Construction method of aptamer modified photonic crystal sensor for visual detection of oxytetracycline

Abstract

The invention discloses a construction method of an aptamer modified photonic crystal sensor for visually detecting oxytetracycline, and belongs to the technical field of sensing. In the invention, a photonic crystal array obtained by a vertical evaporation self-assembly method is combined with polyacrylamide hydrogel to form the photonic crystal hydrogel membrane material. Gold nanoparticles serve as a bridge, one side of each gold nanoparticle is connected with an amide framework of the hydrogel through chelation, the other side of each gold nanoparticle is connected with the oxytetracycline aptamer through covalent bonding, and the aptamer modified photonic crystal sensor is constructed. The oxytetracycline aptamer and oxytetracycline are highly selectively combined through pi-pi stacking and hydrogen bonds, so that the hydrogel swells, the lattice spacing of the photonic crystal is increased, a reflected signal and a structural color are subjected to red shift, and high-selectivity detection of the oxytetracycline is realized. The rapid detection requirements in the fields of food safety, environment monitoring, biological medicine and the like are met. The linear range of oxytetracycline detection is 0.1 mu g / mL-80 mu g / mL, and the lowest detection limit is 0.033 mu g / mL.

Description

Technical Field

[0001] The present invention belongs to the technical field of optical sensing, and relates to a construction method and application of an optical aptamer-modified photonic crystal sensor for oxytetracycline detection. Background Art

[0002] Oxytetracycline, a broad-spectrum antibiotic, is widely used in animal husbandry and aquaculture, but its residues pose a potential threat to both human health and the ecological environment. Currently, traditional detection methods such as chromatography and mass spectrometry, while highly accurate, are complex, costly, and time-consuming, making them unsuitable for rapid on-site testing. Therefore, developing a simple, rapid, and sensitive method for detecting oxytetracycline is of great significance.

[0003] Photonic crystals are widely used in the visual detection of chemical substances because of their unique Bragg diffraction effect that shows bright color changes. Hydrogel is a three-dimensional network polymer material that can absorb a large amount of water and maintain a hydrated state. It has good biocompatibility and adjustability, can respond to changes in external stimuli (such as temperature, pH, ionic strength, etc.), and exhibits certain intelligent behaviors. Therefore, it has shown broad application prospects in drug delivery, wound repair, tissue engineering, etc. Aptamers refer to single-stranded DNA or RNA molecules obtained from a synthetic library through in vitro screening technology and have specific molecular recognition capabilities. They can specifically recognize and bind to target molecules, so they have the advantages of good stability, easy synthesis and modification. Based on the combination of photonic crystals and hydrogels, the regulation of optical signals and the enhancement of biosensing performance can be achieved, and the introduction of aptamers provides a guarantee for the improvement of targeting and sensitivity.

[0004] Based on the stable and adjustable reflected light signal provided by photonic crystals, gold nanoparticles are used as a bridge to introduce oxytetracycline aptamer into the photonic crystal gel skeleton to establish a photochemical sensing platform for the specific optical detection of oxytetracycline, but there have been no related reports. Summary of the Invention

[0005] The present invention aims to provide an aptamer-modified photonic crystal optical sensor that combines high sensitivity, high selectivity, and naked-eye detection. The sensor, with low production cost and simple manufacturing process, enables rapid, visual, semi-quantitative, and optically quantitative detection of OTC.

[0006] The proposed approach is summarized as follows: using photonic crystal arrays as the optical material, polyacrylamide gel as the stimuli-responsive medium, and gold nanoparticles as the bridge, aptamers are bound to the photonic crystal gel backbone, introducing specific binding sites to establish a highly selective optical sensing platform. Π-Π stacking and hydrogen bonding interactions between the aptamers and OTC enable highly selective binding, which causes hydrogel swelling, an increase in the photonic crystal lattice spacing, and a red shift in the reflected signal and structural color. By establishing a relationship between the logarithm of OTC concentration and the spectral peak position, highly sensitive and selective quantitative detection of oxytetracycline is achieved. The relationship between the change in structural color and OTC concentration enables rapid semi-quantitative detection of OTC.

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

[0008] A method for constructing a ligand-modified photonic crystal sensor for optical detection of oxytetracycline comprises the following steps:

[0009] Step 1: Prepare a three-dimensional densely packed photonic crystal array with blue structural color:

[0010] Anhydrous ethanol, tetraethyl orthosilicate, and deionized water were mixed and magnetically stirred to obtain solution A. Solution A was placed in a round-bottom flask, and ammonia water was added to carry out a sol-gel reaction under magnetic stirring. After the reaction was completed, the precipitate was washed by centrifugation with ethanol and water to obtain monodisperse silica microspheres. A monodisperse silica sphere solution was prepared with ethanol, and the silica spheres were self-assembled on a hydrophilic pretreated glass sheet by vertical deposition to form a three-dimensional close-packed photonic crystal array.

[0011] Step 2: Preparation of three-dimensional photonic crystal gel film modified with gold nanoparticles AuNPs:

[0012] The AuNPs solution was mixed with a polyacrylic acid (PAM) prepolymer solution to obtain a prepolymer mixture B, which was then added to the "sandwich" space formed above the three-dimensional close-packed photonic crystal array and polymerized under ultraviolet light to obtain an AuNPs-modified three-dimensional photonic crystal gel film.

[0013] Step 3: Preparation of oxytetracycline aptamer-modified photonic crystal sensor:

[0014] The AuNPs-modified three-dimensional photonic crystal gel membrane was placed in an oxytetracycline aptamer C solution for incubation and then washed with a Tris-HCl buffer solution to finally obtain an oxytetracycline aptamer-modified photonic crystal sensor.

[0015] In step 1, the ratio of anhydrous ethanol, tetraethyl orthosilicate, deionized water, and ammonia water in solution A is 250 mL: 15 mL: 10 mL: 11-16 mL; the temperature of the sol-gel reaction is 25-35° C., and the reaction time is 2-10 hours;

[0016] In step 1, the mass of the monodisperse silicon sphere ethanol solution is 1% to 2%, the self-assembly temperature is 30 to 60° C., and the self-assembly time is 30 to 60 hours, depending on the complete evaporation of the solvent.

[0017] In step 2, the AuNPs solution was prepared by mixing 1% HAuCl4 (1 mL) with deionized water (200 mL) and heating to a slight boil, and then quickly adding 1% sodium citrate (6 mL). After heating for 1 hour, stirring was continued until it cooled to room temperature.

[0018] In step 2, in the solution B, the dosage ratio of AuNPs solution, acrylamide AM, N,N'-methylenebisacrylamide BIS, 2,2-diethoxyacetophenone DEAP solution (DEAP: dimethyl sulfoxide DMSO = 1:9) and water is: 0.5 mL: 0.36 g: 0.04-0.04 g: 14 μL: 1 mL.

[0019] In step 2, the "sandwich" space is composed of an upwardly placed three-dimensional photonic crystal array substrate as the bottom, a clean bare substrate as the top, and 150 μm thick sealing films placed on both sides as spacer layers, forming a "sandwich" space of 2 cm × 1 cm × 150 μm; the volume of the added prepolymer mixture B is 300 μL; the wavelength of the light for the ultraviolet light polymerization reaction is 365 nm; and the polymerization time is 60 to 135 minutes.

[0020] In step 3, in solution C, the oxytetracycline OTC aptamer sequence is 5'-SH-ACG ACA TTC CGT TGATCT CTC CCT TTT GGG TTG GTG TCG T-3'; the OTC aptamer concentration is 1 to 100 μmol / L, the incubation temperature is 4°C, and the incubation time is 4 to 24 hours; and the concentration of the Tris-HCl buffer solution is 50 mmol / L.

[0021] The aptamer-modified photonic crystal sensor constructed by the present invention is used to detect oxytetracycline. The aptamer-modified photonic crystal sensor is immersed in oxytetracycline OTC solutions of different concentrations and is immersed at room temperature for 4 hours. The optical fiber probe of the optical fiber spectrometer is directly irradiated on the flat photonic crystal sensor in a vertical direction, and the reflection spectrum signals generated at different times and different concentrations of oxytetracycline are recorded. A curve is plotted based on the obtained reflection spectrum signal peak position and the logarithm of the oxytetracycline concentration.

[0022] The OTC solution is prepared from a 50 mM Tris-HCl buffer solution with a pH of 8.0, the concentration of the OTC solution is 0.1 μg / mL to 80 μg / mL, the volume of the OTC solution is 10 mL, and the soaking equilibrium time is 4 hours.

[0023] The aptamer-modified photonic crystal sensor constructed by the construction method of the present invention is used to detect oxytetracycline analogs. The aptamer-modified photonic crystal sensor is immersed in a mixture of dihydroartemisinin, fluquinoxaline, kanamycin A, sulfamethoxazole, tetracycline, oxytetracycline and antibiotics at the same concentration and soaked at room temperature for 4 hours; the optical fiber probe of the optical fiber spectrometer is directly irradiated in a vertical direction on the flat photonic crystal sensor, the generated reflection spectrum signal is recorded, and a curve is plotted based on the obtained reflection spectrum signal peak position and the logarithm of the oxytetracycline concentration.

[0024] The concentration of all antibiotic solutions was 40 μg / mL, the solution volume was 10 mL, and the soaking equilibrium time was 4 hours.

[0025] The beneficial effects of the present invention are:

[0026] The present invention successfully established an aptamer-modified photonic crystal optical sensing platform and established an optical and visual detection method for oxytetracycline. Its characteristics and advantages are described as follows:

[0027] (1) The present invention prepares a three-dimensional photonic crystal array based on silicon spheres as an optical platform to provide reflection spectrum and structural color signals, and constructs an aptamer-modified photonic crystal sensor with a green structural base color.

[0028] (2) The present invention designs an aptamer structure that can specifically bind to oxytetracycline, thereby improving the selectivity of the optical sensor for oxytetracycline.

[0029] (3) The present invention realizes the visual semi-quantitative and optical quantitative detection of oxytetracycline by constructing a photonic crystal sensor. In the concentration range of 0.1 μg / mL-80 μg / mL, the logarithm of the OTC concentration (log c OTC ) showed a good linear relationship with the wavelength of the reflectance spectrum, and the detection limit could reach 0.033 μg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Scanning electron microscopy images of three-dimensional photonic crystal arrays and aptamer-modified three-dimensional photonic crystal gel sensors;

[0031] Figure 2 is the UV absorption spectrum of the gold seed solution;

[0032] Figure 3a is the UV absorption spectrum of the aptamer solution before and after immersion in the three-dimensional photonic crystal gel sensor. Figure 3 b is the elemental analysis of the prepared aptamer-modified three-dimensional photonic crystal gel sensor under a scanning electron microscope;

[0033] Figure 4 a is a bar graph comparing the spectral peak red shift of the prepared aptamer-modified three-dimensional photonic crystal gel sensor (ACCH) and the control group (CCH: photonic crystal membrane without aptamers but connected with gold nanoparticles; ACCH without AuNPs: photonic crystal membrane without gold nanoparticles but soaked with aptamers; CCH without AuNPs: photonic crystal membrane without gold nanoparticles and aptamers) detecting 100 μg / mL oxytetracycline solution. Figure 4 b is the kinetic curve of the prepared aptamer-modified photonic crystal sensor in 100 μg / mL oxytetracycline solution;

[0034] Figure 5 The red shift of the spectral peak of the aptamer-modified three-dimensional photonic crystal gel sensor in oxytetracycline solutions with different concentrations and the changes in the structural color of the sensor;

[0035] Figure 6 The spectral red shift of the aptamer-modified three-dimensional photonic crystal gel sensor in different types of antibiotics and blended samples. DETAILED DESCRIPTION

[0036] The present invention will be further described below with reference to the embodiments. The protection scope of the present invention is not limited to the embodiments, and all changes made to the technical solution of the present invention by professionals in this field fall within the protection scope of the present invention.

[0037] Example 1

[0038] (1) 250 mL of anhydrous ethanol, 15 mL of TEOS, and 10 mL of deionized water were added to a flask in sequence. The mixture was stirred magnetically in a water bath at 25°C for 5 min (990 rpm). Subsequently, 12 mL of aqueous ammonia was added to the mixture, the bottle was sealed with a sealing film, and the reaction was carried out at 990 rpm for 10 h to obtain monodisperse silica microspheres. The mixture was washed once with anhydrous ethanol and three times with deionized water. Finally, the mixture was dispersed in 20 mL of deionized water and stored for later use. Monodisperse silica microspheres with a particle size of 200 nm were obtained.

[0039] (2) The coverslip was soaked in piranha solution (concentrated sulfuric acid: hydrogen peroxide = 7:3, V:V) for 12 h, washed and dried for later use, and silica microspheres were dispersed in anhydrous ethanol and adjusted to 1% by mass. The treated coverslip was then placed vertically in the solution and placed in an oven at 60 °C for 30 h. After the solvent was completely evaporated, a monochromatic film grew on the surface of the coverslip, realizing the preparation of a three-dimensional densely packed photonic crystal array.

[0040] (3) 1% HAuCl4 (1 mL) was mixed with deionized water (200 mL) and heated to a slight boil, and then 1% sodium citrate (6 mL) was quickly added. After heating for one hour, stirring was continued until it cooled to room temperature to prepare the AuNPs solution.

[0041] (4) 0.18 g acrylamide AM, 0.005 g N,N'-methylenebisacrylamide BIS, 14 μL 10% 2,2-diethoxyacetophenone DEAP solution (50 μL DEAP: 450 μL dimethyl sulfoxide DMSO) and 0.0202 g acrylic acid AA were dissolved in 1 mL water, and 0.5 mL AuNPs solution was added thereto to obtain a gel prepolymerization mixture.

[0042] (5) A 150 μm thick spacer layer was fixed on both sides of the short side of the three-dimensional photonic crystal slide, and another glass slide was fixed above the array and the spacer layer to form a 2 cm × 6 cm × 150 μm space sandwich above the photonic crystal array. Subsequently, 150 μL of gel prepolymer mixture was poured into the space sandwich and polymerized under ultraviolet light with a wavelength of 365 nm at room temperature for 2 h to obtain an AuNPs-modified three-dimensional photonic crystal gel film.

[0043] (6) After the AuNPs-modified three-dimensional photonic crystal gel membrane was peeled off, it was cut into small pieces of membrane with a size of 0.5 cm × 0.5 cm. The membrane was placed in a 10 μmol / L oxytetracycline aptamer solution and incubated for 12 hours at 4 degrees Celsius. It was then washed with 50 mM Tris-HCl to realize the construction of the oxytetracycline aptamer-modified photonic crystal sensor (ACCH).

[0044] The microstructures of the three-dimensional photonic crystal array and aptamer-modified photonic crystal gel obtained by the method of Example 1 of the present invention were characterized by field emission scanning electron microscopy. Figure 1 ; Figure 1 It shows that the prepared silica microspheres have uniform particle size, and the three-dimensional photonic crystal array obtained by self-assembly arrangement presents a face-centered cubic close-packed structure. After in situ polymerization of the gel and introduction of the aptamer, the modified photonic crystal gel obtained undergoes a swelling effect, and the photonic crystal microstructure becomes a non-close-packed conformation, but still has a good periodic arrangement structure.

[0045] The absorption spectrum of the AuNPs solution obtained in step (3) of the method of Example 1 of the present invention was tested, and the results were shown in FIG. Figure 2 The absorption spectrum showed an obvious single peak at 550 nm, which indicated that the AuNPs were successfully synthesized and well dispersed in water with good dispersibility.

[0046] The absorption spectrum and elemental analysis were used to characterize the introduction of the aptamer modified photonic crystal gel membrane prepared in Example 1. The absorption spectrum of the aptamer solution before and after incubation of the photonic crystal gel membrane was characterized. Figure 3 a. Figure 3 a shows that after incubating the photonic crystal gel membrane, the concentration of the aptamer solution was significantly reduced. The elements of the modified gel membrane were characterized under a scanning electron microscope. The results are shown in Figure 3 b. It can be observed that the Au element representing the presence of AuNPs and the P element representing the presence of the aptamer are evenly distributed on the gel, and the positions of the Au and P elements basically correspond to each other, which indicates that the aptamer has successfully bound to the AuNPs in the gel film, indicating the successful introduction of the aptamer into the photonic crystal gel film.

[0047] Example 2

[0048] (1) 250 mL of anhydrous ethanol, 15 mL of TEOS, and 10 mL of deionized water were added to a flask in sequence. The mixture was stirred magnetically in a water bath at 25°C for 5 min (990 rpm). Subsequently, 12 mL of aqueous ammonia was added to the mixture, the bottle was sealed with a sealing film, and the reaction was carried out at 990 rpm for 10 h to obtain monodisperse silica microspheres. The mixture was washed once with anhydrous ethanol and three times with deionized water. Finally, the mixture was dispersed in 20 mL of deionized water and stored for later use. Monodisperse silica microspheres with a particle size of 200 nm were obtained.

[0049] (2) The coverslip was soaked in piranha solution (concentrated sulfuric acid: hydrogen peroxide = 7:3, V:V) for 12 h, washed and dried for later use, and silica microspheres were dispersed in anhydrous ethanol and adjusted to 1% by mass. The treated coverslip was then placed vertically in the solution and placed in an oven at 60 °C for 30 h. After the solvent was completely evaporated, a monochromatic film grew on the surface of the coverslip, realizing the preparation of a three-dimensional densely packed photonic crystal array.

[0050] (3) 1% HAuCl4 (1 mL) was mixed with deionized water (200 mL) and heated to a slight boil, and then 1% sodium citrate (6 mL) was quickly added. After heating for one hour, stirring was continued until it cooled to room temperature to prepare the AuNPs solution.

[0051] (4) 0.18 g acrylamide AM, 0.005 g N,N'-methylenebisacrylamide BIS, 14 μL 10% 2,2-diethoxyacetophenone DEAP solution (50 μL DEAP: 450 μL dimethyl sulfoxide DMSO), and 0.0202 g acrylic acid AA were dissolved in 1 mL water, and 0.5 mL AuNPs solution was added to obtain a gel prepolymerization mixture. (5) A 150 μm thick spacer layer was fixed on both sides of the short side of the 3D photonic crystal slide, and another glass slide was fixed on top of the array and the spacer layer to form a 2 cm × 6 cm × 150 μm spacer layer above the photonic crystal array. Subsequently, 150 μL of the gel prepolymerization mixture was poured into the spacer layer and polymerized under ultraviolet light with a wavelength of 365 nm at room temperature for 2 h to obtain an AuNPs-modified 3D photonic crystal gel film (CCH).

[0052] Example 3

[0053] (1) 250 mL of anhydrous ethanol, 15 mL of TEOS, and 10 mL of deionized water were added to a flask in sequence. The mixture was stirred magnetically in a water bath at 25°C for 5 min (990 rpm). Subsequently, 12 mL of aqueous ammonia was added to the mixture, the bottle was sealed with a sealing film, and the reaction was carried out at 990 rpm for 10 h to obtain monodisperse silica microspheres. The mixture was washed once with anhydrous ethanol and three times with deionized water. Finally, the mixture was dispersed in 20 mL of deionized water and stored for later use. Monodisperse silica microspheres with a particle size of 200 nm were obtained.

[0054] (2) The coverslip was soaked in piranha solution (concentrated sulfuric acid: hydrogen peroxide = 7:3, V:V) for 12 h, washed and dried for later use, and silica microspheres were dispersed in anhydrous ethanol and adjusted to 1% by mass. The treated coverslip was then placed vertically in the solution and placed in an oven at 60 °C for 30 h. After the solvent was completely evaporated, a monochromatic film grew on the surface of the coverslip, realizing the preparation of a three-dimensional densely packed photonic crystal array.

[0055] (3) 0.18 g of acrylamide AM, 0.005 g of N,N'-methylenebisacrylamide BIS, 14 μL of 10% 2,2-diethoxyacetophenone DEAP solution (50 μL DEAP: 450 μL dimethyl sulfoxide DMSO) and 0.0202 g of acrylic acid AA were dissolved in 1 mL of water, and 0.5 mL of aqueous solution was added thereto to obtain a gel prepolymer mixture.

[0056] (4) A 150 μm thick spacer layer was fixed on both sides of the short side of the three-dimensional photonic crystal slide, and another glass sheet was fixed on top of the array and the spacer layer to form a 2 cm × 6 cm × 150 μm space sandwich above the photonic crystal array. Subsequently, 150 μL of gel prepolymer mixture was poured into the space sandwich and polymerized under ultraviolet light with a wavelength of 365 nm at room temperature for 2 h to obtain a three-dimensional photonic crystal gel film without AuNPs modification.

[0057] (6) After peeling off the three-dimensional photonic crystal gel membrane without AuNPs modification, it was cut into small pieces of membrane with a size of 0.5 cm × 0.5 cm. The membrane was placed in a 10 μmol / L oxytetracycline aptamer solution and incubated for 12 hours at 4 degrees Celsius. It was then washed with 50 mM Tris-HCl to obtain a photonic crystal sensor with oxytetracycline aptamer physically adsorbed (ACCH-without AuNPs).

[0058] Example 4

[0059] (1) 250 mL of anhydrous ethanol, 15 mL of TEOS, and 10 mL of deionized water were added to a flask in sequence. The mixture was stirred magnetically in a water bath at 25°C for 5 min (990 rpm). Subsequently, 12 mL of aqueous ammonia was added to the mixture, the bottle was sealed with a sealing film, and the reaction was carried out at 990 rpm for 10 h to obtain monodisperse silica microspheres. The mixture was washed once with anhydrous ethanol and three times with deionized water. Finally, the mixture was dispersed in 20 mL of deionized water and stored for later use. Monodisperse silica microspheres with a particle size of 200 nm were obtained.

[0060] (2) The coverslip was soaked in piranha solution (concentrated sulfuric acid: hydrogen peroxide = 7:3, V:V) for 12 h, washed and dried for later use, and silica microspheres were dispersed in anhydrous ethanol and adjusted to 1% by mass. The treated coverslip was then placed vertically in the solution and placed in an oven at 60 °C for 30 h. After the solvent was completely evaporated, a monochromatic film grew on the surface of the coverslip, realizing the preparation of a three-dimensional densely packed photonic crystal array.

[0061] (3) 0.18 g of acrylamide AM, 0.005 g of N,N'-methylenebisacrylamide BIS, 14 μL of 10% 2,2-diethoxyacetophenone DEAP solution (50 μL DEAP: 450 μL dimethyl sulfoxide DMSO) and 0.0202 g of acrylic acid AA were dissolved in 1 mL of water, and 0.5 mL of aqueous solution was added thereto to obtain a gel prepolymer mixture.

[0062] (4) A 150 μm thick spacer layer was fixed on both sides of the short side of the three-dimensional photonic crystal slide, and another glass sheet was fixed on top of the array and the spacer layer to form a 2 cm × 6 cm × 150 μm space sandwich above the photonic crystal array. Subsequently, 150 μL of gel prepolymer mixture was poured into the space sandwich and polymerized under ultraviolet light with a wavelength of 365 nm at room temperature for 2 h to obtain a three-dimensional photonic crystal gel film without AuNPs modification (CCH-without AuNPs).

[0063] Example 5

[0064] The oxytetracycline aptamer-modified photonic crystal sensor (ACCH) prepared in Example 1 was immersed in oxytetracycline solutions (pH 8.0, 50 mM Tris-HCl buffer solution) with different concentrations. The optical fiber probe of the optical fiber spectrometer was directly irradiated on the flat photonic crystal sensor in a vertical direction, and the reflection spectrum signals generated at different times and different concentrations of oxytetracycline were recorded. The curve was plotted based on the peak position of the obtained reflection spectrum signal and the logarithm of the oxytetracycline concentration. The obtained curve is shown in FIG. Figure 5 shown.

[0065] Example 6

[0066] A 40 μg / mL mixture of dihydroartemisinin, fluquinoxaline, kanamycin A, sulfadimethoxine, tetracycline, oxytetracycline and antibiotics was prepared using a pH 8.0, 50 mM Tris-HCl buffer solution. The oxytetracycline aptamer-modified photonic crystal sensor (ACCH) prepared in Example 1 was immersed in different antibiotic solutions for 4 hours and the shift of its optical reflection peak was recorded. Figure 6 It can be seen that the oxytetracycline aptamer-modified photonic crystal sensor has high selectivity for oxytetracycline.

Claims

1. A method for constructing an aptamer-modified photonic crystal sensor for optical detection of oxytetracycline, characterized in that: The steps include: Step 1: Prepare a three-dimensional densely packed photonic crystal array with blue structural color: Anhydrous ethanol, tetraethyl orthosilicate, and deionized water were mixed and magnetically stirred to obtain solution A. Solution A was placed in a round-bottom flask, and ammonia water was added to carry out a sol-gel reaction under magnetic stirring. After the reaction was completed, the precipitate was washed by centrifugation with ethanol and water to obtain monodisperse silica microspheres. A monodisperse silica sphere solution was prepared with ethanol, and the silica spheres were self-assembled on a hydrophilic pretreated glass sheet by vertical deposition to form a three-dimensional close-packed photonic crystal array. Step 2: Preparation of three-dimensional photonic crystal gel film modified with gold nanoparticles AuNPs: The AuNPs solution was mixed with a polyacrylic acid (PAM) prepolymer solution to obtain a prepolymer mixture B, which was then added to the "sandwich" space formed above the three-dimensional close-packed photonic crystal array and polymerized under ultraviolet light to obtain an AuNPs-modified three-dimensional photonic crystal gel film. Step 3: Preparation of oxytetracycline aptamer-modified photonic crystal sensor: The AuNPs-modified three-dimensional photonic crystal gel membrane was placed in an oxytetracycline aptamer C solution for incubation and then washed with a Tris-HCl buffer solution to finally obtain an oxytetracycline aptamer-modified photonic crystal sensor.

2. The construction method according to claim 1, wherein In step 1, the ratio of anhydrous ethanol, tetraethyl orthosilicate, deionized water, and ammonia water in solution A is 250 mL: 15 mL: 10 mL: 11-16 mL; the temperature of the sol-gel reaction is 25-35° C., and the reaction time is 2-10 hours.

3. The construction method according to claim 1, wherein In step 1, the mass of the monodisperse silicon sphere ethanol solution is 1% to 2%, the self-assembly temperature is 30 to 60° C., and the self-assembly time is 30 to 60 hours, depending on the complete evaporation of the solvent.

4. The construction method according to claim 1, wherein In step 2, the AuNPs solution was prepared by mixing 1% HAuCl4 (1 mL) with deionized water (200 mL) and heating to a slight boil, and then quickly adding 1% sodium citrate (6 mL). After heating for 1 hour, stirring was continued until it cooled to room temperature.

5. The construction method according to claim 1, wherein: In step 2, in the solution B, the dosage ratio of AuNPs solution, acrylamide AM, N,N'-methylenebisacrylamide BIS, 2,2-diethoxyacetophenone DEAP solution (DEAP: dimethyl sulfoxide DMSO = 1:9) and water is: 0.5 mL: 0.18 g: 0.005-0.02 g: 14 μL: 1 mL.

6. The construction method according to claim 1, wherein: In step 2, the "sandwich" space is formed with an upwardly placed three-dimensional photonic crystal array substrate as the bottom, a clean bare substrate as the top, and 150 μm thick sealing film placed on both sides as a spacer layer, forming a "sandwich" space of 2 cm × 1 cm × 150 μm; the volume of the added prepolymer mixture B is 300 μL; the wavelength of the light for the ultraviolet light polymerization reaction is 365 nm; and the polymerization time is 60 to 135 minutes.

7. The construction method according to claim 1, wherein: In step 3, in solution C, the oxytetracycline OTC aptamer sequence is 5'-SH-ACG ACA TTC CGT TGA TCT CTC CCT TTT GGG TTG GTG TCG T-3'; the OTC aptamer concentration is 1 to 100 μmol / L, the incubation temperature is 4°C, and the incubation time is 4 to 24 hours; and the concentration of the Tris-HCl buffer solution is 50 mmol / L.

8. Use of the aptamer-modified photonic crystal sensor constructed by the construction method according to any one of claims 1 to 7 for detecting oxytetracycline, characterized in that: The detection steps are: (1) The aptamer-modified photonic crystal sensor was immersed in oxytetracycline OTC solutions of different concentrations at room temperature for 4 hours; (2) The optical fiber probe of the optical fiber spectrometer is extended in a vertical direction and directly irradiated onto the flat photonic crystal sensor, and the reflection spectrum signals generated at different times and different concentrations of oxytetracycline are recorded. A curve is drawn based on the obtained reflection spectrum signal peak position and the logarithm of the oxytetracycline concentration.

9. The use according to claim 8, characterized in that In step (1), the OTC solution is prepared from a 50 mM Tris-HCl buffer solution at pH 8.0, the concentration of the OTC solution is 0.1 μg / mL to 80 μg / mL, the volume of the OTC solution is 10 mL, and the soaking equilibrium time is 4 hours.

10. Use of the aptamer-modified photonic crystal sensor constructed by the construction method according to any one of claims 1 to 7 for detecting oxytetracycline analogs, characterized in that: The detection steps are: (1) The aptamer-modified photonic crystal sensor was immersed in a mixture of dihydroartemisinin, fluquinoxaline, kanamycin A, sulfadimethoxine, tetracycline, oxytetracycline, and antibiotics at the same concentration for 4 hours at room temperature; (2) The optical fiber probe of the optical fiber spectrometer is directly irradiated on the flat photonic crystal sensor in a vertical direction, and the generated reflection spectrum signal is recorded. A curve is drawn based on the obtained reflection spectrum signal peak position and the logarithm of the oxytetracycline concentration.

11. The use according to claim 10, characterized in that In step (1), the concentration of all antibiotic solutions was 40 μg / mL, the solution volume was 10 mL, and the soaking equilibrium time was 4 hours.

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