Fluorescent probe synthesized by utilizing expired medicine as well as preparation method and application of fluorescent probe

Abstract

The invention discloses a fluorescent probe synthesized by utilizing expired drugs as well as a preparation method and application of the fluorescent probe, Abz and ethanol are used as raw materials to synthesize crude carbon dots, a fluorescent carbon needle Abz-CDs with a single emission wavelength is obtained through separation and purification, and the fluorescent probe Abz-CDs is used for detecting hypochlorite ions and chloramphenicol. The content of residual hypochlorite ions ClO <-> and chloramphenicol CAP in food is detected, and food safety is guaranteed.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent nanomaterials, and specifically relates to a fluorescent probe synthesized by using expired drugs, a preparation method and an application thereof, and in particular to a fluorescent probe using expired drugs with a benzene ring structure as a carbon source, a preparation method and an application thereof. Background Art

[0002] Salts containing hypochlorite are called hypochlorites. The most widely known household hypochlorite disinfectant is sodium hypochlorite.

[0003] Hypochlorous acid (HClO) is a powerful disinfectant. Its oxidizing ability can effectively destroy the cell membrane and protein structure of microorganisms, thereby killing pathogens such as bacteria, viruses and fungi. Due to its high efficiency and relative safety, hypochlorous acid has been widely used in the disinfection of surfaces, water and food, indoor air and personal hygiene. - When the content exceeds the standard, it will cause arthritis, tumors, cancer and other diseases. Therefore, the national food and drug inspection agency needs to conduct regular and irregular inspections on pesticide residues in vegetables and other foods to protect the health and safety of the public. Therefore, it is urgent to establish an efficient, fast and accurate method for detecting pesticide residues in vegetables.

[0004] Chloramphenicol (CAP) is a broad-spectrum antibiotic that has inhibitory effects on both Gram-positive and Gram-negative bacteria and has been widely used in the prevention and treatment of livestock and poultry diseases. If chloramphenicol remains in animals for human consumption, it may be transmitted to the human body along the food chain, and even at a trace level, it may have a profound impact on human health. At present, the LC-MS determination method of chloramphenicol antibiotics is mostly used for the determination of residues in food. There are few methods for detecting antibiotic residues in special populations such as pregnant women. Therefore, this method is not suitable for routine sampling.

[0005] However, clinical studies have confirmed that chloramphenicol has strong toxic side effects, which can inhibit bone marrow hematopoiesis, cause allergic reactions, and induce aplastic anemia. Long-term consumption of animal foods with chloramphenicol residues may cause intestinal flora imbalance and inhibit antibody formation. Therefore, many countries prohibit the use of chloramphenicol in animal breeding and treatment, and stipulate that chloramphenicol must not be detected in animal foods. Announcement No. 250 of the Ministry of Agriculture and Rural Affairs of my country also makes clear provisions on this. Therefore, the detection of chloramphenicol in food requires accurate detection methods that are applicable to a wide range of people to ensure the health of consumers and food safety.

[0006] In summary, the purpose of this application is to provide a fluorescent sensor with high anti-interference ability, which can effectively distinguish target analytes in complex environments and can specifically detect hypochlorite ions (ClO -) and chloramphenicol (CAP), so as to facilitate the detection of hypochlorite ions in daily water quality and the rapid screening of chloramphenicol residues in meat products, providing a detection method for food safety and environmental protection. Summary of the invention

[0007] To achieve the above purpose, the present application selected expired drugs as the carbon source of the fluorescent probe, and prepared a fluorescent probe for detecting hypochlorite ions and chloramphenicol by treating the expired drugs by hydrothermal method, as follows:

[0008] The invention discloses a fluorescent probe synthesized by using expired drugs. The probe is fluorescent carbon dots Abz-CDs with green fluorescence prepared by using expired drugs albendazole and ethanol as raw materials through a hydrothermal synthesis method.

[0009] Furthermore, a method for preparing a fluorescent probe synthesized using expired drugs comprises the following steps:

[0010] Step 1: Dissolve 1.2 g of albendazole powder in 15 mL of ethanol solution.

[0011] Step 2: Transfer the thoroughly mixed solution in step 1 to a high-temperature reaction vessel made of polytetrafluoroethylene and promote the reaction at 180° C. for 6 hours;

[0012] Step 3: After the reaction is completed, the reactor is naturally cooled to room temperature;

[0013] Step 4: centrifuge to remove coarse precipitate, and then filter through a 0.22 μm microporous filter membrane to obtain purified fluorescent carbon dots Abz-CDs, which are stored at 4°C.

[0014] Application of fluorescent carbon dots Abz-CDs in detection of hypochlorite ions and chloramphenicol.

[0015] Furthermore, the Abz-CDs are used as fluorescent probes to detect hypochlorite ions ClO - .

[0016] Furthermore, the Abz-CDs detects hypochlorite ions ClO - The steps include:

[0017] 1) Dissolve Abz-CDs in water to obtain a 5 μg / L Abz-CDs solution;

[0018] 2) Containing C lO - The solution was mixed with the Abz-CDs solution, and different volumes of 1 mM ClO- solution were added, and the volume was fixed to 2 mL with a pH 8.0 buffer solution to form ClO - Concentration gradient, λ EmDifferent concentrations of ClO were measured at 485 nm. - The fluorescence intensity of the solution was obtained F0 ;

[0019] 3) Mixing the sample to be tested with the Abz-CDs solution at pH = 4.0 to obtain a mixed solution of the sample to be tested, reacting for 10 minutes, and measuring the fluorescence intensity F of the mixed solution of the sample to be tested;

[0020] 4) According to the fluorescence quenching efficiency F / F0 and ClO - The linear relationship between the concentration and the ClO content in the sample to be tested is calculated. - concentration;

[0021] The linear relationship is obtained by obtaining the fluorescence quenching efficiency F / F0 and ClO - Linear relationship with concentration.

[0022] Furthermore, the fluorescence quenching efficiency F / F0 and ClO are obtained. - The linear relationship of concentration includes:

[0023] In the range of 0 to 5 μM, the linear equation is y1 = 0.02678-0.21263x; R1 2 =0.9959; detection limit LOD1 = 0.0141 μM;

[0024] In the range of 5 to 15 μM, the linear equation is y2 = 0.01549x + 0.97667; R2 2 =0.9889;LOD2=0.193μM;

[0025] In the range of 15 to 125 μM, the linear equation is y3 = 1.27538-0.00587x; R3 2 =0.9987; LOD3=0.511μM.

[0026] Furthermore, the Abz-CDs are used as fluorescent probes to quickly detect the chloramphenicol CAP content.

[0027] Furthermore, the step of detecting chloramphenicol CAP by Abz-CDs comprises:

[0028] 1) Dissolve Abz-CDs in water to obtain a 5 μg / L Abz-CDs solution;

[0029] 2) A solution containing chloramphenicol CAP was mixed with 5 μL Abz-CDs solution, and different volumes of 1 mM CAP solution were added, and the volume was fixed to 1.0 mL with a pH = 8 buffer solution to form a CAP concentration gradient. The fluorescence intensity of CAP solutions with different concentrations was measured at λEm at 485 nm to obtain F0;

[0030] 3) Mixing the sample to be tested with the Abz-CDs solution under the condition of pH=4 to obtain a mixed solution of the sample to be tested, reacting for 15 minutes, and measuring the fluorescence intensity F of the mixed solution of the sample to be tested;

[0031] 4) Calculate the concentration of CAP in the sample to be tested based on the linear relationship between the fluorescence quenching efficiency F / F0 and the CAP concentration.

[0032] Furthermore, the linear relationship between the fluorescence quenching efficiency F / F0 and the CAP concentration is used to calculate the concentration of CAP in the sample to be tested, including:

[0033] When the fluorescence linear range is 0-10μM, the linear equation is y1=2.25748-0.128x; R1 2 =0.9907; detection limit LOD1 = 0.0141 μM (S / N = 0.141);

[0034] When the fluorescence linear range is 10-200 μM, the linear equation is y2=0.00312x+0.8578; R2 2 =0.9930; detection limit LOD2 =0.193 μM (S / N = 1.93).

[0035] Working mechanism of the present invention: The present invention uses a hydrothermal method to treat expired drugs to prepare carbon dots. Expired drugs with a benzene ring structure (albendazole) are used as raw materials. After the hydrothermal reaction is completed, crude carbon dots are obtained. Then, purified fluorescent carbon dots Abz-CDs are obtained by centrifugation and microporous membrane filtration. The carbon dots are used to detect ClO - When the content is in the range of 0-5μM and 5-15μM, with the increase of ClO - As the concentration increases, the fluorescence intensity of Abz-CDs at 485 nm gradually increases; in the range of 15-125 μM, with the increase of ClO - When the carbon dots were used to detect the residual CAP in food, the fluorescence intensity of Abz-CDs at 485nm gradually decreased with the increasing concentration of CAP in the range of 0-10μM; and the fluorescence intensity of Abz-CDs at 485nm gradually increased with the increasing concentration of CAP in the range of 10-200μM.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) Using expired drug albendazole as raw material realizes resource recycling and green production;

[0038] (2) The prepared new carbon dots were used to detect chlorate ions (ClO- ) and chloramphenicol (CAP) with good detection effect and high sensitivity;

[0039] (3) Capable of detecting ClO in a targeted manner - and CAP, so as to facilitate the rapid screening of hypochlorite ions in daily water quality and vegetable residues, as well as chloramphenicol residues in meat products, providing detection methods for food safety and environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 TEM image and particle size distribution diagram of Abz-CDs;

[0041] Figure 2 XRD pattern of Abz-CDs

[0042] Figure 3 X-ray photoelectron spectroscopy (XPS) spectra of Abz-CDs, including: (a) full XPS spectrum of Abz-CDs;

[0043] (b) is the C1 s spectrum;

[0044] (c) is the spectrum of N1 s;

[0045] (d) is the O1 s spectrum;

[0046] Figure 4 is the FT IR graph of Abz-CDs;

[0047] Figure 5 UV-Vis, excitation and emission spectra of Abz-CDs;

[0048] Figure 6 is the fluorescence emission spectra of Abz-CDs under different excitations;

[0049] Figure 7 The results of characterization of the stability of Abz-CDs are shown in Figure 2.

[0050] (a) Effect of pH on the emission peak value,

[0051] (b) Effect of NaCl concentration on the emission peak.

[0052] (c) Effect of H2O2 concentration on the emission peak.

[0053] (d) The effect of natural light on the peak emission value,

[0054] (e) The effect of UV light on the peak emission value;

[0055] Figure 8 Abz-CDs as a fluorescent probe for the detection of ClO- The condition optimization results are:

[0056] (a) Effect of pH on the detection of C l O by Abz-CDs - Fluorescence value changes affect

[0057] (b) Effect of reaction time on the detection of C l O by Abz-CDs - Fluorescence value changes;

[0058] Fig. 9 Detection of ClO for Abz-CDs - Interference performance study results;

[0059] Fig.10 Detection of ClO for Abz-CDs - The fluorescence value changes of ClO - The linear fitting of concentration, (a) is the linear fitting of Abz-CDs and ClO - Concentration-dependent fluorescence spectra;

[0060] (b) Abz-CDs and C l O - Fluorescence spectrum at 0-5 μM;

[0061] (c) Abz-CDs and C l O - Fluorescence spectra at 5-15 μM;

[0062] (d) Abz-CDs and C l O - Fluorescence spectrum at 15-125 μM;

[0063] Fig.11 Abz-CDs+C l O - The results of the fluorescence quenching mechanism study,

[0064] (a) Abz-CDs, C l O - , Abz-CDs and C l O - The UV absorption spectrum of the mixture,

[0065] (b) is the Ex-Em-abs diagram of Abz-CDs.

[0066] (c) Abz-CDs, Abz-CDs+C l O - FL decay lifetime diagram,

[0067] (d) Park equation machine theory proof diagram;

[0068] Fig.12 The results of optimization of the conditions for Abz-CDs as a fluorescent probe to analyze and detect CAP.

[0069] (a) Effect of pH value on the change of CAP fluorescence value detected by Abz-CDs.

[0070] (b) Effect of reaction time on the changes of CAP fluorescence value detected by Abz-CDs;

[0071] Fig.13 Study on the performance of Abz-CDs in detecting CAP interference;

[0072] Fig.14 Abz-CDs detects the fluorescence value change of CAP and ClO - The linear fit of the concentration,

[0073] (a) is the fluorescence spectra of Abz-CDs and CAP at 0-10 μM.

[0074] (b) is the fluorescence spectra of Abz-CDs and CAP at 10-200 μM;

[0075] Fig.15 The results of the study on the fluorescence quenching mechanism of Abz-CDs in detecting CAP.

[0076] (a) UV absorption spectra of Abz-CDs, CAP, and Abz-CDs+CAP mixture;

[0077] (b) Ex-Em-abs diagram of Abz-CDs;

[0078] (c) is the life span diagram of Abz-CDs and CAP;

[0079] (d) Theoretical demonstration of Abz-CDs mechanism. DETAILED DESCRIPTION

[0080] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present invention. In addition, in the following invention, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0081] Example 1: Preparation of Abz-CDs

[0082] The preparation method adopts the hydrothermal method. That is, 1.2g of albendazole powder is weighed and dissolved in 15mL of ethanol. After the solution is thoroughly mixed, it is transferred to a high-temperature reaction container of polytetrafluoroethylene, and then placed in a resistive heating furnace at a temperature of 180°C for 6 hours to promote the reaction process. After the reaction is completed, the reactor is allowed to cool down to room temperature naturally with the environment. Then the coarse precipitate is removed by centrifugal separation technology, and then filtered with a 0.22μm microporous filter membrane to achieve a deeper purification effect. Finally, the carbon dot product that has undergone this series of treatments will be stored at 4°C.

[0083] Example 2: Structural Characterization of Abz-CDs Prepared in Example 1

[0084] The prepared Abz-CDs were structurally characterized using TEM, XRD, XPS and FTIR.

[0085] (1) Transmission electron microscopy (TEM)

[0086] Transmission electron microscopy (TEM): observe the morphology and particle size distribution of Abz-CDs, and calculate the average particle size using the statistical average method. Figure 1 As shown, Abz-CDs were spherical in morphology with an average diameter of approximately 2.5 nm, exhibiting good dispersion and no aggregation was observed.

[0087] (2) X-ray powder diffraction pattern (XRD):

[0088] The crystallinity of CDs was studied by XRD. The crystal structure of Abz-CDs was obtained by X-ray powder diffraction (XRD). Figure 2 As shown in the figure, the synthesized fluorescent carbon dots only present a single and prominent diffraction peak in the diffraction pattern, and the corresponding diffraction angle θ is 20.35°, indicating that the carbon dots do not have a typical crystal structure, and their constituent atoms or molecules do not form an orderly, periodically repeated spatial arrangement. This shows that the carbon dots exist in a non-crystalline form, and their internal structure is disordered without a clearly defined lattice.

[0089] (3) X-ray photoelectron spectroscopy (XPS): Qualitative and semi-quantitative analysis of Abz-CDs was performed to obtain information on their surface elemental composition, chemical state, etc. The results are as follows: Figure 3 As shown in a, the full scan XPS spectrum shows four peaks of C1 s (283.2 eV), N1 s (398.4 eV), O1 s (531.2 eV) S2p. The C1 s spectrum shows three peaks: 284.6 eV corresponding to C=C / CC, 285.7 eV corresponding to CN, and 286.6 eV corresponding to CO ( Figure 3 b); Figure 3c shows that the N1s spectrum has two obvious peaks at 399.6 eV (NH) and 401.3 eV (NC); the two peaks of the O1s spectrum appear at 529.7 eV and 530.9 eV, corresponding to COC / C-OH and OH ( Figure 3 d).

[0090] (4) Fourier transform infrared spectroscopy (FTIR): It can be used to infer the surface chemical groups and chemical composition of carbon dots and to identify the structures of unknown compounds.

[0091] The results are as follows Figure 4 As shown, Abz-CDs has a strong absorption peak at 3424.9cm-1, indicating the presence of OH and NH bonds, indicating that Abz-CDs inherit the OH and NH groups of Abz; a peak at 1621.8cm-1 indicates the presence of C=C bonds; a peak at 1425.1cm-1 indicates the presence of CH bonds; and an obvious peak at 1130cm-1 indicates the presence of COC bonds in Abz-CDs. The analysis results of FTIR and XPS are consistent, revealing that the prepared Abz-CDs are rich in hydrophilic groups and amino functional groups on their surfaces, giving it excellent water solubility.

[0092] Example 3: Characterization of optical properties of Abz-CDs prepared in Example 1

[0093] The optical properties of the prepared CDs were characterized. They exhibited a variety of optical phenomena, including efficient photoluminescence, excellent photostability, and fluorescence emission that was closely related to the excitation wavelength. At the same time, they also showed excellent resistance to photobleaching and maintained the intensity and stability of fluorescence even under long-term illumination.

[0094] (1) Ultraviolet absorption spectrum (UV-Vis)

[0095] Abz-CDs UV absorption spectrum ( Figure 5 ) showed that Abz-CDs had an obvious absorption peak at 270 nm, belonging to C=C sp 2 Hybridized π-π* transition.

[0096] (2) Fluorescence emission spectrum

[0097] The fluorescence spectra of Abz-CDs at different excitation wavelengths were measured using a fluorescence spectrophotometer to determine the optimal excitation wavelength and corresponding emission wavelength of Abz-CDs.

[0098] according to Figure 5 The characterization results of Abz-CDs wavelength show that in this experiment, the excitation wavelength of 340nm is selected as the best excitation wavelength of Abz-CDs, and the corresponding emission wavelength is 485nm.

[0099] (3) Excitation wavelength dependence

[0100] Figure 6 The fluorescence emission spectrum of Abz-CDs is recorded every 10nm in the range of 280-380nm. Although the wavelength of the excitation light has changed, the emission peak position of Abz-CDs remains unchanged. The results show that even when the wavelength conditions of external excitation change, Abz-CDs still show dependence on the excitation wavelength, that is, its fluorescence emission characteristics are affected by the excitation wavelength.

[0101] (4) Fluorescence quantum yield

[0102] By comparing the fluorescence emission intensity and absorption characteristics of the reference and carbon dot solutions under the same excitation light conditions, the quantum yield of the carbon dots can be calculated. The experiment adjusted the concentrations of the quinine sulfate solution and the Abz-CDs solution to ensure that the absorbance of both at the measurement wavelength was kept below 0.1, and the fluorescence intensity and UV-visible absorbance of the carbon dots and quinine sulfate were measured, and the corresponding data processing and calculation were performed. The specific formula is: QY = QR (I S / I R )(A R / A S )(ηS2 / ηR2)

[0103] QR—theoretical quantum yield of quinine sulfate;

[0104] I S —The fluorescence intensity of carbon dots under optimal excitation;

[0105] I R —Fluorescence intensity of quinine sulfate under 360nm excitation;

[0106] A R - UV absorbance of quinine sulfate under 360nm excitation;

[0107] A S —Ultraviolet absorbance of carbon dots at the optimal excitation wavelength;

[0108] η S — the refractive index of water;

[0109] η R —The refractive index of 0.1M H2SO4.

[0110] The results are as follows: the best excitation peak is at 340 nm, the two emission peaks are located at 485 nm, and the corresponding quantum yield is 8.26%.

[0111] (5) Fluorescence lifetime

[0112] The decay curve of Abz-CDs at Em of 485nm was calculated by double exponential function fitting to obtain the average fluorescence lifetime of 1.6ns. The nanosecond fluorescence lifetime indicates that it is suitable for optoelectronic and biological applications.

[0113] Example 4: Characterization of the stability of Abz-CDs prepared in Example 1

[0114] (1) pH stability: Take 5 μL of sample from the Abz-CDs solution with a concentration of 0.1 μg / L and dilute it to 2 mL in a 5 mL centrifuge tube. Inject PBS into the centrifuge tube and adjust its pH value to 2.2-12.0 to prepare test samples under different acid-base conditions. Then, vortex on a vortex oscillator for 3 minutes. Measure the fluorescence intensity of Abz-CDs under the conditions of the optimal excitation wavelength (340 nm) and the optimal emission wavelength (485 nm). In order to reduce the experimental error, each sample was measured three times independently to determine the effect of different pH values ​​on the change in the fluorescence intensity of carbon quantum dots.

[0115] The results are as follows Figure 7 As shown in a, when the pH is in the range of 2.2 to 7.0, the fluorescence intensity of Abz-CDs gradually increases, and the F / F0 value continues to rise. When pH = 7.0, the fluorescence intensity of Abz-CDs reaches the highest point, but with the increase of pH, the F / F0 of Abz-CDs under alkaline conditions is higher than that under acidic conditions, so the fluorescence ratio displayed under this pH value is meaningful: this ratio can create a better detection environment. As the pH gradually increases, the fluorescence intensity value of Abz-CDs at the optimal emission wavelength (485nm) is weakened. This shows that the fluorescence characteristics of Abz-CDs are significantly affected by pH.

[0116] Based on the comparison of the fluorescence intensity change trend of Abz-CDs under different pH conditions, it was found that the acid resistance of Abz-CDs was inferior to its alkali resistance. This has guiding significance for the application and property research of Abz-CDs, and also provides a reference for further optimizing its application under different environmental conditions. Because neutral conditions are more convenient for experiments, this experiment was carried out under pH 7.

[0117] (2) Ionic strength stability

[0118] Take 5 μL of the Abz-CDs solution with a concentration of 0.1 μg / L and dilute to 2 mL in a 5 mL centrifuge tube. Add 4M NaCl solution of different concentrations (0-2000 μL), and then dilute the Abz-CDs to 2 mL with a buffer solution of pH 8.0, and then vortex for 3 minutes on a vortex oscillator. Measure the fluorescence intensity of Abz-CDs under the conditions of the optimal excitation wavelength (340 nm) and the optimal emission wavelength (485 nm) to reduce the experimental error.

[0119] The results are shown in 7b. When the concentration of the NaCl solution does not reach 0.8 mol / L, the fluorescence value of Abz-CDs at 485 nm has a slight fluctuation. This phenomenon may be affected by the properties of the precursor, indicating that the stability of Abz-CDs is affected to a certain extent, but relatively unchanged. When the concentration of the NaCl solution is greater than 0.8 mol / L, it is observed that the fluorescence intensity of Abz-CDs increases slightly under different salt concentrations. This may be because Abz-CDs are sensitive to light and are easily affected by light, resulting in instability or oxidation.

[0120] To ensure the stability of Abz-CDs, appropriate storage methods and conditions should be adopted to avoid exposure to light.

[0121] (3) Antioxidant stability

[0122] Take 5 μL of 0.1 μg / L Abz-CDs solution and pour it into a 5 mL centrifuge tube. Add different volumes of 0.5M H2O2 solution (0-800 μL) respectively, then use pH 8.0 buffer solution to make Abz-CDs to 2 mL, and vortex for 3 minutes on a vortex oscillator. Measure the fluorescence intensity of Abz-CDs under the conditions of the optimal excitation wavelength (340 nm) and the optimal emission wavelength (485 nm). In order to reduce the experimental error, each sample was measured three times to examine the antioxidant activity of the point.

[0123] The results are as follows Figure 7 As shown in Figure c, the fluorescence intensity of Abz-CDs under different H2O2 concentrations fluctuates with the increase of H2O2 concentration, but the fluctuation is not large, indicating that Abz-CDs has good antioxidant ability.

[0124] ((4) Stability against photobleaching

[0125] Take 5 μL of 0.1 μg / L Abz-CDs solution and pour it into a 5 mL centrifuge tube. Then use a buffer solution with a pH of 8.0 to make the volume of CDs 2 mL and vortex for 3 minutes. Place them under natural light and ultraviolet light (365 nm) for different irradiation times (0 to 60 min), and measure the fluorescence intensity of CDs under the conditions of the optimal λex of 340 nm and λem of 485 nm (slit: 10 nm, 10 nm). Each sample was measured three times to investigate the changes in the fluorescence intensity of carbon dots under different illumination times.

[0126] The results are as follows Figure 7 d and 7e, the fluorescence intensity of Abz-CDs remained basically stable within 60 min under natural light ( Figure 7 d); UV light box (wavelength 365nm) was continuously irradiated for 60min, and the fluorescence intensity of Abz-CDs at the reflection point remained basically stable ( Figure 7 e), indicating that Abz-CDs have good resistance to photobleaching.

[0127] In summary, as described in Examples 1-4, the drug compound albendazole was used as a precursor, and Abz-CDs was successfully prepared by hydrothermal synthesis. The impurities in the reaction solution were separated, and a bright green fluorescence was obtained under ultraviolet light. The wavelength of 340nm is the best excitation for Abz-CDs, and its corresponding emission wavelength is 485nm. The optimal experimental conditions for Abz-CDs obtained by condition optimization are temperature (180°C), time (6h), and solvent (ethanol), and the measured quantum yield is 8.26%. Abz-CDs were characterized and analyzed: the TEM image shows the uniform distribution and spherical morphology of Abz-CDs, and the XRD spectrum shows that Abz-CDs has a clear broad peak at 20.35°, an amorphous carbon structure, and the comprehensive analysis of XPS and FTIR shows that the surface of Abz-CDs is rich in hydrophilic functional groups. These characteristics lay the foundation for its application in biomedicine, environmental monitoring and other fields. The results show that under 340nm wavelength excitation, Abz-CDs can efficiently emit fluorescence at 485nm, and its quantum yield reaches 8.26%. The average fluorescence lifetime at 485nm is 1.6ns. Although Abz-CDs are sensitive to changes in pH, their excellent performance in ionic strength stability, antioxidant and anti-photobleaching properties provides a solid foundation for the practical application of Abz-CDs in fluorescent labeling, bioimaging and other optoelectronic applications.

[0128] Example 5: Abz-CDs as a fluorescent probe for the analysis and detection of ClO -

[0129] (1) Effect of pH value on fluorescence response:

[0130] Take 5 μL of 5 μg / L Abz-CDs solution and pour it into a 5 mL centrifuge tube. Use a buffer solution with a pH of 8.0 to make the volume of Abz-CDs to 2 mL and vortex on a vortex oscillator for 3 minutes. Measure the fluorescence value at different pH values ​​at an excitation wavelength of 340 nm. Investigate the fluorescence intensity of Abz-CDs under different pH conditions. And use ultrapure water as a blank value for comparison to select the optimal pH.

[0131] Results from Figure 8 It can be seen that the fluorescence quenching effect is higher in the pH range of 2.2-5 and is strongest at pH 4. - It is more stable at pH 4. Therefore, the best pH for detecting hypochlorous acid is 4.

[0132] (2) Impact of reaction time

[0133] Take 5μg / L Abz-CDs and pour it into a 5mL centrifuge tube. Use a buffer solution with a pH of 8.0 to make the volume of Abz-CDs 2mL. The pH value is the optimal value of pH = 4 for Abz-CDs to detect hypochlorous acid and chloramphenicol. Scan the Abz-CDs fluorescence spectrum at an excitation wavelength of 340nm. Add 1mM 100μL hypochlorous acid solution to the Abz-CDs solution and immediately measure the fluorescence intensity value. Measure the fluorescence value every one minute, continue to detect for 15min, and select the best reaction time.

[0134] Results from Figure 8 b shows that the reaction tends to be stable after 10 minutes. Finally, 10 minutes is selected as the optimal reaction time. This time point can ensure that the interaction between the target substance and the carbon dots reaches a fully balanced state. By selecting this optimal reaction time, a stable fluorescence quenching value for Abz-CDs detection of CAP can be obtained, thereby improving the accuracy and reliability of the detection.

[0135] (3) Selectivity

[0136] Take 5 μL of 1 mg / mL Abz-CDs solution and place it in a centrifuge tube, then take 50 μL of 1 mM metal ion (Mg 2+ , Cu 2 + , Sr 3+ , Fe 3+ 、Zn 2+ 、Ni 2+ , Ca 2+ 、Na + , K + ), anion (F - , Cl - Br - ,I- , C lO - 、SO4 2- PO4 3- 、CO3 2- 、HCO3 - ), vitamins (VC, VE, VH, VB2, VB3), amino acids (LG ly, L-Aa l, L-Lse, L-Ser, L-Thr, LG lu) were added to the above solution and measured under the optimal conditions (pH = 4, reaction time 10 min). The fluorescence intensity at the emission wavelength was used to determine whether the probe detected ClO - selectivity.

[0137] The results are as follows Fig. 9 As shown, C10 - It can significantly quench the fluorescence of Abz-CDs.

[0138] (4) Methodological research

[0139] Take 5 μL of 5 μg / L Abz-CDs solution, pour it into a 5 mL centrifuge tube, and add different concentrations of 1 mM ClO - Solution. Abz-CDs was diluted to 2 mL with a pH 8.0 buffer solution to form ClO - The concentration gradient was set, and the reaction conditions were selected to scan the fluorescence spectrum of Abz-CDs at the optimal reaction time of 10 min and the optimal excitation wavelength of 340 nm. The fluorescence value of Abz-CDs at a wavelength of 485 nm was used as the ordinate, C ClO- As the horizontal axis, after linear equation fitting, the obtained equation, R 2 (Correlation coefficient), LOD (Limit of Detection) and LQD (Limit of Quantitation) were calculated.

[0140] The results are as follows Fig.10 As shown, the fluorescence value F / F0 and ClO - The concentration of α-hydroxy-1-hydroxy-2-nitropropene presents a good linear relationship. In the concentration range of 0 to 5 μM, the linear equation is y1=0.02678-0.21263x; R1 2 =0.9959; in the concentration range of 5-15 μM, the linear equation is y2=0.01549x+0.97667; R2 2 =0.9889; in the concentration range of 15-125 μM, the linear equation is y3=1.27538-0.00587x; R3 2 =0.9987; Fig.10 b, 10c: As ClO - As the concentration continued to increase, the fluorescence intensity value of Abz-CDs at 485nm gradually increased. Fig.10d shows that with the increase of ClO - As the concentration of 2-hydroxy-2-nitropropene was gradually increased, the green fluorescence of Abz-CDs gradually weakened. This shows that Abz-CDs can be used as a fluorescent probe to detect ClO - The content.

[0141] (5) Fluorescence quenching mechanism study

[0142] like Fig.11 As shown in the UV-visible absorption spectrum (Figure 11a), the addition of ClO - No new absorption peaks appeared after the reaction, indicating that no new compounds were produced, so it does not belong to the static quenching mechanism (SQ).

[0143] like Fig.11 b, Emission spectra of Abz-CDs and ClO - There is an overlap between the absorption spectra of the two, and it is preliminarily judged that this may be caused by the existence of FRET or IFE.

[0144] According to Abz-CDs, Abz-CDs+ClO - FL lifetime decay diagram ( Fig.11 c) to analyze, after double exponential fitting, Abz-CDs in ClO - The average fluorescence lifetimes under the concentration gradient are 1.69, 1.70, 1.70, and 1.71 ns, respectively. From the experimental results, the fluorescence lifetime of Abz-CDs increases with the concentration of ClO - The increase in concentration will change slightly, but it is almost negligible, so ClO - The quenching mechanism can be determined to be I FE.

[0145] The IFE mechanism is further verified by Park equation:

[0146]

[0147] F obsd —The actual measured fluorescence intensity;

[0148] F cor —Corrected fluorescence intensity after deducting I FE;

[0149] A ex —Absorbance of the solution under optimal excitation after adding hypochlorite ions;

[0150] A em —Absorbance of the solution at the optimal emission wavelength after adding hypochlorite ions;

[0151] s—thickness of the excitation beam (s=0.10 cm);

[0152] g—the distance between the base of the quartz cuvette and the lower edge of the excitation beam (g=0.40 cm);

[0153] d—Width of the quartz colorimetric cell (d=1.00 cm).

[0154] Park equation validation of Abz-CDs for ClO detection - The results are shown in Fig.11 d, ClO - When the concentration increases (15-125 μM), F cor / F obsd The value of increases from 0.7075 to 0.7415, which is consistent with the result of Park equation.

[0155] In summary, the detection of C lO - The optimal pH value was 4, the optimal reaction time was 10 min, and the fluorescent nanoprobe was used to detect ClO - Has good selectivity. - In the concentration range of 0-5μM, 5-15μM, and 15-125μM, ClO - The concentration change shows a good linear relationship with the fluorescence intensity of the fluorescent probe. The linear equation is y1 = 0.02678-0.21263x; R1 2 =0.9959;y2=0.01549x+0.97667;R2 2 =0.9889; y3 = 1.27538-0.00587x; R3 2 =0.9987; LOD1 was 0.0141 μM (S / N=0.141), the limit of quantification was 0.141 μM, LOD2 was 0.193 μM (S / N=1.93), the limit of quantification was 1.93 μM, and LOD3 was 0.511 μM (S / N=5.11), the limit of quantification was 5.11 μM. It can be seen that Abz-CDs can be used as a fluorescent probe for the detection of ClO - The content.

[0156] Example 6: Abz-CDs as a fluorescent probe for the detection of chloramphenicol (CAP)

[0157] The fluorescent carbon dots Abz-CDs prepared in Example 1 were dissolved into an Abz-CDs solution with a concentration of 5 μg / L for use.

[0158] (1) Effect of pH value on fluorescence response:

[0159] Take 5 μL of 5 μg / L Abz-CDs solution and pour it into a 5 mL centrifuge tube. Use a buffer solution with a pH of 8.0 to make the volume of Abz-CDs to 2 mL and vortex on a vortex oscillator for 3 minutes. Measure the fluorescence value at different pH values ​​at an excitation wavelength of 340 nm. Investigate the fluorescence intensity of Abz-CDs under different pH conditions. In this experiment, ultrapure water was used as the blank value, and its fluorescence value intensity at a wavelength of 485 nm was used as a comparison to select the optimal pH with the least effect on the fluorescence intensity value.

[0160] Results from Fig.12 It can be seen from a that the fluorescence quenching effect is higher in the pH range of 2.2 to 5 and is strongest at pH 4. Therefore, pH 4 is the best condition for detecting CAP.

[0161] (2) Impact of response time

[0162] Take 5μL of 5μg / L Abz-CDs solution and pour it into a 5mL centrifuge tube. Use a buffer solution with a pH of 8.0 to make the volume of Abz-CDs 2mL, and the pH value is the optimal pH for Abz-CDs to detect CAP. Scan the Abz-CDs fluorescence spectrum at an excitation wavelength of 340nm. Add 1mM 100μL CAP solution to the Abz-CDs solution and immediately measure the fluorescence intensity value. Measure the fluorescence value every one minute, continue to detect for 15 minutes, and select the best reaction time.

[0163] Results from Fig.12 b shows that the reaction tends to be stable after 15 minutes. Finally, 15 minutes is selected as the optimal reaction time. This time point can ensure that the interaction between the target substance and the carbon dots reaches a fully balanced state. By selecting this optimal reaction time, a stable fluorescence quenching value for Abz-CDs detection of CAP can be obtained, thereby improving the accuracy and reliability of the detection.

[0164] (3) Selectivity

[0165] Take 5 μL of 1 mg / mL Abz-CDs solution and place it in a centrifuge tube, then take 50 μL of 1 mM metal ion (Hg 2+ 、Co 2+ , Cu 2+ , Fe 3+ , Ca 2+ 、Bi 3+ , Ba 2+ 、Na + 、Ag +), antibiotics (CTC·HCl, CAP, AMP, SP, CEF), vitamins (VC, VE, VH, VB2, VB3), amino acids (L-Ser, L-Trp, L-Leu, L-Thr, LG ln, L-Glu, L-Tyr, L-Met, L-Hsp) were added to the above solution and measured under optimal conditions. The selectivity of the probe to detect CAP was determined based on the intensity of its fluorescence value at the emission wavelength.

[0166] Results from Fig.13 As shown: When CAP exists, it can significantly quench the fluorescence of Abz-CDs.

[0167] (4) Methodological research

[0168] Take 5 μL of 5 μg / L Abz-CDs solution, pour it into a 5 mL centrifuge tube, and add different concentrations of 1 mM CAP solution. Use a buffer solution with a pH of 8.0 to make the volume of Abz-CDs 2 mL to form a concentration gradient of CAP. The reaction conditions are selected at the optimal reaction time and the optimal excitation wavelength of 340 nm to scan the Abz-CDs fluorescence spectrum. The fluorescence value of Abz-CDs at a wavelength of 485 nm is the vertical axis, C CAP As the horizontal axis, after linear equation fitting, the obtained equation, R 2 (Correlation coefficient), LOD (Limit of Detection) and LQD (Limit of Quantitation) were calculated.

[0169] The best detection pH of CAP is 4. The best reaction time obtained from the above experiment is 15min. Under this condition, Abz-CDs has good selectivity in detecting CAP. Within the concentration range of 0-10μM and 10-200μM, the concentration change of CAP is linearly related to the fluorescence intensity of the fluorescent probe at the emission wavelength. The linear equation is y1=2.25748-0.128*x; R1 2 =0.9907;y2=0.00312*x+0.8578;R2 2 =0.9930; LOD1 is 0.0141μM (S / N=0.141), and the limit of quantification is 0.141μM; LOD2=0.193μM (S / N=1.93); the limit of quantification is 1.93μM. ( Fig.14 )

[0170] (5) Study on the mechanism of fluorescence sensitization

[0171] The results are as follows Fig.15 As shown in the UV-visible absorption spectrum (Figure 15a), no new absorption peak appeared after the addition of CAP, indicating that no new compounds were produced, so it does not belong to the static quenching mechanism (SQ). Fig.15b shows that the emission spectrum of Abz-CDs overlaps with the absorption spectrum of CAP, which may be caused by the existence of FRET or IFE. According to the FL lifetime decay diagram of Abz-CDs and Abz-CDs+CAP ( Fig.15 c) to analyze, after double exponential fitting, the average fluorescence lifetime of Abz-CDs under CAP concentration gradient is almost unchanged, therefore, the quenching mechanism of CAP can be determined as I FE.

[0172] The results of Park equation for the detection of CAP by Abz-CDs are shown in Fig.15 When the CAP concentration increases (0-10 μM), F cor / F obsd The value of increases from 0.1761 to 0.9748, which is consistent with the result of Park equation.

[0173] In summary, within the concentration range of 0 to 5 μM, the fluorescence value showed a linear relationship with the concentration of CAP. The linear equation was y1 = 0.02678-0.21263*x; R1 2 =0.9959; in the concentration range of 5-15 μM, the linear equation is y2=0.01549*x+0.97667; R2 2 =0.9889; It can be seen that Abz-CDs can be used as a fluorescent probe to detect the content of residual CAP in food.

[0174] According to Examples 1-6, the fluorescent nanoprobe Abz-CDs was prepared by hydrothermal method using expired drugs as carbon source, and the fluorescence quenching effect enabled it to be quenched by ClO - , CAP effectively quenches Cl O - , CAP selectivity, selectivity and interference experiments were performed, and the fluorescence response conditions were optimized to detect Cl O - The optimal conditions were pH 4.0 and reaction time of 10 min; the optimal conditions for detecting CAP were pH 4.0 and reaction time of 15 min. - The fluorescence quenching phenomenon of Abz-CDs and CAP was studied by preliminary mechanism. It was found that the fluorescence emission spectrum (Em) of Abz-CDs was consistent with the ClO - There is a significant overlap between the UV absorption peaks of F and CAP. Through the analysis of Park equation, it is found that cor / F obsd (Ratio of corrected fluorescence intensity to observed fluorescence intensity) As ClO - The results show that Abz-CDs and C lO - The fluorescence quenching mechanism between them is IFE.

[0175] It should be understood that the above specific embodiments of the present invention are only used for illustrative inventions or to explain the principles of the present invention, and do not constitute limitations on the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention. In addition, the appended claims of the present invention are intended to cover all changes and modifications that fall within the scope and boundaries of the appended claims, or the equivalent forms of such scope and boundaries.

Claims

1. A fluorescent probe synthesized using expired drugs, characterized in that: The probe is fluorescent carbon dots Abz-CDs with green fluorescence, which are prepared by hydrothermal synthesis using expired drug albendazole and ethanol as raw materials.

2. A method for preparing a fluorescent probe synthesized using expired drugs according to claim 1, characterized in that: The following steps are involved: Step 1: Dissolve 1.2 g of albendazole powder in 15 mL of ethanol solution. Step 2: Transfer the thoroughly mixed solution in step 1 to a high-temperature reaction vessel made of polytetrafluoroethylene and promote the reaction at 180° C. for 6 hours; Step 3: After the reaction is completed, the reactor is cooled to room temperature naturally; Step 4: centrifuge to remove coarse precipitate, and then filter through a 0.22 μm microporous filter membrane to obtain purified fluorescent carbon dots Abz-CDs, which are stored at 4°C.

3. Use of the fluorescent carbon dots Abz-CDs according to claim 1 or 2 in detecting hypochlorite ions and chloramphenicol.

4. The use according to claim 3, characterized in that: The Abz-CDs are used as fluorescent probes to detect hypochlorite ions ClO - .

5. The application according to claim 4, characterized in that: The Abz-CDs detect hypochlorite ion ClO - The steps include: 1) Dissolve Abz-CDs in water to obtain a 5 μg / L Abz-CDs solution; 2) Containing ClO - The solution was mixed with the Abz-CDs solution and different volumes of 1 mM ClO - The solution was diluted to 2 mL with a pH 8.0 buffer solution to form ClO - The concentration gradient, λ Em Different concentrations of ClO were measured at 485 nm. - The fluorescence intensity of the solution was obtained F0 ; 3) Mixing the sample to be tested with the Abz-CDs solution at pH = 4.0 to obtain a mixed solution of the sample to be tested, reacting for 10 minutes, and measuring the fluorescence intensity F of the mixed solution of the sample to be tested; 4) According to the fluorescence quenching efficiency F / F0 and ClO - The linear relationship between the concentration and the ClO content in the sample to be tested is calculated. - concentration; The linear relationship is obtained by obtaining the fluorescence quenching efficiency F / F0 and ClO - Linear relationship with concentration.

6. The application according to claim 5, characterized in that: The fluorescence quenching efficiency F / F0 and ClO - The linear relationship of concentration includes: In the range of 0 to 5 μM, the linear equation is y1 = 0.02678-0.21263x; R1 2 =0.9959; detection limit LOD1 = 0.0141 μM; In the range of 5 to 15 μM, the linear equation is y2 = 0.01549x + 0.97667; R2 2 =0.9889;LOD2=0.193μM; In the range of 15 to 125 μM, the linear equation is y3 = 1.27538-0.00587x; R3 2 =0.9987; LOD3=0.511μM.

7. The use according to claim 4, characterized in that: The Abz-CDs are used as fluorescent probes to quickly detect the content of chloramphenicol CAP.

8. The application according to claim 7, characterized in that: The step of Abz-CDs detecting chloramphenicol CAP comprises 1) Dissolve Abz-CDs in water to obtain a 5 μg / L Abz-CDs solution; 2) A solution containing chloramphenicol CAP was mixed with 5 μL Abz-CDs solution, and different volumes of 1 mM CAP solution were added, and the volume was fixed to 1.0 mL with a pH = 8 buffer solution to form a CAP concentration gradient. The fluorescence intensity of CAP solutions with different concentrations was measured at λEm at 485 nm to obtain F0; 3) Mixing the sample to be tested with the Abz-CDs solution under the condition of pH=4 to obtain a mixed solution of the sample to be tested, reacting for 15 minutes, and measuring the fluorescence intensity F of the mixed solution of the sample to be tested; 4) Calculate the concentration of CAP in the sample to be tested based on the linear relationship between the fluorescence quenching efficiency F / F0 and the CAP concentration.

9. The application according to claim 8, characterized in that: The linear relationship between the fluorescence quenching efficiency F / F0 and the CAP concentration is used to calculate the concentration of CAP in the sample to be tested, including: When the fluorescence linear range is 0-10μM, the linear equation is y1=2.25748-0.128x; R1 2 =0.9907; Detection limit LOD1 = 0.0141 μM (S / N = 0.141); When the fluorescence linear range is 10-200 μM, the linear equation is y2=0.00312x+0.8578; R2 2 =0.9930; detection limit LOD2 =0.193 μM (S / N = 1.93).

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