A rhodamine B-doped silica / xylan carbon dot composite material and its preparation method and application

Abstract

The present invention discloses a rhodamine B-doped silica / xylan carbon dot composite material and a preparation method thereof. In the method, rhodamine B and tetraethyl silicate are uniformly mixed to form RhB@SiO2. The carbon dots are covalently linked to the RhB@SiO2 to obtain RhB@SiO2 / BCD. A short DNA double helix is ​​formed by grafting a complementary chain onto the surface of the BCD, and a single-stranded aptamer with a quencher attached to one end is coupled to the carbon dots. The single-stranded aptamer specifically targets Vibrio parahaemolyticus. When Vibrio parahaemolyticus is present in the test sample, Apt.D detaches from the BCD surface and binds to a recognition site on the surface of the Vibrio parahaemolyticus, restoring fluorescence. Using the red fluorescence intensity R emitted by RhB@SiO2 as an internal standard, quantitative detection of Vibrio parahaemolyticus is performed based on the ratio of the blue fluorescence intensity B of BCD to R.

Description

Technical Field

[0001] The present invention relates to the technical field of preparation of organic / inorganic nano hybrid composite materials, and in particular to a rhodamine B-doped silica / xylan carbon dot composite material and a preparation method thereof. Background Art

[0002] Vibrio parahaemolyticus is a common pathogen in seafood, contaminating near-shore aquatic organisms such as fish, shrimp, shellfish, and plankton. Food poisoning caused by consuming raw or undercooked seafood can lead to illnesses such as acute gastroenteritis, wound infections, and sepsis. Monitoring seafood for the presence of V. parahaemolyticus is a crucial food safety issue. Among existing V. parahaemolyticus detection technologies, traditional culture remains the standard for qualitative and quantitative analysis. However, culture methods require multiple steps, including bacterial enrichment, isolation, and identification, and are extremely time-consuming. Furthermore, rapid PCR-based detection methods require complex detection systems and expensive instrumentation, relying on laboratories with dedicated infrastructure and well-trained personnel. Therefore, the development of rapid, simple, sensitive, and specific detection methods is urgently needed for routine food safety monitoring, large-scale screening, and emergency response.

[0003] Fluorescence-based detection methods offer convenient measurement, simple operation, high sensitivity, strong reliability, and cost-effectiveness, making them suitable for on-site testing. Carbon dots (CDs) are well-dispersed spherical particles with a size less than 10 nm. As an important fluorescent nanomaterial, they have attracted considerable attention due to their interesting properties, including high stability, water solubility, biocompatibility, antibacterial properties, nontoxicity, excellent electron transfer efficiency, abundant edge defects, and tunable photoluminescence. Lignocellulose, owing to its widespread availability, low cost, and environmentally friendly characteristics, has become a popular carbon source for the low-cost preparation of CDs. Lignocellulose is the most abundant biological resource in nature, composed of cellulose, hemicellulose, and lignin. Hemicellulose is a heteropolysaccharide connecting cellulose and lignin, accounting for 20–35% of lignocellulose. Xylan, the main type of hemicellulose, is often used as a model for hemicellulose. It has the advantages of widespread availability, low cost, renewable nature, biodegradability, good biocompatibility, good solubility, high accessibility, and high hydrothermal carbon yield. By using xylan as a carbon source and undergoing a hydrothermal preparation process, a series of blue fluorescent materials with rich carboxyl groups can be constructed, and fluorescent probe functional materials can be further constructed, thus providing new ideas for the high-value utilization of biomass.

[0004] However, at present, most fluorescence detection platforms for bacterial detection use monochromatic fluorescence as the output signal. Such detection platforms are easily affected by the environment, buffer composition, other interfering compounds, instrument changes, changes in excitation light sources, and even experimental operations, resulting in false positive or false negative results, and reliability cannot be guaranteed. In order to overcome this inherent shortcoming, the number of output signals of the fluorescence detection platform is increased from a single to two, and the self-calibration function is achieved by measuring the dual fluorescence ratio signal, thereby minimizing interference and improving analytical dependence. Therefore, rhodamine B is introduced as an internal standard fluorescence signal. Rhodamine B can emit an orange fluorescence with a wavelength of 575nm when excited. Its wavelength and the blue fluorescence emitted by carbon dots have a good wavelength interval in terms of excitation wavelength, and have spectral decoupling characteristics. However, rhodamine B has the problem of easy quenching in the detection system. At the same time, the step of adding rhodamine B is also prone to increase the systematic error in the detection process. To protect rhodamine B from external influences and systematic errors in the detection system, silica was selected as an optical protectant for rhodamine B due to its advantages such as optical transparency, adjustable size and morphology, easy surface functionalization, good biocompatibility, and high chemical and thermodynamic stability. Silica has enormous application value in bioimaging, chemical sensing, optoelectronic device manufacturing, and anti-counterfeiting technology. Encapsulating fluorescent materials such as fluorescent dyes and carbon dots with silica shells can not only increase the doping level of the fluorescent material and amplify the fluorescence signal, but also protect the fluorescent material from quenching caused by external environmental influences, thereby increasing its stability. Multifunctional silica composites can be constructed by assembling, encapsulating, or integrating one or more different luminescent materials within and on the surface of silica nanoparticles in various ways.

[0005] Therefore, the study used silica as a carrier, integrated fluorescent dyes and fluorescent carbon dots inside and on the surface of silica, and then realized the construction of a dual-fluorescence ratio signal probe to prepare a Rhodamine B-doped silica / xylan carbon dot-aptamer composite material. Summary of the Invention

[0006] The present invention aims to provide a method for the proportional fluorescence detection of Vibrio parahaemolyticus based on silica-loaded rhodamine B and xylan carbon dots. This method involves uniformly mixing rhodamine B with tetraethyl silicate in ethanol to form rhodamine B-doped silica nanospheres (RhB@SiO2). Blue-fluorescing carbon dots (BCD) are covalently linked to RhB@SiO2 to produce SiO2 with dual-color fluorescence emission (RhB@SiO2 / BCD). Complementary strands grafted onto the BCD surface form short DNA double helices, and a single-stranded aptamer (Apt.D) with a quencher attached to one end is coupled to the carbon dots. This single-stranded aptamer specifically targets Vibrio parahaemolyticus. When Vibrio parahaemolyticus is present in the test sample, Apt.D detaches from the BCD surface and binds to a recognition site on the bacterium, restoring BCD fluorescence. Through the above method, a ratiometric fluorescence detection probe was established. The red fluorescence intensity (R) emitted by RhB@SiO2 was used as an internal standard. The ratio of the blue fluorescence intensity (B) of BCD to R was used for quantitative detection of Vibrio parahaemolyticus. The present invention also aims to provide a rhodamine B-doped silica / xylan carbon dot composite prepared by the above method.

[0007] The purpose of the present invention is achieved through the following technical solutions.

[0008] A method for the proportional fluorescence detection of Vibrio parahaemolyticus based on silica-loaded rhodamine B and xylan carbon dots comprises the following steps: first, rhodamine B and tetraethyl silicate are uniformly mixed in ethanol to form rhodamine B-doped silica nanospheres RhB@SiO2; then, the xylan carbon dots are covalently linked to the surface of the RhB@SiO2; and finally, a single-stranded aptamer (Apt.D) with a quencher attached to one end is coupled to BCD via surface-grafted complementary chains to form a DNA double helix short chain. The method specifically comprises the following steps:

[0009] (1) Preparation of RhB@SiO2:

[0010] An ethanol solution of rhodamine B is prepared, tetraethyl silicate is added, and the mixture is stirred to uniformly disperse, and then ammonia water is added to continue the reaction; after the reaction is completed, the mixture is added to the uniformly dispersed ethanol solution of tetraethyl silicate, and after stirring, ammonia water and deionized water are added in sequence, and the reaction is continued by stirring, and tetraethyl silicate and ammonia water are added again, and the reaction is continued; after the reaction is completed, the precipitate is obtained by centrifugation and washed with ethanol multiple times to obtain rhodamine B-doped silica RhB@SiO2;

[0011] (2) Surface amination of RhB@SiO2:

[0012] RhB@SiO2 was resuspended in ethanol solution, and 3-aminopropyltriethoxysilane was added while stirring. The reaction was continued. After the reaction was completed, the precipitate was collected by centrifugation to obtain RhB@SiO2-NH2；

[0013] (3) Preparation of BCD:

[0014] The xylan is prepared in a NaOH / urea aqueous solution, stirred evenly, and reacted under pressurized heating conditions; after the reaction is completed, the precipitate is removed by centrifugation and dialyzed to obtain a xylan carbon dot solution;

[0015] (4) Preparation of RhB@SiO2 / BCD:

[0016] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added to the xylan carbon dot solution in proportion, and after activation, the mixture was evenly mixed with RhB@SiO2-NH2 and stirred for reaction. After the reaction was completed, the precipitate was obtained by centrifugation and washed several times to obtain the RhB@SiO2 / BCD composite material.

[0017] (5) Preparation of RhB@SiO2 / BCD-Apt.D:

[0018] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added in proportion to the redispersed liquid of the RhB@SiO2 / BCD composite material, and after activation, a complementary DNA short chain for Apt.D was added to react. After the reaction, the precipitate was obtained by centrifugation, washed multiple times, and redispersed in water. Apt.D modified with a quencher was added and the reaction was gently stirred. After the reaction, the precipitate was centrifuged and washed multiple times to obtain RhB@SiO2 / BCD-Apt.D.

[0019] Furthermore, in step (1), in the ethanol solution of rhodamine B, the concentration of rhodamine B is 2-6 mg / mL, the concentration of tetraethyl silicate is 0.5-2.5% (volume ratio), and the concentration of ammonia water is 15-25% (wt.).

[0020] Furthermore, in step (1), the reaction temperature is room temperature, about 20 to 30°C.

[0021] Furthermore, in step (1), the temperature of the first-stage reaction is 20-30° C., and the reaction time is 30-90 h; the concentration of the tetraethyl silicate ethanol solution is 1-5%, and the reaction time of the second-stage reaction is 3-12 h; and the reaction time after the final addition of tetraethyl silicate and ammonia water is 6-24 h.

[0022] Furthermore, in step (2), the volume ratio of the RhB@SiO2-NH2 dispersion and ethanol is 5-10:30-35, and the concentration of 3-aminopropyltriethoxysilane is 1-5% (v. / v.).

[0023] Furthermore, in step (2), the reaction time is 12 to 48 hours.

[0024] Furthermore, in step (3), the weight average molecular weight of the xylan is 4.5×10 4 ~5.5×10 4 g / mol, and by weight percentage, the sugar groups are: 85.00-90.00% xylose, 7.50-12.00% arabinose, 0.50-1.00% glucose, 0.20-0.70% galactose, and 1.05-2.95% glucuronic acid.

[0025] Furthermore, in step (3), in the NaOH / urea aqueous solution of xylan, the concentration of xylan is 20-50 g / L, the concentration of NaOH is 5-10% (wt.), and the concentration of urea is 10-15% (wt.).

[0026] Furthermore, in step (3), the hydrothermal temperature is 200-260°C.

[0027] Furthermore, in step (3), the reaction time is 12 to 24 hours.

[0028] Furthermore, in step (3), the concentration of the xylan solution is 0.1-0.2 mg / ml.

[0029] Furthermore, in step (3), the weight average molecular weight of the xylan is 4.5×10 4 ~5.5×10 4 g / mol, and by weight percentage, the sugar groups are: 85.00-90.00% xylose, 7.50-12.00% arabinose, 0.50-1.00% glucose, 0.20-0.70% galactose, and 1.05-2.95% glucuronic acid.

[0030] Furthermore, in step (4), the concentration of the xylan carbon dots is 0.1 to 0.2 mg / mL.

[0031] Furthermore, in step (4), the concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution is 30 to 50 mg / mL.

[0032] Furthermore, in step (4), the concentration of the N-hydroxysuccinimide solution is 20 to 40 mg / mL.

[0033] Furthermore, in step (4), the activation time is 15 to 60 minutes, preferably 15 to 30 minutes, and the reaction time is 2 to 4 hours.

[0034] Furthermore, in step (5), the concentration of the rhodamine B-doped silica / xylan carbon dot composite redispersion liquid is 10 to 20 mg / mL; the concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 30 to 50 mg / mL; the concentration of the N-hydroxysuccinimide is 20 to 40 mg / mL; the concentration of the fixed DNA short chain primer is 25 to 100 μmol / L, the volume is 100 μL; and the activation time is 15 to 60 min. ; The binding reaction time of the DNA short-chain primer and the rhodamine B-doped silica / xylan carbon dots is 1 to 3 hours; the binding reaction of Apt.D and the DNA short-chain primer has a reaction time of 2 to 4 hours as described below; the concentration of the Apt.D is 25 to 100 μmol / L, and the reaction time after adding Apt.D is 2 to 4 hours; the concentration of the Vibrio parahaemolyticus aptamer primer modified with a quencher is 100 μM, and the volume is 100 μL; the reaction time during the gentle stirring process is 1 to 3 hours.

[0035] A method for detecting Vibrio parahaemolyticus ratio fluorescence based on silica-loaded rhodamine B and xylan carbon dots, prepared by any of the preparation methods described above.

[0036] The application of the rhodamine B-doped silica / xylan carbon dot composite material as a fluorescent sensor; specifically, when Vibrio parahaemolyticus is present in the test sample, Apt.D detaches from the BCD surface and binds to the recognition site on the surface of Vibrio parahaemolyticus, so that the fluorescence of BCD is restored. Through the above method, a detection probe based on ratio fluorescence is established, and the red fluorescence intensity R emitted by RhB@SiO2 is used as an internal standard. Vibrio parahaemolyticus is quantitatively detected according to the ratio of the blue fluorescence intensity B of BCD to R.

[0037] Compared with the prior art, the present invention has the following advantages:

[0038] (1) The present invention prepares a ratio fluorescence detection method for Vibrio parahaemolyticus based on silica-loaded rhodamine B and xylan carbon dots. The ratio fluorescence model with built-in standards and the response model of fluorescence recovery are used to avoid the systematic error caused by the complex detection environment on the single fluorescence signal. The fluorescence sensor constructed of the prepared nanocomposite material realizes the specific recognition of Vibrio parahaemolyticus based on DNA base matching and competition. It has the advantages of high sensitivity, high specificity, low cost, greenness, good biocompatibility, etc. It has important research and application value in the synthesis of bacterial detection probes based on ratio fluorescence, and can realize the detection of Vibrio parahaemolyticus in seawater and clam samples. It has broad application prospects in the detection and monitoring of foodborne bacteria.

[0039] (2) The present invention introduces rhodamine B and adds optically inert silica as a protection for rhodamine B, thereby reducing the system errors caused by changes in the external environment and experimental operations, and generating a stable and reliable internal standard fluorescence signal. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1a The infrared absorption spectra of SiO2, RhB@SiO2, and RhB@SiO2-NH2 in Example 1;

[0041] Figure 1b The infrared absorption spectra of BCD, RhB@SiO2 / BCD, and RhB@SiO2 / BCD-Apt.D in Example 1 are shown;

[0042] Figure 2a Fluorescence spectra of SiO2, RhB@SiO2, RhB@SiO2 / BCD, and RhB@SiO2 / BCD-Apt.D in Example 1;

[0043] Figure 2b The zeta potential diagrams of SiO2, RhB@SiO2, RhB@SiO2 / BCD, and RhB@SiO2 / BCD-Apt.D in Example 1 are shown;

[0044] Figure 3 This is the sensitivity performance diagram of the RhB@SiO2 / BCD-Apt.D composite material in the detection of Vibrio parahaemolyticus in Example 1;

[0045] Figure 4a Thermogravimetric diagrams of RhB@SiO2 and pure rhodamine B in Example 1;

[0046] Figure 4b This is the photobleaching trace of RhB@SiO2 and pure Rhodamine B in Example 1. DETAILED DESCRIPTION

[0047] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings, but the implementation manner and protection scope of the present invention are not limited thereto.

[0048] The xylan used in the specific embodiment of the present invention is extracted from bagasse, and the weight average molecular weight of xylan is 4.5×10 4 ~5.5×10 4 g / mol, and by weight percentage, the sugar groups are: 85.00-90.00% xylose, 7.50-12.00% arabinose, 0.50-1.00% glucose, 0.20-0.70% galactose, and 1.05-2.95% glucuronic acid.

[0049] Example 1

[0050] The preparation of the Rhodamine B-doped silica / xylan carbon dot composite material and its fluorescence sensor specifically includes the following steps:

[0051] (1) 4.79 mg of rhodamine B and 1 mL of tetraethyl silicate were dispersed in 10 mL of anhydrous ethanol and magnetically stirred for 15 min. Then, 800 μL of ammonia water was slowly added dropwise and the stirring was continued for 80 min to obtain a RhB@SiO2 precursor solution. Meanwhile, another tetraethyl silicate dispersion solution was prepared: 1 mL of tetraethyl silicate was dispersed in 30 mL of anhydrous ethanol and stirred for 30 min to obtain a tetraethyl silicate dispersion solution. The RhB@SiO2 precursor solution was slowly added to the tetraethyl silicate dispersion solution and magnetically stirred for 30 min. Then, 1 mL of ammonia water and 3 mL of water were added and the reaction was allowed to proceed for 6 h. Then, 3 mL of tetraethyl silicate and 4 mL of ammonia water were added and the magnetic stirring was continued for 18 h to obtain RhB@SiO2.

[0052] (2) RhB@SiO2 was dispersed in 40 mL of ethanol solution, 1 mL of 3-aminopropyltriethoxysilane was added, and the mixture was stirred at room temperature (about 24 °C) for 24 hours.

[0053] The infrared spectra of silica nanospheres, RhB@SiO2 and RhB@SiO2-NH2 are shown in Figure 1. Figure 1a It can be seen that the three silica nanospheres are all at 1400 cm -1 , 1095cm -1 , 955cm -1 , 800cm -1 The Si-O stretching vibration peak, the Si-O-Si bond antisymmetric stretching vibration peak, and the Si-OH bending vibration peak are shown at 1232 cm -1 There is an obvious CN stretching vibration peak enhancement at 3460 cm -1 The broad peaks of symmetric and asymmetric stretching vibrations of amino groups (-NH2) appearing at the pores confirm that the surface of the silica nanospheres has been successfully modified with amino groups.

[0054] (3) Preparation of BCD

[0055] A NaOH / urea aqueous solution of xylan was prepared, wherein the concentration of NaOH was 12% (w / w) and the concentration of urea was 8% (w / w), stirred evenly, and reacted at 260° C. for 24 hours. After the reaction was completed, the precipitate was removed by centrifugation and dialyzed to obtain BCD.

[0056] (4) To 5 mL of BCD solution, 38.34 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 23.02 mg of N-hydroxysuccinimide were added and activated under magnetic stirring for 30 min. After activation, 5 mL of aminated rhodamine B-doped silica nanospheres were added and stirred at room temperature for 2 h to obtain RhB@SiO2 / BCD carbon dots.

[0057] (5) Then, 40 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 40 mg of N-hydroxysuccinimide were added to 3 mL of RhB@SiO2 / BCD dispersion and activated with magnetic stirring in a 37°C water bath for 30 min. Subsequently, 100 μL of 50 μmol / L short DNA chain (fssDNA) for immobilization (5'-ATT TTT AGA CGAAT TTTTT-NH2-3', purchased from Beijing Qingke Biotechnology Co., Ltd.) was added and the reaction was continued with stirring for 12 hours. Finally, 100 μL of 50 μmol / L Apt.D (5'-ATTCG TCT AAA AAT GGG CAA AGA AAC AGT GAC TCG TTG AGA TACT-Dabcyl-3', purchased from Beijing Qingke Biotechnology Co., Ltd.) was added and the reaction was continued at 37°C for 2 hours to obtain the RhB@SiO2 / BCD-Apt.D composite material.

[0058] Figure 1b The infrared absorption spectra of BCD, RhB@SiO2 / BCD, and RhB@SiO2 / BCD-Apt.D. Figure 1b It can be seen from the infrared spectrum curve of BCD that 1630cm -1 The strong signal characteristic absorption peak at 1232cm is attributed to the C=O bond of the abundant carboxyl groups on BCD. In the infrared spectrum curves of RhB@SiO2 / BCD and RhB@SiO2 / BCD-Apt.D, there are Si-O stretching and bending vibration peaks and antisymmetric stretching vibration peaks of Si-O-Si bond belonging to silica. In addition, the RhB@SiO2 / BCD composite material is located at 1232cm -1 The signal of the CN bond at 1600 cm-1 decreased after the BCD was connected, and the content of CN bonds belonging to primary amine groups decreased, indicating that the amino groups on the surface of the silica spheres were amidated during the BCD connection process. Since the quencher used in the RhB@SiO2 / BCD-Apt.D composite material is Dabcyl, which contains a CN bond and a carbonyl C=O bond connected to the benzene ring in its molecular structure, the quencher at 1600 cm-1 decreased after the BCD was connected. -1 The CN bond characteristic peak at 1630 cm -1 The carbonyl characteristic peak signals at the positions are enhanced.

[0059] Figure 2a The fluorescence spectra of rhodamine B, xylan carbon dots, RhB@SiO2, RhB@SiO2 / BCD, and RhB@SiO2 / BCD-Apt.D are shown. As can be seen, doping rhodamine B into silica nanospheres only slightly reduces its fluorescence intensity at 575nm, but the protection provided by silica protects rhodamine B from quenching caused by environmental changes. Under ultraviolet excitation, the RhB@SiO2 / BCD composite simultaneously emits blue fluorescence from BCD and orange fluorescence from RhB@SiO2. After coupling with Apt.D, the blue fluorescence emission intensity from BCD is significantly reduced, providing a fluorescence recovery window for constructing a restorative fluorescent probe.

[0060] Figure 2b Zeta potential plots of rhodamine B, xylan carbon dots, RhB@SiO2, RhB@SiO2-NH2, RhB@SiO2 / BCD, and RhB@SiO2 / BCD-Apt.D. The zeta potentials of rhodamine B and RhB@SiO2 are -2.69 mV and -14.4 mV, respectively. After surface amino modification, the zeta potential of RhB@SiO2 increased to +30.3 mV, indicating successful surface amino modification. Because xylan carbon dots have a negative surface charge of -14.3 mV, the incorporation of xylan carbon dots reduces the surface charge of the RhB@SiO2 / BCD material to -34.7 mV. Further incorporation of Apt.D increases the surface zeta potential of the RhB@SiO2 / BCD-Apt.D material to -16.7 mV.

[0061] Figure 3 The sensitivity of the RhB@SiO2 / BCD-Apt.D composite fluorescent probe in the detection of Vibrio parahaemolyticus was investigated. As the concentration of Vibrio parahaemolyticus increased exponentially, the RhB@SiO2 / BCD-Apt.D composite fluorescent probe showed that the blue fluorescence from BCD gradually recovered, while the orange fluorescence intensity from RhB@SiO2 remained roughly unchanged. The ratio of blue fluorescence intensity to red fluorescence intensity was calculated, and this ratio showed a good linear relationship with the concentration of Vibrio parahaemolyticus: Y (ratio of blue and red fluorescence) = 2.37X (Log10 of bacterial concentration) + 0.031 (R 2 =0.9827), and a detection limit of 1.14 CFU / mL (S / N=3) and 8-8×10 5 Detection range of CFU / mL.

[0062] Figure 4aThermogravimetry was used to investigate the effect of an optically inert silica protective layer on rhodamine B. Rhodamine B began to lose weight at 250°C and completely lost weight by 550°C, with a total weight loss of approximately 70%. By doping rhodamine B within silica nanospheres to protect it, RhB@SiO2 exhibited a normal weight loss trend with increasing temperature, but the total weight loss was only approximately 10%. Rhodamine B is embedded within the silica network and bound by the nano-silica, making it less susceptible to weight loss. Between 40°C and 150°C, weight loss primarily stems from the evaporation of free water within the silica pores and from the pure rhodamine.

[0063] Figure 4b The photobleaching traces of Rhodamine B and RhB@SiO2 under full-band xenon lamp irradiation are shown. Under xenon lamp irradiation, Rhodamine B photobleaches, with the fluorescence intensity of its characteristic fluorescence peak at 575 nm decreasing gradually from 758 to 441.9, a 41.7% decrease. However, the Rhodamine B doped in the nano-silica spheres, embedded within the pores of the silica mesh and protected by the silica, shows a decrease in the fluorescence intensity of its characteristic fluorescence peak at 575 nm from 789.5 to 605.2, a decrease of only 23.34%. This demonstrates that silica plays a significant role in protecting the Rhodamine B internal standard from photobleaching.

[0064] Example 2

[0065] The preparation of the Rhodamine B-doped silica / xylan carbon dot composite material and its fluorescence sensor specifically includes the following steps:

[0066] (1) Disperse 10 mg of rhodamine B and 1.5 mL of tetraethyl silicate in 10 mL of anhydrous ethanol, stir magnetically for 15 min, then slowly add 1 mL of ammonia water and continue stirring for 60 min to obtain a rhodamine B-doped silica nanosphere precursor solution; at the same time, prepare another tetraethyl silicate dispersion solution: disperse 3 mL of tetraethyl silicate in 30 mL of anhydrous ethanol and stir for 30 min to obtain a tetraethyl silicate dispersion solution; slowly add the rhodamine B-doped silica nanosphere precursor solution to the tetraethyl silicate dispersion solution, stir magnetically for 30 min, then add 2 mL of ammonia water and 5 mL of water, and react for 12 hours; then, add 3 mL of tetraethyl silicate and 4 mL of ammonia water and continue magnetic stirring for 24 hours to obtain rhodamine B-doped silica nanospheres.

[0067] (2) Rhodamine B-doped silica nanospheres were dispersed in 30 mL of ethanol solution, 0.5 mL of 3-aminopropyltriethoxysilane was added, and the mixture was stirred at room temperature (about 24° C.) for 24 hours.

[0068] (3) Preparation of Xylan Carbon Dots

[0069] A NaOH / urea aqueous solution of xylan was prepared, wherein the concentration of NaOH was 8% (w / w) and the concentration of urea was 8% (w / w), stirred evenly, and reacted at 200°C for 24 hours. After the reaction was completed, the precipitate was removed by centrifugation and dialyzed to obtain xylan carbon dots.

[0070] (4) To 2.5 mL of the xylan carbon dot solution, 38.34 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 23.02 mg of N-hydroxysuccinimide were added and activated under magnetic stirring for 30 min. After activation, 5 mL of aminated rhodamine B-doped silica nanospheres were added and stirred at room temperature for 4 h to obtain rhodamine B-doped silica / xylan carbon dots.

[0071] (5) Then, 30 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 30 mg of N-hydroxysuccinimide were added to 3 mL of the Rhodamine B-doped silica / xylan carbon dot dispersion, and the mixture was activated by magnetic stirring in a 37°C water bath for 30 min. Subsequently, 100 μL of 50 μmol / L short DNA chain (fssDNA) for immobilization was added, and the reaction was continued with stirring for 12 hours. Finally, 100 μL of 50 μmol / L of the Vibrio parahaemolyticus aptamer modified with the quencher Dabcyl was added, and the reaction was continued at 37°C for 4 hours to obtain the Rhodamine B-doped silica / xylan carbon dot / Vibrio parahaemolyticus aptamer composite material.

[0072] Example 3

[0073] The preparation of the Rhodamine B-doped silica / xylan carbon dot composite material and its fluorescence sensor specifically includes the following steps:

[0074] (1) 2.39 mg of rhodamine B and 1 mL of tetraethyl silicate were dispersed in 10 mL of anhydrous ethanol, magnetically stirred for 15 min, and then 400 μL of ammonia water was slowly added dropwise, and the stirring was continued for 60 min to obtain a rhodamine B-doped silica nanosphere precursor solution; at the same time, another tetraethyl silicate dispersion solution was prepared: 1 mL of tetraethyl silicate was dispersed in 30 mL of anhydrous ethanol, and stirred for 30 min to obtain a tetraethyl silicate dispersion solution; the rhodamine B-doped silica nanosphere precursor solution was slowly added to the tetraethyl silicate dispersion solution, magnetically stirred for 30 min, and then 0.5 mL of ammonia water and 1 mL of water were added, and the reaction was carried out for 12 hours; then, 1.5 mL of tetraethyl silicate and 1 mL of ammonia water were added, and the magnetic stirring was continued for 24 hours to obtain rhodamine B-doped silica nanospheres.

[0075] (2) Rhodamine B-doped silica nanospheres were dispersed in 20 mL of ethanol solution, 1 mL of 3-aminopropyltriethoxysilane was added, and the mixture was stirred and reacted at room temperature (about 24° C.) for 24 hours.

[0076] (3) Preparation of Xylan Carbon Dots

[0077] A NaOH / urea aqueous solution of xylan was prepared, wherein the concentration of NaOH was 10% (w / w) and the concentration of urea was 8% (w / w), stirred evenly, and reacted at 240°C for 24 hours. After the reaction was completed, the precipitate was removed by centrifugation and dialyzed to obtain xylan carbon dots.

[0078] (4) To 1 mL of the xylan carbon dot solution, 38.34 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 23.02 mg of N-hydroxysuccinimide were added and activated under magnetic stirring for 30 min. After activation, 2.5 mL of aminated rhodamine B-doped silica nanospheres were added and stirred at room temperature for 2 h to obtain rhodamine B-doped silica / xylan carbon dots.

[0079] (5) Then, 40 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 40 mg of N-hydroxysuccinimide were added to 3 mL of the Rhodamine B-doped silica / xylan carbon dot dispersion, and the mixture was activated by magnetic stirring in a 37°C water bath for 30 min. Subsequently, 100 μL of 10 μmol / L short DNA chain (fssDNA) for immobilization was added, and the reaction was continued with stirring for 12 hours. Finally, 100 μL of 10 μmol / L of the Vibrio parahaemolyticus aptamer modified with the quencher Dabcyl was added, and the reaction was continued at 37°C for 2 hours to obtain the Rhodamine B-doped silica / xylan carbon dot / Vibrio parahaemolyticus aptamer composite material.

[0080] Example 4

[0081] The preparation of the Rhodamine B-doped silica / xylan carbon dot composite material and its fluorescence sensor specifically includes the following steps:

[0082] (1) 40.7 mg of rhodamine B and 1 mL of tetraethyl silicate were dispersed in 10 mL of anhydrous ethanol, magnetically stirred for 15 min, and then 1.6 mL of ammonia water was slowly added dropwise, and the stirring was continued for 80 min to obtain a rhodamine B-doped silica nanosphere precursor solution; at the same time, another tetraethyl silicate dispersion solution was prepared: 1 mL of tetraethyl silicate was dispersed in 30 mL of anhydrous ethanol, and stirred for 30 min to obtain a tetraethyl silicate dispersion solution; the rhodamine B-doped silica nanosphere precursor solution was slowly added to the tetraethyl silicate dispersion solution, magnetically stirred for 30 min, and then 1 mL of ammonia water and 3 mL of water were added, and the reaction was carried out for 6 hours; then, 3 mL of tetraethyl silicate and 4 mL of ammonia water were added, and the magnetic stirring was continued for 18 hours to obtain rhodamine B-doped silica nanospheres.

[0083] (2) Rhodamine B-doped silica nanospheres were dispersed in 40 mL of ethanol solution, 2 mL of 3-aminopropyltriethoxysilane was added, and the mixture was stirred and reacted at room temperature (about 24° C.) for 24 hours.

[0084] (3) Preparation of Xylan Carbon Dots

[0085] A NaOH / urea aqueous solution of xylan was prepared, wherein the concentration of NaOH was 15% (w / w) and the concentration of urea was 10% (w / w), stirred evenly, and reacted at 260°C for 24 hours. After the reaction was completed, the precipitate was removed by centrifugation and dialyzed to obtain xylan carbon dots.

[0086] (4) To 2.5 mL of the xylan carbon dot solution, 383.4 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 230.2 mg of N-hydroxysuccinimide were added and activated under magnetic stirring for 30 min. After activation, 5 mL of aminated rhodamine B-doped silica nanospheres were added and stirred at room temperature for 4 h to obtain rhodamine B-doped silica / xylan carbon dots.

[0087] (5) Then, 40 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 40 mg of N-hydroxysuccinimide were added to 3 mL of the Rhodamine B-doped silica / xylan carbon dot dispersion, and the mixture was activated by magnetic stirring in a 37°C water bath for 30 min. Subsequently, 100 μL of 50 μmol / L short DNA chain (fssDNA) for immobilization was added, and the reaction was continued with stirring for 12 hours. Finally, 100 μL of 50 μmol / L of the Vibrio parahaemolyticus aptamer modified with the quencher Dabcyl was added, and the reaction was continued at 37°C for 4 hours to obtain the Rhodamine B-doped silica / xylan carbon dot / Vibrio parahaemolyticus aptamer composite material.

[0088] Example 5

[0089] The preparation of the Rhodamine B-doped silica / xylan carbon dot composite material and its fluorescence sensor specifically includes the following steps:

[0090] (1) Disperse 10 mg of rhodamine B and 2 mL of tetraethyl silicate in 10 mL of anhydrous ethanol, stir magnetically for 15 min, then slowly add 1 mL of ammonia water and continue stirring for 80 min to obtain a rhodamine B-doped silica nanosphere precursor solution; at the same time, prepare another tetraethyl silicate dispersion solution: disperse 3 mL of tetraethyl silicate in 30 mL of anhydrous ethanol and stir for 30 min to obtain a tetraethyl silicate dispersion solution; slowly add the rhodamine B-doped silica nanosphere precursor solution to the tetraethyl silicate dispersion solution, stir magnetically for 30 min, then add 1 mL of ammonia water and 3 mL of water, and react for 12 hours; then, add 3 mL of tetraethyl silicate and 4 mL of ammonia water and continue magnetic stirring for 24 hours to obtain rhodamine B-doped silica nanospheres.

[0091] (2) Rhodamine B-doped silica nanospheres were dispersed in 40 mL of ethanol solution, 4 mL of 3-aminopropyltriethoxysilane was added, and the mixture was stirred and reacted at room temperature (about 24° C.) for 24 hours.

[0092] (3) Preparation of Xylan Carbon Dots

[0093] A NaOH / urea aqueous solution of xylan was prepared, wherein the concentration of NaOH was 10% (w / w) and the concentration of urea was 12% (w / w), stirred evenly, and reacted at 260°C for 24 hours. After the reaction was completed, the precipitate was removed by centrifugation and dialyzed to obtain xylan carbon dots.

[0094] (4) To 0.5 mL of the xylan carbon dot solution, 38.34 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 23.02 mg of N-hydroxysuccinimide were added and activated under magnetic stirring for 30 min. After activation, 5 mL of aminated rhodamine B-doped silica nanospheres were added and stirred at room temperature for 3.5 h to obtain rhodamine B-doped silica / xylan carbon dots.

[0095] (5) Then, 40 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 40 mg of N-hydroxysuccinimide were added to 3 mL of the Rhodamine B-doped silica / xylan carbon dot dispersion, and the mixture was activated by magnetic stirring in a 37°C water bath for 30 min. Subsequently, 50 μL of 10 μmol / L short DNA chain (fssDNA) for immobilization was added, and the reaction was continued with stirring for 4 hours. Finally, 50 μL of 10 μmol / L of the Vibrio parahaemolyticus aptamer modified with the quencher Dabcyl was added, and the reaction was continued at 37°C for 2 hours to obtain the Rhodamine B-doped silica / xylan carbon dot / Vibrio parahaemolyticus aptamer composite material.

[0096] The above embodiments are only preferred implementation modes of the present invention and are only used to explain the present invention rather than to limit the present invention. Any changes, substitutions, combinations, simplifications, modifications, etc. made by those skilled in the art without departing from the spirit and principles of the present invention shall be considered as equivalent replacement methods and shall be included in the scope of protection of the present invention.

Claims

1. A method for preparing a rhodamine B-doped silica / xylan carbon dot composite material, characterized in that: The steps include: (1) Preparation of RhB@SiO2: An ethanol solution of rhodamine B is prepared, tetraethyl silicate is added, and the mixture is stirred to uniformly disperse, and then ammonia water is added to continue the reaction; after the reaction is completed, the mixture is added to the uniformly dispersed ethanol solution of tetraethyl silicate, and after stirring, ammonia water and deionized water are added in sequence, and the reaction is continued by stirring, and tetraethyl silicate and ammonia water are added again, and the reaction is continued; after the reaction is completed, the precipitate is obtained by centrifugation and washed with ethanol multiple times to obtain rhodamine B-doped silica RhB@SiO2; (2) Surface amination of RhB@SiO2: RhB@SiO2 was resuspended in an ethanol solution, and 3-aminopropyltriethoxysilane was added while stirring. The reaction was continued. After the reaction was completed, the precipitate was collected by centrifugation to obtain RhB@SiO2-NH2. (3) Preparation of blue fluorescent carbon dots BCD: The xylan was prepared in a NaOH / urea aqueous solution, stirred evenly, and reacted under pressurized and heated conditions. After the reaction, the precipitate was removed by centrifugation and dialyzed to obtain a blue fluorescent carbon dot BCD solution. (4) Preparation of RhB@SiO2 / BCD: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added to the xylan carbon dot solution in proportion, and after activation, the mixture was evenly mixed with RhB@SiO2-NH2 and stirred for reaction. After the reaction was completed, the precipitate was obtained by centrifugation and washed several times to obtain the RhB@SiO2 / BCD composite material. (5) Preparation of RhB@SiO2 / BCD-Apt.D: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added in proportion to the redispersed liquid of the RhB@SiO2 / BCD composite material, and after activation, a complementary DNA short chain for Apt.D is added to react; after the reaction, the precipitate is obtained by centrifugation, washed multiple times, and redispersed in water, and Apt.D modified with a quencher is added, and the reaction is gently stirred; after the reaction, the precipitate is centrifuged and washed multiple times to obtain RhB@SiO2 / BCD-Apt.D; the Apt.D is a single-chain adaptor with a fluorescence quencher connected to one end.

2. The method for preparing a Rhodamine B-doped silica / xylan carbon dot composite material according to claim 1, characterized in that: In step (1), in the ethanol solution of rhodamine B, the concentration of rhodamine B is 2-6 mg / mL, the volume percentage concentration of tetraethyl silicate is 0.5-2.5%, and the mass percentage concentration of ammonia water is 15-25 wt%; the temperature of the first stage reaction is 20-30° C., and the reaction time is 30-90 h; the concentration of the ethanol solution of tetraethyl silicate is 1-5%, and the reaction time of the second stage is 3-12 h; and the reaction time after the final addition of tetraethyl silicate and ammonia water is 6-24 h.

3. The method for preparing a Rhodamine B-doped silica / xylan carbon dot composite material according to claim 1, characterized in that: In step (2), the volume percentage concentration of 3-aminopropyltriethoxysilane is 1 to 5%; and the reaction time is 12 to 48 hours.

4. The method for preparing a Rhodamine B-doped silica / xylan carbon dot composite material according to claim 1, wherein: In step (3), the weight average molecular weight of the xylan is 4.5×10 4 ~5.5×10 4 g / mol, and by weight percentage, the sugar groups are: 85.00-90.00% xylose, 7.50-12.00% arabinose, 0.50-1.00% glucose, 0.20-0.70% galactose, and 1.05-2.95% glucuronic acid.

5. The method for preparing a Rhodamine B-doped silica / xylan carbon dot composite material according to claim 1, characterized in that: In step (3), the concentration of xylan is 20-50 g / L, the mass percentage concentration of NaOH is 5-10% (wt.), the mass percentage concentration of urea is 10-15% (wt.), the hydrothermal temperature is 200-260° C., and the reaction time is 12-24 h.

6. The method for preparing a Rhodamine B-doped silica / xylan carbon dot composite material according to claim 1, wherein: In step (4), the concentration of the xylan carbon dot solution is 0.1-0.2 mg / mL; the concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 30-50 mg / mL; the concentration of the N-hydroxysuccinimide is 20-40 mg / mL; the activation time is 15-60 min; and the reaction time is 2-4 h.

7. The method for preparing a Rhodamine B-doped silica / xylan carbon dot composite material according to claim 1, characterized in that: In step (5), the concentration of the rhodamine B-doped silica / xylan carbon dot composite redispersion liquid is 10-20 mg / mL; the concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 30-50 mg / mL; the concentration of the N-hydroxysuccinimide is 20-40 mg / mL; the concentration of the fixed DNA short chain primer is 25-100 μmol / L, and the volume is 100 μL; the activation time is 15-60 min; D The binding reaction time of the NA short-chain primer and the rhodamine B-doped silica / xylan carbon dots is 1 to 3 hours; the binding reaction of Apt.D and the DNA short-chain primer has a reaction time of 2 to 4 hours as described below; the concentration of the Apt.D is 25 to 100 μmol / L, and the reaction time after adding Apt.D is 2 to 4 hours; the concentration of the Vibrio parahaemolyticus aptamer primer modified with a quencher is 100 μM, and the volume is 100 μL; the reaction time during the gentle stirring process is 1 to 3 hours.

8. A rhodamine B-doped silica / xylan carbon dot composite material prepared by the preparation method according to any one of claims 1 to 5.

9. Use of the rhodamine B-doped silica / xylan carbon dot composite material according to claim 8 as a fluorescence sensor.

10. The use according to claim 9, characterized in that: When Vibrio parahaemolyticus is present in the test sample, Apt.D detaches from the BCD surface and binds to the recognition site on the surface of Vibrio parahaemolyticus, restoring fluorescence. Through the above method, a detection probe based on ratiometric fluorescence was established. The red fluorescence intensity R emitted by RhB@SiO2 was used as the internal standard, and the quantitative detection of Vibrio parahaemolyticus was performed based on the ratio of the blue fluorescence intensity B of BCD to R.

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