Construction of three-color photonic crystal sensor for environmental detection

Abstract

The application provides a construction method of a three-color photonic crystal sensor for detecting terramycin in an environment. The steps are as follows: step one, preparing monodisperse silica microspheres with different sizes; and step two, constructing the three-color photonic crystal sensor on the same substrate by a step-by-step vertical sedimentation method. The novel three-color photonic crystal sensor constructed by the application can make the characteristic wavelengths shift to different degrees (R: 867 nm to 921 nm; G: 723 nm to 771 nm; and B: 548 nm to 601 nm), thereby generating three-color transitions (R: light red to red; G: indigo to brick yellow; and B: purple to blue) that can be distinguished by naked eyes, and realizing high-precision visual detection of terramycin in a high ionic strength water environment. Meanwhile, in a logarithmic linear range of 0.1-80 mu g / mL, the R / G / B channels of the three-color photonic crystal sensor have R 2 respectively, and a field structural color change threshold (0.5 mu g / mL) meets water product safety standards. The multi-color photonic crystal sensor provides a new idea for constructing an on-site environment sensor with both laboratory-level quantification and instant visual reading.

Description

Technical Field

[0001] This invention belongs to the field of optical biosensing technology, and relates to a method for constructing a photonic crystal sensor device based on a three-color photonic crystal array to achieve multi-color synergistic detection of the environmental pollutant oxytetracycline. Background Technology

[0002] Tetracycline antibiotics, characterized by their typical tetracyclic molecular skeleton, are widely used antibacterial agents in global aquaculture and animal husbandry. Oxytetracycline (OTC), a representative drug, poses a serious threat to the ecological environment and human health due to its overuse and residue problems, potentially causing 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 arranged nanoparticles can achieve colorimetric sensing without complex instruments through visible light diffraction effects. In existing technologies, colloidal crystal antibiotic detection systems mainly employ two approaches: one is the molecularly imprinted photonic crystal sensing strategy, which mainly achieves multi-site recognition of template molecules or their structural analogs by binding template molecules to optical hydrogels. However, this method suffers from uneven distribution of binding sites and insufficient rebinding kinetics. The other strategy is the design of enzyme-functionalized photonic crystal sensors, which mainly triggers the optical hydrogel to shift diffraction wavelengths and change structural colors through enzymatic reactions. Despite the high selectivity of this enzyme response system, the sensitivity of the enzyme to physiological conditions and the complexity of the immobilization process limit its practical application.

[0003] Aptamers, as a novel biometric element, possess advantages such as high affinity, programmable conformation, and excellent thermal stability. Currently, the combination of aptamers and photonic crystal gel sensors is being gradually applied to thrombin, Ag... + The detection of targets such as cysteine ​​is possible. However, this monochromatic photonic crystal sensor still faces two major technical bottlenecks: 1) aptamer fixation requires complex intermediate steps; 2) due to the limitations of the microstructure of the photonic crystal in regulation, the monochromatic signal system cannot simultaneously achieve quantitative detection and visualization output, especially in complex biological matrices where it cannot meet the comprehensive requirements of specificity, sensitivity, and rapid reading.

[0004] This invention employs a controllable stepwise vertical sedimentation method to integrate red, green, and blue colors into a single unit, constructing a three-color photonic crystal sensor with a synergistic color-changing effect. By combining an aptamer with specific responsiveness to oxytetracycline onto the photonic crystal gel, it enables common multicolor sensing and detection of oxytetracycline, thereby improving the structural color response accuracy of the photonic crystal sensor to oxytetracycline. It has also successfully achieved color-changing detection of oxytetracycline at a threshold of 0.5 μmol / mL, meeting the safety standards for aquatic product testing. Summary of the Invention

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

[0006] The method for constructing a three-color photonic crystal sensor for detecting oxytetracycline in this invention includes 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. Then, ammonia is added to start 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: Fabrication of a three-color photonic crystal array:

[0010] First, silica nanoparticles with diameters of 238 nm, 203 nm, and 312 nm were dispersed in ethanol to prepare silica gel ethanol suspensions. A uniform dispersion system was obtained by ultrasonic treatment. Subsequently, a three-dimensional photonic crystal array with a height of 3 cm was deposited sequentially on the same hydrophilic glass slide. After the solvent evaporated, a three-color photonic crystal array that gradually changed from green to blue and then to red was finally formed on the substrate.

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

[0012] First, using a substrate on one side of the tri-color photonic crystal array as a base plate, adhesive tape layers are fixed at the short sides of both sides of the base plate, and a blank glass slide is placed on top of the sample to form a spatial interlayer. Then, a pre-prepared gold nanoparticle (AuNPs) solution C is added to the prepolymer solution B to form a hydrogel precursor solution. Next, the precursor solution is injected into the interlayer gap, and a polymerization reaction is carried out under ultraviolet light. After the reaction is completed, a tri-color photonic crystal gel film is obtained. After soaking in deionized water to peel off the substrate, it is cut to a certain size. Finally, it is incubated and equilibrated in an oxytetracycline aptamer solution, washed with buffer solution, and the tri-color photonic crystal sensor device is obtained.

[0013] In step 1, the ratio of ethanol, tetraethyl orthosilicate, deionized water and ammonia in solution A is 250 mL: 15 mL: 10 mL: 12~16 mL; the temperature of the sol-gel reaction is 25℃ and the reaction time is 6 hours.

[0014] In step 2, the mass of the monodisperse silicon sphere ethanol solution is 1%, the self-assembly temperature is 60℃, and the self-assembly time is 30~40 hours, with the array height on the substrate being 3 cm.

[0015] In step 3, the size of the interlayer formed in the space is 2 cm × 1 cm × 150 μm (length × width × height).

[0016] 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.

[0017] In step 3, in solution C, 0.5 mL of 1% chloroauric acid solution is mixed with 100 mL of deionized water and heated to a gentle boil. Then, 3 mL of 1% sodium citrate solution is quickly added. After heating for 1 hour, the mixture is naturally cooled to room temperature to obtain a dark pink AuNPs solution, which is then stored for later use. 1 mL of solution C is then added to the prepolymer solution.

[0018] In step 3, the ultraviolet light wavelength is 365 nm and the light focusing time is 60 minutes.

[0019] In step 3, the aptamer solution contains 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℃; and the incubation time is 12 hours. The aptamer solution is prepared using Tris-HCl buffer solution with a concentration of 50 mmol / L and a pH of 8.0.

[0020] The application of the tricolor photonic crystal sensor prepared in this invention for detecting oxytetracycline involves the following steps:

[0021] (1) The three-color photonic crystal sensor was immersed in Tris-HCl buffer solution to equilibrate as a control group, and the reflected spectral peaks were recorded by the fiber optic spectrometer.

[0022] (2) The three-color photonic crystal sensor was transferred to different concentrations of oxytetracycline buffer solution. After reaching equilibrium, its reflection spectrum peak was recorded again using a fiber optic spectrometer. The positions of the spectral peaks of the three independent colors were plotted against the logarithm of the oxytetracycline concentration to form a standard curve.

[0023] (3) The three-color photonic crystal sensor was transferred to different concentrations of oxytetracycline buffer solution. After reaching equilibrium, its structural color was recorded again using a fiber optic spectrometer to establish the relationship between the sensor structural color with red, green and blue as primary colors and the concentration of oxytetracycline.

[0024] (4) Use Photoshop software to extract the colors of the image, and plot the values ​​of hue (H) in the HSV color model extracted from the image against the concentration of oxytetracycline to obtain a concentration relationship curve based on color changes.

[0025] 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 NaCl and 2.5 mol / L MgCl2·6H2O are added. In steps (2) and (3), the concentration range of oxytetracycline buffer is 0.1-100 μg / mL.

[0026] This invention successfully constructs a three-color photonic crystal sensor with multi-color synergistic color-changing effect through a stepwise vertical sedimentation method, realizing multi-color synergistic detection of oxytetracycline. Its features and advantages are described below:

[0027] (1) The present invention constructs a red, green and blue three-color photonic crystal sensor by stepwise vertical sedimentation method using monodisperse silica microspheres of different particle sizes. The three color regions exhibit independent structural color changes under the same volume change, providing a new method for collaborative detection.

[0028] (2) The three-color photonic crystal sensor constructed in this invention improves the visualization detection accuracy of oxytetracycline concentration through structural color. Compared with the monochromatic photonic crystal sensor, its concentration detection accuracy is doubled.

[0029] (3) The three-color photonic crystal sensor constructed in this invention realizes digital quantification of oxytetracycline in the concentration range of 0.5 μg / mL-80 μg / mL through HSV color difference analysis. It can distinguish oxytetracycline solution with a minimum concentration of 0.5 μg / mL, which is on the same order of magnitude as the safety standard threshold (0.2 μg / mL) for aquatic product testing. Attached Figure Description

[0030] Figure 1 Scanning electron microscope (SEM) images and optical microscope images of the fabricated purple, green, and red monochromatic photonic crystal arrays;

[0031] Figure 2 a is a schematic diagram of the construction method of the prepared three-color photonic crystal sensor. Figure 2 b shows the structural color image and optical microscope image of the fabricated three-color photonic crystal array. Figure 2 c shows the structural color image and optical microscope image of the fabricated three-color photonic crystal sensor;

[0032] Figure 3 The reflection spectra of the fabricated three-color photonic crystal array and the three-color photonic crystal sensor are shown.

[0033] Figure 4 The variation of spectral peak wavelengths of the three color domains (R: red; G: green; B: blue) of the fabricated tricolor photonic crystal sensor in oxytetracycline solutions of different concentrations was investigated.

[0034] Figure 5 The structural color of the fabricated tricolor photonic crystal sensor varies in three color domains (R: red; G: green; B: blue) in oxytetracycline solutions of different concentrations. Detailed Implementation

[0035] The present invention will now be described in detail with reference to examples and accompanying drawings, but the present invention is not limited to these embodiments; Example 1

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

[0037] 203 nm silica nanoparticles were dispersed in ethanol to prepare a 1% (v / v) colloidal suspension. The system was then ultrasonically treated to obtain a uniform dispersion. Subsequently, a hydrophilic glass slide was vertically placed in the dispersion and placed in a 60-degree oven for 3-7 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 in Example 1 of this invention was characterized in terms of its microstructure and structural color using field emission scanning electron microscopy and dark-field optical microscopy, respectively. The results are shown in […]. Figure 1 (i); Figure 1 (i) shows that a close-packed photonic crystal array with hexagonal packing can be obtained by 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 and excellent optical performance. Example 2

[0039] 250 mL of ethanol, 15 mL of tetraethyl orthosilicate and 10 mL of deionized water were mixed to form a homogeneous solution. After stirring magnetically for 5 minutes, 15 mL of ammonia was added and the reaction was carried out at room temperature for 6 hours. The particle size of silica nanoparticles was controlled within 238 nm by adjusting the volume of ammonia. After the reaction was completed, the nanoparticles were separated by centrifugation, washed once with ethanol and three times with deionized water, and finally dispersed in deionized water for later use.

[0040] 238 nm silica nanoparticles were dispersed in ethanol to prepare a 1% (v / v) colloidal suspension. The system was then ultrasonically treated to obtain a uniform dispersion. Subsequently, a hydrophilic glass slide was vertically placed in the dispersion and placed in a 60-degree oven for 3 to 7 days until the solution was completely evaporated, forming a green three-dimensional photonic crystal array on the substrate.

[0041] The green three-dimensional photonic crystal array obtained by the method in Example 2 of this invention was characterized in terms of its microstructure and structural color using field emission scanning electron microscopy and dark-field optical microscopy, respectively. The results are shown in […]. Figure 1 (ii); Figure 1 (ii) shows that a close-packed photonic crystal array with hexagonal packing can be obtained by assembly, and it appears uniformly green under an optical microscope without obvious cracks or defects, indicating that the obtained green photonic crystal array has a regular periodic arrangement and excellent optical performance. Example 3

[0042] (1) Mix 250 mL of ethanol, 15 mL of tetraethyl orthosilicate and 10 mL of deionized water to form a homogeneous solution. After stirring magnetically for 5 minutes, add 16 mL of ammonia water and react at room temperature for 6 hours. The particle size of silica nanoparticles is controlled within the range of 312 nm by adjusting the volume of ammonia water. After the reaction is completed, centrifuge to separate the nanoparticles, wash them once with ethanol and three times with deionized water, and finally disperse them in deionized water for later use.

[0043] (2) 312 nm silica nanoparticles were dispersed in ethanol to prepare a colloidal suspension with a volume fraction of 1% (v / v). The system was uniformly dispersed by ultrasonic treatment. Then, a hydrophilic glass slide was vertically placed in the dispersion and placed in a 60-degree oven for 3 to 7 days until the solution was completely evaporated, forming a red three-dimensional photonic crystal array on the substrate.

[0044] The red three-dimensional photonic crystal array obtained by the method in Example 3 of this invention was characterized in terms of its microstructure and structural color using field emission scanning electron microscopy and dark-field optical microscopy, respectively. The results are shown in […]. Figure 1 (iii); Figure 1 (iii) indicates that a close-packed photonic crystal array with hexagonal packing can be obtained by assembly, and it appears uniformly red under an optical microscope without any obvious defects, indicating that the obtained red photonic crystal array has a regular periodic arrangement and excellent optical performance. Example 4

[0045] (1) Select silica nanoparticles with particle sizes of 238 nm, 203 nm and 312 nm and disperse them in ethanol to prepare a colloidal suspension with a volume fraction of 1% (v / v). The system is uniformly dispersed by ultrasonic treatment. Then, a three-dimensional photonic crystal array with a height of 3 cm is deposited on the same hydrophilic glass slide in sequence. After the solvent evaporates, a three-color photonic crystal array that gradually changes from green to blue and then to red is finally formed on the substrate.

[0046] (2) Mix 0.5 mL of 1% chloroauric acid solution with 100 mL of deionized water and heat gently to boiling. Quickly add 3 mL of 1% sodium citrate solution and continue heating for 1 hour. Then cool naturally to room temperature to obtain a dark pink AuNPs solution, which can be stored directly for later use.

[0047] (3) A tape layer was fixed on the short sides of both sides of the green-blue-red three-color photonic crystal array substrate, and a blank glass slide was placed on top of the array to form a space interlayer with a thickness of 150 μm. Then, 0.36 g acrylamide, 0.01 g N,N'-methylenebisacrylamide and 28 μL DEAP solution (volume fraction 10%, solvent DMSO) were dissolved in 2 mL deionized water to form a mixture, and then 0.5 mL 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. After soaking in deionized water and peeling off the substrate, it was cut into 1 cm × 1 cm size. Finally, it was incubated in 10 μM oxytetracycline aptamer solution at 4 °C for 12 hours to obtain a three-color photonic crystal sensor that can be used for oxytetracycline detection.

[0048] The fabrication process of the green-blue-red transitional tri-color photonic crystal array and the red-green-blue transitional tri-color photonic crystal sensor obtained by the method in Example 4 of this invention is as follows: Figure 2 As shown in figure a, the structural color characterization of the interface was performed using dark-field optical microscopy and a camera, and the results are shown in figure a. Figure 2 bc; Figure 2 bc indicates that after the responsive gel is injected into the tri-color photonic crystal array, the period of the tri-color photonic crystal undergoes a redshift due to the water absorption and swelling effect of the gel in the aqueous solution and the gel modification process, resulting in a redshift of the overall structural color. The original green-blue-red tri-color photonic crystal array is ultimately transformed into a red-green-blue transition tri-color photonic crystal sensor; its specific reflection spectrum changes are as follows: Figure 3 As shown. Example 5

[0049] The tricolor photonic crystal sensor from Example 4 was immersed in Tris-HCl buffer (50 mM Tris-HCl, 0.5 M NaCl, 2.5 M MgCl2·6H2O, pH 8.0) to equilibrate as a control group; subsequently, the tricolor photonic crystal sensors were transferred to buffers containing 0.1-100 μg / mL oxytetracycline, and after equilibration, the reflectance spectra of the red, green, and blue regions of the photonic crystal were recorded again using a fiber optic spectrometer.

[0050] The reflectance spectrum results tested using the method in Example 5 of this invention are as follows: Figure 4 As shown, as the concentration of oxytetracycline increased from 0.1 μg / mL to 100 μg / mL, the three-color photonic crystal sensor showed a redshift trend in the spectra of the red (R), green (G), and blue (B) regions, and all reached equilibrium in an 80 μg / mL oxytetracycline solution. Example 6

[0051] (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) to equilibrate as a control group. The sample was photographed with a smartphone to directly record the color image of the sensor under natural light. Subsequently, the three-color photonic crystal sensor was transferred to buffer containing 0.1-100 μg / mL oxytetracycline. After equilibration, its color image was recorded under the same light environment to compare the color changes of the sensor before and after the transfer.

[0052] The structural color change results tested using the method in Embodiment 6 of this invention are as follows: Figure 5 As shown, as the concentration of oxytetracycline increased from 0.1 μg / mL to 80 μg / mL, the three-color photonic crystal sensor showed a gradual red shift trend in the red (R), green (G), and blue (B) color regions.

Claims

1. A method for constructing a three-color photonic crystal sensor for detecting oxytetracycline in the environment, characterized in that, Includes the following steps: Step 1: Preparation of monodisperse silica nanoparticles: First, ethanol, tetraethyl orthosilicate, and deionized water are mixed to form a homogeneous solution A. Then, ammonia is added to start 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: Fabrication of a three-color photonic crystal array: First, monodisperse silica microspheres with particle sizes of 238 nm, 203 nm, and 312 nm were dispersed in ethanol to prepare silica-ethanol suspensions. A uniform dispersion system was obtained through ultrasonic treatment. Then, a hydrophilic glass slide was vertically placed into the dispersion system, and a tricolor photonic crystal array with a height of 3 cm was sequentially deposited on the same hydrophilic glass slide. After the solvent evaporated, a tricolor photonic crystal array that gradually changes from green to blue and then to red was finally formed on the substrate. Step 3: Fabrication of a three-color photonic crystal sensor: First, using the substrate on one side of the three-color photonic crystal array as the base plate, fix the adhesive tape layer on both short sides of the base plate, and cover the top of the sample with a blank glass slide to form a spatial interlayer. Subsequently, a pre-prepared gold nanoparticle (AuNPs) solution C was added to prepolymer solution B to form a hydrogel precursor solution. The ratio of acrylamide (AM), N,N'-methylenebisacrylamide (BIS), 2,2-diethoxyacetophenone (DEAP) solution, and water in prepolymer solution B was 0.4 g:0.01 g:28 μL:2 mL, with a DEAP solution volume ratio of DEAP:dimethyl sulfoxide (DMSO) = 1:

9. The precursor solution was then injected into the interlayer voids, and polymerization was carried out under ultraviolet light. After the reaction, a three-color photonic crystal gel film was obtained. After soaking in deionized water to remove the substrate, the film was cut to a specific size. Finally, the film was incubated in an oxytetracycline aptamer solution to achieve equilibrium, and then washed with buffer to obtain a three-color photonic crystal sensor. Under the synergistic color change of the three colors, this three-color photonic crystal sensor can achieve significant structural color changes within the oxytetracycline concentration range of 0.5 μg / mL to 80 μg / mL, therefore it can be directly used for naked-eye monitoring of changes in oxytetracycline solution concentration.

2. The construction method as described in claim 1, characterized in that, In step 1, the ratio of ethanol, tetraethyl orthosilicate, deionized water and ammonia in solution A is 250 mL: 15 mL: 10 mL: 12~16 mL; the reaction time is 6 hours.

3. The construction method as described in claim 1, characterized in that, In step 2, the mass fraction of the silica gel ethanol suspension is 1%, the self-assembly temperature is 60℃, and the self-assembly time is 30~40 hours, with the array height on the substrate being 3 cm.

4. The construction method as described in claim 1, characterized in that, In step 3, the space interlayer formed has a length of 2 cm, a width of 1 cm, and a height of 150 μm.

5. The construction method as described in claim 1, characterized in that, In step 3, in solution C, 0.5 mL of 1% chloroauric acid solution is mixed with 100 mL of deionized water and heated to a gentle boil. Then, 3 mL of 1% sodium citrate solution is quickly added. After heating for 1 hour, the mixture is naturally cooled to room temperature to obtain a dark pink AuNPs solution, which is then stored for later use. 1 mL of solution C is added to prepolymer solution B.

6. The construction method as described in claim 1, characterized in that, In step 3, the ultraviolet light wavelength is 365 nm and the light focusing time is 60 minutes.

7. The construction method as described in claim 1, characterized in that, In step 3, the oxytetracycline aptamer solution contains 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℃; and the incubation time is 12 hours. The aptamer solution is prepared using Tris-HCl buffer solution with a concentration of 50 mmol / L and a pH of 8.0.

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