Construction of three-color photonic crystal sensor for environment detection

Abstract

The invention provides a construction method of a three-color photonic crystal sensor for environmental detection of oxytetracycline. The preparation method comprises the following steps: step 1, preparing monodisperse silicon dioxide microspheres with different sizes; and 2, constructing the three-color photonic crystal sensor on the same substrate through a step-by-step vertical sedimentation method. According to the novel three-color photonic crystal sensor constructed by the invention, the characteristic wavelength can be shifted to different degrees (R: 867-921nm; g: 723 to 771 nm; b: 548 to 601 nm), thereby generating three-color transition (R: light red to red; g: indigo blue-brick yellow; b: purple-blue), and high-precision visual detection of oxytetracycline in the high-ionic-strength water environment is realized. Meanwhile, within the logarithmic linear range of 0.1-80 [mu] g / mL, R2 of R / G / B channels is 0.966 / 0.956 / 0.992, and the on-site structural color change threshold (0.5 [mu] g / mL) meets the aquatic product safety standard. The multicolor photonic crystal sensor provides a new thought for constructing a field environment sensor with laboratory-level quantitative and instant visual reading functions.

Description

Technical Field

[0001] The present invention belongs to the technical field of optical biosensors and relates to a method for constructing a photonic crystal sensor device based on a three-color photonic crystal array to realize multi-color coordinated detection of the environmental pollutant oxytetracycline. Background Art

[0002] Tetracycline antibiotics, characterized by their typical four-ring molecular skeleton, are widely used antimicrobial agents in aquaculture and animal husbandry worldwide. Oxytetracycline (OTC), a representative example, poses a serious threat to the ecological environment and human health due to its residual effects caused by overuse, potentially leading to gastrointestinal toxicity, allergic reactions, and the spread of antibiotic resistance. In recent years, photonic crystals, with their unique optical properties, have become an ideal platform for visual detection. These periodically arrayed nanoparticles can achieve colorimetric sensing through visible light diffraction without the need for complex instrumentation. Currently, colloidal crystal antibiotic detection systems primarily employ two approaches: one is the molecularly imprinted photonic crystal sensing strategy, which primarily combines a template molecule with an optical hydrogel to achieve multi-site recognition of the template molecule or its structural analogs. However, this approach suffers from uneven binding site distribution and insufficient rebinding kinetics. Another strategy involves the design of enzyme-functionalized photonic crystal sensors, which primarily trigger a shift in the diffraction wavelength and a change in the structural color of the optical hydrogel through an enzymatic reaction. Although this enzyme-responsive system has high selectivity, the sensitivity of the enzyme to physiological conditions and the complexity of the immobilization process limit its practical application.

[0003] As a new type of biorecognition element, aptamers have the advantages of high affinity, programmable conformation and excellent thermal stability. At present, the combination of aptamers and photonic crystal gel sensors has been gradually applied to thrombin, Ag + However, these monochromatic photonic crystal sensors still face two major technical bottlenecks: 1) the immobilization of the aptamer requires complex intermediate steps; 2) the monochromatic signal system, due to the limitations of the photonic crystal microstructure in regulation, is difficult to achieve both quantitative detection and visual output. In particular, it cannot meet the combined requirements of specificity, sensitivity, and rapid readout in complex biological matrices.

[0004] The present invention adopts a controllable step-by-step vertical sedimentation method to integrate red, green and blue colors into one, and constructs a three-color photonic crystal sensor with a synergistic color-changing effect. By combining an aptamer with a specific response capability to oxytetracycline on the photonic crystal gel, the common multi-color sensing detection of oxytetracycline can be achieved, thereby improving the response accuracy of the photonic crystal sensor to oxytetracycline in structural color, and successfully realizing the color change detection of oxytetracycline at the threshold of 0.5μmol / mL, meeting the safety standards for aquatic product detection. Summary of the Invention

[0005] The present invention aims to provide a three-color photonic crystal sensor with higher-precision visualization capability for use in the detection of oxytetracycline.

[0006] The method for constructing a three-color photonic crystal sensor for detecting oxytetracycline in the present invention comprises the following steps:

[0007] Step 1: Preparation of monodisperse silica nanoparticles:

[0008] First, ethanol, tetraethyl orthosilicate, and deionized water are mixed to form a homogeneous solution A. Subsequently, ammonia water is added to initiate the reaction. After the reaction is completed, the nanoparticles are separated by centrifugation, washed sequentially with ethanol and deionized water, and finally dispersed in deionized water to obtain monodisperse silica microspheres for later use.

[0009] Step 2: Prepare a three-color photonic crystal array:

[0010] First, silica nanoparticles with particle sizes of 238nm, 203nm, and 312nm were dispersed in ethanol to prepare a silica colloidal ethanol suspension. A uniform dispersion was then achieved through ultrasonic treatment. Subsequently, a three-dimensional photonic crystal array with a height of 3cm was deposited sequentially on the same hydrophilic-treated glass slide. After the solvent evaporated, a three-color photonic crystal array with a gradual transition from green to blue and then to red was formed on the substrate.

[0011] Step 3: Prepare a three-color photonic crystal sensor:

[0012] First, the substrate on one side of the three-color photonic crystal array is used as the bottom plate, and a tape layer is fixed on the short sides of both sides of the bottom plate, and a blank glass slide is covered on the top of the sample to form a spatial interlayer; then, a prefabricated gold nanoparticle (AuNPs) solution C is added to the prepolymer solution B to form a hydrogel precursor solution; then, the precursor solution is injected into the interlayer gap and polymerized under ultraviolet light. After the reaction is completed, a three-color photonic crystal gel membrane is obtained, which is then soaked in deionized water, peeled off the substrate, and cut to a certain size; finally, it is incubated in a oxytetracycline aptamer solution for equilibrium and washed with buffer to obtain a three-color photonic crystal sensor device.

[0013] In step 1, the amount ratio of ethanol, tetraethyl orthosilicate, deionized water and ammonia water in solution A is 250 mL: 15 mL: 10 mL: 12-16 mL; the temperature of the sol-gel reaction is 25° C., and the reaction time is 6 hours; in step 2, the mass of the monodisperse silica sphere ethanol solution is 1%, the self-assembly temperature is 60° C., and the self-assembly time is 30-40 hours, based on an array height of 3 cm on the substrate.

[0014] In step 3, the interlayer formed in the space has a size of 2 cm × 1 cm × 150 μm (length × width × height). In step 3, the ratio of acrylamide AM, N,N'-methylenebisacrylamide BIS, 2,2-diethoxyacetophenone DEAP solution (DEAP: dimethyl sulfoxide DMSO = 1:9) and water in solution B is: 0.4 g: 0.01 g: 28 μL: 2 mL.

[0015] In step 3, in the solution C, 0.5 mL of chloroauric acid solution (1%) was mixed with 100 mL of deionized water and heated to a slight boil, 3 mL of sodium citrate solution (1%) was quickly added, and the mixture was heated for 1 hour and then naturally cooled to room temperature to obtain a dark pink AuNPs solution, which was directly stored for later use; 1 mL of solution C was added to the prepolymer solution.

[0016] In step 3, the wavelength of the ultraviolet light is 365 nm, and the light focusing time is 60 minutes.

[0017] In step 3, the oxytetracycline aptamer solution has an aptamer sequence of 5'-SH-ACG ACA TTC CGT TGATCT CTC CCT TTT GGG TTG GTG TCG T-3'; the aptamer concentration is 10 μmol / L, the incubation temperature is 4°C, and the incubation time is 12 hours; the aptamer solution is prepared with Tris-HCl buffer solution with a concentration of 50 mmol / L and a pH of 8.0.

[0018] The three-color photonic crystal sensor prepared by the present invention is used to detect oxytetracycline, and the specific steps are as follows:

[0019] (1) The three-color photonic crystal sensor was immersed in Tris-HCl buffer for equilibrium as a control group, and the reflection spectrum peak was recorded using a fiber optic spectrometer;

[0020] (2) The three-color photonic crystal sensors were transferred to different concentrations of oxytetracycline buffer solutions, and after reaching equilibrium, their reflection spectrum peaks were recorded again using a fiber optic spectrometer. The positions of the spectrum peaks of the three independent colors were plotted against the logarithmic values of the oxytetracycline concentrations to create standard curves.

[0021] (3) The three-color photonic crystal sensors were transferred to different concentrations of oxytetracycline buffer solutions. After reaching equilibrium, their structural colors were recorded again using a fiber optic spectrometer to establish the relationship between the structural colors of the sensors with primary colors of red, green, and blue and the oxytetracycline concentrations.

[0022] (4) The image color was extracted using Photoshop software, and the hue (H) value in the HSV color model extracted from the image was plotted against the concentration of oxytetracycline to obtain a concentration relationship curve based on color change.

[0023] Wherein, in step (1), the concentration of Tris-HCl buffer is 50 mmol / L and the pH is 8.0. In addition, 0.5 mol / L of NaCl and 2.5 mol / L of MgCl2·6H2O are added; in steps (2) and (3), the concentration range of oxytetracycline buffer is 0.1-100 μg / mL.

[0024] The beneficial effects of the present invention are:

[0025] The present invention successfully constructed a three-color photonic crystal sensor with a multi-color synergistic color-changing effect by using a step-by-step vertical sedimentation method, realizing multi-color synergistic detection of oxytetracycline. Its characteristics and advantages are described as follows:

[0026] (1) The present invention uses monodisperse silica microspheres of different particle sizes to construct red, green, and blue photonic crystal sensors through a step-by-step vertical sedimentation method. The three color regions exhibit independent structural color changes under the same volume change, providing a new method for collaborative detection.

[0027] (2) The three-color photonic crystal sensor constructed by the present invention improves the visual detection accuracy of oxytetracycline concentration through structural color. Compared with the single-color photonic crystal sensor, its concentration detection accuracy is doubled;

[0028] (3) The three-color photonic crystal sensor constructed in the present invention realizes the digital quantification of oxytetracycline in the concentration range of 0.5μg / mL-80μg / mL through HSV color difference analysis, and can distinguish oxytetracycline solutions with a minimum concentration of 0.5μg / mL, which is at the same order of magnitude as the safety standard threshold for aquatic product testing (0.2μg / mL). BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Scanning electron microscope images and optical microscope images of the prepared purple, green and red monochromatic photonic crystal arrays;

[0030] Figure 2 a is a schematic diagram of the construction method of the prepared three-color photonic crystal sensor. Figure 2 b is the structural color image and optical microscope image of the prepared three-color photonic crystal array. Figure 2 c is the structural color image and optical microscope image of the prepared three-color photonic crystal sensor;

[0031] Figure 3 The reflection spectra of the prepared three-color photonic crystal array and three-color photonic crystal sensor are shown in Figure 2.

[0032] Figure 4 The changes of the spectral peak wavelengths of the three color domains (R: red; G: green; B: blue) of the prepared three-color photonic crystal sensor in different concentrations of oxytetracycline solution;

[0033] Figure 5 The changes in structural color of the prepared three-color photonic crystal sensor in three color domains (R: red; G: green; B: blue) in oxytetracycline solutions of different concentrations. DETAILED DESCRIPTION

[0034] The present invention will be described in detail below with reference to examples and accompanying drawings, but the present invention is not limited to these embodiments;

[0035] Example 1

[0036] (1) 250 mL of ethanol, 15 mL of tetraethyl orthosilicate, and 10 mL of deionized water were mixed to form a homogeneous solution. After magnetic stirring for 5 minutes, 12 mL of ammonia water was added and the mixture was reacted at room temperature for 6 hours. The particle size of the silica nanoparticles was controlled to be within the range of 203 nm by adjusting the volume of ammonia water. After the reaction, the nanoparticles were separated by centrifugation, washed once with ethanol and three times with deionized water, and finally dispersed in deionized water for use.

[0037] (2) 203 nm silica nanoparticles were dispersed in ethanol to prepare a colloidal suspension with a volume fraction of 1% (v / v), and a uniform dispersion system was obtained by ultrasonic treatment. Subsequently, a hydrophilic glass slide was placed vertically in the dispersion and allowed to stand in an oven at 60 degrees for 3 to 7 days until the solution was completely evaporated, forming a blue three-dimensional photonic crystal array on the substrate.

[0038] The blue three-dimensional photonic crystal array obtained by the method of Example 1 of the present invention was characterized by field emission scanning electron microscopy and dark field optical microscopy for its microstructure and structural color. Figure 1 (i); Figure 1 (i) shows that a densely packed photonic crystal array with hexagonal stacking can be obtained after assembly, and it appears uniformly blue under an optical microscope, without obvious cracks or defects, indicating that the obtained blue photonic crystal array has a regular periodic arrangement conformation and excellent optical properties.

[0039] Example 2

[0040] (1) 250 mL of ethanol, 15 mL of tetraethyl orthosilicate, and 10 mL of deionized water were mixed to form a homogeneous solution. After magnetic stirring for 5 minutes, 15 mL of ammonia water was added and the mixture was reacted at room temperature for 6 hours. The particle size of the silica nanoparticles was controlled to be within the range of 238 nm by adjusting the volume of ammonia water. After the reaction, the nanoparticles were separated by centrifugation, washed once with ethanol and three times with deionized water, and finally dispersed in deionized water for use.

[0041] (2) 238 nm silica nanoparticles were dispersed in ethanol to prepare a colloidal suspension with a volume fraction of 1% (v / v), and a uniform dispersion system was obtained by ultrasonic treatment. Subsequently, a hydrophilic glass slide was placed vertically in the dispersion and allowed to stand in an oven at 60 degrees for 3 to 7 days until the solution was completely evaporated, forming a green three-dimensional photonic crystal array on the substrate.

[0042] The green three-dimensional photonic crystal array obtained by the method of Example 2 of the present invention was characterized by field emission scanning electron microscopy and dark field optical microscopy for its microstructure and structural color. Figure 1 (ii); Figure 1 (ii) shows that a densely packed photonic crystal array with hexagonal stacking can be obtained after assembly, and it appears uniform green under an optical microscope, without obvious cracks or defects, indicating that the obtained green photonic crystal array has a regular periodic arrangement conformation and excellent optical properties.

[0043] Example 3

[0044] (1) 250 mL of ethanol, 15 mL of tetraethyl orthosilicate, and 10 mL of deionized water were mixed to form a homogeneous solution. After magnetic stirring for 5 minutes, 16 mL of ammonia water was added and the mixture was reacted at room temperature for 6 hours. The particle size of the silica nanoparticles was controlled to be within the range of 312 nm by adjusting the volume of ammonia water. After the reaction, the nanoparticles were separated by centrifugation, washed once with ethanol and three times with deionized water, and finally dispersed in deionized water for use.

[0045] (2) 312 nm silica nanoparticles were dispersed in ethanol to prepare a colloidal suspension with a volume fraction of 1% (v / v), and a uniform dispersion system was obtained by ultrasonic treatment. Subsequently, a hydrophilic glass slide was placed vertically in the dispersion and allowed to stand in a 60°C oven for 3 to 7 days until the solution was completely evaporated, forming a red three-dimensional photonic crystal array on the substrate.

[0046] The red three-dimensional photonic crystal array obtained by the method of Example 3 of the present invention was characterized by field emission scanning electron microscopy and dark field optical microscopy for its microstructure and structural color. Figure 1 (iii); Figure 1 (iii) shows that a densely packed photonic crystal array with hexagonal stacking can be obtained after assembly, and it appears uniform red under an optical microscope and has no obvious defects, indicating that the obtained red photonic crystal array has a regular periodic arrangement conformation and excellent optical properties.

[0047] Example 4

[0048] (1) Silica nanoparticles with particle sizes of 238 nm, 203 nm, and 312 nm were dispersed in ethanol to prepare a colloidal suspension with a volume fraction of 1% (v / v). A uniform dispersion system was obtained by ultrasonic treatment. Subsequently, a three-dimensional photonic crystal array with a height of 3 cm was deposited on the same hydrophilic treated glass slide in order. After the solvent evaporated, a three-color photonic crystal array was finally formed on the substrate, with the color gradually changing from green to blue and then to red.

[0049] (2) 0.5 mL of 1% chloroauric acid solution was mixed with 100 mL of deionized water and heated to a slight boil. 3 mL of 1% sodium citrate solution was quickly added. The mixture was heated for 1 hour and then cooled to room temperature to obtain a dark pink AuNPs solution, which was directly stored for later use.

[0050] (3) A tape layer was fixed on the short sides of the green-blue-red transition three-color photonic crystal array substrate, and a blank glass slide was covered on the top of the array to form a space sandwich with a thickness of 150 μm; then, 0.36 g acrylamide, 0.01 g N,N'-methylenebisacrylamide and 28 μL of DEAP solution (volume fraction 10%, solvent DMSO) were dissolved in 2 mL of deionized water to form a mixed solution, and then 0.5 mL of AuNPs solution was added to form a hydrogel precursor; then, 150 μL of the precursor was injected into the interlayer gap and polymerized under 365 nm ultraviolet light for 1 hour to obtain a three-color photonic crystal gel film, which was then soaked in deionized water and peeled off the substrate and cut into 1 cm × 1 cm size; finally, it was incubated at 4 ° C for 12 hours in a 10 μM oxytetracycline aptamer solution to obtain a three-color photonic crystal sensor that can be used for oxytetracycline detection;

[0051] The preparation process of the green-blue-red transition type three-color photonic crystal array and the red-green-blue transition type three-color photonic crystal sensor obtained by the method of Example 4 of the present invention is as follows: Figure 3 As shown in a, the structural color at the interface was characterized using a dark field optical microscope and a camera. Figure 3 bc; Figure 3 bc shows that after the three-color photonic crystal array is filled with responsive gel, the period of the three-color photonic crystal is red-shifted due to the water absorption and swelling of the gel in the aqueous solution and the gel modification process, thereby causing the red shift of the overall structural color. The original green-blue-red three-color photonic crystal array finally obtains a three-color photonic crystal sensor with red-green-blue transition; the specific changes in its reflection spectrum are shown in Figure 2. Figure 3 shown.

[0052] Example 5

[0053] (1) The three-color photonic crystal sensor in Example 4 was immersed in Tris-HCl buffer (50 mM Tris-HCl, 0.5 M NaCl, 2.5 M MgCl2·6H2O, pH 8.0) for equilibrium as a control group; then, the three-color photonic crystal sensors were transferred to a buffer containing 0.1-100 μg / mL oxytetracycline, and after reaching equilibrium, the reflectance spectra of the photonic crystal in the red, green, and blue regions were recorded again using a fiber optic spectrometer;

[0054] The reflectance spectrum results tested by the method of Example 5 of the present invention are as follows Figure 4 As shown in the figure, as the concentration of oxytetracycline increases from 0.1 μg / mL to 100 μg / mL, the spectra of the three-color photonic crystal sensor in the red (R), green (G), and blue (B) color regions all show a red-shift trend, and all reach equilibrium in 80 μg / mL oxytetracycline solution.

[0055] Example 6

[0056] (1) The three-color photonic crystal sensor in Example 4 was immersed in Tris-HCl buffer (50 mM Tris-HCl, 0.5 M NaCl, 2.5 M MgCl2·6H2O, pH 8.0) for equilibrium as a control group. The sample was photographed with a smartphone, and the color image of the sensor under natural light was directly recorded. Subsequently, the three-color photonic crystal sensor was transferred to a buffer containing 0.1-100 μg / mL oxytetracycline. After reaching equilibrium, its color image was recorded under the same light environment, and the color change of the sensor before and after transfer was compared.

[0057] The structural color change results tested by the method of Example 6 of the present invention are as follows Figure 5 As shown in the figure, as the concentration of oxytetracycline increases from 0.1μg / mL to 80μg / mL, the colors of the three-color photonic crystal sensor in the red (R), green (G), and blue (B) color regions all show a trend of gradual red shift, and under the effect of the three-color synergistic color change, the three-color photonic crystal sensor can achieve obvious structural color changes in the oxytetracycline concentration range of 0.5μg / mL-80μg / mL. Therefore, it can be directly used for naked eye monitoring of the concentration changes of oxytetracycline solution and has great potential in field tests.

Claims

1. A method for constructing a three-color photonic crystal sensor for environmental oxytetracycline detection, characterized in that: The steps include: Step 1: Preparation of monodisperse silica nanoparticles: First, ethanol, tetraethyl orthosilicate, and deionized water are mixed to form a homogeneous solution A. Subsequently, ammonia water is added to initiate the reaction. After the reaction is completed, the nanoparticles are separated by centrifugation, washed sequentially with ethanol and deionized water, and finally dispersed in deionized water to obtain monodisperse silica microspheres for later use. Step 2: Prepare a three-color photonic crystal array: First, silica nanoparticles with particle sizes of 238nm, 203nm, and 312nm were dispersed in ethanol to prepare a silica colloidal ethanol suspension. A uniform dispersion was then achieved through ultrasonic treatment. Subsequently, a three-dimensional photonic crystal array with a height of 3cm was deposited sequentially on the same hydrophilic-treated glass slide. After the solvent evaporated, a three-color photonic crystal array with a gradual transition from green to blue and then to red was formed on the substrate. Step 3: Prepare a three-color photonic crystal sensor: First, the substrate on one side of the three-color photonic crystal array is used as the bottom plate, a tape layer is fixed on the short sides of both sides of the bottom plate, and a blank glass slide is placed on top of the sample to form a space sandwich; Subsequently, a prefabricated gold nanoparticle (AuNPs) solution C was added to the prepolymer solution B to form a hydrogel precursor solution. The precursor solution was then injected into the interlayer gaps and polymerized under ultraviolet light. After the reaction, a three-color photonic crystal gel membrane was obtained. After being soaked in deionized water and peeled off from the substrate, it was cut to a certain size. Finally, it was incubated in a oxytetracycline aptamer solution for equilibrium and washed with buffer to obtain a three-color photonic crystal sensor device.

2. The construction method according to claim 1, wherein In step 1, in the solution A, the ratio of ethanol, ethyl orthosilicate, deionized water, and ammonia water is 250 mL: 15 mL: 10 mL: 12-16 mL; the temperature of the sol-gel reaction is 25° C., and the reaction time is 6 hours.

3. The construction method according to claim 1, wherein In step 2, the mass of the monodisperse silicon sphere ethanol solution is 1%, the self-assembly temperature is 60° C., the self-assembly time is 30 to 40 hours, and the height of the array on the substrate is 3 cm.

4. The construction method according to claim 1, wherein In step 3, the size of the interlayer formed in the space interlayer is 2 cm×1 cm×150 μm (length×width×height).

5. The construction method according to claim 1, wherein: In step 3, in the solution B, the usage ratio of acrylamide AM, N,N'-methylenebisacrylamide BIS, 2,2-diethoxyacetophenone DEAP solution (DEAP: dimethyl sulfoxide DMSO = 1:9) and water is: 0.4 g: 0.01 g: 28 μL: 2 mL.

6. The construction method according to claim 1, wherein: In step 3, in the solution C, 0.5 mL of chloroauric acid solution (1%) was mixed with 100 mL of deionized water and heated to a slight boil, 3 mL of sodium citrate solution (1%) was quickly added, and the mixture was heated for 1 hour and then naturally cooled to room temperature to obtain a dark pink AuNPs solution, which was directly stored for later use; 1 mL of solution C was added to the prepolymer solution.

7. The construction method according to claim 1, wherein: In step 3, the wavelength of the ultraviolet light is 365 nm, and the light focusing time is 60 minutes.

8. The construction method according to claim 1, wherein: In step 3, the oxytetracycline aptamer solution has an aptamer sequence of 5'-SH-ACG ACA TTC CGT TGA TCT CTC CCT TTT GGG TTG GTG TCG T-3'; the aptamer concentration is 10 μmol / L, the incubation temperature is 4°C, and the incubation time is 12 hours; the aptamer solution is prepared with Tris-HCl buffer solution with a concentration of 50 mmol / L and a pH of 8.

0.

9. Use of the three-color photonic crystal sensor constructed by the construction method according to claims 1-8 for detecting oxytetracycline, characterized in that: The detection steps are: (1) The three-color photonic crystal sensor was immersed in Tris-HCl buffer for equilibrium as a control group, and the reflection spectrum peak was recorded using a fiber optic spectrometer; (2) The three-color photonic crystal sensors were transferred to different concentrations of oxytetracycline buffer solutions, and after reaching equilibrium, their reflection spectrum peaks were recorded again using a fiber optic spectrometer. The positions of the spectrum peaks of the three independent colors were plotted against the logarithmic values of the oxytetracycline concentrations to create standard curves. (3) The three-color photonic crystal sensors were transferred to different concentrations of oxytetracycline buffer solutions. After reaching equilibrium, their structural colors were recorded again using a fiber optic spectrometer to establish the relationship between the structural colors of the sensors with primary colors of red, green, and blue and the oxytetracycline concentrations. (4) The image color was extracted using Photoshop software, and the hue (H) value in the HSV color model extracted from the image was plotted against the concentration of oxytetracycline to obtain a concentration relationship curve based on color change.

10. The use according to claim 9, characterized in that In step (1), the concentration of Tris-HCl buffer is 50 mmol / L, the pH is 8.0, and in addition, 0.5 mol / L of NaCl and 2.5 mol / L of MgCl2·6H2O are added; In steps (2) and (3), the concentration range of oxytetracycline buffer is 0.1-100 μg / mL.

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