Tri-emission ratio fluorescent nanosensor for tetracycline detection and its preparation method

Abstract

This invention relates to a three-emission ratio fluorescence nanosensor for detecting tetracycline and its preparation method. The sensor uses a carbon dot-gold cluster nanocomposite material as the fluorescence signal. The synthesis steps and detection process of the carbon dot-gold cluster nanocomposite material in this invention are simple, convenient, and rapid, exhibiting rich fluorescence color changes. It does not require the assistance of complexes or coupling reagents. By constructing a three-emission ratio fluorescence sensor, the goal of highly sensitive and selective detection of tetracycline can be achieved.

Description

Technical Field

[0001] This invention relates to a three-emission ratio fluorescent nanosensor for detecting tetracycline and its preparation method. Background Technology

[0002] Tetracycline antibiotics are broad-spectrum antibiotics, represented by tetracycline (TC), oxytetracycline (OTC), and chlortetracycline (CTC). They are effective against Gram-positive or Gram-negative bacteria, as well as many pathogens resistant to cell wall inhibitors. Tetracyclines are commonly used antibiotics, primarily for the prevention and treatment of diseases in humans and animals, and in aquaculture and livestock farming. However, the continuous accumulation of tetracycline in water can disrupt the balance of the ecosystem and, through bioaccumulation in the food chain, harm the entire ecological environment and human health. Therefore, it is essential to develop a sensitive, rapid, and efficient method for detecting trace amounts of tetracycline.

[0003] Common methods for tetracycline analysis include chromatographic methods such as high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS), as well as other methods such as enzyme-linked immunosorbent assay (ELISA) and biological methods. Chromatographic methods provide accurate analysis, but are generally time-consuming and require complex sample pretreatment and specialized personnel. Biological or ELISA methods are relatively simpler than the former, but they have high requirements for reagent types and suffer from drawbacks such as high cost, susceptibility to mutation, and significant susceptibility to interference from complex matrices. Fluorescence analysis methods, on the other hand, have seen rapid development in chemical sensing and bioanalysis due to their high sensitivity, ease of operation, and shorter processing time. Ratio fluorescence sensors have attracted widespread attention due to their effective improvement in signal-to-noise ratio and reliable quantitative analysis.

[0004] Currently, most ratio fluorescence sensors used for tetracycline analysis are dual-emission modes. Their color composition modes mainly involve blue-red, green-red, and green-blue in analyte identification, but the color change window is narrow and the color visual range is not wide enough. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides a method for preparing a three-emission ratio fluorescent nanosensor for tetracycline detection. This method enables the design of a rapid and highly sensitive tetracycline detection system, where the blue emission of carbon dots, the red emission of gold clusters, and the green emission of ovalbumin-sensitized tetracycline can be clearly distinguished. The synthesis route and detection process are simple and rapid, providing a new approach for the visual detection of antibiotics in actual water samples.

[0006] A method for preparing a tri-emission ratio fluorescent nanosensor for detecting tetracycline includes the following steps:

[0007] Step 1: Dissolve 3-aminophenylboronic acid in water, add sodium hydroxide while stirring continuously, transfer to a high-pressure reactor, heat at 170-190℃ for 3-5 hours using hydrothermal method, filter through a microporous membrane and dialyze to obtain 3-aminophenylboronic acid carbon dots;

[0008] Step 2: Add ovalbumin to the 3-aminophenylboronic acid carbon dots described in Step 1 to form carbon dot-functionalized ovalbumin, which acts as a reducing agent and stabilizer in the synthesis of gold clusters, to obtain carbon dot-gold cluster nanocomposite material, namely the three-emission ratio fluorescent nanosensor of the present invention.

[0009] Preferably, step 1 is as follows: Weigh 0.05-0.15g of 3-aminophenylboronic acid and dissolve it in 5-15mL of water. Add 0.1-0.3mL of 0.05-0.15mol / L sodium hydroxide while stirring continuously. Transfer the solution to a high-pressure reactor with a polytetrafluoroethylene liner. Heat the solution at 170-190℃ for 3-5h. Cool the solution to room temperature and filter it through a 0.22μm microporous membrane. Dialyze the supernatant for 20-28h to obtain 3-aminophenylboronic acid carbon dots.

[0010] Preferably, step 2 is as follows: 10-30 mg of ovalbumin is added to 1-5 mL of 3-aminophenylboronic acid carbon dots, and the mixture is reacted at room temperature for 0.5-1.5 h. Then, 340-370 μL of 5-15 mmol / L chloroauric acid solution is added to the mixture, and the mixture is stirred continuously at room temperature for 1-5 min. Subsequently, 0.5-1.5 mol / L sodium hydroxide is added to adjust the pH of the solution to 8-10, and the mixture is reacted continuously at 90-100 °C for 0.5-1.5 h. After the reaction is complete, the crude product is dialyzed for 20-28 h to obtain the carbon dot-gold cluster nanocomposite material.

[0011] A three-emission ratio fluorescent nanosensor for detecting tetracycline was prepared using the method described above for preparing a three-emission ratio fluorescent nanosensor.

[0012] Preferably, the sensor uses a carbon dot-gold cluster nanocomposite material as the fluorescence signal.

[0013] Compared with the prior art, the present invention has the following advantages:

[0014] (1) The ovalbumin involved in this invention is a glycoprotein that can interact with boric acid-functionalized carbon dots through boron affinity, resulting in a gold cluster nanocomposite material based on carbon dot hybridized ovalbumin.

[0015] (2) This invention uses carbon dot-gold cluster nanocomposite materials as a fluorescence signal source. It creatively constructs a three-emission ratio fluorescence nanosensor by utilizing the dynamic quenching mechanism of the red fluorescence peak and the quenching of the blue fluorescence peak caused by the internal filtering effect, and the enhancement of the inherent green fluorescence peak of tetracycline by the sensitization effect of ovalbumin and tetracycline. The resulting nanosensor exhibits rich fluorescence color variations, providing an effective approach for rapid and visual identification of tetracycline. Specifically, the three-emission ratio fluorescence sensor designed in this method can visually and rapidly identify tetracycline. Visual fluorescence detection enables immediate detection by naked-eye judgment through color signal (color intensity or hue) output, providing a reliable new platform for rapid on-site analysis. The ratiometric fluorescence sensor records two or more independent fluorescence signals under the influence of the analyte, effectively improving the signal-to-noise ratio and enabling reliable quantitative analysis of the analyte.

[0016] (3) This invention does not require the help of complexes and coupling reagents, and has significant advantages such as simplicity, speed, high selectivity, high sensitivity, good reliability and strong practicality; the detection range of this method is relatively wide in the range of 0.5-40μM. Attached Figure Description

[0017] Figure 1 This is a schematic diagram illustrating the preparation and tetracycline detection process of the carbon dot-gold cluster nanocomposite material involved in the embodiments of the present invention;

[0018] Figure 2 The images shown are transmission electron microscopy (A) and Fourier transform infrared (B) images of the carbon dot-gold cluster nanocomposite material involved in the embodiments of the present invention.

[0019] Figure 3 The graph shows the change in fluorescence emission peak intensity and color change under ultraviolet light at 365 nm for the three-emission ratio fluorescent nanosensor involved in the embodiments of the present invention as the amount of tetracycline added increases.

[0020] Figure 4 This is an experimental diagram of the selectivity of the three-emission ratio fluorescent nanosensor involved in the embodiments of the present invention relative to different antibiotic analogs; the concentration of tetracycline and other analogs is 10 μmol / L. Detailed Implementation

[0021] The present invention will now be described in detail with reference to specific embodiments. It should be noted that the following embodiments are merely further illustrations of the present invention, but the scope of protection of the present invention is not limited to the following embodiments.

[0022] Example 1

[0023] A method for preparing a three-emission ratio fluorescent nanosensor for detecting tetracycline, see [link to documentation]. Figure 1 As shown, the steps are as follows:

[0024] Step 1, Preparation of 3-aminophenylboronic acid carbon dots: Weigh 0.1g of 3-aminophenylboronic acid and dissolve it in 10mL of water. Add 0.2mL of 0.1mol / L sodium hydroxide while stirring continuously. Transfer the solution to a high-pressure reactor with a polytetrafluoroethylene liner. Heat at 180℃ for 4h. Cool to room temperature and filter through a 0.22μm microporous membrane. Dialyze the supernatant for 24h to obtain 3-aminophenylboronic acid carbon dots.

[0025] Step 2, Preparation of the three-emission ratio fluorescent nanosensor: 20 mg of ovalbumin was added to 3 mL of 3-aminophenylboronic acid carbon dots obtained in Step 1, and the mixture was reacted at room temperature for 1 h. Then, 355 μL of 10 mmol / L chloroauric acid solution was added to the mixture, and the mixture was stirred continuously at room temperature for 3 min. Subsequently, 1 mol / L sodium hydroxide was added to adjust the pH of the solution to 9, and the mixture was reacted continuously at 98 °C for 1 h. After the reaction was completed, the crude product was dialyzed for 24 h to obtain the carbon dot-gold cluster nanocomposite material, which is the three-emission ratio fluorescent nanosensor of the present invention.

[0026] 70 μL of carbon dot-gold cluster nanocomposite material was diluted in 920 μL of Tris-HCl buffer (10 mmol / L, pH 8.5), and 10 μL of the test solution was added for spectral determination.

[0027] The prepared carbon dot-gold cluster nanocomposite material was vacuum dried and then measured using a transmission electron microscope. Figure 2 A demonstrates that the nanomaterial has a near-spherical morphology, good dispersibility, and an average diameter of approximately 3-4 nm. The lattice interstices, measured from high-resolution transmission electron microscopy images, are 0.217 nm, consistent with d. 1100 The hexagonal structure of graphene is close to that of graphene. Figure 2 In B, the carbon point is 3376 cm⁻¹ -1 The peak values ​​at these locations correspond to the tensile vibrations of OH. (1581, 1618, 1490 cm) -1 The characteristic peaks at these locations are vibrational peaks for C=C, C=O, and NN, respectively. Furthermore, at 1371 and 1430 cm⁻¹... -1 Vibrations corresponding to the asymmetric BO stretching mode were observed at 1030 and 1167 cm. -1 The absorption peaks are attributed to the stretching vibrations of CB and BOH, respectively. Therefore, it can be determined that the carbon dot surface contains abundant boric acid groups, which provides the conditions for the subsequent synthesis of carbon dot-gold cluster nanocomposites. Infrared spectroscopy analysis of the gold clusters shows clear, broad characteristic peaks ~3398 cm⁻¹. -1 OH groups were observed at 1541 and 1644 cm. -1The two distinct characteristic peaks at λ correspond to amide groups, which is consistent with the structure of ovalbumin. Therefore, the carbon dot-gold cluster nanocomposite material possesses characteristic peaks of both carbon dots and gold clusters. Furthermore, the carbon dot-gold cluster nanocomposite material can be clearly observed at 1167 cm⁻¹ in the infrared spectrum. -1 The disappearance of the characteristic peak (BOH) indicates that the hybridization of this nanocomposite material is indeed assembled through the interaction of borate groups with the cis-diol bonds on ovalbumin.

[0028] 70 μL of carbon dot-gold cluster nanocomposite material was diluted in 920 μL of Tris-HCl buffer (10 mmol / L, pH 8.5), and then 10 μL of a series of tetracycline solutions of different concentrations (final tetracycline concentrations of 0.5, 2, 3, 5, 7, 8, 10, 15, 25, and 40 μmol / L) were added. The mixture was shaken well for a period of time, and then the fluorescence intensity of each sample was measured using a fluorescence spectrometer.

[0029] This method not only has a wide detection range in the 0.5-40μM range, but also provides obvious visualization of color changes.

[0030] like Figure 3As shown, with increasing tetracycline concentration, the intensities of the blue (423 nm) and red (620 nm) emission peaks of the carbon dot-gold cluster nanocomposite gradually decrease, while the intensity of the green emission peak (520 nm) increases. By establishing a linear relationship between the fluorescence intensity ratio change and the tetracycline concentration, highly sensitive detection of tetracycline can be achieved based on the fluorescence intensity changes of the test solution. Simultaneously, a rich color change is observed, with the solution color clearly changing from blue-purple to pink, orange-red, yellow, and finally yellow-green. Specifically, before the addition of tetracycline, this method exhibits two emission peaks at 423 nm and 660 nm, representing the fluorescence emission peaks of the carbon dots and gold clusters, respectively. After the addition of tetracycline, the fluorescence peaks at 423 nm and 660 nm are continuously quenched by TC, while the fluorescence emission peak of TC itself at 520 nm undergoes protein sensitization with OVA, resulting in a significant enhancement of fluorescence. One of the key features of this invention is the simultaneous change in fluorescence intensity across three emission peaks. Under 365nm UV light irradiation, a richer color spectrum emerges, transitioning from initial blue-violet to purplish-red, orange-red, yellow, and finally yellow-green, significantly improving visualization. The principle is as follows: Tetracycline itself has weak fluorescence, but when it interacts with proteins, tetracycline molecules enter the hydrophobic cavity of ovalbumin, causing them to aggregate and restrict their rotational movement, resulting in significant fluorescence enhancement—that is, sensitization by the protein. After the addition of tetracycline, the fluorescence peak at 423nm is continuously quenched by the tetracycline due to the internal filtration effect, and the fluorescence peak at 660nm is continuously quenched by the dynamic quenching process. Meanwhile, the tetracycline's own fluorescence emission peak at 520nm interacts with ovalbumin and is sensitized by the protein, resulting in significant fluorescence enhancement. Therefore, a three-emission fluorescence sensor was designed for tetracycline detection.

[0031] 70 μL of carbon dot-gold cluster nanocomposite material was diluted in 920 μL of Tris-HCl buffer (10 mmol / L, pH 8.5). Then, 10 μL of solutions of tetracycline, oxytetracycline, chlortetracycline, serine, threonine, glutathione, clindamycin, ciprofloxacin, enrofloxacin, sulfonamide, and sulfamethazine (final concentration 10 μmol / L) were added, mixed and shaken for a period of time, and then the fluorescence intensity of each sample was measured using a fluorescence spectrometer.

[0032] like Figure 4 The figure shows the selectivity experiment of the three-emission ratio fluorescent nanosensor involved in the embodiments of the present invention relative to different antibiotic analogs. Figure 4 The substances involved are: tetracycline, oxytetracycline, chlortetracycline, serine, threonine, glutathione, clindamycin, ciprofloxacin, enrofloxacin, sulfonamides, and sulfamethazine. The structural formulas of the above substances are as follows:

[0033]

[0034] exist Figure 4 Among the analytes, tetracycline showed the best fluorescence enhancement to the sensor, followed by oxytetracycline, which was also significantly higher than other antibiotics and amino acids. Since oxytetracycline and tetracycline have similar chemical structures, oxytetracycline also exhibited higher fluorescence enhancement. This phenomenon suggests that the cavity structure of ovalbumin plays a crucial role in the fluorescence enhancement process. These results demonstrate that the prepared fluorescent nanosensor exhibits excellent selectivity for tetracycline detection.

[0035] This invention employs a one-pot hydrothermal method to prepare boron-doped carbon dots. Based on the boron affinity interaction between the ortho-dihydroxyl groups on the surface of the glycoprotein ovalbumin and the borate groups on the carbon dot surface, the modified ovalbumin further acts as a reducing agent and stabilizer in the synthesis of gold clusters, ultimately yielding a carbon dot-gold cluster nanocomposite material. The prepared carbon dot-gold cluster nanocomposite material is used as a fluorescence signal source. The fluorescence intensity of the solution is measured using a fluorescence spectrophotometer (i.e., a fluorescence spectrophotometer). Quantitative detection is performed by utilizing the correlation between the changes in the ratio of red, green, and blue fluorescence intensities and the tetracycline concentration.

[0036] In this invention, the sensitization of ovalbumin with tetracycline molecules significantly enhances the weak fluorescence of tetracycline itself. During tetracycline detection, tetracycline molecules enter the hydrophobic cavity of ovalbumin, causing them to aggregate and restrict their rotational movement, resulting in significant fluorescence enhancement. The blue fluorescence peak at 423 nm of the carbon dot-gold cluster nanocomposite overlaps with the excitation wavelength of tetracycline, and the fluorescence lifetime remains approximately unchanged before and after the addition of tetracycline, indicating a quenching process based on the internal filtration effect. For the red fluorescence at 660 nm, the fluorescence lifetime of the carbon dot-gold cluster nanocomposite significantly decreases after the addition of tetracycline, indicating a dynamic quenching mechanism for the red fluorescence peak. The synthesis steps of the carbon dot-gold cluster nanocomposite and the tetracycline detection process in this invention are simple, convenient, and rapid, with rich fluorescence color changes. Without the aid of complexes or coupling reagents, a three-emission ratio fluorescent nanosensor can be constructed to achieve visualized, highly sensitive, and selective detection of tetracycline.

[0037] This invention can be summarized in other specific forms that do not depart from the spirit or essential features of the invention. Therefore, in all respects, the above embodiments of the invention should be considered illustrative only and not limiting, while the claims define the scope of the invention. The foregoing description does not define the scope of the invention; therefore, any changes within the meaning and scope equivalent to the claims should be considered to be included within the scope of the claims.

Claims

1. A method for preparing a three-emission ratio fluorescent nanosensor for detecting tetracycline, characterized in that, Includes the following steps: Step 1: Dissolve 3-aminophenylboronic acid in water, add sodium hydroxide while stirring continuously, transfer to a high-pressure reactor, heat at 170-190 ℃ for 3-5 h using hydrothermal method, filter through a microporous membrane and dialyze to obtain 3-aminophenylboronic acid carbon dots; Step 2: 20 mg of ovalbumin was added to 1-5 mL of 3-aminophenylboronic acid carbon dots to form carbon dot-functionalized ovalbumin, which participated in the synthesis of gold clusters as a reducing agent and stabilizer. The reaction was carried out at room temperature for 0.5-1.5 h. Then, 340-370 μL of 5-15 mmol / L chloroauric acid solution was added to the mixed product, and the mixture was stirred continuously at room temperature for 1-5 min. Subsequently, 0.5-1.5 mol / L sodium hydroxide was added to adjust the pH of the solution to 8-10, and the reaction was carried out at 90-100 ℃ for 0.5-1.5 h. After the reaction was completed, the crude product was dialyzed for 20-28 h to obtain the carbon dot-gold cluster nanocomposite material.

2. The method for preparing the three-emission ratio fluorescent nanosensor as described in claim 1, characterized in that, The specific steps of step 1 are as follows: Weigh 0.05-0.15 g of 3-aminophenylboronic acid and dissolve it in 5-15 mL of water. Add 0.1-0.3 mL of 0.05-0.15 mol / L sodium hydroxide while stirring continuously. Transfer the solution to a high-pressure reactor with a polytetrafluoroethylene liner. Heat the solution at 170-190 ℃ for 3-5 h. Cool the solution to room temperature and filter it through a 0.22 μm microporous membrane. Dialyze the supernatant for 20-28 h to obtain 3-aminophenylboronic acid carbon dots.

3. A three-emission ratio fluorescent nanosensor for detecting tetracycline, characterized in that, The nanosensor was prepared using the method described in any one of claims 1-2.

4. The three-emission ratio fluorescent nanosensor as described in claim 3, characterized in that, The sensor uses carbon dot-gold cluster nanocomposite material as the fluorescence signal.

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