Hollow silica photonic crystal sensing film based on covalently immobilized fluorescent donor and preparation method and application thereof

Abstract

This invention discloses a hollow silica photonic crystal sensing film based on a covalently fixed fluorescence donor, its preparation method, and its application, belonging to the field of nanomaterials and sensor technology. The sensing film includes a substrate and a photonic crystal film disposed on the substrate. The photonic crystal film is composed of periodically arranged hollow silica nanoparticles with silanized acridine orange derivatives covalently fixed on their surfaces. In preparation, a silica shell layer is first formed on the surface of polystyrene nanoparticles. After calcination to remove the polystyrene core layer, the silanized acridine orange derivative is grafted onto the surface of the hollow silica nanoparticles, and then the photonic crystal film is formed by vertical deposition. During detection, the film is contacted with the sample to be tested, rhodamine 6G, and ammonium molybdate in an acidic medium. The complex formed by the reaction of EGCG and ammonium molybdate alters the fluorescence state and energy transfer process of rhodamine 6G, and quantitative analysis of EGCG is achieved through changes in fluorescence intensity.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials and sensor technology, and specifically relates to a hollow silica photonic crystal sensing film based on a covalently fixed fluorescent donor, its preparation method and application. Background Technology

[0002] Epigallocatechin gallate (EGCG) is the most abundant and biologically active catechin monomer in tea, exhibiting significant antioxidant, antitumor, anti-inflammatory, and cardiovascular protective functions. EGCG also holds important value in food nutritional evaluation and drug mechanism research. Therefore, establishing a rapid, sensitive, and highly selective method for EGCG detection is of significant practical importance for the quality evaluation of tea and its products and related pharmacological research.

[0003] Currently, quantitative analysis of EGCG mainly relies on traditional techniques such as high-performance liquid chromatography (HPLC), mass spectrometry (MS), electrochemical analysis, and spectrophotometry. While these methods offer advantages in accuracy and reliability, they generally suffer from complex sample pretreatment, long detection cycles, expensive equipment, and high operator skill requirements, making them unsuitable for rapid on-site detection and real-time analysis. Furthermore, in complex matrices such as tea extracts or biological samples, coexisting tea polyphenols, flavonoids, and small-molecule metabolites can easily interfere with EGCG determination, further increasing the difficulty of detection.

[0004] In recent years, fluorescent probe technology and fluorescence resonance energy transfer (FRET) systems have been widely used in the detection of bioactive molecules due to their advantages such as high sensitivity, fast response speed, and relatively simple operation. FRET systems achieve changes in fluorescence signals through energy transfer between donor and acceptor molecules, thereby enabling the identification and quantification of target molecules. However, traditional FRET systems still have significant limitations in practical applications: First, FRET efficiency is extremely sensitive to the donor-acceptor distance, typically requiring the distance to be controlled within the range of 1–10 nm. Stable and precise control is difficult to achieve in complex detection systems, leading to large fluctuations in energy transfer efficiency and affecting the repeatability and reliability of detection results. Second, under low-concentration detection conditions, due to limited fluorescence quenching efficiency and insufficient signal amplification, the detection sensitivity of traditional FRET systems for trace EGCG remains unsatisfactory, with a high detection limit. Third, in complex systems such as tea extracts or biological samples, strong background fluorescence and sample autofluorescence can easily interfere with the FRET signal, reducing the signal-to-noise ratio and detection sensitivity.

[0005] Against the backdrop of the aforementioned technological developments, photonic crystal sensing technology has gradually attracted attention due to its tunable, periodically ordered micro / nano structures, which can intuitively reflect changes in the external environment through structural color variations. It also offers advantages such as visual detection and the elimination of the need for complex instruments. Li et al. (Chemical Engineering Journal, 2026, 539, 177069) reported an EGCG detection method based on the synergistic enhancement of Rhodamine 6G (Rh6G) fluorescence using hollow spherical photonic crystals (HSPCs) and β-cyclodextrin (β-CD). This study achieved synergistic enhancement of the Rh6G fluorescence signal by leveraging the photonic band-edge effect and photolocalization effect of HSPCs, combined with the host-guest inclusion microenvironment modulation of Rh6G by β-CD, achieving a theoretical detection limit of 5.1 × 10⁻⁶. -16 mol·L -1 However, this system still has room for further optimization. First, the fluorescence microenvironment is mainly regulated by non-covalent host-guest interactions between β-CD and Rh6G. The immobility and spatial distribution of fluorescent molecules are affected by dynamic equilibrium, and the lack of stable binding between the fluorescence enhancement unit and the photonic crystal framework limits the structural controllability and long-term stability of the sensing interface. Second, this system mainly relies on β-CD host-guest inclusion to regulate the Rh6G microenvironment for fluorescence enhancement. It lacks independent FRET donor molecules and energy transfer mechanisms based on donor-acceptor distance regulation, resulting in a single signal amplification pathway that limits further expansion of the signal amplification pathway.

[0006] In summary, existing photonic crystal sensing systems still suffer from poor structural stability, a single signal amplification path, and difficulty in further improving detection sensitivity. Summary of the Invention

[0007] The purpose of this invention is to provide a hollow silica photonic crystal sensing film based on a covalently fixed fluorescent donor, its preparation method, and its application, in order to solve the problems of insufficient stability of fluorescent molecules, single signal amplification path, and insufficient sensitivity for detecting trace target molecules in existing photonic crystal fluorescence sensing systems.

[0008] This invention uses hollow silica nanoparticles as photonic crystal building blocks. By introducing functionalized fluorescent donor molecules onto the surface of the silica shell, the fluorescent donors are covalently fixed to the photonic crystal framework, thereby constructing a FRET nanosensing interface with a stable spatial structure.

[0009] The specific technical solution is as follows: This invention discloses a hollow silica photonic crystal sensing film based on a covalently fixed fluorescent donor, comprising a substrate and a photonic crystal film disposed on the substrate; The photonic crystal film is composed of periodically arranged hollow silicon dioxide nanoparticles; The hollow silica nanoparticles have fluorescent donor molecules fixed on their surface. The fluorescent donor molecules are covalently linked to the silica shell through silicon-oxygen bonds or silane coupling groups. The fluorescent donor molecules are silanized acridine orange. The fluorescent donor molecule is used to form a fluorescence resonance energy transfer system with an external fluorescent acceptor molecule to enhance the sensing signal.

[0010] Furthermore, the hollow silica nanoparticles have a hollow shell structure, and the hollow silica photonic crystal sensing film based on a covalently fixed fluorescence donor has a face-centered cubic ordered stacking structure, which is used to generate photonic bandgap and optical localization enhancement effect.

[0011] Furthermore, the substrate is a light-transmitting substrate used for fluorescence signal detection.

[0012] This invention also discloses a method for preparing a hollow silica photonic crystal sensing thin film based on a covalently fixed fluorescence donor, comprising the following steps: (1) Synthesis of monodisperse PS nanoparticles; (2) A silica shell was coated on the surface of the PS nanoparticles using the sol-gel method to obtain PS@SiO2 core-shell nanospheres; (3) The PS@SiO2 core-shell nanoparticles are calcined to remove the PS core layer and obtain hollow silica nanoparticles. (4) The hollow silica nanoparticles are brought into contact with silanized fluorescent donor molecules, and the silanized fluorescent donor molecules are covalently fixed to the shell surface of the hollow silica nanoparticles through Si-O-Si bonds, so as to obtain hollow silica nanoparticles covalently fixed with fluorescent donor molecules. (5) The hollow silica nanoparticles with covalently fixed fluorescent donor molecules are redispersed in a dispersion medium and assembled by vertical deposition to form a photonic crystal film with a periodic ordered structure, thereby obtaining the hollow silica photonic crystal film based on covalently fixed fluorescent donor.

[0013] Furthermore, in step (1), the monodisperse polystyrene nanospheres are synthesized by dispersion polymerization: using polyvinylpyrrolidone as a stabilizer and azobisisobutyramidine hydrochloride as an initiator, the reaction is carried out at 50-70°C for 5-8 h under nitrogen protection.

[0014] Further, step (2) specifically involves dispersing PS nanoparticles in ethanol, adding ammonia as a catalyst, adding tetraethyl orthosilicate to carry out a hydrolysis-condensation reaction, and forming a silica shell on the surface of the polystyrene nanoparticles; the calcination temperature in step (3) is 400-600℃, and the holding time is 1-3 h.

[0015] Further, in step (4), the silanized fluorescent donor molecule is silanized acridine orange; the hydrolyzable silane group in the silanized acridine orange undergoes a hydrolysis-condensation reaction with the silanol groups on the surface of the hollow silica nanoparticles to form Si-O-Si bonds, thereby covalently fixing the silanized acridine orange to the surface of the hollow silica nanoparticles.

[0016] Further, step (5) includes washing the hollow silica nanoparticles after the grafting reaction to remove unreacted or physically adsorbed fluorescent donor molecules, redispersing the washed hollow silica nanoparticles in ethanol or an ethanol-water mixed solvent, and assembling them on the surface of a transparent substrate to form a photonic crystal film by vertical deposition.

[0017] This invention also discloses a method for detecting EGCG using the hollow silica photonic crystal sensing film based on a covalently fixed fluorescence donor, comprising the following steps: The hollow silica photonic crystal sensing film based on covalently fixed fluorescent donor is contacted with the fluorescent acceptor molecule rhodamine 6G and a surfactant. The surfactant enriches rhodamine 6G near the silica shell with acridine orange fixed through micellar enrichment and interfacial assembly, promoting the formation of a fluorescence resonance energy transfer system between the fluorescent donor molecule and rhodamine 6G. Add the test sample containing EGCG and ammonium molybdate solution. The EGCG in the test sample reacts with ammonium molybdate to form an EGCG-Mo complex. The EGCG-Mo complex interacts with rhodamine 6G, changing the fluorescence state of rhodamine 6G and the energy transfer efficiency from acridine orange to rhodamine 6G, thereby causing a change in the fluorescence intensity of the system. The fluorescence intensity change of the hollow silica photonic crystal sensing film based on the covalently fixed fluorescence donor is detected, and the content of EGCG in the sample is determined according to the quantitative relationship between the fluorescence intensity change and the EGCG concentration.

[0018] Furthermore, the excitation wavelength of the fluorescence detection is 350~380 nm, the emission wavelength is 550~600 nm, and the surfactant is sodium dodecylbenzenesulfonate.

[0019] Compared with the prior art, the present invention has the following beneficial technical effects: This invention provides a hollow silica photonic crystal sensing film based on a covalently fixed fluorescence donor. The photonic crystal framework is constructed by periodically arranging hollow silica nanoparticles. Its hollow structure endows the film with a high specific surface area and strong light localization capability, providing a physical basis for fluorescence signal enhancement. At the same time, acridine orange (AO) is covalently bonded to the surface of the silica shell. This not only stably fixes AO as a FRET donor on the photonic crystal framework, avoiding the problems of easy leakage and uneven distribution of donor molecules in physical adsorption or inclusion methods, but also ensures the long-term stability of AO in repeated use and complex detection environments through covalent bonding.

[0020] Furthermore, the thin film is defined as having a face-centered cubic ordered stacking structure, which can generate significant photonic bandgap and photolocalization effect, effectively enhancing the fluorescence emission intensity of AO and providing a stronger donor signal for subsequent FRET energy transfer.

[0021] This invention also discloses a method for preparing a hollow silica photonic crystal sensing film based on a covalently immobilized fluorescent donor. The method involves first preparing hollow silica nanoparticles, followed by fluorescent donor grafting. First, a silica shell is formed on the surface of polystyrene nanoparticles. Then, the polystyrene core layer is removed by calcination to obtain hollow silica nanoparticles. Next, under low-temperature and light-protected conditions, a silanized acridine orange derivative undergoes a hydrolytic condensation reaction with the silanol groups on the surface of the hollow silica nanoparticles, achieving covalent immobilization of the fluorescent donor through Si-O-Si bonds. This preparation sequence avoids high-temperature calcination of the fluorescent donor and reduces the possibility of elution due to the fluorescent donor existing only in a physical adsorption manner.

[0022] This invention also discloses a method for detecting EGCG using a hollow silica photonic crystal sensing film based on a covalently fixed fluorescent donor. The method uses an acridine orange fluorescent core fixed on the surface of hollow silica nanoparticles as the fluorescent donor, and introduces rhodamine 6G, sodium dodecylbenzenesulfonate, and ammonium molybdate during the detection process. The EGCG-Mo complex formed by the reaction of EGCG with ammonium molybdate under acidic conditions mainly acts on the Rh6G acceptor state, affecting the AO→Rh6G energy transfer efficiency by changing the acceptor fluorescence quantum efficiency, thereby causing a change in the fluorescence signal and achieving EGCG detection. Specifically, Rh6G, as a FRET acceptor, receives energy transfer from the AO donor. Its fluorescence signal is synergistically enhanced by the photonic crystal light field enhancement and FRET amplification, enabling the detection of EGCG at an amolar concentration level. SDBS, as an anionic surfactant, helps regulate the distribution of Rh6G at the photonic crystal interface, increasing the effective collision probability of the donor and acceptor, and shortening the average spatial distance between the AO donor and the Rh6G acceptor, thereby promoting AO→Rh6G fluorescence resonance energy transfer. SDBS also helps reduce signal fluctuations caused by disordered Rh6G aggregation, improving the stability and repeatability of the detection system. The acidic medium provides a suitable pH environment for the reaction of EGCG and ammonium molybdate, ensuring the efficient formation of the EGCG-Mo complex; and the FRET energy transfer pathway from AO to Rh6G exhibits higher energy transfer efficiency and better structural stability compared to the β-CD physical inclusion of Rh6G. Attached Figure Description

[0023] Figure 1 Example 1 added 1×10 -18 Fluorescence spectra of M EGCG solution compared with solution without EGCG; Figure 2 Example 1 added 1×10 -16 Fluorescence spectra of M EGCG solution compared with solution without EGCG; Figure 3 Example 1 added 1×10 -14 Fluorescence spectra of M EGCG solution compared with solution without EGCG; Figure 4 Example 1 added 1×10 -12 Fluorescence spectra of M EGCG solution compared with solution without EGCG; Figure 5 Example 1 added 1×10 -10 Fluorescence spectra of M EGCG solution compared with solution without EGCG; Figure 6 Example 1 added 1×10 -6Fluorescence spectra of M EGCG solution compared with solution without EGCG; Figure 7 Example 1 added 1×10 -4 Fluorescence spectra of M EGC solution compared with solution without EGCG; Figure 8 Example 1 added 1×10 -4 Fluorescence spectra of M in EC solution compared to solution without EGCG; Figure 9 Example 1 added 1×10 -12 M's EGCG solution with 1×10⁻⁶ caffeine interferon added -12 Fluorescence spectra of M compared to EGCG solution.

[0024] Figure 10 Example 1 added 1×10 -12 M's EGCG standard solution with 1×10⁻⁶ ascorbic acid interferon added -12 Fluorescence spectra of M compared with EGCG solution; Figure 11 The effects of AO-Si grafting feed amount and the fraction of the suspended liquid in the film-forming process of hollow SiO2-AO nanoparticles on the fluorescence intensity of HSAPCs films were investigated. Examples 1, 2, and 3 were used to compare the effect of AO-Si grafting feed amount, while Examples 1, 4, and 5 were used to compare the effect of the fraction of the suspended liquid in the film-forming process. Detailed Implementation

[0025] The following abbreviations have the following meanings in this invention: EGCG is epigallocatechin gallate; EGC is epigallocatechin; EC is epicatechin; PS is polystyrene; PVP is polyvinylpyrrolidone; AIBA is azobisisobutyramidine hydrochloride; TEOS is tetraethyl orthosilicate; AO is acridine orange. EDC·HCl is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; NHS is N-hydroxysuccinimide; DMF is N,N-dimethylformamide; APTES is 3-aminopropyltriethoxysilane; AO-Si is an acridine orange derivative containing a triethoxysilyl group obtained by reacting a carboxyethylated acridine orange derivative with an aminosilane coupling agent; Rh6G is rhodamine 6G; SDBS is sodium dodecylbenzenesulfonate; AM is ammonium molybdate; SAPCs are close-packed photonic crystals; HSAPCs are hollow SiO2-AO photonic crystals; FCC is face-centered cubic; FRET is fluorescence resonance energy transfer.

[0026] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0027] Example 1 This invention discloses a method for preparing a hollow silica photonic crystal sensing thin film based on a covalently fixed fluorescence donor, comprising the following steps: Step 1: Synthesize monodisperse polystyrene (PS) nanoparticles.

[0028] Dissolve 1.50 g of PVP in 80 mL of deionized water in a 250 mL three-necked flask and sonicate until fully dissolved. Then, heat at 50 °C and 250 rpm. -1 The mixture was mechanically stirred under the following conditions, while N2 was continuously introduced to remove dissolved oxygen from the water. 7 mL of styrene was added in batches and stirred for 2 h. Then, 0.20 g of AIBA dissolved in 20 mL of deionized water was slowly added and stirred for another 6 h. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 45 min, the product was collected, washed three times with ethanol, and then redispersed in ethanol for later use.

[0029] Step 2: Preparation of PS@SiO2 core-shell nanoparticles.

[0030] The previously prepared PS emulsion was ultrasonically dispersed to ensure uniform particle dispersion. According to the solution dilution law, an appropriate amount of ethanol was added to dilute the PS suspension to 5% (v / v), and the solution was transferred to a single-necked flask. Subsequently, 2.0 mL of ammonia was added as a catalyst to promote the hydrolysis and condensation reaction of TEOS. The system temperature was set to 50℃, and the reaction was continued for 2 h. Then, 2.0 mL of TEOS was added dropwise, and the reaction was continued for another 5 h, allowing TEOS to undergo hydrolysis and condensation, forming a silica shell on the surface of the PS nanoparticles. After the reaction was complete, the product was collected by centrifugation at 6000 rpm for 10 min, washed three times with ethanol, and dried at 30℃ for 48 h to obtain PS@SiO2 core-shell nanoparticle powder.

[0031] Step 3: Prepare hollow SiO2 nanoparticles.

[0032] The PS@SiO2 composite nanoparticle powder prepared in step two was calcined in a muffle furnace at 2℃·min. -1 The temperature was increased to 500℃ and held for 2 hours to allow the PS core layer to decompose and be removed. After calcination, the mixture was allowed to cool naturally to room temperature, and the resulting powder was collected to obtain hollow SiO2 nanoparticles with a hollow core-shell structure.

[0033] Step 4: Prepare carboxyethylated acridine orange (AO-COOH).

[0034] 0.50 g of acridine orange (approximately 1.8 mmol) was dissolved in 50 mL of anhydrous acetonitrile, and 0.50 g of 3-bromopropionic acid (approximately 3.6 mmol, twice the molar amount) was added. The mixture was reacted at 80 °C under reflux in the dark for 12 h. Nitrogen gas was maintained during the reaction to minimize oxidation side reactions. After the reaction was complete, the solvent was removed under reduced pressure, and ethyl acetate was added for precipitation. The solid product was collected by filtration and washed with an ethyl acetate / ethanol system to obtain the purified carboxyethylated acridine orange derivative (AO-COOH).

[0035] Step 5: Preparation of silanized acridine orange (AO-Si) The AO-COOH prepared in step four was added to anhydrous N,N-dimethylformamide (DMF) in a molar ratio of 1:1.5:1.5 to carry out a carboxyl activation reaction with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS).

[0036] Specifically, 0.50 g of AO-COOH (approximately 1.43 mmol) was dissolved in 20 mL of anhydrous DMF, and 0.41 g of EDC·HCl (2.15 mmol) and 0.25 g of NHS (2.15 mmol) were added. The mixture was activated at room temperature under light-protected and nitrogen-protected conditions with stirring for 2 h. Subsequently, 3-aminopropyltriethoxysilane (APTES) was added, and the activated AO-COOH was coupled to APTES at a carboxyl to amino molar ratio of 1:1.5. 0.47 g of APTES (2.15 mmol, approximately 0.50 mL) was added, and the reaction was continued under light-protected and nitrogen-protected conditions for 9 h to form silanized acridine orange (AO-Si) containing hydrolyzable silane groups. After the reaction was complete, the product was purified to remove unreacted starting materials and byproducts, and the purified AO-Si was dissolved in anhydrous ethanol for later use.

[0037] Step 6: AO-Si grafted hollow SiO2 nanoparticles Weigh 1.00 g of the hollow SiO2 nanoparticles obtained in step three, add 60 mL of anhydrous ethanol and 2.0 mL of deionized water, and ultrasonically disperse for 30 min to form a uniform suspension. Add 20 mL of a 10 mg / mL solution. -1An AO-Si ethanol solution was prepared, with 0.20 g of AO-Si added. The total volume of the reaction system was then brought to 100 mL with anhydrous ethanol, and 0.50 mL of ammonia was added as a hydrolysis condensation catalyst. The reaction was carried out at 40 °C for 8 h under nitrogen protection and in the dark. After adding AO-Si to the hollow SiO2 nanoparticle dispersion system, the triethoxysilyl group in the AO-Si molecule hydrolyzed under water and ammonia catalysis to generate silanol groups (Si–OH). Subsequently, the silanol groups underwent a dehydration condensation reaction with the silanol groups on the surface of the hollow SiO2 nanoparticles to form Si–O–Si bonds, thereby covalently grafting AO-Si containing acridine orange fluorescent groups onto the surface of the hollow SiO2 nanoparticles to obtain hollow SiO2-AO nanoparticles. After the reaction was completed, the mixture was stirred at 8000 r·min⁻¹. -1 The product was collected by centrifugation for 10 min, and redispersed and washed three times with 30 mL of anhydrous ethanol each time to remove unreacted or physically adsorbed AO-Si. The washed hollow SiO2-AO nanoparticles were then redispersed in anhydrous ethanol and centrifuged at 3000 r·min. -1 Centrifuge at low speed for 5 min to remove obvious agglomerates and obtain a stable suspension of hollow SiO2-AO nanoparticles for vertical deposition.

[0038] Step 7: Preparation of hollow SiO2-AO photonic crystal (HSAPCs) thin films The hollow SiO2-AO nanoparticle suspension obtained in step six was ultrasonically dispersed in anhydrous ethanol to prepare a 5% (v / v) stable emulsion. A glass slide and a 5 mL beaker were pretreated with piranha water (H2O2:H2SO4 = 3:7, v / v) for 24 h, followed by repeated rinsing and ultrasonication to remove organic impurities and improve hydrophilicity. The etched glass slide was placed vertically in a similarly treated 5 mL beaker, and the diluted hollow SiO2-AO emulsion was injected. The beaker was then transferred to a constant temperature drying oven at 30°C for 48 h. As the solvent slowly evaporated, the hollow SiO2-AO nanoparticles self-assembled on the glass substrate surface to form a photonic crystal film with a face-centered cubic (FCC) ordered structure, resulting in hollow silica photonic crystal sensing films (HSAPCs) based on covalently immobilized acridine orange.

[0039] Performance verification: Prepare 1×10 -2 mol L 1 Rh6G stock solution, diluted to 2.5 × 10⁻⁶ before use. -4 M; Prepare 0.1 M SDBS solution, 2.0 M H2SO4 solution, and 0.1 M AM stock solution, diluting to 1×10⁻⁶ before use. -3 M. The EGCG stock solution was prepared into 1×10⁻⁶ solutions using a stepwise dilution method.-6 ~1×10 -18 mol·L -1 A series of standard working solutions.

[0040] The HSAPCs prepared in this example were immersed in glass petri dishes. Equal volumes and concentrations of SDBS solution, Rh6G solution, and ammonium molybdate solution were added to the petri dishes, followed by different concentrations of EGCG standard working solution in a volume ratio of 1:1:3:4:4. The pH of the reaction system was adjusted to 3.0±0.1 using dilute sulfuric acid working solution, and the total volume of each group was adjusted to be the same using ultrapure water. After shaking and standing for 5 min, the fluorescence intensity was measured at an excitation wavelength of 365 nm and an emission wavelength of 572 nm. The blank group was identical to the experimental group in all reagent concentrations, added volumes, pH, total volume, standing time, and detection conditions except for the absence of EGCG. The measured fluorescence intensity was considered the initial intensity (100%), and subsequent fluorescence changes in the experimental groups were compared to it.

[0041] The fluorescence intensity of each experimental group was compared with that of the blank group (without EGCG) to calculate the fluorescence quenching rate: quenching rate (%) = (F0 - F) / F0 × 100%, where F0 is the fluorescence intensity of the blank group and F is the fluorescence intensity of the experimental group.

[0042] It should be noted that in the above detection system, the H2SO4 solution is used to maintain a stable acidic reaction environment (pH 3.0). Rh6G, as a fluorescent acceptor molecule, forms a FRET energy transfer system with AO fixed on the photonic crystal framework. SDBS, as an anionic surfactant, regulates the spatial distribution of Rh6G on the photonic crystal surface through micellar enrichment and interfacial assembly, causing Rh6G to accumulate near the silica shell where AO is covalently fixed, shortening the average spatial distance between the AO donor and the Rh6G acceptor, thereby promoting AO→Rh6G fluorescence resonance energy transfer. SDBS also helps reduce signal fluctuations caused by disordered Rh6G aggregation, improving the stability and repeatability of the detection system. AM reacts with EGCG to form an EGCG-Mo complex. This complex interacts with the FRET detection system, changing the luminescence state of the fluorescent acceptor Rh6G, altering the AO→Rh6G energy transfer process, and thus changing the fluorescence intensity of the detection system, thereby achieving indirect quantitative detection of EGCG.

[0043] like Figure 1 As shown, without EGCG, the fluorescence intensity of rhodamine 6G reached 19372.97, and with the addition of 1×10⁻⁶ EGCG, the fluorescence intensity increased significantly. -18After being treated with EGCG solution M, the quenching rate reached 14.93%, indicating that the detection system still exhibited a identifiable fluorescence response, demonstrating its potential for trace EGCG detection. Furthermore, the system demonstrated high sensitivity and high stability at low concentrations, overcoming the problems of insufficient sensitivity and poor stability in traditional detection methods.

[0044] like Figure 2 As shown, add 1×10 -16 After reacting with EGCG solution M, a quenching rate of 23.78% was detected, indicating that HSAPCs films can efficiently detect extremely low concentrations of EGCG solution. Furthermore, this system exhibits high sensitivity and high stability at low concentrations, overcoming the problems of insufficient sensitivity and poor stability in traditional detection methods.

[0045] like Figure 3 As shown, add 1×10 -14 After reacting with EGCG solution M, a quenching rate of 33.98% was detected, indicating that HSAPCs films can efficiently detect extremely low concentrations of EGCG solution. Furthermore, this system exhibits high sensitivity and high stability at low concentrations, overcoming the problems of insufficient sensitivity and poor stability in traditional detection methods.

[0046] like Figure 4 As shown, add 1×10 -12 After reacting with EGCG solution M, the detected quenching rate reached 47.61%, indicating that HSAPCs films can efficiently detect extremely low concentrations of EGCG solution. Furthermore, this system exhibits high sensitivity and high stability at low concentrations, overcoming the problems of insufficient sensitivity and poor stability in traditional detection methods.

[0047] like Figure 5 As shown, add 1×10 -10 After reacting with EGCG solution M, a quenching rate of 53.18% was detected, indicating that HSAPCs films can efficiently detect low concentrations of EGCG solution. Furthermore, this system exhibits high sensitivity and high stability at low concentrations, overcoming the problems of insufficient sensitivity and poor stability in traditional detection methods.

[0048] like Figure 6 As shown, add 1×10 -6 After reacting with EGCG solution M, the detected quenching rate reached 75.31%, indicating that HSAPCs films can efficiently detect low concentrations of EGCG solution. Furthermore, this system exhibits high sensitivity and high stability at low concentrations, overcoming the problems of insufficient sensitivity and poor stability in traditional detection methods.

[0049] Conclusion: Figures 1-6As shown, the fluorescence quenching rate of the detection system generally increases with increasing EGCG concentration, indicating that the HSAPCs film has good concentration response characteristics to EGCG. Within the experimental concentration range set in this embodiment, even when the EGCG concentration is reduced to the minimum test concentration of 1×10⁻⁶, the fluorescence quenching rate of the detection system generally increases. -18 M. The system still showed a identifiable fluorescence change relative to the blank group, indicating that the detection system can effectively respond to the concentration of EGCG and has the potential to detect trace amounts of EGCG, overcoming the problems of insufficient sensitivity and poor stability of traditional detection methods.

[0050] like Figure 7 As shown, add 1×10 -4 After M was treated with epigallocatechin (EGC) solution, the quenching rate was measured to be 13.46%, which was still significantly lower than 1×10⁻⁶. -6 The response of M EGCG indicates that the system has a strong differentiated response to EGCG and excellent selectivity, which solves the problem that traditional photonic crystal sensing systems are not able to distinguish structurally similar compounds.

[0051] like Figure 8 As shown, add 1×10 -4 After M was treated with an epicatechin (EC) solution, the quenching rate was measured to be 4.71%, which is far lower than 1×10⁻⁶. -6 The response value of M EGCG is >75%. This result further confirms that the present sensing system has a high specific recognition ability for EGCG, and can effectively distinguish EGCG from other catechin structural analogs, overcoming the defect of poor selectivity in traditional photonic crystal sensing systems.

[0052] like Figure 7 and Figure 8 As shown, to further verify the system's anti-interference ability, structural analogs with concentrations higher than those detected by EGCG were selected for testing. Under the experimental conditions, 1×10 -4 The fluorescence quenching responses generated by MEGC and EC were both below 1×10⁻⁶. -6 The response generated by MEGCG indicates that the detection system exhibits a certain degree of differentiated response capability and excellent selectivity to EGCG, EGC and EC, solving the problem of insufficient ability of traditional photonic crystal sensing systems to distinguish structurally similar compounds.

[0053] like Figure 9 As shown, 1×10⁻⁶ caffeine interferon was added. -12 After treatment with MEG EGCG solution, the quenching rate reached 45.00%, compared to 1×10⁻⁶ without the interferon. -12Compared to EGCG solution, the quenching rate decreased by only 2.61 percentage points. This result indicates that caffeine, as a common coexisting substance in tea, has negligible interference with the detection of EGCG in this sensing system. This demonstrates that the HSAPCs film exhibits good anti-interference performance when measuring EGCG solution, solving the problems of low sensitivity and poor anti-interference ability of traditional detection methods.

[0054] like Figure 10 As shown, 1×10⁻⁶ of ascorbic acid interferon was added. -12 After treatment with MEG EGCG solution, the quenching rate reached 42.66%, compared to 1×10⁻⁶ without the interferon. -12 Compared to EGCG solution, the quenching rate decreased by only 4.95 percentage points. This result indicates that ascorbic acid has minimal interference with the detection of EGCG in this sensing system, and the HSAPCs film exhibits good anti-interference performance when measuring EGCG solution, solving the problems of low sensitivity and poor anti-interference ability of traditional detection methods.

[0055] like Figure 9 and Figure 10 As shown, after adding a final concentration of 1×10 -3 After M caffeine or ascorbic acid, 1×10 -12 The fluorescence quenching rate of the MEGCG detection system showed little change compared to the system without added interfering substances, indicating that at the concentrations of interfering substances used in this experiment, caffeine and ascorbic acid had limited impact on the EGCG detection response, and the detection system exhibited a certain degree of anti-interference ability.

[0056] Example 2 The same preparation process as in Example 1 was used, except that 10 mL of a 10 mg·mL⁻¹ solution was added during the AO-Si grafting process in step six. -1 An AO-Si ethanol solution was prepared, with an AO-Si feed amount of 0.10 g. After grafting, the resulting hollow SiO2-AO nanoparticles were formulated into a 5% (v / v) suspension for vertical deposition. All other preparation and detection conditions were the same as in Example 1.

[0057] The performance verification is described in Example 1, and will not be repeated here. The conclusions are shown in Table 1: Table 1

[0058] As shown in Table 1, the HSAPCs thin film obtained in this embodiment exhibits corresponding fluorescence quenching responses to different concentrations of EGCG, and the quenching rate generally increases with increasing EGCG concentration. Under the experimental conditions, the responses generated by EGC and EC are lower than those generated by lower concentrations of EGCG, indicating that the system has a certain differentiated response capability to the above-mentioned catechin substances. After adding caffeine or ascorbic acid, the system response changes relatively little, with the quenching rate decreasing by only 2.20 percentage points and 3.86 percentage points, respectively, indicating that it has a certain anti-interference capability. This shows that the sensor successfully overcomes the problems of low sensitivity, poor specificity recognition, weak anti-interference capability, and poor stability of traditional photonic crystals.

[0059] Example 3 The same preparation process as in Example 1 was used, except that 30 mL of a 10 mg·mL⁻¹ solution was added during the AO-Si grafting process in step six. -1 An AO-Si ethanol solution was used to prepare an AO-Si feed amount of 0.30 g. After grafting, the resulting hollow SiO2-AO nanoparticles were formulated into a 5% (v / v) suspension for vertical deposition. All other preparation and testing conditions were the same as in Example 1. Performance verification was performed as described in Example 1, and will not be repeated here. The conclusions are shown in Table 2. Table 2

[0060] As shown in Table 2, the HSAPCs films obtained in this embodiment exhibited corresponding fluorescence quenching responses to different concentrations of EGCG, and the quenching rate generally increased with increasing EGCG concentration. Under the experimental conditions, the responses generated by EGC and EC were lower than those generated by lower concentrations of EGCG, indicating that the system has a certain differentiated response capability to the above-mentioned catechin substances. After adding caffeine or ascorbic acid, the changes in the system response were relatively small, with the quenching rate decreasing by only 1.63 percentage points and 0.74 percentage points, respectively, indicating that it has a certain anti-interference ability.

[0061] Example 4 The same preparation process as in Example 1 was used, with AO-Si grafting feed amount of 0.20 g. The only difference was that in step seven, the grafted and washed hollow SiO2-AO nanoparticles were prepared into a suspension with a volume fraction of 3% (v / v) before vertical deposition. All other preparation and testing conditions were the same as in Example 1. Performance verification was performed as described in Example 1, and will not be repeated here. The conclusions are shown in Table 3. Table 3

[0062] As shown in Table 3, the HSAPCs films obtained in this embodiment exhibited corresponding fluorescence quenching responses to different concentrations of EGCG, and the quenching rate generally increased with increasing EGCG concentration. Under the experimental conditions, the responses generated by EGC and EC were lower than those generated by lower concentrations of EGCG, indicating that the system has a certain differentiated response capability to the above-mentioned catechin substances. After adding caffeine or ascorbic acid, the changes in the system response were relatively small, with the quenching rate decreasing by 1.33 percentage points and 2.67 percentage points, respectively, indicating that it has a certain anti-interference capability.

[0063] Example 5 The same preparation process as in Example 1 was used, with AO-Si grafting feed amount of 0.20 g. The only difference was that in step seven, the grafted and washed hollow SiO2-AO nanoparticles were prepared into a suspension with a volume fraction of 7% (v / v) before vertical deposition. All other preparation and testing conditions were the same as in Example 1. Performance verification was performed as described in Example 1, and will not be repeated here. The conclusions are shown in Table 4. Table 4

[0064] As shown in Table 4, the HSAPCs films obtained in this embodiment exhibited corresponding fluorescence quenching responses to different concentrations of EGCG, and the quenching rate generally increased with increasing EGCG concentration. Under the experimental conditions, the responses generated by EGC and EC were lower than those generated by lower concentrations of EGCG, indicating that the system has a certain differentiated response capability to the above-mentioned catechin substances. After adding caffeine or ascorbic acid, the changes in the system response were relatively small, with the quenching rate decreasing by 2.05 percentage points and 0.93 percentage points, respectively, indicating that it has a certain anti-interference capability.

[0065] like Figure 11 As shown, both the liquid fraction of SiO2-AO nanoparticles and the amount of AO-Si grafting material affect the structure and fluorescence enhancement performance of HSAPCs films. Example 1 exhibited the highest fluorescence intensity, indicating that the AO-Si grafting amount and particle film formation conditions were suitable when the AO-Si feeding amount was 0.20 g and the liquid fraction was 5% (v / v). This indicates that these conditions effectively balance AO grafting density, particle dispersion stability, and photonic crystal assembly performance. If the AO-Si feeding amount is too low, the amount of fluorescence donor fixation is insufficient; if the feeding amount is too high, self-quenching or particle agglomeration may occur. If the liquid fraction is too low, film formation is insufficient; if it is too high, particles tend to accumulate disorderly, both of which reduce the fluorescence enhancement effect. Therefore, under the experimental conditions described in Example 1, the experimental parameters exhibited a superior overall fluorescence enhancement effect.

[0066] This invention discloses a method for preparing photonic crystal thin films for EGCG detection. Addressing the issues of insufficient sensitivity and poor selectivity in existing technologies, it innovatively constructs a high-sensitivity detection system based on hollow SiO2-AO photonic crystals (HSAPCs). By utilizing the large specific surface area and localized light field enhancement effect of hollow SiO2 nanoparticles, the bottleneck of low sensitivity in traditional photonic crystals is overcome. Furthermore, by immobilizing AO-Si containing acridine orange fluorescent cores onto the surface of hollow SiO2 nanoparticles through a low-temperature post-grafting method, the fixation of fluorescent molecules (AO) on the nanoparticle surface is effectively stabilized, significantly improving the mechanical strength and reusability of the detection system. By controlling the amount of AO-Si grafting material and the integral number of the liquid suspension in the particle film formation, the fluorescence response performance of the resulting HSAPCs thin film can be adjusted, solving the problems of easy collapse or signal attenuation during use of traditional fluorescent probes and photonic crystals. By optimizing FRET efficiency, this invention achieves significant amplification of the Rh6G fluorescence signal, enabling the system to perform high-sensitivity detection of EGCG at the ammolar level or even lower concentrations. The enhanced anti-interference performance improved the system's stability and molecular recognition specificity in complex environments. In EGCG detection, the HSAPCs film, Rh6G, SDBS, and ammonium molybdate together constituted the fluorescence detection system. Experimental results showed that the overall fluorescence quenching response of the system increased with increasing EGCG concentration, reaching a higher level at 1×10⁻⁶ ppm. -18 Even under low-concentration EGCG conditions, identifiable fluorescence changes were observed in M. Furthermore, the system exhibited differentiated responses to EGC and EC, and maintained relatively stable fluorescence responses in the presence of caffeine and ascorbic acid. These results demonstrate that the HSAPCs thin film described in this invention has the potential for trace EGCG detection. This technology overcomes the problems of low sensitivity and poor stability associated with traditional photonic crystals, providing a novel technical solution for the rapid trace detection of EGCG and other small molecules. Its superior performance makes this method widely applicable in food, pharmaceutical, and environmental monitoring fields, possessing significant industrialization potential and market value.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A hollow silica photonic crystal sensing film based on a covalently fixed fluorescence donor, characterized in that, Includes a substrate and a photonic crystal thin film disposed on the substrate; The photonic crystal film is composed of periodically arranged hollow silicon dioxide nanoparticles; The hollow silica nanoparticles have fluorescent donor molecules fixed on their surface. The fluorescent donor molecules are covalently linked to the silica shell through silicon-oxygen bonds or silane coupling groups. The fluorescent donor molecules are silanized acridine orange. The fluorescent donor molecule is used to form a fluorescence resonance energy transfer system with an external fluorescent acceptor molecule to enhance the sensing signal.

2. The hollow silica photonic crystal sensing film based on a covalently fixed fluorescence donor according to claim 1, characterized in that, The hollow silica nanoparticles have a hollow shell structure, and the hollow silica photonic crystal sensing film based on a covalently fixed fluorescence donor has a face-centered cubic ordered stacking structure, which is used to generate photonic bandgap and optical localization enhancement effect.

3. The hollow silica photonic crystal sensing film based on a covalently fixed fluorescence donor according to claim 1, characterized in that, The substrate is a light-transmitting substrate used for fluorescence signal detection.

4. A method for preparing a hollow silica photonic crystal sensing thin film based on a covalently fixed fluorescence donor, characterized in that, Includes the following steps: (1) Synthesis of monodisperse PS nanoparticles; (2) A silica shell was coated on the surface of the PS nanoparticles using the sol-gel method to obtain PS@SiO2 core-shell nanospheres; (3) The PS@SiO2 core-shell nanoparticles are calcined to remove the PS core layer and obtain hollow silica nanoparticles. (4) The hollow silica nanoparticles are brought into contact with silanized fluorescent donor molecules, and the silanized fluorescent donor molecules are covalently fixed to the shell surface of the hollow silica nanoparticles through Si-O-Si bonds, so as to obtain hollow silica nanoparticles covalently fixed with fluorescent donor molecules. (5) The hollow silica nanoparticles with covalently fixed fluorescent donor molecules are redispersed in a dispersion medium and assembled by vertical deposition to form a photonic crystal film with a periodic ordered structure, thereby obtaining the hollow silica photonic crystal film based on covalently fixed fluorescent donor.

5. The preparation method according to claim 4, characterized in that, In step (1), the monodisperse polystyrene nanospheres are synthesized by dispersion polymerization: polyvinylpyrrolidone is used as a stabilizer and azobisisobutyramidine hydrochloride is used as an initiator, and the reaction is carried out at 50-70°C for 5-8 h under nitrogen protection.

6. The preparation method according to claim 4, characterized in that, Step (2) specifically involves dispersing PS nanoparticles in ethanol, adding ammonia as a catalyst, and adding tetraethyl orthosilicate to carry out a hydrolysis-condensation reaction to form a silica shell on the surface of the polystyrene nanoparticles; the calcination temperature in step (3) is 400-600℃, and the holding time is 1-3 h.

7. The preparation method according to claim 4, characterized in that, In step (4), the silanized fluorescent donor molecule is silanized acridine orange; the hydrolyzable silane group in the silanized acridine orange undergoes a hydrolysis-condensation reaction with the silanol groups on the surface of the hollow silica nanoparticles to form Si-O-Si bonds, thereby covalently fixing the silanized acridine orange to the surface of the hollow silica nanoparticles.

8. The preparation method according to claim 4, characterized in that, Step (5) includes washing the hollow silica nanoparticles after the grafting reaction to remove unreacted or physically adsorbed fluorescent donor molecules, redispersing the washed hollow silica nanoparticles in ethanol or an ethanol-water mixed solvent, and assembling them on the surface of a transparent substrate to form a photonic crystal film by vertical deposition.

9. A method for detecting EGCG using a hollow silica photonic crystal sensing film based on a covalently fixed fluorescence donor as described in any one of claims 1 to 3, characterized in that, Includes the following steps: The hollow silica photonic crystal sensing film based on covalently fixed fluorescent donor is contacted with the fluorescent acceptor molecule rhodamine 6G and a surfactant. The surfactant enriches rhodamine 6G near the silica shell with acridine orange fixed through micellar enrichment and interfacial assembly, promoting the formation of a fluorescence resonance energy transfer system between the fluorescent donor molecule and rhodamine 6G. Add the test sample containing EGCG and ammonium molybdate solution. The EGCG in the test sample reacts with ammonium molybdate to form an EGCG-Mo complex. The EGCG-Mo complex interacts with rhodamine 6G, changing the fluorescence state of rhodamine 6G and the energy transfer efficiency from acridine orange to rhodamine 6G, thereby causing a change in the fluorescence intensity of the system. The fluorescence intensity change of the hollow silica photonic crystal sensing film based on the covalently fixed fluorescence donor is detected, and the content of EGCG in the sample is determined according to the quantitative relationship between the fluorescence intensity change and the EGCG concentration.

10. The method for detecting EGCG based on a hollow silica photonic crystal sensing film with a covalently fixed fluorescent donor according to claim 9, characterized in that, The excitation wavelength for fluorescence detection is 350-380 nm, the emission wavelength is 550-600 nm, and the surfactant is sodium dodecylbenzenesulfonate.

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