiber coupled by 1x2Y optical fiber coupler, the tail end of the synergistically modified optical fiber SPR sensor is connected with the 1x2Y optical fiber jumper through the SMA905 optical fiber adapter, the two ends of the 1x2Y optical fiber jumper are connected with the broadband light source and the marine optical fiber spectrometer respectively, and the marine optical fiber spectrometer is connected with the computer display; wherein the synergistically modified optical fiber SPR sensor comprises a multimode optical fiber 1 and a single-mode optical fiber 2, and the surface of the sensing area of the single-mode optical fiber 2 is sequentially modified with a gold film 5, AuNPs / CNTs 4 and DNA aptamer 3.

[0058] The application of an SPR sensing system is used for rapid and high-sensitivity detection of the concentration of vancomycin. The synergistically modified optical fiber SPR sensor is used for selective capture of vancomycin molecules in a biological sample. After the DNA aptamer on the surface of the sensing area is combined with the vancomycin molecules, the conformation of the aptamer changes, thereby causing a change in the refractive index of the sensing interface of the single-mode optical fiber. After being transmitted to the spectrometer, the change in wavelength on the reflection spectrum is displayed on the computer terminal. The corresponding wavelength is obtained by using standard concentration vancomycin for detection. The function relationship between the concentration of vancomycin and the wavelength is determined by fitting. In the actual detection of the concentration of the vancomycin sample, the concentration of vancomycin in the sample is calculated by the shift of the resonance valley on the reflection spectrum. The specific steps are as follows:

[0059] S1: Use TBS buffer (10 mM Tris, 150 mM NaCl, pH = 7.0-7.4) to prepare vancomycin standard concentration solutions at 25°C, with concentrations of 0 μM, 30 μM, 60 μM, 120 μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM;

[0060] S2: Build the above-mentioned SPR sensing system. The light emitted by the broadband light source reaches the synergistically modified optical fiber SPR sensor through the 1x2Y optical fiber, excites SPR at the sensing area, and the reflected light is transmitted to the marine optical fiber spectrometer again through the 1x2Y optical fiber and then to the computer terminal.

[0061] S3: Fix the co-modified fiber SPR sensor on the dip-coating machine to maintain the stability of the optical path during the detection process. Then, insert the sensing areas of the co-modified fiber SPR sensor into the vancomycin standard solution prepared in step S1 in order of increasing concentration. Collect and display the corresponding SPR spectrum through the computer terminal in S2. Obtain the offset of the wavelength λ of the vancomycin standard solution at other concentrations from the wavelength λ0 of the 0μM vancomycin standard solution. After fitting, obtain the functional relationship between vancomycin concentration and wavelength.

[0062] S4: Insert the sensing area of ​​the co-modified fiber SPR sensor into the serum sample to be tested. Collect and display the corresponding SPR spectrum through the computer terminal in S2. Using the wavelength λ0 of the 0μM vancomycin standard solution as a reference, calculate the wavelength offset of the serum sample to be tested relative to λ0. Select the corresponding function relationship between vancomycin concentration and wavelength based on the offset to obtain the concentration of the serum sample to be tested.

[0063] Unless otherwise specified, the experimental methods used in this embodiment of the invention are conventional methods. Unless otherwise specified, the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0064] The preparation of AuNPs solution in step 3 of the preparation method of the synergistic modified fiber SPR sensor in various embodiments of the present invention is as follows: 1 mL of 1 wt% HAuCl4 solution is diluted with 100 mL of deionized water, heated to boiling in a water bath, and then 7 mL of 1 wt% Na3C6H5O7 solution is quickly added. The mixture is stirred and reacted for 15 min-20 min until the solution changes from purplish-black to wine-red. Heating is then stopped and the solution is allowed to cool naturally to obtain the AuNPs solution.

[0065] The DNA aptamer sequence used in step 6 of the preparation method of the co-modified fiber optic SPR sensor in various embodiments of the present invention is 5'-COOH-CGA GGG TAC CGC AAT AGT ACT TAT TGT TCG CCT ATT GTG GGT CGG-3', and the nucleotide sequence involved is shown in SEQ ID NO.1.

[0066] The experimental equipment used in the various embodiments of this invention is as follows:

[0067] The welding machine model is: S179C;

[0068] The cutting blade model is: S326;

[0069] The ion sputtering instrument model is: JS-1600M;

[0070] The model of the light source is HL-2000;

[0071] The model of the dip coating machine is SC-DP-I.

[0072] In the embodiments of the present application, the experimental material is obtained from:

[0073] The multi-mode optical fiber (core diameter 105 μm) and the single-mode optical fiber (core diameter 8.2 μm);

[0074] The amino-functionalized multi-walled carbon nanotube (outer diameter 7 nm-15 nm, inner diameter 2 nm-5 nm, length 7 nm-15 nm) is purchased from Nanjing Carbon Nanomaterials Technology Co., Ltd.

[0075] The TBS dry powder (10 mM Tris, 150 mM NaCl, pH=7.0-7.4) is purchased from Sevine (Beijing) Biotech Co., Ltd.

[0076] DTT and sodium citrate (C6H5Na3O7·H2O) are purchased from Shanghai Macklin Biochemical Reagent Co., Ltd.

[0077] MUA, EDC and NHS are all purchased from Aladdin Biochem Technology Co., Ltd.

[0078] The sequence of the DNA aptamer chain is purchased from Shenguo Bioengineering (Shanghai) Co., Ltd.

[0079] Chloroauric acid (HAuCl4·xH2O) is purchased from Tianjin Hengxing Chemical Reagent Co., Ltd.

[0080] Anhydrous ethanol is used to prepare the MUA solution and is purchased from Tianjin Kaitong Chemical Reagent Co., Ltd.

[0081] Example 1

[0082] A synergistically modified optical fiber SPR sensor, the optical fiber SPR sensor is a multi-mode-single-mode optical fiber cascade structure; the single-mode optical fiber serves as a sensing area, and the surface is sequentially modified with a gold film, AuNPs, CNTs and DNA aptamer; the thickness of the gold film on the surface of the single-mode optical fiber is 50 nm, and the length of the single-mode optical fiber is 3 mm, thereby forming an optical fiber SPR sensor based on AuNPs / CNTs / DNA aptamer synergistic modification.

[0083] A preparation method of a synergistically modified optical fiber SPR sensor, comprising the following steps:

[0084] Step 1: The coated layer of 15 mm of the multimode optical fiber and the single mode optical fiber end face region was axially stripped, and the end face was cleaned with a lens cleaning paper, then a cutting knife was used to cut a flat end face, and the distance from the coated layer to the end face was 5 mm. Then, a fusion machine was used to observe whether the end faces of the two optical fibers were flat. If not, the cutting was repeated. After the end faces were flat, fusion was performed. After the fusion was completed, a cutting knife was used to cut off a certain length of the single mode optical fiber, and the length of the single mode optical fiber was controlled to be 3 mm.

[0085] Step 2: The end of the fused optical fiber was cleaned with ultrapure water, and after drying, the optical fiber was vertically placed in an ion sputtering instrument to plate a gold film. The magnetron sputtering current was controlled to be 18 mA, and the single mode optical fiber sensing area was plated for 120 s to form a gold film with a thickness of 50 nm, thereby obtaining a probe.

[0086] Step 3: In a dark environment, the single mode optical fiber sensing area of the probe was immersed in a 10 mM DTT solution for 24 h, so that the thiol group at one end of the DTT molecule was combined with the gold film. The surface of the gold film has high affinity and can form a covalent bond with the sulfur atom on the thiol group of the DTT structure.

[0087] The AuNPs solution was prepared by the sodium citrate reduction method, and then the single mode optical fiber sensing area of the probe was immersed in a 5 mg / mL AuNPs solution for 12 h after the thiol group was combined with the gold film. The other end of the DTT molecule was combined with AuNPs, and a stable covalent structure was formed between AuNPs and the gold film through self-assembly.

[0088] Step 4: After the treatment in step 3, the probe was washed with ultrapure water, and then the single mode optical fiber sensing area was immersed in a 50 mM MUA solution for 12 h. The sulfur atom of the thiol group in the MUA structure formed a covalent bond with AuNPs, and the carboxyl group was combined on the surface of the gold nanoparticles. Then, the single mode optical fiber sensing area was immersed in a mixed solution formed by mixing equal volumes of 20 mM EDC and 50 mM NHS for 40 min to activate the carboxyl group on the surface of the gold nanoparticles.

[0089] Step 5: After the activation in step 4 was completed, the probe was washed with ultrapure water to remove physically adsorbed molecules. Then, the single mode optical fiber sensing area was immersed in a 4 mg / mL CNTs solution for 4 h, so that the amino group of CNTs was covalently combined with the carboxyl group on the surface of the gold nanoparticles, thereby being fixed on the probe.

[0090] Step 6: The single mode optical fiber sensing area of the probe after step 5 was immersed in a 1 μM DNA aptamer solution for 6 h, so that the carboxyl group of the DNA aptamer was covalently combined with the amino group of CNTs, thereby uniformly and firmly modifying the DNA aptamer on the surface of the probe, thereby obtaining a synergistically modified optical fiber SPR sensor.

[0091] An SPR sensing system, comprising: the collaborative modification fiber SPR sensor of embodiment 1, a 1x2Y fiber jumper, a broadband light source, an Ocean Optics fiber spectrometer and a computer, the 1x2Y fiber jumper is composed of three sections of optical fiber coupled by a 1x2Y fiber coupler, the tail end of the collaborative modification fiber SPR sensor is connected with the 1x2Y fiber jumper through an SMA905 fiber adapter, the two ends of the 1x2Y fiber jumper are connected with the broadband light source and the Ocean Optics fiber spectrometer respectively, and the Ocean Optics fiber spectrometer is connected with the computer display.

[0092] An application of the SPR sensing system is used for rapid and high-sensitivity detection of the concentration of vancomycin. The vancomycin molecules in the biological sample are selectively captured by the collaborative modification fiber SPR sensor of embodiment 1. After the DNA aptamer on the surface of the sensing area is combined with the vancomycin molecules, the conformation of the aptamer changes, thereby causing the change of the refractive index of the sensing interface of the single-mode optical fiber. After being transmitted to the spectrometer, the change of the wavelength on the reflection spectrum is displayed on the computer terminal. The corresponding wavelength is detected by using the standard concentration of vancomycin. The function relationship between the concentration of vancomycin and the wavelength is determined by fitting. In the actual detection of the concentration of the vancomycin sample, the concentration of vancomycin in the sample is calculated by the shift of the resonance valley on the reflection spectrum. The specific steps are as follows:

[0093] S1: A vancomycin standard concentration solution is prepared at 25°C using a TBS buffer (10 mM Tris, 150 mM NaCl, pH = 7.0-7.4), and the concentrations are 0 μM, 30 μM, 60 μM, 120 μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 500 μM, 600 μM, 700 μM and 800 μM respectively.

[0094] S2: The above-mentioned SPR sensing system is built. The light emitted by the broadband light source reaches the collaborative modification fiber SPR sensor through the 1x2Y fiber, excites SPR in the sensing area, and the reflected light is transmitted to the Ocean Optics fiber spectrometer through the 1x2Y fiber again and then to the computer terminal.

[0095] S3: The collaborative modification fiber SPR sensor is fixed on an immersion pull-off coating machine to maintain the stability of the optical path during the detection process. Then the sensing area of the collaborative modification fiber SPR sensor is sequentially inserted into the vancomycin standard solutions with concentrations from low to high according to step S1. The corresponding SPR spectrum is collected and displayed on the computer terminal of step S2. The shift amount of the wavelength λ of the vancomycin standard solution with other concentrations from the wavelength λ0 of the 0 μM vancomycin standard solution is obtained. After fitting processing, the function relationship between the concentration of vancomycin and the wavelength is obtained.

[0096] S4: Insert the sensing area of ​​the co-modified fiber SPR sensor into the serum sample to be tested. Collect and display the corresponding SPR spectrum through the computer terminal in S2. Using the wavelength λ0 of the 0μM vancomycin standard solution as a reference, calculate the wavelength offset of the serum sample to be tested relative to λ0. Select the corresponding function relationship between vancomycin concentration and wavelength based on the offset to obtain the concentration of the serum sample to be tested.

[0097] Performance Analysis:

[0098] (1) Detection sensitivity analysis

[0099] like Figure 3 The reflectance spectrum shown indicates that the co-modified fiber optic SPR sensor of Example 1 exhibits ultra-high sensitivity to vancomycin concentrations, with a redshift in wavelength within the vancomycin concentration range of 0 μM-800 μM. Furthermore, the SPR resonance peak redshifts with increasing vancomycin concentration gradient, indicating continuous binding of vancomycin to the sensor surface. The sensing response is divided into two linear intervals: within the vancomycin concentration range of 30 μM-400 μM, the wavelength shift compared to λ0 is 18.85 nm, with a sensitivity of 0.0527 nm / μM; within the vancomycin concentration range of 400 μM-800 μM, the wavelength shift compared to the 400 μM vancomycin wavelength is 7.52 nm, with a sensitivity of 0.0188 nm / μM. Figure 4 As shown, the relationship between vancomycin concentration (c) and resonance wavelength (λ) is:

[0100]

[0101] (2) Specificity analysis

[0102] Chloramphenicol (CPL), tetracycline (TCY), cysteine ​​(CYS), oxytetracycline (OXY), histidine (HIS), doxorubicin (DOX), glucose (GLU), and urea (URE) solutions were prepared at 25°C using TBS buffer (10 mM Tris, 150 mM NaCl, pH = 7.0-7.4) at concentrations of 0 μM, 30 μM, 60 μM, 120 μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 500 μM, 600 μM, 700 μM, and 800 μM, respectively. The SPR sensing system of Example 1 was then used to detect these substances at concentrations of 0 μM-800 μM. The detection results of vancomycin at concentrations of 0 μM-800 μM were compared with those of vancomycin at concentrations of 0 μM-800 μM. The trends of wavelength shift and concentration change are shown below. Figure 5 As shown, after being converted into a more intuitive comparative bar chart, as... Figure 6As shown, the wavelength shift of each interfering substance is less than 2.5 nm, which is significantly lower than the response generated by vancomycin, indicating that the synergistically modified optical fiber SPR sensor of the present application has high specificity for vancomycin.

[0103] (3) Stability analysis

[0104] The synergistically modified optical fiber SPR sensor of Example 1 was immersed in TBS buffer (10 mM Tris, 150 mM NaCl, pH = 7.0-7.4) for 60 min, and as shown in Figure 7 the resonance wavelength remained highly stable, with a maximum shift of only 0.886 nm, indicating that the synergistically modified optical fiber SPR sensor of the present application has ultra-high stability, and the standard deviation in the stability experiment was 0.31363.

[0105] According to the sensitivity and stability analysis, combined with the following calculation formula:

[0106] LOD = 3 × σ / S

[0107] S is the sensitivity of the calibration curve, and σ is the standard deviation in the experiment;

[0108] The detection lower limit in Example 1 is 3 × 0.31363 / 0.0527 ≈ 17.85 μM.

[0109] Example 2

[0110] A synergistically modified optical fiber SPR sensor, the optical fiber SPR sensor is a multi-mode-single-mode optical fiber cascade structure; the single-mode optical fiber serves as a sensing area, and the surface is sequentially modified with a gold film, AuNPs, CNTs, and DNA aptamer; the thickness of the gold film on the surface of the single-mode optical fiber is 50 nm, and the length of the single-mode optical fiber is 2 mm, forming an optical fiber SPR sensor based on AuNPs / CNTs / DNA aptamer synergistic modification.

[0111] A preparation method of a synergistically modified optical fiber SPR sensor, comprising the following steps:

[0112] Step 1: The coated layer of 15 mm is removed from the end face area of the multi-mode optical fiber and the single-mode optical fiber in the axial direction, and then wiped clean with a mirror paper, and then a cutting knife is used to cut a flat end face, so that the distance from the coated layer to the end face is 5 mm, then a fusion splicer is used to observe whether the end faces of the two optical fibers are flat, if not, re-cut, and then fuse after flattening, and then a cutting knife is used to cut off a certain length of single-mode optical fiber, and the length of the single-mode optical fiber is controlled to be 3 mm.

[0113] Step 2: The end of the fusion completed optical fiber is cleaned with ultrapure water, and after drying, the optical fiber is vertically placed in an ion sputtering instrument to coat a gold film, the magnetron sputtering current is controlled at 18 mA, and the single-mode optical fiber sensing area is coated for 120 s to form a gold film with a thickness of 50 nm, thereby obtaining a probe.

[0114] Step 3: In a dark environment, the single-mode optical fiber sensing area of the probe is immersed in a 10 mM DTT solution for 24 h, so that the thiol group at one end of the DTT molecule is combined with the gold film, and the gold film has high affinity and can form a covalent bond with the sulfur atom on the structural thiol group of DTT;

[0115] The AuNPs solution is prepared by a sodium citrate reduction method, and then the thiol group is combined with the gold film. The single-mode optical fiber sensing area of the probe is immersed in a 5 mg / mL AuNPs solution for 12 h, and the thiol group at the other end of the DTT molecule is combined with AuNPs. A stable covalent structure is formed between AuNPs and the gold film through self-assembly.

[0116] Step 4: After the treatment in step 3, the probe is washed with ultrapure water, and then the single-mode optical fiber sensing area is immersed in a 50 mM MUA solution for 12 h. The sulfur atom of the thiol group in the MUA structure forms a covalent bond with AuNPs, and the carboxyl group is combined on the surface of the gold nanoparticles. Then, the single-mode optical fiber sensing area is immersed in a mixed solution formed by mixing equal volumes of 20 mM EDC and 50 mM NHS for 30 min to activate the carboxyl group on the surface of the gold nanoparticles.

[0117] Step 5: After the activation in step 4 is completed, the probe is washed with ultrapure water to remove physically adsorbed molecules. Then, the single-mode optical fiber sensing area is immersed in a 4 mg / mL CNTs solution for 1 h, so that the amino group of CNTs is covalently combined with the carboxyl group on the surface of the gold nanoparticles, thereby being fixed on the probe.

[0118] Step 6: The single-mode optical fiber sensing area of the probe after step 5 is immersed in a 1 μM DNA aptamer solution for 7 h, so that the carboxyl group of the DNA aptamer is covalently combined with the amino group of CNTs, thereby uniformly and firmly modifying the DNA aptamer on the surface of the probe, thereby obtaining a synergistically modified optical fiber SPR sensor.

[0119] Example 3

[0120] A synergistically modified optical fiber SPR sensor, wherein the optical fiber SPR sensor has a multi-mode-single-mode optical fiber cascade structure; the single-mode optical fiber serves as a sensing area and is sequentially modified with a gold film, AuNPs, CNTs, and a DNA aptamer; the thickness of the gold film on the surface of the single-mode optical fiber is 50 nm; the length of the single-mode optical fiber is 3 mm; and the single-mode optical fiber forms an optical fiber SPR sensor based on AuNPs / CNTs / DNA aptamer synergistic modification.

[0121] A method for preparing a synergistically modified optical fiber SPR sensor, comprising the following steps:

[0122] Step 1: The coated layer of the multimode optical fiber and the single mode optical fiber was stripped axially by 15 mm in the end face area, and then cleaned with a mirror paper. Then, a cutting knife was used to cut a flat end face, so that the distance between the coated layer and the end face was 5 mm. Then, a fusion machine was used to observe whether the end faces of the two optical fibers were flat. If not, the cutting was repeated. After the end faces were flat, fusion was performed. After the fusion was completed, a cutting knife was used to cut off a certain length of the single mode optical fiber, and the length of the single mode optical fiber was controlled to be 3 mm.

[0123] Step 2: The end of the fused optical fiber was cleaned with ultrapure water, and after drying, the optical fiber was vertically placed in an ion sputtering instrument to plate a gold film. The magnetron sputtering current was controlled to be 18 mA, and the single mode optical fiber sensing area was plated for 120 s to form a gold film with a thickness of 50 nm, thereby obtaining a probe.

[0124] Step 3: In a dark environment, the single mode optical fiber sensing area of the probe was immersed in a 10 mM DTT solution for 24 h, so that the thiol group at one end of the DTT molecule was combined with the gold film. The surface of the gold film has high affinity and can form a covalent bond with the sulfur atom on the thiol group of the DTT structure.

[0125] The AuNPs solution was prepared by the sodium citrate reduction method, and then the single mode optical fiber sensing area of the probe was immersed in a 5 mg / mL AuNPs solution for 12 h after the thiol group was combined with the gold film. The other end of the DTT molecule was combined with AuNPs, and a stable covalent structure was formed between AuNPs and the gold film through self-assembly.

[0126] Step 4: After the treatment in step 3, the probe was washed with ultrapure water, and then the single mode optical fiber sensing area was immersed in a 50 mM MUA solution for 10 h. The sulfur atom of the thiol group in the MUA structure formed a covalent bond with AuNPs, and the carboxyl group was combined on the surface of the gold nanoparticles. Then, the single mode optical fiber sensing area was immersed in a mixed solution formed by mixing equal volumes of 20 mM EDC and 50 mM NHS for 50 min to activate the carboxyl group on the surface of the gold nanoparticles.

[0127] Step 5: After the activation in step 4 was completed, the probe was washed with ultrapure water to remove physically adsorbed molecules. Then, the single mode optical fiber sensing area was immersed in a 4 mg / mL CNTs solution for 2 h, so that the amino group of CNTs was covalently combined with the carboxyl group on the surface of the gold nanoparticles, thereby being fixed on the probe.

[0128] Step 6: The single mode optical fiber sensing area of the probe treated in step 5 was immersed in a 1 μM DNA aptamer solution for 5 h, so that the carboxyl group of the DNA aptamer was covalently combined with the amino group of CNTs, thereby uniformly and firmly modifying the DNA aptamer on the surface of the probe, thereby obtaining a synergistically modified optical fiber SPR sensor.

[0129] Example 4

[0130] A synergistically modified optical fiber SPR sensor, the optical fiber SPR sensor is a multi-mode-single-mode optical fiber cascade structure; the single-mode optical fiber is used as a sensing area, and the surface is sequentially modified with a gold film, AuNPs, CNTs and DNA aptamer; the thickness of the gold film on the surface of the single-mode optical fiber is 50 nm, and the length of the single-mode optical fiber is 3 mm, thereby forming an optical fiber SPR sensor based on AuNPs / CNTs / DNA aptamer synergistic modification.

[0131] A preparation method of a synergistically modified optical fiber SPR sensor, comprising the following steps:

[0132] Step 1: The coating layer of 15 mm of the multi-mode optical fiber and the single-mode optical fiber is removed from the end face area in an axial direction, and then the end face is wiped clean with a mirror paper, and then a cutting knife is used to cut a flat end face, so that the distance from the coating layer to the end face is 5 mm, then a fusion splicer is used to observe whether the end faces of the two optical fibers are flat, if not, the cutting is re-performed, and after being flat, the fusion is performed, and then the cutting knife is used to cut off a certain length of the single-mode optical fiber, and the length of the single-mode optical fiber is controlled to be 3 mm.

[0133] Step 2: The end of the fused optical fiber is cleaned with ultrapure water, and after being dried, the optical fiber is vertically placed in an ion sputtering instrument to plate a gold film, the magnetron sputtering current is controlled to be 18 mA, the single-mode optical fiber sensing area is plated for 120 s to form a gold film with a thickness of 50 nm, and a probe is obtained.

[0134] Step 3: In a dark environment, the single-mode optical fiber sensing area of the probe is soaked in a 10 mM DTT solution for 24 h, so that the thiol group at one end of the DTT molecule is combined with the gold film, the gold film has high affinity, and can form a covalent bond with the sulfur atom on the thiol group of the DTT structure;

[0135] AuNPs solution is prepared by a sodium citrate reduction method, and then the thiol group is combined with the gold film, and the single-mode optical fiber sensing area of the probe is soaked in a 5 mg / mL AuNPs solution for 10 h, and the thiol group at the other end of the DTT molecule is combined with AuNPs, and a stable covalent structure is formed between AuNPs and the gold film through self-assembly.

[0136] Step 4: After the treatment in step 3, the probe is washed with ultrapure water, and then the single-mode optical fiber sensing area is soaked in a 50 mM MUA solution for 14 h, the sulfur atom of the thiol group in the MUA structure forms a covalent bond with AuNPs, so that the carboxyl group is combined on the surface of the gold nanoparticles, and then the mixed solution formed by mixing equal volumes of 20 mM EDC and 50 mM NHS is soaked for 40 min, and the carboxyl group on the surface of the gold nanoparticles is activated.

[0137] Step 5: After the activation of step 4 is completed, the probe is cleaned with ultrapure water to remove physically adsorbed molecules, and then the single-mode optical fiber sensing area is immersed in a 4 mg / mL CNTs solution for 3 h, so that the amino groups of the CNTs are covalently combined with the carboxyl groups on the surface of the gold nanoparticles to be fixed on the probe.

[0138] Step 6: The single-mode optical fiber sensing area of the probe after step 5 is treated is immersed in a 1 μM DNA aptamer solution for 6 h, so that the carboxyl groups of the DNA aptamer are covalently combined with the amino groups of the CNTs, thereby uniformly and firmly modifying the DNA aptamer on the surface of the probe to obtain a synergistically modified optical fiber SPR sensor.

[0139] Example 5

[0140] A synergistically modified optical fiber SPR sensor, wherein the optical fiber SPR sensor has a multi-mode-single-mode optical fiber cascade structure; the single-mode optical fiber serves as a sensing area and is sequentially modified with a gold film, AuNPs, CNTs, and DNA aptamer; the thickness of the gold film on the surface of the single-mode optical fiber is 45 nm; the length of the single-mode optical fiber is 3 mm; and the single-mode optical fiber forms an optical fiber SPR sensor based on AuNPs / CNTs / DNA aptamer synergistic modification.

[0141] A method for preparing a synergistically modified optical fiber SPR sensor, comprising the following steps:

[0142] Step 1: The coated layer of 15 mm is axially removed from the end face area of a multi-mode optical fiber and a single-mode optical fiber, and then the end face is wiped clean with a mirror cloth, and then a cutting knife is used to cut a flat end face, so that the distance from the coated layer to the end face is 5 mm, and then a fusion splicer is used to observe whether the end faces of the two optical fibers are flat, and if not, the cutting is re-performed, and after being flat, the fusion is performed, and after the fusion is completed, the single-mode optical fiber of a certain length is cut off again, and the length of the single-mode optical fiber is controlled to be 3 mm.

[0143] Step 2: The end of the fused optical fiber is cleaned with ultrapure water, and after being dried, the optical fiber is vertically placed in an ion sputtering instrument to be plated with a gold film, and the magnetron sputtering current is controlled to be 18 mA, and the single-mode optical fiber sensing area is plated for 100 s to form a gold film with a thickness of 45 nm, thereby obtaining a probe.

[0144] Step 3: The single-mode optical fiber sensing area of the probe is immersed in a 10 mM DTT solution in a dark environment for 20 h, so that the thiol group at one end of the DTT molecule is combined with the gold film, and the surface of the gold film has high affinity and can form a covalent bond with the sulfur atom on the thiol group of the DTT structure.

[0145] An AuNPs solution is prepared by a sodium citrate reduction method, and then the single-mode optical fiber sensing area of the probe after the thiol group is combined with the gold film is immersed in a 5 mg / mL AuNPs solution for 14 h, and the thiol group at the other end of the DTT molecule is combined with the AuNPs, thereby forming a stable covalent structure between the AuNPs and the gold film through self-assembly.

[0146] Step 4: After the step 3 treatment, the probe was washed with ultrapure water, and then the single-mode optical fiber sensing area was immersed in a 50 mM MUA solution for 12 h, and the sulfur atom of the thiol group in the MUA structure formed a covalent bond with the AuNPs, so that the carboxyl group was combined on the surface of the gold nanoparticles, and then the single-mode optical fiber sensing area was immersed in a mixed solution formed by mixing equal volumes of 20 mM EDC and 50 mM NHS for 40 min to activate the carboxyl group on the surface of the gold nanoparticles.

[0147] Step 5: After the activation of step 4 was completed, the probe was washed with ultrapure water to remove physically adsorbed molecules, and then the single-mode optical fiber sensing area was immersed in a 4 mg / mL CNTs solution for 4 h to covalently bond the amino group of the CNTs to the carboxyl group on the surface of the gold nanoparticles, thereby fixing the CNTs on the probe.

[0148] Step 6: The single-mode optical fiber sensing area of the probe after step 5 was immersed in a 1 μM DNA aptamer solution for 6 h to covalently bond the carboxyl group of the DNA aptamer to the amino group of the CNTs, thereby uniformly and firmly modifying the DNA aptamer on the surface of the probe, and obtaining a synergistically modified optical fiber SPR sensor.

[0149] Example 6

[0150] A synergistically modified optical fiber SPR sensor, the optical fiber SPR sensor has a multi-mode-single-mode optical fiber cascade structure; the single-mode optical fiber serves as a sensing area and is sequentially modified with a gold film, AuNPs, CNTs, and DNA aptamer; the thickness of the gold film on the surface of the single-mode optical fiber is 55 nm; the length of the single-mode optical fiber is 3 mm; and the single-mode optical fiber forms an optical fiber SPR sensor based on AuNPs / CNTs / DNA aptamer synergistic modification.

[0151] A method for preparing a synergistically modified optical fiber SPR sensor, comprising the following steps:

[0152] Step 1: The coated layer of 15 mm was removed from the end face area of the multi-mode optical fiber and the single-mode optical fiber in an axial direction, and the end face was wiped clean with a mirror wiping paper, then a cutting knife was used to cut a flat end face, so that the distance from the coated layer to the end face was 5 mm, then a fusion splicer was used to observe whether the end faces of the two optical fibers were flat, and if not, the cutting was repeated, after the end faces were flat, the fusion splicing was performed, and then a cutting knife was used to cut off a certain length of the single-mode optical fiber, and the length of the single-mode optical fiber was controlled to be 3 mm.

[0153] Step 2: The end of the fused optical fiber was cleaned with ultrapure water, and after drying, the optical fiber was vertically placed in an ion sputtering instrument to coat a gold film, and the magnetron sputtering current was controlled to be 18 mA, the single-mode optical fiber sensing area was coated for 140 s to form a gold film with a thickness of 55 nm, and a probe was obtained.

[0154] Step 3: The single-mode optical fiber sensing area of the probe is immersed in a 10 mM DTT solution for 26 h in a dark environment, so that the thiol group at one end of the DTT molecule is combined with the gold film, and the gold film has high affinity and can form a covalent bond with the sulfur atom on the structure thiol group of DTT;

[0155] The AuNPs solution is prepared by a sodium citrate reduction method, and then the single-mode optical fiber sensing area of the probe is immersed in a 5 mg / mL AuNPs solution for 12 h after the thiol group is combined with the gold film, and the other end of the DTT molecule is combined with the AuNPs, and a stable covalent structure is formed between the AuNPs and the gold film through self-assembly.

[0156] Step 4: After the treatment in step 3, the probe is washed with ultrapure water, and then the single-mode optical fiber sensing area is immersed in a 50 mM MUA solution for 12 h, so that the sulfur atom of the thiol group in the MUA structure forms a covalent bond with the AuNPs, and the carboxyl group is combined on the surface of the gold nanoparticles, and then the single-mode optical fiber sensing area is immersed in a mixed solution formed by mixing equal volumes of 20 mM EDC and 50 mM NHS for 40 min, to activate the carboxyl group on the surface of the gold nanoparticles.

[0157] Step 5: After the activation in step 4 is completed, the probe is washed with ultrapure water to remove physically adsorbed molecules, and then the single-mode optical fiber sensing area is immersed in a 4 mg / mL CNTs solution for 5 h, so that the amino group of the CNTs is covalently combined with the carboxyl group on the surface of the gold nanoparticles to be fixed on the probe.

[0158] Step 6: The single-mode optical fiber sensing area of the probe after step 5 is immersed in a 1 μM DNA aptamer solution for 6 h, so that the carboxyl group of the DNA aptamer is covalently combined with the amino group of the CNTs, so that the DNA aptamer is uniformly and firmly modified on the surface of the probe, and a synergistically modified optical fiber SPR sensor is obtained.

[0159] Example 7

[0160] A synergistically modified optical fiber SPR sensor, the optical fiber SPR sensor has a multi-mode-single-mode optical fiber cascade structure; the single-mode optical fiber serves as a sensing area, and the surface of the single-mode optical fiber is sequentially modified with a gold film, AuNPs, CNTs, and a DNA aptamer; the thickness of the gold film on the surface of the single-mode optical fiber is 48 nm, the length of the single-mode optical fiber is 3 mm, and an optical fiber SPR sensor based on AuNPs / CNTs / DNA aptamer synergistic modification is formed.

[0161] A method for preparing a synergistically modified optical fiber SPR sensor, comprising the following steps:

[0162] Step 1: The coated layer of the multimode optical fiber and the single mode optical fiber was stripped axially by 15 mm in the end face area, and then cleaned with a mirror paper. Then, a cutting knife was used to cut a flat end face, so that the distance between the coated layer and the end face was 5 mm. Then, a fusion machine was used to observe whether the end faces of the two optical fibers were flat. If not, the cutting was repeated. After the end faces were flat, fusion was performed. After the fusion was completed, a cutting knife was used to cut off a certain length of the single mode optical fiber, and the length of the single mode optical fiber was controlled to be 3 mm.

[0163] Step 2: The end of the fused optical fiber was cleaned with ultrapure water. After drying, the optical fiber was vertically placed in an ion sputtering instrument to plate a gold film. The magnetron sputtering current was controlled to be 15 mA. The single mode optical fiber sensing area was plated for 120 s to form a gold film with a thickness of 48 nm, and a probe was obtained.

[0164] Step 3: In a dark environment, the single mode optical fiber sensing area of the probe was soaked in a 10 mM DTT solution for 24 h, so that the thiol group at one end of the DTT molecule was combined with the gold film. The surface of the gold film has high affinity and can form a covalent bond with the sulfur atom on the thiol group of the DTT structure.

[0165] The AuNPs solution was prepared by the sodium citrate reduction method. Then, the single mode optical fiber sensing area of the probe was soaked in a 5 mg / mL AuNPs solution for 12 h after the thiol group was combined with the gold film. The other end of the DTT molecule was combined with AuNPs, and a stable covalent structure was formed between AuNPs and the gold film through self-assembly.

[0166] Step 4: After the treatment in step 3, the probe was washed with ultrapure water. Then, the single mode optical fiber sensing area was soaked in a 50 mM MUA solution for 12 h. The sulfur atom of the thiol group in the MUA structure formed a covalent bond with AuNPs, so that the carboxyl group was combined on the surface of the gold nanoparticles. Then, the single mode optical fiber sensing area was soaked in a mixed solution formed by mixing equal volumes of 20 mM EDC and 50 mM NHS for 40 min to activate the carboxyl group on the surface of the gold nanoparticles.

[0167] Step 5: After the activation in step 4 was completed, the probe was washed with ultrapure water to remove physically adsorbed molecules. Then, the single mode optical fiber sensing area was soaked in a 4 mg / mL CNTs solution for 6 h, so that the amino group of CNTs was covalently combined with the carboxyl group on the surface of the gold nanoparticles to be fixed on the probe.

[0168] Step 6: The single mode optical fiber sensing area of the probe after the treatment in step 5 was soaked in a 1 μM DNA aptamer solution for 6 h, so that the carboxyl group of the DNA aptamer was covalently combined with the amino group of CNTs, so that the DNA aptamer was uniformly and firmly modified on the surface of the probe to obtain a synergistically modified optical fiber SPR sensor.

[0169] Example 8

[0170] A synergistically modified optical fiber SPR sensor, the optical fiber SPR sensor is a multi-mode-single-mode optical fiber cascade structure; the single-mode optical fiber is used as a sensing area, and the surface is sequentially modified with a gold film, AuNPs, CNTs and DNA aptamer; the thickness of the gold film on the surface of the single-mode optical fiber is 52 nm, and the length of the single-mode optical fiber is 4 mm, thereby forming an optical fiber SPR sensor based on AuNPs / CNTs / DNA aptamer synergistic modification.

[0171] A preparation method of a synergistically modified optical fiber SPR sensor, comprising the following steps:

[0172] Step 1: The coating layer of 15 mm of the multi-mode optical fiber and the single-mode optical fiber is removed axially from the end face area, and then the end face is wiped clean with a mirror paper, and then a cutting knife is used to cut a flat end face, so that the distance from the coating layer to the end face is 5 mm, then a fusion machine is used to observe whether the end faces of the two optical fibers are flat, if not, the cutting is re-performed, and after being flat, the fusion is performed, and after the fusion is completed, the single-mode optical fiber of a certain length is cut off again, and the length of the single-mode optical fiber is controlled to be 4 mm.

[0173] Step 2: The end of the fused optical fiber is cleaned with ultrapure water, and after being dried, the optical fiber is vertically placed in an ion sputtering instrument to plate a gold film, the magnetron sputtering current is controlled to be 20 mA, the single-mode optical fiber sensing area is plated for 120 s, a gold film with a thickness of 52 nm is formed, and a probe is obtained.

[0174] Step 3: In a dark environment, the single-mode optical fiber sensing area of the probe is soaked in a 10 mM DTT solution for 24 h, so that the thiol group at one end of the DTT molecule is combined with the gold film, the gold film has high affinity, and can form a covalent bond with the sulfur atom on the thiol group of the DTT structure;

[0175] AuNPs solution is prepared by a sodium citrate reduction method, and then the thiol group is combined with the gold film, and the single-mode optical fiber sensing area of the probe is soaked in a 5 mg / mL AuNPs solution for 12 h, the thiol group at the other end of the DTT molecule is combined with AuNPs, and a stable covalent structure is formed between AuNPs and the gold film through self-assembly.

[0176] Step 4: After the treatment in step 3, the probe is washed with ultrapure water, and then the single-mode optical fiber sensing area is soaked in a 50 mM MUA solution for 12 h, the sulfur atom of the thiol group in the MUA structure forms a covalent bond with AuNPs, so that the carboxyl group is combined on the surface of the gold nanoparticles, and then the mixed solution formed by mixing equal volumes of 20 mM EDC and 50 mM NHS is soaked for 40 min, and the carboxyl group on the surface of the gold nanoparticles is activated.

[0177] Step 5: After the activation of step 4, the probe was washed with ultrapure water to remove the physically adsorbed molecules, and then the sensing region of the single-mode optical fiber was immersed in a 4 mg / mL CNTs solution for 7 h to covalently bind the amino groups of the CNTs to the carboxyl groups on the surface of the gold nanoparticles, thereby fixing the CNTs on the probe.

[0178] Step 6: The sensing region of the single-mode optical fiber of the probe treated in step 5 was immersed in a 1 μM DNA aptamer solution for 6 h to covalently bind the carboxyl groups of the DNA aptamer to the amino groups of the CNTs, thereby uniformly and firmly modifying the DNA aptamer on the surface of the probe to obtain a synergistically modified fiber SPR sensor.

[0179] Comparative Example 1

[0180] The DNA aptamer-based fiber SPR sensor of this comparative example 1 was used to detect vancomycin solution after being composed into an SPR sensing system.

[0181] As shown in Figure 8 , it can be seen that the DNA aptamer-based fiber SPR sensor of this comparative example 1 has a weak response to the concentration of vancomycin, and the wavelength shift is only 1.77 nm in the range of 60 μM-400 μM vancomycin concentration, and the sensitivity is only 0.00531 nm / μM, as shown in Figure 9 , the relationship between the concentration (c) of vancomycin and the resonance wavelength (λ) is:

[0182]

[0183] Compared with the synergistically modified fiber SPR sensor of Example 1, the sensitivity of the sensor of Example 1 is about 9.92 times that of the sensor of Comparative Example 1 when detecting vancomycin with a concentration below 400 μM.

[0184] Comparative Example 2

[0185] The CNTs / DNA aptamer-based fiber SPR sensor differs from Example 1 in that, in addition to the gold film, the surface of the sensing region of the single-mode optical fiber is modified with CNTs / DNA aptamer, and the sensor is used to detect vancomycin solution after being composed into an SPR sensing system.

[0186] As shown in Figure 10 , it can be seen that the DNA aptamer-based fiber SPR sensor of this comparative example 1 has a weak response to the concentration of vancomycin, and the wavelength shift is only 1.77 nm in the range of 60 μM-400 μM vancomycin concentration, and the sensitivity is only 0.00531 nm / μM, as shown in Figure 11 , the relationship between the concentration (c) of vancomycin and the resonance wavelength (λ) is:

[0187]

[0188] In comparison with the co-modified optical fiber SPR sensor of Example 1, the sensitivity of the sensor of Example 1 was about 1.37 times that of the sensor of Comparative Example 2 in detecting vancomycin at a concentration of 400 μM or less.

Claims

1. A co-modified fiber optic SPR sensor, characterized in that, The synergistically modified fiber SPR sensor is a multimode-single-mode fiber cascade structure; the single-mode fiber serves as the sensing region, and its surface is sequentially modified with a gold film, AuNPs, CNTs, and DNA aptamers to form a fiber SPR sensor based on the synergistic modification of AuNPs / CNTs / DNA aptamers.

2. The co-modified fiber optic SPR sensor as described in claim 1, characterized in that, The gold film thickness on the surface of the single-mode fiber is 45nm-55nm, and the length of the single-mode fiber is 2mm-4mm.

3. A method for fabricating a synergistically modified fiber optic SPR sensor as described in claim 1, characterized in that, Includes the following steps: Step 1: Remove part of the coating along the axial direction of one end face area of ​​the multimode fiber and single-mode fiber, wipe it clean, then cut out a flat end face, and then use a fusion splicer to perform fusion splicing, controlling the length of the single-mode fiber. Step 2: Clean one end of the fused fiber, dry it, and place it vertically in the ion sputtering instrument to deposit a gold film on the single-mode fiber sensing area to obtain the probe. Step 3: In a dark environment, immerse the single-mode fiber sensing area of ​​the probe in a dithiothreitol solution, so that the thiol group at one end of the dithiothreitol molecule combines with the gold film. Prepare an AuNPs solution by sodium citrate reduction. Then immerse the single-mode fiber sensing area of ​​the probe after the thiol group has been combined with the gold film in the AuNPs solution. The thiol group at the other end of the dithiothreitol molecule combines with the AuNPs, forming a stable covalent structure between the AuNPs and the gold film through self-assembly. Step 4: Clean the probe after the treatment in Step 3, then immerse the single-mode fiber sensing area in 11-mercaptoundecanoic acid solution to bind carboxyl groups to the surface of gold nanoparticles, and then immerse it in a mixed solution of 1-ethyl-(3-dimethoxyaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to activate the carboxyl groups on the surface of gold nanoparticles. Step 5: After the probe is activated in Step 4, clean it thoroughly and then immerse the single-mode fiber sensing area in an aminated CNT solution to covalently bind the amino groups of the CNTs with the carboxyl groups on the surface of the gold nanoparticles. Step 6: Immerse the single-mode fiber sensing area of ​​the probe after step 5 into the DNA aptamer solution, so that the carboxyl group of the DNA aptamer covalently binds to the amino group of CNTs. The DNA aptamer is uniformly and firmly modified on the probe surface, resulting in a synergistically modified fiber SPR sensor.

4. The method for fabricating a synergistically modified fiber optic SPR sensor as described in claim 3, characterized in that, In step 1, the length of the coating layer removed from the multimode fiber and the single-mode fiber is 15mm-21mm, and a flat end face is cut so that the distance from the coating layer to the end face is 4mm-6mm, and the length of the single-mode fiber is controlled to be 2mm-4mm. In step 2, the thickness of the gold film is 45nm-55nm, the magnetron sputtering current is 15mA-20mA, and the time is 100s-140s.

5. The method for fabricating a synergistically modified fiber optic SPR sensor as described in claim 3, characterized in that, In step 3, the concentration of dithiothreitol is 10 mM, and the probe is immersed in the dithiothreitol solution for 20-26 hours. The preparation of AuNPs solution by sodium citrate reduction involves diluting a 1 wt% chloroauric acid solution with deionized water at a volume ratio of 1:

100. After heating to boiling in a water bath, a 1 wt% sodium citrate solution is quickly added at a volume ratio of 1:

7. The mixture is stirred for 15-20 minutes until the solution changes from purplish-black to wine-red. Heating is then stopped and the solution is allowed to cool naturally to obtain the AuNPs solution. The AuNPs solution concentration was 5 mg / mL, and the probe was immersed in the AuNPs solution for 10-14 hours.

6. The method for fabricating a synergistically modified fiber optic SPR sensor as described in claim 3, characterized in that, In step 4, the concentration of 11-mercaptoundecanoic acid is 50 mM, and the probe is immersed in 11-mercaptoundecanoic acid for 10-14 hours; the concentration of ethyl-(3-dimethoxyaminopropyl)carbodiimide hydrochloride is 20 mM, and the concentration of N-hydroxysuccinimide is 50 mM. The two are mixed in equal volumes to form a solution, and the probe is immersed in the mixed solution for 30-50 minutes. In step 5, the concentration of the CNTs solution is 4 mg / mL, and the probe is immersed in the CNTs solution for 1-7 hours.

7. The method for fabricating a synergistically modified fiber optic SPR sensor as described in claim 3, characterized in that, In step 6, the concentration of the DNA aptamer solution is 1 μM, and the probe is immersed in the DNA aptamer solution for 5-7 hours. The DNA aptamer sequence is 5'-COOH-CGA GGG TAC CGC AAT AGT ACT TAT TGT TCG CCT ATTGTG GGT CGG-3', and the nucleotide sequence involved is shown in SEQ ID NO.

1.

8. An SPR sensing system, characterized in that, include: The co-modified fiber SPR sensor, Y-type fiber optic patch cord, light source, spectrometer, and computer described in claim 1 are as follows: the tail end of the co-modified fiber SPR sensor is connected to one end of the Y-type fiber optic patch cord via an SMA905 fiber optic adapter; the other two ends of the Y-type fiber optic patch cord are respectively connected to the light source and the spectrometer; and the spectrometer is connected to the computer.

9. The SPR sensing system as described in claim 8, characterized in that, The Y-type fiber optic patch cord is 1×2Y type, the light source is a broadband light source, and the spectrometer is a marine optical fiber spectrometer.

10. An application of the SPR sensing system according to claim 8, characterized in that, This method is used for rapid and highly sensitive detection of vancomycin concentrations in a low concentration range. A co-modified fiber optic SPR sensor selectively captures vancomycin molecules in biological samples. After the DNA aptamer on the sensing area surface binds to the vancomycin molecule, the aptamer conformation changes, leading to a change in the refractive index of the single-mode fiber optic sensing interface. This change is transmitted to a spectrometer and displayed as a change in wavelength on the reflectance spectrum on a computer terminal. The corresponding wavelength is obtained using a standard concentration of vancomycin, and a functional relationship between vancomycin concentration and wavelength is determined by fitting the data. In actual vancomycin sample concentration detection, the concentration of vancomycin in the sample is calculated by the shift of the resonance trough in the reflectance spectrum.

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