A method for preparing p-toluenesulfonic acid assisted des carbon dots and detecting tetracycline

Abstract

The application discloses a kind of p-toluenesulfonic acid auxiliary DES carbon dot preparation and tetracycline detection method, comprising: the synthesis of deep eutectic solvent DES;Extraction of lignin;The preparation of fluorescent TLD-CDs;Tetracycline detection.The present application uses deep eutectic solvent DES as green extraction medium, and efficiently extracts the lignin in bamboo powder;With the DES extraction of bamboo powder lignin as the core precursor, continue to use DES as the reaction medium, and add p-toluenesulfonic acid as auxiliary reagent, adopt hydrothermal method to synthesize the carbon quantum dots CQDs with high fluorescent performance;When carbon quantum dots contact with tetracycline, obvious fluorescence quenching phenomenon will occur, and then realize the rapid, sensitive detection of tetracycline residue in environment;The present application realizes the high-value utilization of bamboo powder lignin, and the fluorescent sensing system constructed is simple in operation and environment-friendly, which provides a new technical scheme for the rapid quantitative detection of tetracycline residue in natural water body.

Description

Technical Field

[0001] This invention relates to the field of p-toluenesulfonic acid-assisted DES carbon dot preparation and tetracycline detection, and particularly to a p-toluenesulfonic acid-assisted DES carbon dot preparation and tetracycline detection method. Background Technology

[0002] With increasing global focus on energy sustainability and environmental protection, the efficient conversion and high-value utilization of renewable biomass resources has become an important research direction. Lignocellulose biomass, as the most abundant renewable resource on Earth, is mainly composed of three components: cellulose, hemicellulose, and lignin. Among them, lignin, as the second most abundant natural biomass, has a high carbon content and rich oxygen-containing functional groups, making it a high-quality precursor for the preparation of carbon-based materials. While the stubborn structure of lignin limits the bioconversion efficiency of lignocellulose, it also endows it with potential application value in the field of materials science.

[0003] Bamboo powder, as a typical lignocellulosic biomass resource, is characterized by its wide availability, high renewability, and high lignin content. Achieving efficient separation and high-value conversion of lignin from bamboo powder is of great significance for improving the utilization efficiency of biomass resources and promoting green development.

[0004] Traditional lignin extraction methods generally suffer from drawbacks such as severe equipment corrosion, highly toxic solvents, and low extraction efficiency, which limit their large-scale application.

[0005] Deep eutectic solvent (DES), as a novel green and environmentally friendly solvent, has rapidly become a research frontier in the field of lignin extraction due to its outstanding advantages such as non-toxicity, low preparation cost, excellent lignin solubility, and good environmental compatibility.

[0006] Carbon quantum dots (CQDs), as a novel class of fluorescent nanomaterials, possess advantages such as strong optical stability, low biotoxicity, simple preparation process, and wide range of applications, demonstrating promising prospects in fields such as environmental monitoring and bioimaging. Preparing lignin-based CQDs using lignin extracted from bamboo powder as a precursor can effectively realize the high-value utilization of bamboo powder lignin, breaking through the traditional predicament of low added value utilization of lignin, and fully aligning with the concept of green sustainable development and the development trend of biomass resource recycling.

[0007] At the same time, antibacterial drugs are among the most commonly used drugs in the world. Antibiotics, as a class of complex molecular compounds, can destroy or slow down bacterial growth. Tetracycline is one of the most commonly used antibiotics.

[0008] Since its isolation from Streptomyces in the late 1940s, tetracycline has been widely used in animal husbandry, agriculture, and medicine due to its broad-spectrum activity, low cost, and high efficacy. It can inhibit the activity of most Gram-positive and Gram-negative bacterial strains and protozoan parasites, making it a first-line treatment option for many infectious diseases. However, overuse of tetracycline not only leads to the emergence of antibiotic resistance genes (ARGs), weakening the drug's antibacterial activity, but also spreads horizontally to other bacteria, causing serious negative impacts on human health.

[0009] More concerning is that tetracycline has a low metabolic rate in humans and animals, and its residues have been widely found in soil, surface water, marine environments, and even biological samples. These residues accumulate along the food chain, exert toxicity on microbial communities, damage aquatic ecosystems, and pollute drinking water and irrigation water, as well as disrupt the human gut microbiota, thus becoming a major threat to the environment and human health.

[0010] Traditional tetracycline detection methods have limitations such as expensive instruments and complex operation (similar to the drawbacks of traditional methods for detecting moisture in organic solvents). Fluorescence sensing has become an ideal choice for tetracycline detection due to its advantages of simple operation, rapid response, and low cost. Among them, the internal filtration effect (IFE) does not require complex modification of the fluorescent probe, has a simple detection principle, and is widely used.

[0011] Therefore, in view of the problems existing in the prior art, it is an urgent technical problem to be solved by those skilled in the art to provide a rapid quantitative detection method for tetracycline residues in natural water bodies. Summary of the Invention

[0012] In view of this, the present invention provides a method for preparing DES carbon dots assisted by p-toluenesulfonic acid and for detecting tetracycline.

[0013] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A method for preparing DES carbon dots with p-toluenesulfonic acid and detecting tetracycline includes the following steps: (1) Synthesis of deep eutectic solvent DES: Oxalic acid OA and choline chloride ChCl are mixed in a specified molar ratio, heated and stirred, and stirred intermittently to promote complete melting of the solid and the formation of a stable hydrogen bond network structure. After the system forms a transparent liquid, it is placed in a desiccator and cooled to room temperature to obtain DES. (2) Extraction of lignin: The DES prepared in step (1) is mixed with bamboo powder at a specified mass ratio and reacted in an oil bath to obtain a brown solution; After the reaction was completed, the flask was transferred to an ice-water bath to cool to room temperature. Then the reaction mixture was filtered through a Buchner funnel lined with filter paper. After centrifugation of the filtrate, the solid precipitate at the bottom was collected and washed 4-5 times with deionized water until the pH of the washing solution was neutral. The precipitate was then freeze-dried to obtain pure lignin. (3) Preparation of fluorescent TLD-CDs: Take the DES from step (1), the lignin from step (2), p-toluenesulfonic acid TsOH and deionized water, add them to the reaction vessel and mix evenly, and heat at a constant temperature; After the reaction was completed, the mixture was cooled to room temperature, filtered through a microporous membrane, and freeze-dried to obtain fluorescent TLD-CDs powder. (4) Tetracycline detection: Prepare tetracycline aqueous solutions with different concentration gradients. Transfer 100 μL of fluorescent TLD-CDs aqueous solution to 1000 μL of tetracycline solutions of different concentrations. After mixing evenly, measure and record the fluorescence intensity of the solution at the optimal excitation wavelength. Quantitative detection of tetracycline can be achieved by measuring the change in fluorescence intensity.

[0014] Preferably, in step (1), oxalic acid OA and choline chloride ChCl are mixed in a molar ratio of 1:1 and heated and stirred at 60°C for 3 hours.

[0015] Preferably, in step (2), the DES prepared in step (1) is mixed with bamboo powder at a mass ratio of 1:15 and reacted in an oil bath at 120°C for 1 hour; finally, it is freeze-dried for 12 hours to obtain pure lignin.

[0016] Preferably, in step (3), 3 mL of DES from step (1), 0.03 g of lignin from step (2), 0.03 g of p-toluenesulfonic acid TsOH, and 2 mL of deionized water are added to a reaction vessel and mixed evenly, and heated at 200°C for 12 hours.

[0017] Preferably, in step (3), the reaction mixture is filtered through a 0.22 μm microporous membrane and freeze-dried for 24 hours to obtain fluorescent TLD-CDs powder.

[0018] Preferably, step (4) further includes a stability test of the fluorescent TLD-CDs, specifically: (a) Time stability test: 100 μL of fluorescent TLD-CDs aqueous solution was dispersed in 1000 μL of ultrapure water, and the fluorescence intensity was continuously detected and recorded at 6-minute intervals over 30 minutes; (b) pH stability test: 100 μL of fluorescent TLD-CDs aqueous solution was added to 1000 μL of aqueous solution with pH value of 7-9, mixed well and the fluorescence intensity was measured and recorded. (c) Temperature stability test: 100 μL of fluorescent TLD-CDs aqueous solution was added to 1000 μL of aqueous solution and heated for 30 minutes under different temperature conditions. The fluorescence intensity was detected and recorded.

[0019] Preferably, step (4) further includes ion selectivity testing: 100 μL of fluorescent TLD-CDs aqueous solution is transferred to 1000 μL of aqueous solution containing different ions, mixed evenly, and the fluorescence intensity of each system is measured and recorded.

[0020] Preferably, step (4) further includes an anti-interference test: adding aqueous solutions of different ions to an aqueous system containing the target tetracycline, transferring 100 μL of fluorescent TLD-CDs aqueous solution to it, mixing it evenly, measuring the fluorescence intensity, comparing the fluorescence intensity change when no coexisting ions are present, and evaluating the anti-interference ability.

[0021] Preferably, in step (4), all fluorescence tests are set up in three parallel experiments, and the average value of the three sets of data is used as the final experimental result.

[0022] Preferably, the method also includes actual water sample testing: tap water, Siyuan Lake water and Yongjiang River water are selected as actual water samples. After the water samples are filtered and pretreated, tetracycline standard solution is added to prepare spiked water samples. The tetracycline spiking concentration is set to 25 μM, 50 μM, 70 μM and 100 μM, and its fluorescence spectrum is tested.

[0023] The present invention achieves the following technical effects compared to the prior art: This invention first uses a DES system to efficiently extract lignin from bamboo powder. Characterization confirms that it retains a complete aromatic skeleton and abundant oxygen-containing functional groups, with uniform molecular weight distribution and moderate particle size. Subsequently, using lignin as the carbon source, DES as the reaction medium, and p-toluenesulfonic acid as the auxiliary reagent, fluorescent TLD-CDs were synthesized via a hydrothermal method. These CDs are rich in oxygen- and nitrogen-containing polar functional groups, exhibiting a near-spherical shape with a particle size of approximately 5.7 nm, good dispersibility, and excellent optical, thermal, and pH stability. Their optimal excitation wavelength is 370 nm, and the mechanism of tetracycline quenching is an internal filtration effect. These carbon dots demonstrated good selectivity and anti-interference ability for tetracycline detection, with a good linear relationship between fluorescence intensity and tetracycline concentration. Actual water sample spiked recovery experiments showed that they can be reasonably applied to water sample detection. This invention realizes the high-value utilization of bamboo powder lignin, and the constructed fluorescence sensing system is simple to operate and environmentally friendly, providing a new technical solution for the rapid quantitative detection of tetracycline residues in natural water bodies. Attached Figure Description

[0024] Figure 1 A flowchart illustrating the simplified synthesis of fluorescent TLD-CDs for tetracycline detection; Figure 2 (A) Comparison of FT-IR spectra of lignin extracted by different DES systems (4000-500 cm⁻¹) -1 (B) UV-Vis absorption spectrum of lignin; Figure 3 (A) Comparison of FT-IR spectra of lignin extracted by different DES systems (4000-500 cm⁻¹) -1 (B) UV-Vis absorption spectrum of lignin; Figure 4 (A) GPC auto-scaled chromatogram of lignin; (B) GPC calibration curve; Figure 5 (A) Fourier transform infrared spectrum of fluorescent TLD-CDs; (B) Ultraviolet-Vis absorption spectrum; Figure 6 (A) Full XPS spectrum of TLD-CDs, (B) High-resolution XPS spectrum of C1s, (C) High-resolution XPS spectrum of N1s and (D) High-resolution XPS spectrum of O1s; Figure 7 (A) Full XPS spectrum of TLD-CDs, (B) High-resolution XPS spectrum of C1s, (C) High-resolution XPS spectrum of N1s and (D) High-resolution XPS spectrum of O1s; Figure 8 (A) XPS full spectrum of TLD-CDs, (B) C1s high-resolution XPS spectrum, (C) N1s high-resolution XPS spectrum and (D) O1s high-resolution XPS spectrum; Figure 9 The UV-Vis absorption spectrum of tetracycline (TC), and the excitation spectrum (Ex = 371 nm) and emission spectrum (Em = 486 nm) of TLD-CDs are shown. Figure 10 (A) Effects of temperature, (B) pH and (C) time on the fluorescence intensity of TLD-CDs; Figure 11 (A) Fluorescence emission spectra of TLD-CDs at different tetracycline concentrations; (B) Selective response of TLD-CDs to tetracycline; (C) Comparison of fluorescence response of TLD-CDs to tetracycline and coexisting interfering substances; (D) Linear calibration curve of TLD-CD fluorescence intensity versus tetracycline concentration. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] like Figure 1 As shown, this invention discloses a method for preparing DES carbon dots with p-toluenesulfonic acid and detecting tetracycline, specifically including: 1. Experimental reagents The reagents required for the experiment are shown in Table 1; Table 1: Experimental Reagents and Materials

[0027] 2. Experimental apparatus The instruments required for the experiment are shown in Table 2.

[0028] Table 2: List of Experimental Instruments

[0029] 3. Lignin Extraction First, oxalic acid (OA) and choline chloride (ChCl) were mixed in a 1:1 molar ratio and heated and stirred at 60 °C for 3 h to gradually form a homogeneous and transparent deep eutectic solvent (DES).

[0030] During the reaction, intermittent stirring is necessary to promote complete melting of the solid and the formation of a stable hydrogen bond network structure. After the system has completely formed a transparent liquid, it should be placed in a desiccator and cooled to room temperature to avoid moisture absorption.

[0031] The prepared DES and bamboo powder were reacted in an oil bath at 120°C for 1 hour at a mass ratio of 1:15 to obtain a brown solution. After the reaction was completed, the flask was placed in an oil bath and cooled to room temperature in an ice-water bath to terminate the reaction. The cooled reaction mixture was then slowly transferred to a Buchner funnel lined with filter paper for filtration. After centrifugation, the solid precipitate at the bottom was collected and washed 4-5 times with deionized water until the pH of the washing solution reached neutral to completely remove residual DES and small molecule impurities from the precipitate. Finally, the solid precipitate was transferred to a freeze dryer and freeze-dried for 12 hours to obtain pure lignin.

[0032] 4. Preparation of fluorescent TLD-CDs Take 3 mL of the prepared DES, 0.03 g of lignin, 0.03 g of p-toluenesulfonic acid (TsOH) and 2 mL of deionized water, add them together to the reaction vessel, mix thoroughly, and then heat the reaction vessel at 200℃ for 12 h. After the reaction is completed, wait for the reaction vessel to cool to room temperature, filter the reaction mixture through a 0.22 μm microporous membrane, and freeze-dry for 24 h until sample powder is obtained, thus obtaining fluorescent TLD-CDs.

[0033] 5. Optical testing of fluorescent TLD-CDs The stability test was conducted using the following methods: 100 μL of fluorescent TLD-CDs aqueous solution was dispersed in 1000 μL of ultrapure water. The fluorescence intensity of the solution was continuously detected and recorded at 6-minute intervals over a 30-minute period to complete the time stability test. 100 μL of fluorescent TLD-CDs aqueous solution was then transferred to 1000 μL of aqueous solutions with different pH values ​​(7-9), mixed thoroughly, and the fluorescence intensity was measured and recorded to conduct the pH stability test. 100 μL of fluorescent TLD-CDs aqueous solution was then added to 1000 μL of aqueous solution, and heated at different temperatures for 30 minutes. After heating, the fluorescence intensity of the solution was measured and recorded to conduct the temperature stability test.

[0034] 6. Test of the responsiveness of fluorescent TLD-CDs to tetracycline First, tetracycline aqueous solutions with different concentration gradients were prepared. 100 μL of fluorescent TLD-CDs aqueous solution was transferred to 1000 μL of tetracycline solutions of different concentrations and mixed thoroughly. The fluorescence intensity of the solution was then measured and recorded under the optimal excitation wavelength.

[0035] The ion selectivity test procedure is as follows: Take 100 μL of fluorescent TLD-CDs aqueous solution and add it to 1000 μL of aqueous solution containing different ions. After mixing thoroughly, measure and record the fluorescence intensity of each system.

[0036] Anti-interference test: In an aqueous system containing the target tetracycline, different ion aqueous solutions were added separately. 100 μL of fluorescent TLD-CDs aqueous solution was transferred and added to each solution. After mixing thoroughly, the fluorescence intensity was measured. The fluorescence intensity change was compared with that without coexisting ions to evaluate the anti-interference ability of the fluorescent carbon dots for tetracycline detection. Three parallel experiments were set up for the test, and the average value was used for analysis.

[0037] All of the above fluorescence tests were conducted in triplicate, and the average of the three sets of data was used as the final experimental result for subsequent calculations and discussions.

[0038] 7. Actual water sample testing To explore the practical application value of fluorescent TLD-CDs, this experiment selected tap water, Siyuan Lake water, and Yongjiang River water as actual water samples. After filtration and pretreatment, tetracycline standard solution was added to the water samples to prepare spiked water samples. The tetracycline spiking concentrations were set to 25 μM, 50 μM, 70 μM, and 100 μM, respectively, and then their fluorescence spectra were tested.

[0039] Example 1: Fourier Transform Infrared Spectroscopy (FT-IR) Analysis The functional group composition of lignin was characterized by Fourier transform infrared spectroscopy (FTIR), and the results are as follows: Figure 2 As shown in Figure A.

[0040] To further verify the accuracy of the extracted lignin structure, the deep eutectic solvent (DES) ratio was replaced with lactic acid and choline chloride (molar ratio 3:1). Lignin was prepared under the condition of 6 h oil bath time and other unchanged operating conditions. Its infrared characteristic peaks were completely consistent with those of the lignin obtained by the oxalic acid-choline chloride system, proving that the lignin extracted by different DES systems all maintained typical chemical structures.

[0041] At 3442 cm -1 The presence of a broad and strong absorption peak at 2939 cm⁻¹ indicates the abundance of phenolic and aliphatic hydroxyl groups in the sample, which provide active sites for subsequent chemical modification of lignin; -1 The absorption peak at that point is attributed to the CH stretching vibration of the methyl or methylene group, reflecting the presence of alkyl structures in the lignin side chain.

[0042] At 1727 cm -1 A distinct absorption peak was observed at the point, corresponding to the stretching vibration of the C=O double bond in non-conjugated ketones, carbonyl groups, and ester groups, reflecting the structural characteristics of carbonyl functional groups in lignin molecules.

[0043] In addition, 1519 cm -1 The characteristic peaks are closely related to the vibrations of the lignin aromatic ring skeleton and are typical indicators of the lignin aromatic structure, indicating that the sample retains a complete lignin aromatic skeleton. (1336 cm⁻¹) -1 and 1220 cm -1 The absorption peaks at 1160 cm⁻¹ correspond to the stretching vibrations of CH and CO in the syringyl (S) structural unit, further confirming the syringyl unit structure of lignin; -1 The absorption peak is attributed to the stretching vibration of the ether bond (COC) in the lignin molecule, reflecting the ether bond connection mode between lignin molecules.

[0044] The aforementioned characteristic peaks collectively indicate that the obtained sample retains the typical aromatic skeleton structure of lignin and a variety of oxygen-containing functional groups, demonstrating good chemical structural integrity.

[0045] Ultraviolet-visible absorption spectroscopy (UV-Vis) can be used to perform qualitative analysis of chromophores and conjugated systems in the lignin structure. The results are as follows: Figure 2 As shown in Figure B, a distinct absorption peak is observed in the 260-375 nm region. This is mainly attributed to the π-π* electronic transitions in the aromatic ring structure of the lignin molecule, as well as the π-π* electron transitions of substituents (such as hydroxyl and methoxy groups) and conjugated carbonyl groups on the aromatic ring. or n-π Electronic transitions indicate that the sample contains typical aromatic structural units (such as guaiacol and syringyl), which is one of the core structural features of lignin.

[0046] Example 2: Thermogravimetric Analysis (TGA) and Particle Size Distribution like Figure 3 Thermogravimetric analysis results indicate that lignin exhibits obvious staged thermal decomposition characteristics during the heating process.

[0047] As the temperature rises, a significant weight loss peak appears in the range of approximately 203-234℃. The weight loss near 234℃ is mainly attributed to the degradation of the lignin side chain structure. At the same time, some small molecular components in the lignin extracted by DES undergo cleavage, generating gaseous products such as CO, CO2, and CH4.

[0048] As the temperature rises further, another significant weight loss stage occurs around 361℃. This stage is mainly related to the breakage of β-O-4 bonds and other bonds within the lignin molecule, leading to the formation of a large number of small molecule volatile products, which in turn causes further mass loss.

[0049] This result indicates that the obtained lignin exhibits typical thermal decomposition behavior and shows a relatively obvious structural cleavage process in the medium and high temperature range.

[0050] Statistical analysis of the particle size distribution of lignin particles, such as... Figure 3 As shown in Figure B, the results indicate that the sample particle size is mainly distributed in the range of 0-3 μm, with particles of approximately 1-2 μm accounting for the highest proportion. The particle size distribution exhibits a unimodal skewed distribution, with an average particle size of approximately 1.5 μm. Simultaneously, only a small number of aggregated particles larger than 5 μm exist, accounting for less than 5%, resulting in a relatively uniform overall particle size distribution. The smaller and more uniform particle size can effectively increase the specific surface area of ​​lignin, enhancing its reactivity and dispersion properties in subsequent composite or functionalization processes, thus providing a favorable morphological basis for the preparation of lignin-based functional materials.

[0051] Example 3: Molecular Weight Analysis To further investigate the structural characteristics of the prepared lignin, it was characterized by gel permeation chromatography (GPC), and the molecular weight and distribution of the lignin were determined.

[0052] According to the calculation results of the GPC auto-scaling chromatogram and calibration curve, the number-average molecular weight (Mn) of this lignin is 636 Da, the weight-average molecular weight (Mw) is 736 Da, and the polydispersity index (PDI) is 1.16. Figure 4 As shown in Figure A.

[0053] The lower molecular weight indicates that the lignin polymer structure underwent a certain degree of depolymerization under the action of the DES system, forming relatively smaller molecular structural units. Simultaneously, the narrower molecular weight distribution suggests that the resulting lignin molecules have a more uniform structure. The GPC calibration curve exhibits a good linear relationship, indicating that the test results have good reliability. Figure 4 As shown in B.

[0054] In summary, this lignin has a low molecular weight and a narrow molecular weight distribution, which provides a good structural basis for its subsequent material modification and functionalization applications.

[0055] Example 4: Characterization of fluorescent TLD-CDs like Figure 5 As shown in Figure A, the FT-IR spectrum indicates that at approximately 3400 cm⁻¹... -1 The presence of a broad absorption peak at approximately 1700 cm⁻¹ is mainly attributed to the stretching vibrations of OH and NH, indicating the abundance of oxygen- and nitrogen-containing functional groups such as hydroxyl and amino groups on the carbon dot surface; -1 The presence of a distinct absorption peak nearby corresponds to the C=O stretching vibration, indicating that the carbon dot surface contains carboxyl or amide structures. In addition, at 1500-1200 cm -1 Vibrational peaks of CN and CO bonds were observed within the range, further indicating the introduction of nitrogen- and oxygen-containing functional groups onto the carbon dot surface. The presence of these polar functional groups not only enhances the hydrophilicity of the carbon dots but also provides important surface states for their optical properties. UV-Vis absorption spectroscopy results show that... Figure 5 As shown in Figure B, the carbon dot exhibits a significant absorption peak in the wavelength range of approximately 220-280 nm, which is mainly attributed to the π-π transition of C=C in the sp² carbon structure. At the same time, a weak absorption tail band appears in the longer wavelength region, which is usually related to the n-π transition caused by surface functional groups such as C=O or CN, and is consistent with the functional group information observed in the infrared spectrum.

[0056] The above results show that the carbon dots prepared with lignin as the carbon source and DES as the dopant have abundant oxygen- and nitrogen-containing functional groups on their surface and exhibit typical optical absorption characteristics of carbon-based nanomaterials, laying a structural foundation for their subsequent applications in fields such as fluorescence sensing.

[0057] like Figure 6 As shown in A-6D, the XPS spectrum clearly confirms that the carbon dots prepared using the lignin / deep eutectic solvent (DES) system and p-toluenesulfonic acid as raw materials are mainly composed of three elements: C, N, and O. The full spectrum shows characteristic peaks of C1s, N1s, and O1s at approximately 284 eV, 400 eV, and 531 eV, respectively, which intuitively demonstrates that the carbon dot surface is rich in oxygen- and nitrogen-containing functional groups. This is consistent with the functional groups reflected in the FTIR and UV-Vis spectra, and fully reveals the elemental composition characteristics of the carbon dots.

[0058] After fitting, the high-resolution C1s spectrum can be divided into two main components at approximately 284.6 eV and 286 eV, corresponding to C / C=C and CO / CN bonds, respectively. This indicates that the carbon dots have formed a carbon framework with a certain degree of graphitization, and also shows that the surface is loaded with abundant oxygen- and nitrogen-containing functional groups, which directly reflects the core structure and surface characteristics of the carbon dots. The N1s spectrum shows a single significant characteristic peak at approximately 400 eV, further confirming that nitrogen is mainly doped into the surface or framework of the carbon dots in the form of CN and NH, which not only enhances the polar characteristics of the carbon dots, but also provides support for their optical properties and application potential. The O1s spectrum can be decomposed into two sub-peaks around 531 eV and 532-533 eV, which belong to C=O and CO / OH bonds, respectively, completely covering the existence forms of various oxygen-containing functional groups on the carbon dot surface.

[0059] Depend on Figure 7 As can be seen from Figure A, the prepared carbon dots are generally uniformly dispersed without obvious agglomeration, indicating good dispersibility and stability. The carbon dots exhibit a near-spherical or quasi-spherical structure with a relatively regular morphology.

[0060] according to Figure 7 As indicated in B, the particle size is approximately 5.7 nm, falling within the typical nanoscale range. The smaller and more uniform particle size not only improves the dispersion stability of the carbon dots but also enhances their quantum confinement effect, thus imparting excellent fluorescence properties to the material.

[0061] Example 5: Optical characterization of fluorescent TLD-CDs like Figure 8As shown in Figure A, the carbon dots exhibit obvious fluorescence emission peaks at different excitation wavelengths from 300 to 400 nm, and show excitation-dependent luminescence characteristics. The emission peak position gradually redshifts with the excitation wavelength, and the fluorescence intensity first increases and then decreases, reaching its maximum value at 370 nm excitation. Therefore, 370 nm is determined to be the optimal excitation wavelength for this carbon dot.

[0062] like Figure 8 The fluorescence lifetime test results of B show that the decay curve of the carbon point conforms to the single exponential fitting law, and the average fluorescence lifetime is about 1.30 μs, indicating that its luminescence process mainly comes from the radiative recombination of uniform surface states or defect states.

[0063] Example 6: Feasibility Analysis of Fluorescence Response of TLD-CDs like Figure 9 As shown, the spectral relationship between the prepared TLD-CDs and tetracycline (TC) is illustrated to elucidate its fluorescence detection mechanism: the blue curve represents the optimal excitation spectrum of the carbon dots, with the excitation peak located at 371 nm, corresponding to the maximum emission wavelength of 486 nm in the red emission curve; the black TC UV absorption curve shows a strong absorption band in the 300-400 nm range, which significantly overlaps with the carbon dot excitation peak (371 nm), indicating that TC can effectively compete for the absorption of excitation light and weaken the fluorescence emission of the carbon dots.

[0064] This phenomenon confirms that the fluorescence response of carbon dots to TC mainly originates from the internal filtering effect (IFE), that is, TC suppresses the fluorescence signal of carbon dots by absorbing excitation light (or partially emitting light), rather than through energy transfer or electron transfer processes, thereby enabling the fluorescence detection of tetracycline.

[0065] Example 7: Stability and Selectivity Detection of TLD-CDs To evaluate the stability of the carbon dots in fluorescent TLD-CDs, the changes in their fluorescence intensity under different temperature, pH, and time conditions were tested.

[0066] like Figure 10 As shown in Figure A, the fluorescence intensity of the carbon dots remained basically stable within the temperature range of 0-40℃, with only slight fluctuations, indicating that the carbon dots have good thermal stability.

[0067] The pH stability test results are shown in Figure 10B. The carbon dots can maintain a high and relatively stable fluorescence intensity in the pH range of 4-8. The fluorescence intensity is slightly enhanced at pH≈5, but the overall change is small, indicating that the carbon dots still have good fluorescence stability in a wide range of acid and alkaline environments.

[0068] Furthermore, the time stability experiment results show that... Figure 10As shown in Figure C, the fluorescence intensity of the carbon dots remained basically constant during the 0-30 min test, without any obvious attenuation, indicating that they have good photostability.

[0069] To evaluate the fluorescence detection performance of the prepared lignin-based carbon dots for tetracycline (TC), its fluorescence response, selectivity, anti-interference ability, and concentration response relationship were systematically studied.

[0070] like Figure 11 As shown in A-11D, the fluorescence intensity of the carbon dot decreased significantly at approximately 470 nm after the addition of tetracycline, indicating that tetracycline effectively quenched the fluorescence signal of the carbon dot, demonstrating that the carbon dot exhibits good fluorescence response characteristics to tetracycline. Selectivity experiments showed that the fluorescence intensity of the carbon dot changed little in the presence of various common metal ions and amino acids (such as Na⁺, Al³⁺, Ba²⁺, Mg²⁺, Fe³⁺, Glu, Try, etc.), while the fluorescence decreased significantly after the addition of tetracycline, indicating that the carbon dot has high selectivity for tetracycline.

[0071] Meanwhile, in the anti-interference experiment, when tetracycline coexisted with multiple interfering substances, the system fluorescence still showed a significant quenching effect, indicating that these coexisting substances had little impact on the detection results, demonstrating the system's good anti-interference ability. Furthermore, as the tetracycline concentration increased from 0 μmol / L to 100 μmol / L, the fluorescence intensity of the carbon dots gradually decreased, exhibiting a clear concentration dependence.

[0072] A linear fitting curve plotted based on the relationship between fluorescence intensity and tetracycline concentration shows a good linear relationship between the two within this concentration range. The fitting equation is y = -12.67x + 1635.8, and the correlation coefficient R0 is [value missing]. 2 = 0.987. The above results indicate that the lignin-based carbon dots have good detection performance for tetracycline, enabling sensitive detection of tetracycline and showing potential application value in environmental monitoring and food safety testing.

[0073] Example 8: Actual water sample testing To verify the reliability and accuracy of the constructed fluorescent TLD-CDs system in actual samples, spiked recovery experiments were conducted on samples of tap water, Siyuan Lake water, and Yongjiang River water.

[0074] The results showed that the recoveries of each water sample at different spiking levels ranged from 99.04% to 110.80%, and the relative standard deviations (RSDs) of each group of experiments were all below 2%, with the lowest reaching 0.04%. These results indicate that the prepared fluorescent TLD-CDs system exhibits good anti-interference ability and detection accuracy for tetracycline in actual water matrices, demonstrating excellent method precision. It is suitable for the quantitative detection of tetracycline in natural water bodies, meeting the needs of practical environmental monitoring, and the experimental results are stable and reliable.

[0075] Table 3 shows the detection of tetracycline in actual water samples by optical TLD-CDs. Table 3: Detection of tetracycline in real water samples by fluorescent TLD-CDs

[0076] This study successfully prepared TLD-CDs with good fluorescence properties using lignin extracted by p-toluenesulfonic acid-assisted deep eutectic solvent extraction as a carbon source. The average fluorescence lifetime was approximately 1.30 μs, the optimal excitation wavelength was 370 nm, the maximum emission wavelength was 486 nm, the particle size was uniform, and the dispersion was good. In actual water sample detection, the recovery rate reached 99.04%-110.80%, and the relative standard deviation was less than 2%, demonstrating excellent detection accuracy and precision.

[0077] In summary, the lignin-based carbon dots prepared by this method can efficiently and sensitively detect tetracycline in water, providing a simple and feasible new approach for the high-value utilization of natural biomass and the detection of tetracycline pollutants in the aquatic environment.

[0078] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing DES carbon dots with p-toluenesulfonic acid assistance and detecting tetracycline, characterized in that, Includes the following steps: (1) Synthesis of deep eutectic solvent DES: Oxalic acid OA and choline chloride ChCl are mixed in a specified molar ratio, heated and stirred, and stirred intermittently to promote complete melting of the solid and the formation of a stable hydrogen bond network structure. After the system forms a transparent liquid, it is placed in a desiccator and cooled to room temperature to obtain DES. (2) Extraction of lignin: The DES prepared in step (1) is mixed with bamboo powder at a specified mass ratio and reacted in an oil bath to obtain a brown solution; After the reaction was completed, the flask was transferred to an ice-water bath to cool to room temperature. Then the reaction mixture was filtered through a Buchner funnel lined with filter paper. After centrifugation of the filtrate, the solid precipitate at the bottom was collected and washed 4-5 times with deionized water until the pH of the washing solution was neutral. The precipitate was then freeze-dried to obtain pure lignin. (3) Preparation of fluorescent TLD-CDs: Take the DES from step (1), the lignin from step (2), p-toluenesulfonic acid TsOH and deionized water, add them to the reaction vessel and mix evenly, and heat at a constant temperature; After the reaction was completed, the mixture was cooled to room temperature, filtered through a microporous membrane, and freeze-dried to obtain fluorescent TLD-CDs powder. (4) Tetracycline detection: Prepare tetracycline aqueous solutions with different concentration gradients. Transfer 100 μL of fluorescent TLD-CDs aqueous solution to 1000 μL of tetracycline solutions of different concentrations. After mixing evenly, measure and record the fluorescence intensity of the solution at the optimal excitation wavelength. Quantitative detection of tetracycline can be achieved by measuring the change in fluorescence intensity.

2. The method for preparing DES carbon dots with p-toluenesulfonic acid and detecting tetracycline according to claim 1, characterized in that, In step (1), oxalic acid OA and choline chloride ChCl are mixed in a molar ratio of 1:1 and heated and stirred at 60°C for 3 hours.

3. The method for preparing DES carbon dots with p-toluenesulfonic acid and detecting tetracycline according to claim 1, characterized in that, In step (2), the DES prepared in step (1) is mixed with bamboo powder at a mass ratio of 1:15 and reacted in an oil bath at 120°C for 1 hour; finally, it is freeze-dried for 12 hours to obtain pure lignin.

4. The method for preparing DES carbon dots with p-toluenesulfonic acid and detecting tetracycline according to claim 1, characterized in that, In step (3), take 3 mL of DES from step (1), 0.03 g of lignin from step (2), 0.03 g of p-toluenesulfonic acid TsOH, and 2 mL of deionized water, add them to the reaction vessel and mix them evenly. Heat the mixture at 200°C for 12 hours.

5. The method for preparing DES carbon dots with p-toluenesulfonic acid and detecting tetracycline according to claim 1, characterized in that, In step (3), the reaction mixture is filtered through a 0.22 μm microporous membrane and freeze-dried for 24 hours to obtain fluorescent TLD-CDs powder.

6. The method for preparing DES carbon dots with p-toluenesulfonic acid and detecting tetracycline according to claim 1, characterized in that, Step (4) also includes a stability test of the fluorescent TLD-CDs, specifically: (a) Time stability test: 100 μL of fluorescent TLD-CDs aqueous solution was dispersed in 1000 μL of ultrapure water, and the fluorescence intensity was continuously detected and recorded at 6-minute intervals over 30 minutes; (b) pH stability test: 100 μL of fluorescent TLD-CDs aqueous solution was added to 1000 μL of aqueous solution with pH value of 7-9, mixed well and the fluorescence intensity was measured and recorded. (c) Temperature stability test: 100 μL of fluorescent TLD-CDs aqueous solution was added to 1000 μL of aqueous solution and heated for 30 minutes under different temperature conditions. The fluorescence intensity was detected and recorded.

7. The method for preparing DES carbon dots with p-toluenesulfonic acid and detecting tetracycline according to claim 1, characterized in that, In step (4), the ion selectivity test is also included: 100 μL of fluorescent TLD-CDs aqueous solution is transferred to 1000 μL of aqueous solution containing different ions, mixed evenly, and the fluorescence intensity of each system is measured and recorded.

8. The method for preparing DES carbon dots with p-toluenesulfonic acid and detecting tetracycline according to claim 1, characterized in that, The step (4) also includes an anti-interference test: adding aqueous solutions of different ions to an aqueous system containing the target tetracycline, transferring 100 μL of fluorescent TLD-CDs aqueous solution to it, mixing it evenly and measuring the fluorescence intensity, comparing the fluorescence intensity change when no coexisting ions are present, and evaluating the anti-interference ability.

9. The method for preparing DES carbon dots with p-toluenesulfonic acid and detecting tetracycline according to claim 1, characterized in that, In step (4), all fluorescence tests are set up in three parallel experiments, and the average value of the three sets of data is used as the final experimental result.

10. The method for preparing DES carbon dots with p-toluenesulfonic acid and detecting tetracycline according to claim 1, characterized in that, It also includes actual water sample testing: tap water, Siyuan Lake water and Yongjiang River water were selected as actual water samples. After the water samples were filtered and pretreated, tetracycline standard solution was added to prepare spiked water samples. The tetracycline spiking concentration was set to 25μM, 50μM, 70μM and 100μM, and its fluorescence spectrum was tested.

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