A method for detecting amoxicillin concentration based on optical fiber

Abstract

The application discloses a kind of amoxicillin concentration detection methods based on optical fiber, using device includes: broadband light source, single-mode optical fiber, polarization controller, flow cell, TFBG-SPR sensor and spectrum analyzer.First by broadband light source to emit the light of wavelength range between 1420~1620nm, the polarization state of light is adjusted to P state by polarization controller, then through polydopamine, polyaniline and beta-lactamase layer-by-layer modified sensing probe, finally form spectrum on spectrum analyzer.Bio-identification film based on beta-lactamase modified on the surface of optical fiber will be combined with amoxicillin molecule to cause the change of refractive index, and the drift of transmission spectrum is shown on spectrum analyzer, the relationship between different amoxicillin solution concentration and transmission spectrum drift is compared, to realize the rapid, accurate, trace detection of amoxicillin concentration.The application provides a new method for the measurement of amoxicillin and other beta-lactam antibiotics concentration, which has simple structure, high accuracy and fast response, and has very important potential application value in environmental pollution and food health monitoring.

Description

Technical Field

[0001] This invention relates to the field of fiber optic sensing technology, and specifically to a method for detecting amoxicillin concentration based on a gold-plated tilted fiber grating combined with a β-lactamase-based biorecognition film. Background Technology

[0002] Amoxicillin, a broad-spectrum β-lactam antibiotic, has been widely used in clinical medicine and animal husbandry due to its excellent antibacterial activity and good oral bioavailability. However, the environmental and health risks it brings are becoming increasingly prominent.

[0003] Amoxicillin, a representative penicillin antibiotic, exerts its bactericidal effect by inhibiting bacterial cell wall synthesis. It exhibits excellent antibacterial efficacy against both Gram-positive and some Gram-negative bacteria, and has become a first-line drug for treating respiratory, urinary tract, and digestive tract infections. However, during its production and use, amoxicillin enters the environment through industrial wastewater, aquaculture waste, and excrement, leading to the continuous accumulation of antibiotic residues in aquatic ecosystems. Long-term exposure of aquatic organisms to these low-concentration antibiotic environments may not only lead to the accumulation and spread of drug-resistant genes but also disrupt the balance of the microbial community, thereby affecting the stability of the entire ecosystem. In animal husbandry, improper use or abuse of amoxicillin can result in drug residues in animal-derived foods, which can then enter the human body through the food chain. Long-term intake may cause health problems such as allergies, intestinal flora imbalance, and drug resistance transfer, seriously threatening public health and safety.

[0004] Currently, the most commonly used methods for detecting amoxicillin residues in food or solutions include microbiological methods, immunoassay, liquid chromatography, liquid chromatography-tandem mass spectrometry, and electrochemiluminescence. However, these methods have drawbacks such as high requirements for environmental conditions and equipment, high cost, and complex sample pretreatment processes, making it difficult to meet practical application needs for amoxicillin concentration detection.

[0005] In recent years, fiber optic sensors have demonstrated great application potential in many fields such as biomedicine and chemical detection due to their advantages of high accuracy, fast detection speed, strong resistance to electromagnetic interference, and high sensitivity.

[0006] As a type of fiber optic grating, TFBG possesses high sensitivity, high robustness, and low loss due to its unique fiber structure design, making it widely applicable in the fields of physical quantity, chemical substance, and biological detection.

[0007] On the other hand, enzyme membrane bioreceptors are a biosensing technology that immobilizes biorecognition elements such as enzymes, antibodies, and receptor proteins on a membrane carrier material for the specific recognition and detection of target analytes. This method not only retains the high specificity of biorecognition but also improves the stability and reusability of the recognition elements by utilizing the membrane structure, maintaining good detection performance even in complex matrices.

[0008] The combination of fiber optic sensors and enzyme membrane biological receptors makes it possible to achieve specific recognition of veterinary drugs. Summary of the Invention

[0009] To overcome the shortcomings of existing technologies, this study proposes a trace amoxicillin sensor based on a β-lactamase-based biorecognition film combined with TFBG-SPR. Surface plasmon resonance (SPR) is achieved by sputtering a 50 nm thick gold film onto a tilted fiber Bragg grating surface, followed by layer-by-layer modification of the gold film surface with a polydopamine biocompatible immobilization platform. This amplifies the polyaniline signal and enables specific recognition by β-lactamase, thus achieving the specific detection of amoxicillin. The specific steps include: S1. Fabrication of tilted fiber gratings and sputtering of gold films; S2. Preparation and layered modification of β-lactamase-based biorecognition membranes; Connection of S3, broadband light source (1), polarization controller (2), TFBG-SPR sensor (5), and spectrometer (6); S4, detection of amoxicillin concentration and specificity.

[0010] The tilted fiber Bragg grating in step S1 is etched using a phase mask. After fabrication, it is placed in a magnetron sputtering deposition chamber for gold film deposition, ultimately depositing a gold film with a thickness of about 50 nm on the surface of the tilted fiber Bragg grating.

[0011] In step S2, the bottom layer polydopamine, the middle layer polyaniline, and the top layer β-lactamase in the β-lactamase-based biorecognition membrane were prepared by using dopamine hydrochloride, Tris-HCl buffer at pH 8.5, 1M phosphate, aniline, ammonium persulfate, β-lactamase lyophilized powder, and PBS buffer at pH 7.4, respectively.

[0012] Specifically, polydopamine undergoes oxidative self-polymerization under weakly alkaline conditions, forming a dense polydopamine film on the gold membrane surface. This polydopamine layer, rich in catechol and amino functional groups, exhibits excellent adhesion and biocompatibility, enabling it to firmly adhere to the gold membrane surface while providing a high density of active binding sites for subsequent enzyme molecule immobilization. This layer acts like a bio-glue, achieving a stable connection between the enzyme membrane and the inorganic sensing interface, and its hydrophilicity reduces interference from non-specific adsorption in the sample.

[0013] Based on polydopamine modification, a polyaniline layer was introduced via in-situ polymerization. Polyaniline, a typical conductive polymer, possesses excellent electron transport capabilities and good biocompatibility. Its introduction has a dual effect: firstly, the porous structure of polyaniline increases the specific surface area, enabling it to load more β-lactamase molecules and improve recognition site density; secondly, polyaniline can regulate the interfacial electron distribution. When enzyme-substrate binding triggers a conformational change, polyaniline translates this biorecognition into a change in the interfacial dielectric constant, thereby amplifying the SPR signal response amplitude.

[0014] A fiber optic sensor modified with polydopamine@polyaniline was immersed in a phosphate buffer containing β-lactamase. The enzyme molecules were immobilized on the polyaniline surface through physical adsorption, covalent cross-linking, or affinity interactions. The catechol groups in the polydopamine layer can undergo Michael addition or Schiff base reactions with the amino groups on the enzyme molecules, achieving stable covalent immobilization of the enzyme. The porous structure of polyaniline provides a suitable microenvironment for the enzyme molecules, helping to maintain their native conformation and biological activity. This ultimately forms a complete β-lactamase-functionalized TFBG-SPR sensor.

[0015] When the sensor is immersed in a sample solution containing amoxicillin, the amoxicillin molecules diffuse to the sensing interface and specifically bind to the β-lactamase immobilized in the enzyme membrane.

[0016] In step S3, the broadband light source (1), polarization controller (3), TFBG-SPR sensor (5), and spectrometer (6) are connected as follows: the broadband light source (1) is connected to the left end of the polarization controller (3) through a single-mode fiber (2), the polarization controller (3) is connected to the TFBG-SPR sensor (5), the TFBG-SPR sensor (5) is fixed in the flow cell (4), and the right end is connected to the spectrometer (6); the broadband light source (1) is used to provide light source, the polarization controller (3) is used to adjust p-state polarization, the flow cell (4) is used to add the amoxicillin solution to be tested, and the spectrometer (6) is used to monitor and record the spectral changes; when the β-lactamase modified on the surface of TFBG-SPR (5) binds to the amoxicillin in the solution to be tested, the refractive index changes, which is manifested as a shift in the transmission spectrum on the spectrometer (6). By comparing the relationship between different amoxicillin concentrations and the transmission spectrum shift, the concentration of amoxicillin can be detected.

[0017] Preferably, the wavelength of the broadband light source (1) is 1420~1620nm.

[0018] The amoxicillin concentration detected in step S4 is adjusted to: 10 -13 M, 10 -12 M, 10-11 M, 10 -10 M, 1 0-9 M, 10 - 8 M, 10 -7 M.

[0019] The biomolecules included in the specific detection in step S4 are: chloramphenicol, oxytetracycline hydrochloride, doxycycline, and cephalexin.

[0020] The working principle of the trace amoxicillin sensor based on a β-lactamase-infused biorecognition film combined with TFBG-SPR prepared in this invention is as follows: When light waves enter an optical fiber and are completely reflected by the fiber sidewalls, the tilted fiber grating can also excite evanescent surface plasmon resonance waves on the gold surface. When the p-polarized component of the light enters the gold film, the free electrons of the gold film interact with the p-polarized component of the light, generating plasma. At the interface between the optical fiber and the gold film, plasma vibration forms surface plasmon waves. When the horizontal component of the evanescent wave vector matches the wave vector of the surface plasmon wave and energy transfer occurs, the evanescent wave and the surface plasmon wave resonate, generating surface plasmon resonance. Light energy near the resonance wavelength is absorbed, resulting in a resonance valley in the output spectrum. The β-lactamase on the surface of the tilted fiber grating binds to amoxicillin in the test solution, causing a change in the local refractive index, and the resonance valley and resonance wavelength also change. Therefore, the concentration of amoxicillin can be inferred by monitoring the shift of the resonance wavelength.

[0021] In summary, this invention provides a TFBG-SPR sensor capable of specifically detecting amoxicillin concentration. This sensor is based on a β-lactamase biorecognition membrane that can specifically bind to amoxicillin, altering the effective refractive index and causing a shift in the transmission spectrum, thereby enabling the detection of amoxicillin. It has advantages such as simple structure, low detection limit, and fast response time.

[0022] The beneficial effects of this invention are as follows: First, a thin gold film is deposited on the surface of a tilted fiber grating to excite the SPR effect. Then, a polydopamine adhesion layer, a polyaniline signal amplification layer, and a β-lactamase recognition layer are sequentially modified onto the gold film surface, constructing a β-lactamase-based composite thin-film sensing interface. The precise binding of amoxicillin molecules to the β-lactamase active sites on the sensor surface causes a local change in the refractive index of the sensor surface, thereby achieving highly sensitive detection of amoxicillin. The dynamic detection range of this sensor is 10... -13 M-10 -7 The response sensitivity is 0.462 dB / (M), and the linear correlation coefficient R² is 0.98011. The detection limit is 2.95 × 10⁻⁶. -15With a response time of approximately 4.9 minutes and good stability, this sensor, as an amoxicillin sensor with extremely low detection limits and fast response speed, undoubtedly has significant potential application value in areas such as environmental pollution and food health monitoring. Attached Figure Description

[0023] Figure 1 A schematic diagram of an experimental setup for amoxicillin-sensing TFBG-SPR spectral measurement.

[0024] Figure 2 Transmission spectra of the TFBG-SPR sensor in amoxicillin solutions of different concentrations.

[0025] Figure 3 Specific response diagram of the TFBG-SPR sensor for amoxicillin detection. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to specific embodiments:

[0027] Example 1: Fabrication of tilted fiber Bragg grating and sputtering of gold film.

[0028] In this embodiment, see Figure 1 First, a 1 cm photosensitive fiber (51) was fused into a single-mode fiber, and the fiber structure was placed in a reactor at 15.2 MPa and 20 °C, where hydrogen was injected for 14 days of hydrogen loading treatment. Then, the hydrogen-loaded fiber structure was placed in front of a phase mask with an 8° tilt angle, and a precision displacement stage was used to write high-energy pulsed ultraviolet laser light generated by a frequency-doubled argon-ion laser onto the photosensitive fiber in the fiber structure through the phase mask. Finally, the prepared tilted fiber grating was annealed in a constant temperature oven at 120 °C for 12 hours to release residual hydrogen in the photosensitive fiber. The prepared tilted fiber grating was then cleaned with anhydrous alcohol and placed in a magnetron sputtering deposition chamber for gold film deposition, ultimately depositing a gold film with a thickness of approximately 50 nm on the surface of the tilted fiber grating.

[0029] Example 2: Preparation and layered modification of biorecognition membranes based on β-lactamase.

[0030] In this embodiment, the preparation and modification of the biorecognition film that can specifically bind to amoxicillin are carried out through the following specific steps: a) Fiber pretreatment. Place the TFBG in an ethanol solution, shake and wash for 10 minutes, then rinse thoroughly with plenty of deionized water and dry for later use. b) Preparation of the polydopamine substrate. Dopamine hydrochloride was dissolved in 10 mM Tris-HCl buffer to prepare a 2 mg / mL solution. The pretreated TFBG probe was completely immersed in the polydopamine solution and allowed to stand at room temperature in the dark for 2 h. Finally, the TFBG was removed and rinsed with deionized water to remove the physically adsorbed polydopamine particles on the surface. After drying, TFBG-SPR@polydopamine was obtained. c) Preparation of the polyaniline interlayer. Aniline was dissolved in 1M phosphoric acid solution to prepare a 50mM aniline solution. Ammonium persulfate was dissolved in 1M phosphoric acid solution to prepare a 50mM ammonium persulfate solution. 5 mL of each solution was mixed and allowed to stand for 15 minutes. The TFBG-SPR@polydopamine probe was completely immersed in the mixture and allowed to stand at room temperature for 8 minutes. Finally, the probe was removed and rinsed with deionized water, followed by PBS buffer, and dried to obtain TFBG-SPR@polydopamine@polyaniline.

[0031] d) β-Lactamase Immobilization. A 120 IU / mL β-lactamase solution was prepared using pre-cooled PBS buffer (4°C). The TFBG-SPR@polydopamine@polyaniline probe was immersed in the enzyme solution and incubated at 4°C for 2 hours. The probe was then removed and rinsed with pre-cooled PBS buffer to obtain the TFBG-SPR@polydopamine@polyaniline@β-lactamase sensor. The prepared probe was then immersed in PBS buffer and stored at 4°C.

[0032] Example 3: Construction of the detection platform.

[0033] In this embodiment, see Figure 1 The detection platform is a single-mode optical fiber (2) connecting a broadband light source (1), a polarization controller (3), a TFBG-SPR sensor (5) placed in a flow cell (4), and a spectrometer (6). The wavelength range of the broadband light source (1) is 1420~1620nm, which is used to provide light source. The polarization controller (3) is used to obtain greater stripe contrast. The flow cell (4) is used to add the amoxicillin solution to be tested. The spectrometer (6) is used to monitor and record the spectral changes. When the β-lactamase modified on the surface of TFBG-SPR (5) binds to the amoxicillin in the solution to be tested, the refractive index changes, which is reflected in the drift of the transmission spectrum on the spectrometer (6). By comparing the relationship between different amoxicillin concentrations and the transmission spectrum drift, the concentration of amoxicillin can be detected.

[0034] Example 4: Detection of amoxicillin concentration and specificity.

[0035] Amoxicillin solutions and specificity tests were performed; all drug solutions were prepared with deionized water.

[0036] Amoxicillin concentration detection: The β-lactamase-functionalized TFBG-SPR sensor was placed in the flow cell (4), and amoxicillin solutions of different concentrations were prepared using deionized water, with a concentration of 10... -13 M, 10 -12 M, 10 -11 M, 10 -10 M, 1 0 -9 M, 10 -8 M, 10 -7 M. Deionized water was added to the flow cell (4), and the initial spectrum was recorded using a spectrometer (6). Then, amoxicillin solution was added to the flow cell (4) and allowed to stand. The transmission spectrum was then recorded using a spectrometer (6). After the detection was completed, the liquid in the flow cell (4) was drained, and deionized water was introduced to rinse the surface of the optical fiber and the flow cell (4). Then, another test solution was added, and the operation was repeated to complete the spectral acquisition in sequence. The transmission spectra of amoxicillin solutions of different concentrations were obtained, differential fitting was performed, the intensity difference and fitting curve were plotted, and the detection limit was calculated. Figure 2 As shown.

[0037] Specific detection: 10 were prepared respectively. -10 The test solutions for chloramphenicol, oxytetracycline hydrochloride, doxycycline, and cephalexin were used. First, the transmission spectrum of the TFBG-SPR sensor in deionized water was measured. The optical fiber was rinsed multiple times with deionized water. The test solutions were added to the flow cell and allowed to stand. The transmission spectra were then recorded using a spectrometer (6). After the test, the liquid in the flow cell (4) was drained, and deionized water was introduced to rinse the surface of the optical fiber and the flow cell (4). Different test solutions were added sequentially, and the above steps were repeated to evaluate the specificity of the TFBG-SPR sensor for different drugs, such as… Figure 3 As shown.

[0038] The embodiments described above provide a detailed explanation of the technical solution of the present invention, but the scope of protection of this disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of the present invention.

Claims

1. A fiber optic method for detecting amoxicillin concentration based on β-lactamase functionalization, characterized in that: The detection device consists of a broadband light source (1), a single-mode fiber (2), a polarization controller (3), a flow cell (4), a TFBG-SPR sensor (5), and a spectrometer (6); the broadband light source (1) is connected to the input end of the polarization controller (3) through the single-mode fiber (2); the output end of the polarization controller (3) is connected to the left end of the TFBG-SPR sensor (5), and the right end of the TFBG-SPR sensor (5) is connected to the spectrometer (6); The fabrication steps of the TFBG-SPR sensor (5) are as follows: First, hydrogen is loaded onto the photosensitive fiber (51) and then a tilted fiber grating (52) with a tilt angle of 8 degrees is formed by ultraviolet etching and annealing. Then, the tilted fiber grating (52) is placed in a magnetron sputtering deposition chamber and a gold film with a thickness of 50 nm is deposited on its surface. A bio-recognition film (53) based on β-lactamase is modified on the gold film on the surface of the tilted fiber grating (52). The preparation steps of the β-lactamase-based biorecognition film (53) are as follows: a biocompatible immobilization platform is constructed on the TFBG-SPR sensing interface by utilizing the self-polymerization properties of polydopamine, and the signal is amplified by polyaniline. β-lactamase is introduced as a recognition enzyme and modified layer by layer onto the surface of the tilted fiber grating (52) to form a biorecognition film (53) that can specifically recognize amoxicillin β-lactamase. The steps of the fiber-optic amoxicillin concentration detection method are as follows: First, fix the TFBG-SPR sensor (5) in the flow cell (4), then add the amoxicillin test solution to the flow cell (4). The amoxicillin binds to the β-lactamase biorecognition film (53) modified on the surface of the TFBG-SPR sensor (5). The light emitted by the broadband light source (1) passes through the TFBG-SPR sensor (5) and the transmission spectrum is recorded on the spectrometer (6). The flow cell (4) is cleaned with deionized water. The data recorded on the spectrometer (6) with amoxicillin solutions of different concentrations added to the flow cell (4) are fitted to obtain the relationship between different amoxicillin concentrations and transmission spectrum drift, thereby realizing the concentration detection of amoxicillin.

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