Melamine-modified carbon dots and their application as a dual-signal fluorescent sensor for salbutamol detection

Abstract

The application discloses a kind of based on melamine modified carbon dot's dual signal fluorescence sensor construction and its application in sotalol detection.The sotalol molecule can be gathered by hydrogen bond interaction to cause originally dispersed melamine modified carbon dot, and cause energy resonance transfer to cause melamine modified carbon dot under the irradiation of 365 nanometer excitation light Blue fluorescence quenching, under the irradiation of 460 nanometer excitation light Yellow fluorescence enhancement.The intensity change of two color fluorescences is linear with the concentration of sotalol, whereby the change of different color fluorescences under two excitation lights realizes low cost, visual quantitative analysis to sotalol residue.This dual signal fluorescence sensor has self-correcting function, improves the accuracy of detection result.

Description

Technical Field

[0001] This invention relates to melamine-modified carbon dots and their application as a dual-signal fluorescence sensor in the detection of salbutamol, belonging to the technical fields of material synthesis, visualization detection, and food quality monitoring. Background Technology

[0002] Salbutamol is a common bronchodilator used to treat chronic lung diseases and asthma. However, it has been misused as a "lean meat enhancer" added to pork, beef, and other foods. Accidental ingestion can cause palpitations, diarrhea, nervousness, and headaches, raising growing concerns about its effects. Salbutamol is currently either banned or used under restricted conditions as a feed additive in many countries. Furthermore, in sporting events, salbutamol is considered a low-dose stimulant. Its use is limited to specific amounts over a certain period; otherwise, it is considered "improper" use. Therefore, the detection of salbutamol, especially in edible meat and drinking water, is of significant practical importance.

[0003] The detection of salbutamol residues requires extremely high sensitivity and strict timeliness. Traditional laboratory methods are unsuitable for rapid detection because they typically require bulky equipment and time-consuming operation by highly trained professionals. In recent years, researchers have utilized fluorescence sensors to achieve sensitive and selective identification of drug residues, biotoxins, and heavy metal ions. Therefore, there is an urgent need for more sensitive, selective, and efficient analytical methods for the rapid trace detection of salbutamol. Summary of the Invention

[0004] To address the shortcomings of existing technologies for detecting salbutamol residues, this invention provides a melamine-modified carbon dot-based dual-signal fluorescence sensor for salbutamol detection. The linear relationship between fluorescence intensity values ​​at wavelengths of 460 nm and 520 nm and salbutamol concentration was investigated. This melamine-modified carbon dot-based dual-signal fluorescence sensor can be used for highly sensitive, visualized, and quantitative detection of salbutamol residues. This sensing system enables reliable, convenient, and on-site detection of salbutamol residues. Specifically, this invention adopts the following technical solution:

[0005] Firstly, the melamine-modified carbon dots provided by this invention are prepared by the following method:

[0006] Step 1: Preparation of carbon dots

[0007] A solution of citric acid and cysteine ​​is heated by microwave to induce a carbonization reaction. After the reaction is complete, the resulting solution is cooled to room temperature, water is added, and the solution is centrifuged. The solution is purified by dialysis and the precipitate is removed by microfiltration. The resulting clear liquid is the carbon dot solution.

[0008] Step 2: Melamine-modified carbon dots

[0009] The buffer solution and carbon dot solution were mixed, and then melamine was added. The mixture was stirred excessively at room temperature to allow the reaction to proceed fully, thus obtaining melamine-modified carbon dots.

[0010] As a preferred technical solution, the melamine-modified carbon dots of the present invention can be prepared by the following method:

[0011] Step 1: Preparation of carbon dots

[0012] Add 2.5–3.0 g of citric acid monohydrate and 1.5–2.0 g of L-cysteine ​​to 18–20 mL of ultrapure water, sonicate until fully dissolved and transparent, and then heat under microwave at 300–400 watts for 4–5 minutes. After the reaction is complete, cool the resulting solution to room temperature, add ultrapure water, centrifuge at 4000–6000 rpm for 2–8 minutes, and purify the resulting solution by dialysis for 5–6 hours. Then filter through a 0.22 μm microporous membrane to remove the precipitate, and the clear liquid obtained is the carbon dot solution.

[0013] Step 2: Melamine-modified carbon dots

[0014] Mix 45–50 mL of buffer solution and 45–50 mL of carbon dot solution, then add 0.03–0.04 mg of melamine and stir vigorously at room temperature for about 15–20 minutes to obtain a melamine-modified carbon dot solution.

[0015] The melamine-modified carbon dots described above are subjected to intermittent microwave heating in step 1, with 3 to 4 pauses during the process, in order to avoid explosive boiling and to prevent uneven carbonization caused by rapid heating.

[0016] The melamine-modified carbon dots described above have a molecular weight cutoff of 800-2000 Da, preferably 1000-1200 Da, for dialysis in step 2. The buffer solution can be a phosphate buffer with pH = 6.0-6.5, preferably a phosphate buffer with pH = 6.5.

[0017] Secondly, this invention also provides the application of the melamine-modified carbon dots in the fluorescence visualization detection of salbutamol. Specifically, the melamine-modified carbon dots can be used as a dual-signal fluorescence sensor for the fluorescence visualization detection of salbutamol.

[0018] The application described in this invention, in the qualitative detection of salbutamol, involves mixing melamine-modified carbon dots with a buffer solution of pH=6, adding the mixture to a quartz colorimetric tube, then adding a salbutamol solution, mixing and shaking, and observing obvious fluorescence color changes at excitation wavelengths of 365nm and 460nm, thereby achieving visual qualitative detection of salbutamol.

[0019] In the quantitative detection of salbutamol described in this invention, 18–20 μL of melamine-modified carbon dot solution is first thoroughly mixed with 1.8–2 mL of pH 6 buffer solution and added to a 3 mL quartz colorimetric tube. Then, 2–3 μL of salbutamol solutions of different concentrations are added sequentially, and the mixture is shaken for 4–5 minutes. Fluorescence spectra in the 400–700 nm range at excitation wavelengths of 365 nm and 460 nm are recorded. By establishing the relationship between the change in fluorescence peak intensity and the concentration of salbutamol, quantitative detection of salbutamol is achieved. The pH 6.0 buffer solution can be a phosphate buffer solution.

[0020] The novel rapid visual dual-signal fluorescence sensor, the core of this invention, achieves selective quantitative detection of salbutamol by integrating melamine-modified carbon dots. Upon contact with the melamine-modified carbon dot-based dual-signal fluorescence sensing system, salbutamol induces the carbon dots to aggregate from dispersion through hydrogen bonding. With increasing salbutamol concentration, the fluorescence intensity of blue emission under 365nm excitation decreases, while the fluorescence intensity of yellow emission under 460nm excitation continuously increases. The detection limits for salbutamol under 365nm and 460nm excitation sources are 7.12 and 16.67 nM, respectively. Therefore, visual fluorescence qualitative detection of salbutamol using MA-CDs (melamine-modified carbon dots) can be achieved, and the content of salbutamol in the sample can be detected by the degree of fluorescence change in the dual-signal fluorescence sensing system, thus achieving quantitative detection of salbutamol. Furthermore, the dual-signal fluorescence detection provides a built-in self-calibration function for salbutamol detection, ensuring high detection sensitivity and accuracy. Furthermore, this dual-signal fluorescence sensing system has been successfully applied to the detection of salbutamol in actual samples, providing a new method for constructing a visualized quantitative system for the detection of trace hazardous substances. This dual-signal fluorescence sensor has a self-correcting function; the blue fluorescence detection results at 365 nm and the yellow fluorescence detection results under 460 nm excitation mutually corroborate each other, improving the accuracy of the detection results.

[0021] The principle of detecting salbutamol using a fluorescence sensor in this invention is based on an aggregation-induced energy resonance transfer strategy. Specifically, salbutamol is enriched due to hydrogen bonding between it and the prepared melamine-modified carbon dots. Appropriate energy levels promote the transfer of excited-state electrons from dispersed carbon dots to aggregated carbon dots, resulting in quenching of blue emission fluorescence under 365 nm excitation and enhancement of yellow fluorescence under 460 nm excitation. The reaction is completed within 5 minutes, enabling visual detection of salbutamol. By establishing the relationship between the change in fluorescence peak intensity and the concentration of salbutamol, quantitative detection of salbutamol is achieved.

[0022] Compared with existing detection technologies, the beneficial effects of this invention are reflected in:

[0023] 1. The dual-signal fluorescence sensing system of this invention detects salbutamol and shows better visual detection effect compared with monochromatic fluorescence detection. It effectively avoids the problem of instability of monochromatic fluorescence intensity. Compared with other dual-signal fluorescence detection, this invention does not require the introduction of a reference fluorescence unit, is simple to operate, and achieves reliable and accurate visual detection.

[0024] 2. On the fluorescence spectrometer of this invention, under excitation light sources of 365 nm and 460 nm, the detection limits of salbutamol are 7.12 and 16.67 nM, respectively, which are lower than the permissible limit for salbutamol residue.

[0025] 3. The fluorescence system of the dual-signal sensor based on melamine-modified carbon dots prepared in this invention has good selectivity and sensitivity to salbutamol, can effectively avoid interference from other impurities, and has a fast response. Attached Figure Description

[0026] Figure 1 The images are transmission electron microscopy (TEM) images: (A) TEM image of the prepared carbon dots (CDs), (B) TEM image of melamine-modified carbon dots (MA-CDs), and (C) TEM image of carbon dots (MA-CDs+Sal) after the addition of salbutamol (Sal). The average particle size of CDs is 4-6 nm, the average particle size of MA-CDs is 5-7 nm, and the average particle size of MA-CDs+Sal is 10-14 nm.

[0027] Figure 2 This is the infrared spectrum of the fluorescent system. The broad absorption band near 3420 cm⁻¹ is related to the stretching vibration of the -OH group of the carboxyl group, and the C=O vibration absorption band near 1651 cm⁻¹ is clearly visible, indicating that the CDs contain a carboxyl group. The bands at 3466 cm⁻¹, 3417 cm⁻¹, and 3219 cm⁻¹ correspond to the -NH vibration of melamine, while the higher wavenumber region of 3000-3500 cm⁻¹ is completely broadened due to the stretching vibration of the -OH and -NH groups of salbutamol.

[0028] Figure 3 This is the X-ray photoelectron spectrum of the prepared carbon dots. The C 1s region spectrum can be divided into three peaks: C=O (288.56 eV), CO / CN (286.35 eV), and C=C (284.66 eV). The N 1s spectrum shows two peaks at 1165.2 eV and 1134.2 eV, belonging to graphic-N and pyrollic-N, respectively. The X-ray photoelectron spectrum further confirms the presence of C=C and CO.

[0029] Figure 4 These are zeta potential diagrams for different samples. The zeta potentials of CDs, MA, MA-CDs, and Sal are -20.14, 10.27, 6.83, and -27.5 mV, respectively, indicating that MA-CDs were successfully formed. The reverse charge effect between MA-CDs and Sal can shorten the distance, making hydrogen bonding interactions possible.

[0030] Figure 5 (A) is the optimal excitation wavelength diagram of the 460nm fluorescence emission peak of the dual-signal fluorescence sensing system, and (B) is the optimal excitation wavelength diagram of the 520nm fluorescence emission peak of the dual-signal fluorescence sensing system.

[0031] Figure 6 This is the optimal reaction time diagram for the dual-signal fluorescence sensing system.

[0032] Figure 7 This is the optimal pH profile for a dual-signal fluorescence sensing system.

[0033] Figure 8 This is the optimal operating temperature diagram for the dual-signal fluorescence sensing system.

[0034] Figure 9 (A) shows the fluorescence spectrum and color change of a melamine-modified carbon dot-based dual-signal fluorescence sensor with different concentrations of salbutamol. (B) shows the relationship between fluorescence intensity and salbutamol concentration under excitation at 365 nm and 460 nm, respectively.

[0035] Figure 10 (A) is the selective spectrum of the fluorescent probe under 365nm excitation, i.e., the dual-signal fluorescent sensor of this invention; (B) is the anti-interference diagram of the fluorescent probe under 365nm excitation; (C) is the fluorescence photograph corresponding to the selectivity of the fluorescent probe under 365nm excitation; (D) is the selective spectrum of the fluorescent probe under 460nm excitation; (E) is the anti-interference diagram of the fluorescent probe under 460nm excitation; (F) is the fluorescence photograph corresponding to the selectivity of the fluorescent probe under 460nm excitation. 1-12 represent K respectively. + Ca 2+ Na + Mg 2+The test included glucose, glycine, L-tyrosine, L-arginine, L-glutamic acid, vitamin C, salbutamol (Sal), and a mixture of all substances. Selectivity tests were performed using 1-10 individual substances, while anti-interference tests were performed by adding Sal to each of the 1-10 individual substances. Detailed Implementation

[0036] The following embodiments are further illustrations of the present invention and serve as explanations of the technical content of the present invention. However, the essence of the present invention is not limited to the embodiments described below. Those skilled in the art can and should know that any simple changes or substitutions based on the spirit of the present invention should fall within the protection scope claimed by the present invention.

[0037] Example 1:

[0038] Fabrication of dual-signal fluorescent nanosensors

[0039] 1. Preparation of carbon dots

[0040] Add 3.0 g of citric acid monohydrate as the carbon source and 2.0 g of L-cysteine ​​to a 50 mL beaker, then mix with 20 mL of ultrapure water. The solution is then sonicated until fully dissolved and transparent. The solution is transferred to a microwave oven and heated at 350 watts (medium power) for 5 minutes. Three pauses are made during heating to avoid explosive boiling and uneven carbonization caused by rapid heating. After the reaction is complete, the resulting solution is cooled to room temperature. An appropriate amount of ultrapure water is added, and the mixture is centrifuged at 5000 rpm for 5 minutes. The final solution is purified by dialysis (1000 Da) for 6 hours, and then filtered through a 0.22 μm microporous membrane to remove the precipitate. The resulting clear liquid is the carbon dot solution, which is stored at 4 °C for later use.

[0041] 2. Preparation of a dual-signal fluorescence sensing system based on melamine-modified carbon dots

[0042] Mix 50 mL of phosphate buffer solution (pH 6.5) with 50 mL of the prepared carbon dot solution, then add 0.037 mg of melamine. Stir thoroughly at room temperature for about 20 minutes to ensure that the melamine is modified on the carbon dot surface. This simple experiment prepared melamine-modified carbon dots, which were then stored at 4 °C for later use, thus obtaining a dual-signal fluorescent nanosensor system.

[0043] Example 2:

[0044] Dual-signal fluorescence sensing mechanism, component characterization, and optimization of detection conditions

[0045] 1. The principle of detecting salbutamol using a fluorescence sensor in this invention is based on an aggregation-induced energy resonance transfer strategy. Salbutamol is enriched due to hydrogen bonding between it and the prepared melamine-modified carbon dots. Appropriate energy levels promote the transfer of excited-state electrons from dispersed carbon dots to aggregated carbon dots, resulting in quenching of blue fluorescence emission under 365nm excitation and enhancement of yellow fluorescence under 460nm excitation. The reaction is completed within 5 minutes, achieving visual detection of salbutamol. Quantitative detection of salbutamol is achieved by establishing the relationship between the change in fluorescence peak intensity and the concentration of salbutamol. Specifically, melamine-modified carbon dots are mixed with ultrapure water and added to a quartz colorimetric tube, followed by the addition of a salbutamol solution. After mixing and shaking, obvious changes in fluorescence color are observed at excitation wavelengths of 365nm and 460nm, achieving visual qualitative detection of salbutamol.

[0046] 2. Component characterization of the dual-signal fluorescence sensing system

[0047] Considering that the detection sensitivity of a probe (sensor) to an analyte is related to its own properties, the structural features and spectral properties of the dual-signal fluorescent nanoprobe were investigated using FT-IR, UV-vis, and fluorescence spectroscopy, respectively. Furthermore, TEM and XPS were used to determine the morphology and elemental composition of the dual-signal fluorescent nanoprobe.

[0048] 3. Effects of excitation wavelength, reaction time, pH, and temperature on dual-signal fluorescent probes

[0049] Excitation wavelength, reaction time, pH, and temperature all have a certain impact on the fluorescence intensity and detection performance of dual-signal fluorescent probes.

[0050] from Figure 5 It can be seen that the blue fluorescence intensity at 460 nm and the yellow fluorescence intensity at 520 nm differ under different excitation wavelengths, indicating that the excitation wavelength affects the fluorescence intensity of the dual-signal fluorescent probe. Taking all factors into consideration, in this invention, 365 nm is selected as the optimal excitation wavelength for blue fluorescence, and 460 nm as the optimal excitation wavelength for yellow fluorescence.

[0051] Reaction time, such as Figure 6 As shown, fluorescence no longer changes when the reaction time exceeds 5 minutes. Taking all factors into consideration, we selected 5 minutes as the optimal reaction time.

[0052] from Figure 7 It can be seen that alkaline conditions have no effect on fluorescence. As the pH value gradually decreases, the fluorescence intensity at 460 nm in the dual-signal fluorescence system also decreases, while the fluorescence intensity at 520 nm increases. This indicates that acidic conditions affect fluorescence intensity. The optimal pH for detection is 6, and a buffer solution with the corresponding pH can be used directly in actual detection.

[0053] Reaction temperature plays a crucial role in the detection efficiency of salbutamol. For example... Figure 8 As shown, when the temperature rises to 40 degrees Celsius, the fluorescence intensity change of the dual-signal fluorescence system can be observed. Taking all factors into consideration, we selected room temperature (25 degrees Celsius) as the optimal reaction temperature.

[0054] Example 3:

[0055] 1. Detection of salbutamol using dual-signal fluorescent probe solution

[0056] To detect salbutamol, using the aforementioned dual-signal fluorescence sensor as the detection reagent, we first thoroughly mixed 20 μL of MA-CDs solution with 2 mL of pH=6 phosphate buffer solution and added it to a 3 mL quartz colorimetric tube. Then, we added 2 μL of salbutamol solutions of different concentrations sequentially, mixed and shaken for 5 minutes, and recorded the fluorescence spectra in the 400-700 nm range at excitation wavelengths of 365 nm and 460 nm, respectively. By establishing the relationship between the change in fluorescence peak intensity and the concentration of salbutamol, we achieved quantitative detection of salbutamol.

[0057] 2. Drawing the standard curve

[0058] Different concentrations of salbutamol solution were added to the dual-signal fluorescent probe solution, and the fluorescence intensity was measured after mixing. The results showed that the blue fluorescence emission peak under 365 nm excitation gradually weakened, while the yellow fluorescence emission peak under 460 nm excitation gradually strengthened. By establishing the relationship between fluorescence intensity and salbutamol concentration, quantitative detection of salbutamol can be achieved. When the excitation light was 365 nm and 460 nm, the fluorescence spectrum of the mixed system in the wavelength range of 400–700 nm was recorded. Figure 9 The relationship between fluorescence intensity and salbutamol concentration is shown, with a linear relationship between the fluorescence intensity change and the concentration. The horizontal axis represents the salbutamol concentration, and the vertical axis represents the fluorescence intensity values ​​at 460 nm and 520 nm.

[0059] Example 4:

[0060] 1. Preparation of carbon dots

[0061] The preparation process in this step is the same as in Example 1.

[0062] 2. Preparation of a dual-signal fluorescence sensing system based on melamine-modified carbon dots

[0063] The preparation process in this step is the same as in Example 1.

[0064] 3. Selectivity and interference testing of fluorescent nanoprobes

[0065] We investigated the effects of MD-CD probes on other possible interfering substances (K). +Ca 2+ Na + Mg 2+ The response was influenced by the concentrations of glucose, glycine, L-tyrosine, L-arginine, L-glutamic acid, and vitamin C (which were 10 times higher than those of salbutamol). Figure 10 As can be seen, the response of interfering substances is much lower than that of salbutamol. The fluorescence response images taken under both excitation conditions are shown in the figure. Experiments demonstrate that this sensing system has high selectivity for the detection of salbutamol. A possible mechanism is that the β-agonist contains a large number of -OH and -NH- groups that form hydrogen bonds with melamine. To assess potential interference, we also studied the fluorescence response of the sensing system to various coexisting substances. These coexisting substances did not cause significant interference to the detection, indicating that the system has strong anti-interference capabilities.

[0066] It should be noted that the above-described technical content of this invention is merely an explanation and clarification to enable those skilled in the art to understand the technical essence of this invention, and therefore is not intended to limit the scope of protection of this invention. The scope of protection of this invention should be determined by the claims. Those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made based on the essential spirit of this invention should be within the scope of protection of this invention.

Claims

1. The application of melamine-modified carbon dots in the fluorescence visualization detection of salbutamol; wherein the melamine-modified carbon dots are prepared by the following method: Step 1: Preparation of carbon dots A solution of citric acid and cysteine ​​is heated by microwave to induce a carbonization reaction. After the reaction is complete, the resulting solution is cooled to room temperature, water is added, and the solution is centrifuged. The solution is purified by dialysis and the precipitate is removed by microfiltration. The resulting clear liquid is the carbon dot solution. Step 2: Melamine-modified carbon dots The buffer solution and carbon dot solution were mixed, and then melamine was added. The mixture was stirred excessively at room temperature to allow the reaction to proceed fully, thus obtaining melamine-modified carbon dots.

2. The application as described in claim 1, characterized in that, The melamine-modified carbon dots were prepared using the following method: Step 1: Preparation of carbon dots Add 2.5-3.0 g of citric acid monohydrate and 1.5-2.0 g of L-cysteine ​​to 18-20 mL of ultrapure water, sonicate until fully dissolved and transparent, and then heat under microwave at 300-400 watts for 4-5 minutes. After the reaction is complete, cool the resulting solution to room temperature, add ultrapure water, centrifuge at 4000-6000 rpm for 2-8 minutes, and purify the resulting solution by dialyzing for 5-6 hours. Then filter through a 0.22 μm microporous membrane to remove the precipitate, and the clear liquid obtained is the carbon dot solution. Step 2: Melamine-modified carbon dots Mix 45-50 mL of buffer solution and 45-50 mL of carbon dot solution, then add 0.03-0.04 mg of melamine, and stir thoroughly at room temperature for 15-20 minutes to obtain melamine-modified carbon dot solution.

3. The application as described in claim 2, characterized in that, Step 1: Microwave heating is performed intermittently.

4. The application as described in claim 2, characterized in that, Step 2: Dialysis cutoff molecular weight 800-2000 Da.

5. The application as described in claim 1, characterized in that, The application of melamine-modified carbon dots as a dual-signal fluorescence sensor in the fluorescence visualization detection of salbutamol.

6. The application as described in claim 5, characterized in that, Melamine-modified carbon dots were mixed with a buffer solution at pH 6 and added to a quartz colorimetric tube. Salbutamol solution was then added, and the mixture was shaken. Obvious fluorescence color changes were observed at excitation wavelengths of 365 nm and 460 nm, enabling the visual qualitative detection of salbutamol.

7. The application as described in claim 5, characterized in that, First, 18-20 μL of melamine-modified carbon dot solution was thoroughly mixed with 1.8-2 mL of pH=6 buffer solution and added to a 3 mL quartz colorimetric tube. Then, 2-3 μL of salbutamol solution of different concentrations was added sequentially, and the mixture was shaken for 4-5 minutes. The fluorescence spectra in the range of 400-700 nm under excitation wavelengths of 365 nm and 460 nm were recorded respectively. By establishing the relationship between the change in fluorescence peak intensity and salbutamol concentration, the quantitative detection of salbutamol was achieved.

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