A 5-Nitropyrimidine Functionalized Carbon Dots Fluorescent Probe and Its Preparation Method and Application

Abstract

The present invention discloses a 5-nitropyrimidine-functionalized carbon dot fluorescent probe, its preparation method and application, belonging to the technical field of nanomaterials, and comprising the following steps: first, CDs are prepared by a high-temperature reflux method, and then p-chloro-4-nitropyrimidine and triethylamine are successively added to the DMF solution of the obtained CDs for reaction. After the reaction is completed, the solvent is removed under reduced pressure, and then purified by column chromatography to obtain the 5-nitropyrimidine-functionalized carbon dots. The present invention also discloses the 5-nitropyrimidine-functionalized carbon dot fluorescent probe prepared by the above preparation method and its application in the detection of thiols. The probe obtained by the present invention has the advantages of high selectivity, high sensitivity and strong anti-interference ability for biological thiols, and shows good detection ability for total biological thiols in food.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterials, and particularly relates to a 5-nitropyrimidine-functionalized carbon dot fluorescent probe, a preparation method thereof, and an application thereof. Background Art

[0002] Thiol is an organic compound containing a sulfhydryl group (-SH), which plays an important role in organisms and participates in many biological processes. For example, changes in the redox state of the thiol / disulfide pair can affect protein conformation, enzyme activity, ligand-receptor binding, protein-protein interaction, and protein-DNA interaction. Biological thiols also play an important role in food. For example, many fresh foods contain biological thiols, such as fish, eggs, meat, etc. Once the food starts to deteriorate, the thiols in it will gradually decompose, and the freshness of the food can be determined by detecting the change in their content. Furthermore, thiols can be used to detect the freshness of food. Secondly, thiols can be used to detect whether there are harmful chemical substances in food, such as organic pollutants, heavy metals, etc. Such harmful substances will affect or destroy the biological thiol molecules in food, thereby changing their content, and then the safety of food can be determined by detecting the change in thiol content. In addition, thiols can be used to identify different varieties of food. Different types of food contain different concentrations of thiols. Therefore, different varieties or the same variety of food from different origins and sources can be distinguished by detecting the difference in their content, such as different types of wine, cheese, fish, etc. In addition, the concentration of thiols will be affected during food processing and storage, and the process of food processing and storage can be analyzed by detecting the change in their content. It can be seen that the detection of thiols plays a very wide role not only in the human body, but also in drugs and food. Accurately and rapidly detecting thiols has important significance.

[0003] In the prior art, the methods for detecting thiols are mainly traditional instrument methods, including gas chromatography, liquid chromatography, mass spectrometry, electrochemistry, etc. However, the cost of instrument methods is generally relatively high, and trained operators are required. In recent years, the method based on fluorescent probes has received extensive attention from researchers because of its advantages such as simple operation, controllable price, high sensitivity and accuracy, and strong selectivity. The structure of a fluorescent probe contains a fluorophore and a recognition group, but the fluorophores in the prior art are generally organic-structured fluorophores, which have problems such as complex synthesis and photobleaching.

[0004] Therefore, how to provide a fluorescent probe for detecting thiols with a simple preparation method, low cost, high sensitivity and high accuracy is a technical problem that those skilled in the art urgently need to solve. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a 5-nitropyrimidine-functionalized carbon dot fluorescent probe, a preparation method thereof, and an application thereof.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A preparation method of a 5-nitropyrimidine-functionalized carbon dot fluorescent probe, comprising the following steps:

[0008] First, prepare CDs by the high-temperature reflux method, and then sequentially add 2-chloro-5-nitropyrimidine and triethylamine to the DMF (N,N-dimethylformamide) solution of the obtained CDs for reaction. After the reaction is completed, remove the solvent under reduced pressure, and then purify by column chromatography to obtain the 5-nitropyrimidine-functionalized carbon dots.

[0009] More preferably, the solvent used in the column chromatography purification process is a mixture of dichloromethane and methanol in a volume ratio of (50 - 20):1.

[0010] Beneficial effects: As Figure 1 shown, the fluorescent probe obtained in the present invention uses CDs as the fluorophore and the p-nitropyrimidine group as the quencher to quench the fluorescence of CDs. When biological thiols are present in the system, the mercapto group of the thiol will break the ether bond in CDs-CNPMD, restoring the fluorescence of CDs, thereby realizing the detection of biological thiols.

[0011] Preferably, the preparation of CDs by the high-temperature reflux method comprises the following steps:

[0012] Add concentrated phosphoric acid to the ethylene glycol solution of resorcinol, carry out a reflux reaction in air, remove the solvent after the reaction is completed, then add water and adjust the pH to precipitate, and after purifying the obtained precipitate, the CDs are obtained.

[0013] Beneficial effects: The present invention can obtain carbon dots in air, but it is difficult to obtain carbon dots under protective gases such as nitrogen, which may be related to the formation mechanism of carbon dots. Condensation and coupling are more likely to occur in the presence of oxygen.

[0014] Preferably, the addition ratio of resorcinol, ethylene glycol, and concentrated phosphoric acid is 500 mg:50 mL:150 μL.

[0015] Preferably, the temperature of the reflux reaction is 180 °C and the time is 5 h.

[0016] Preferably, the pH adjustment is to adjust to pH = 13 and then adjust to pH = 6.

[0017] Beneficial effects: The temperature and reaction time in the present invention are also important conditions for the formation of carbon dots. Carbon dots can be formed at a temperature of 180 °C and a time of 5 h. During post-treatment, when pH > 7, the phenolic hydroxyl groups on the surface of the carbon dots are in the state of phenolate anions and are easily soluble in water. However, when pH < 7 and approaches 6, carbon dot precipitates can be precipitated, which is related to the transformation of phenolate anions into phenolic hydroxyl groups and the deterioration of solubility.

[0018] Preferably, the solvent used in the column chromatography purification process for preparing CDs by the high-temperature reflux method is a mixture of dichloromethane and methanol in a volume ratio of (10 - 5):1.

[0019] Preferably, the addition ratio of the CDs, DMF, 2-chloro-5-nitropyrimidine, and triethylamine is 100 mg:5 mL:50 mg:50 μL.

[0020] Preferably, the temperature of the reaction is 40 °C and the time is 24 h.

[0021] Beneficial effects: The carbon dots formed in the present invention dissolve well in DMF, and in the presence of triethylamine, they are transformed into strongly nucleophilic phenolate anions, which undergo a nucleophilic substitution reaction with 2-chloro-5-nitropyrimidine to form ether bonds. A reaction temperature of 40 °C and a time of 24 h can ensure that all phenolic hydroxyl groups on the surface of the carbon dots react completely with 2-chloro-5-nitropyrimidine.

[0022] A 5-nitropyrimidine-functionalized carbon dot fluorescent probe prepared by a preparation method of a 5-nitropyrimidine-functionalized carbon dot fluorescent probe.

[0023] Beneficial effects: The functionalized carbon dots obtained in the present invention can further broaden the application fields and scope of carbon dots, endowing them with more functionality. For example, in this study, biological thiols are detected in a fluorescence-turn-on manner.

[0024] An application of a 5-nitropyrimidine-functionalized carbon dot fluorescent probe in the detection of thiols.

[0025] Beneficial effects: The probe provided by the present invention can be used for the detection of biological thiols in foods with high selectivity and sensitivity, and the raw materials are cheap, the preparation is simple, and the cost is low.

[0026] The present invention discloses a 5-nitropyrimidine-functionalized carbon dot fluorescent probe, its preparation method and application. Based on carbon quantum dots with high biocompatibility and high quantum yield, the present invention designs and synthesizes a fluorescent probe CDs-CNPMD capable of detecting biological thiols. This fluorescent probe uses CDs as the fluorophore and the p-nitropyrimidine group as the quencher to quench the fluorescence of CDs. When biological thiols exist in the system, the mercapto group of the thiol will break the ether bond in CDs-CNPMD, restoring the fluorescence of CDs, thereby realizing the detection of biological thiols. The probe obtained in the present invention has the advantages of high selectivity, high sensitivity and strong anti-interference ability for biological thiols, and shows good detection ability for total biological thiols in food. Brief Description of the Drawings

[0027] The drawings constituting a part of this application are used to provide a further understanding of this application. The schematic embodiments of this application and their descriptions are used to explain this application and do not constitute an improper limitation to this application. In the drawings:

[0028] Figure 1 It is the flow chart of CDs-CNPMD detecting biological thiols in the present invention;

[0029] Figure 2 They are the TEM, HRTEM images and XRD pattern of the CDs obtained in Example 1;

[0030] Figure 3 They are the full-spectrum XPS spectra, high-resolution XPS spectra and high-resolution XPS spectra of CDs and CDs-CNPMD;

[0031] Among them, a is the full-spectrum XPS spectrum of CDs; b is the high-resolution XPS spectrum C1s of CDs; c is the high-resolution XPS spectrum O1s of CDs-CNPMD; d is the full-spectrum XPS spectrum of CDs-CNPMD; e is the high-resolution XPS spectrum C1s of CDs-CNPMD; f is the high-resolution XPS spectrum N1s of CDs-CNPMD;

[0032] Figure 4 They are the FT-IR spectra of CDs and CDs-CNPMD;

[0033] Figure 5 They are the excitation, emission spectra and 3D fluorescence spectra of the UV-visible absorption spectra of CDs, CDs-CNPMD and CDs-CNPMD + Cys;

[0034] Among them, a is the excitation, emission spectra of the UV-visible absorption spectra of CDs, CDs-CNPMD and CDs-CNPMD + Cys; b is the 3D fluorescence spectrum of CDs; c is the 3D fluorescence spectrum of CDs-CNPMD + Cys;

[0035] Figure 6 Fluorescence intensity diagrams of CDs-CNPMD and CDs-CNPMD + Cys at different pH values;

[0036] Figure 7 Fluorescence intensity vs. time diagrams and reaction kinetics diagrams of CDs-CNPMD and the reaction products of CDs-CNPMD with Cys, Hcy, and GSH;

[0037] Among them, a is the fluorescence intensity vs. time diagram, and b is the reaction kinetics diagram based on a;

[0038] Figure 8 Fluorescence spectra diagrams and fluorescence intensity diagrams after reaction with CDs-CNPMD;

[0039] Among them, a is the fluorescence spectra diagram and fluorescence intensity diagram after reaction of CDs-CNPMD with different concentrations of Cys; b is the fluorescence spectra diagram and fluorescence intensity diagram after reaction of CDs-CNPMD with different concentrations of Hcy; c is the fluorescence spectra diagram and fluorescence intensity diagram after reaction of CDs-CNPMD with different concentrations of GSH;

[0040] Figure 9 Diagrams of the reaction selectivity of CDs-CNPMD with various amino acids and ions and the interference test results of detecting biological thiols by CDs-CNPMD in the presence of various amino acids and ions.

[0041] Among them, (a) is the selectivity and interference diagram of the probe for detecting Cys; (b) is the selectivity and interference diagram of the probe for detecting Hcy; (c) is the selectivity and interference diagram of the probe for detecting GSH; Detailed implementation manners

[0042] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0043] To make the above objects, features, and advantages of the present invention more obvious and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific implementation manners.

[0044] The raw materials in the present invention are all obtained through commercial channels.

[0045] Example 1

[0046] A preparation method of a 5-nitropyrimidine-functionalized carbon dot fluorescent probe, comprising the following steps:

[0047] (1) First, dissolve 500 mg of resorcinol in 50 mL of ethylene glycol, then add 150 μL of concentrated phosphoric acid, and reflux at 180 °C in air for 5 h. During this process, the color of the solution turns brownish-black. After the reaction is completed, remove the ethylene glycol, then add 10 mL of deionized water, adjust the pH value of the solution to 13 and then to pH = 6 to precipitate, and then filter the obtained precipitate and purify it by column chromatography to obtain green carbon quantum dots CDs (yield 12.6%);

[0048] Among them, the solvent used in the column chromatography purification process is a mixture of dichloromethane and methanol with a volume ratio of 10:1;

[0049] (2) Dissolve 100 mg of the CDs obtained in step (1) in 5 ml of N,N-dimethylformamide (DMF), then add 50 mg of 2-chloro-5-nitropyrimidine, stir evenly, then add 50 μL of triethylamine, and react at 40 °C for 24 hours. After the reaction is completed, remove the DMF under reduced pressure and purify it by column chromatography to obtain a dark brown solid powder, namely the 5-nitropyrimidine-functionalized carbon dot fluorescent probe, denoted as CDs-CNPMD.

[0050] Among them, the solvent used in the column chromatography purification process is a mixture of dichloromethane and methanol with a volume ratio of 50:1.

[0051] Example 2

[0052] A preparation method of a 5-nitropyrimidine-functionalized carbon dot fluorescent probe, comprising the following steps:

[0053] (1) First, dissolve 500 mg of resorcinol in 50 mL of ethylene glycol, then add 150 μL of concentrated phosphoric acid, and reflux at 180 °C under nitrogen protection for 5 h. During this process, the color of the solution turns brownish-black. After the reaction is completed, remove the ethylene glycol, then add 10 mL of deionized water, adjust the pH value of the solution to 13 and then to pH = 6 to precipitate, and then filter the obtained precipitate and purify it by column chromatography to obtain green carbon quantum dots CDs (yield 12.6%);

[0054] Among them, the solvent used in the column chromatography purification process is a mixture of dichloromethane and methanol with a volume ratio of 7:1;

[0055] (2) Dissolve 100 mg of the CDs obtained in step (1) in 5 ml of N,N-dimethylformamide (DMF), then add 50 mg of 2-chloro-5-nitropyrimidine. After stirring evenly, add 50 μL of triethylamine and react at 40 °C for 24 hours. After the reaction is completed, remove DMF under reduced pressure and purify by column chromatography to obtain a dark brown solid powder, namely the 5-nitropyrimidine-functionalized carbon dot fluorescent probe, denoted as CDs-CNPMD.

[0056] Among them, the solvent used in the column chromatography purification process is a mixture of dichloromethane and methanol with a volume ratio of 30:1.

[0057] Example 3

[0058] A preparation method of a 5-nitropyrimidine-functionalized carbon dot fluorescent probe, comprising the following steps:

[0059] (1) First, dissolve 500 mg of resorcinol in 50 mL of ethylene glycol, then add 150 μL of concentrated phosphoric acid, and reflux at 180 °C for 5 h under nitrogen protection. During this process, the color of the solution shows brownish black. After the reaction is completed, remove ethylene glycol, then add 10 mL of deionized water, adjust the pH value of the solution to 13 and then adjust it to pH = 6 to precipitate, and then filter the obtained precipitate and purify it by column chromatography to obtain green carbon quantum dots CDs (yield 12.6%);

[0060] Among them, the solvent used in the column chromatography purification process is a mixture of dichloromethane and methanol with a volume ratio of 5:1;

[0061] (2) Dissolve 100 mg of the CDs obtained in step (1) in 5 ml of N,N-dimethylformamide (DMF), then add 50 mg of 2-chloro-5-nitropyrimidine. After stirring evenly, add 50 μL of triethylamine and react at 40 °C for 24 hours. After the reaction is completed, remove DMF under reduced pressure and purify by column chromatography to obtain a dark brown solid powder, namely the 5-nitropyrimidine-functionalized carbon dot fluorescent probe, denoted as CDs-CNPMD.

[0062] Among them, the solvent used in the column chromatography purification process is a mixture of dichloromethane and methanol with a volume ratio of 20:1.

[0063] Technical effects

[0064] 1. Characterization of CDs and CDs-CNPMD

[0065] Using resorcinol as a raw material, adding H3PO4, and preparing CDs by high-temperature reflux method to obtain the CDs in step (1) of Example 1. Characterize the synthesized CDs by transmission electron microscopy (TEM) and XRD powder diffraction, and the results are as Figure 2 and 3 shown.

[0066] The TEM image ( Figure 2 part a) shows that the CDs are monodispersed, Figure 2 part b) shows that the size of the CDs is about 5 nm and the particle size is uniform. Figure 2 Part c) clearly shows the parallel lattice fringes of the carbon core of the CDs. The X-ray diffraction (XRD) pattern of R-CDs ( Figure 2 part d) shows a broad diffraction peak centered at 2θ = 26°, which is related to the (002) lattice spacing of graphite, indicating that the CDs have a carbon core with a graphite-like structure.

[0067] X-ray photoelectron spectroscopy (XPS) characterizes the surface elemental composition of CDs and CDs-CNPMD. The full XPS spectrum of CDs ( Figure 3 part a) clearly shows two peaks at 285.12 and 532.78 eV, which are attributed to C1s and O1s, respectively. In the high-resolution spectrum of C1s ( Figure 3 part b), three characteristic peaks appear at 284.8, 286.4, and 287.1 eV, which are attributed to C-C / C═C, C-O, and O═C groups, respectively. In the high-resolution spectrum of O1s ( Figure 3 part c), it is split into two characteristic peaks at 532.9 eV and 530.9 eV, corresponding to C-OH and C═O, respectively. These data show that the surface of CDs contains rich hydroxyl groups (-OH) and carbonyl groups (C═O), and it can be inferred that CDs are composed of graphene cores with highly oxidized groups on the surface. From the full XPS spectrum of CDs-CNPMD ( Figure 3 part d), it can be seen that a peak of N1s appears at 400.1 eV, and the atomic percentage content is 8.09%. The high-resolution XPS spectrum of N 1s is as shown in Figure 3 part f. It can be seen that it is split into three peaks at 399.2, 401.5, and 406.3 eV, corresponding to C═N, C-NH2, and NO2 groups, respectively. This indicates that the p-nitropyrimidine group has been successfully connected to the CDs.

[0068] 2. Spectra of CDs and CDs-CNPMD probes

[0069] Figure 4 are the FT-IR spectra of the CDs and CDs-CNPMD obtained in Example 1. The broad absorption band at 3100 - 3500 cm -1 in the CDs is attributed to the stretching vibration of O-H. The absorption band at 1600 cm -1 is the characteristic absorption of aromatic ring C═C. In CDs-CNPMD, the absorption band at 1650 cm -1 corresponds to the stretching vibration of C═N, and 1330 cm-1 The absorption peak at corresponds to the stretching vibration of the nitro group, which further proves that the p-nitropyrimidine group has been successfully linked to the carbon quantum dots.

[0070] Figure 5 In part a of , the UV-visible absorption spectra and emission spectra of the CDs, CDs-CNPMD, and CDs-CNPMD fluorescence probe solution (10 μg / mL, 2 mL) obtained in Example 1 with a final concentration of 50 μmol / L Cys (CDs-CNPMD + Cys) added were shown. It can be seen from the figure that the UV absorption of CDs is at 488 nm, and the maximum emission wavelength is at 508 nm, indicating that the emission peak at 508 nm originates from the absorption of light at 488 nm. Figure 5 In part b of , the three-dimensional excitation-emission spectrum of CDs was shown. It can be seen from the figure that the position of the emission peak of CDs is independent of the excitation wavelength. After the p-nitropyrimidine group was linked to the CDs, the UV absorption spectrum of CDs-CNPMD changed greatly, and a new absorption peak appeared at 350 nm, indicating that there were significant changes in the electron cloud and energy level orbital distribution in the molecular structure, and there was likely a large degree of electron transfer; the fluorescence emission intensity of CDs-CNPMD at 508 nm was very low, showing an obvious fluorescence quenching phenomenon, while after adding the biothiol Cys, the fluorescence intensity at 508 nm was significantly restored. Figure 5 In part c of , the three-dimensional excitation-emission spectrum of CDs-CNPMD + Cys was shown. It can be seen from the figure that the position of the emission peak is similar to that of CDs and is still independent of the excitation wavelength.

[0071] 3. pH Exploration Experiment for Detecting Thiols by CDs-CNPMD

[0072] The specific experimental steps are as follows:

[0073] (1) Prepare a stock solution of CDs-CNPMD with a concentration of 1 mg / mL for later use.

[0074] (2) Prepare a stock solution of Cys with a concentration of 1 mmol / L for later use.

[0075] (3) Prepare PBS solutions with pH values of 6, 6.5, 7, 7.4, 8, 9, and 10 and a concentration of 20 mmol / L for later use.

[0076] (4) Take 2 mL of each of the above PBS solutions with different pH values, add 20 μL of the 1 mg / mL CDs-CNPMD stock solution, mix well, and perform fluorescence spectral analysis after standing for 120 min.

[0077] (5) Take 2 mL of the above-mentioned PBS solutions with different pH values, add 20 μL of the 1 mg / mL CDs-CNPMD mother liquor, then add 20 μL of the 1 mmol / L Cys mother liquor, mix them, and place for 120 min before performing fluorescence spectrum analysis.

[0078] The results are as Figure 6 shown.

[0079] Figure 6 Figure shows the fluorescence intensity diagrams of CDs-CNPMD and CDs-CNPMD + Cys at different pH values. It can be seen from the figure that as the pH value increases, the intensity of fluorescence recovery gradually increases. This may be because under alkaline conditions, the sulfhydryl groups of biothiols gradually form sulfhydryl anions, with enhanced nucleophilicity and being more likely to react with the probe. On the other hand, as the pH value increases, the fluorescence intensity of the CDs-CNPMD probe itself also continuously increases. Considering both factors, 7.4 is finally selected as the reaction pH value.

[0080] 4. Experiment on exploring the reaction time for CDs-CNPMD to detect thiols

[0081] (1) Prepare the 1 mg / mL CDs-CNPMD mother liquor for use.

[0082] (2) Prepare the 1 mmol / L Cys, Hcy, and GSH mother liquors for use respectively.

[0083] (3) Take 4 portions of 2 mL of the above-mentioned PBS solution with pH = 7.4 numbered 1 - 4. Add 20 μL of the 1 mg / mL CDs-CNPMD mother liquor to the solution numbered 1, and record the changes in its fluorescence spectrum at different times after mixing.

[0084] (4) Take 3 portions of 2 mL of the above-mentioned PBS solution with pH = 7.4. After adding 20 μL of the 1 mg / mL CDs-CNPMD mother liquor to each portion, then add 20 μL of the 1 mmol / L Cys, Hcy, and GSH mother liquors respectively, and record the changes in their fluorescence spectra at different times after mixing.

[0085] The results are as Figure 7 shown.

[0086] Figure 7 Part a in shows the change diagram of fluorescence intensity with reaction time. It can be seen from the figure that the fluorescence intensity gradually increases with the increase of reaction time, and the increase of fluorescence intensity begins to slow down when the reaction time reaches 120 minutes. The fluorescence intensity is still slowly increasing when recorded at 160 minutes. Considering the time cost, the reaction time is finally selected as 120 minutes. Figure 7The b part in the figure is the reaction rate diagram of the reaction of three kinds of biothiols with the probe calculated from the a part. The results show that the initial reaction rate of Cys is the highest, with a rate constant of 0.0196. The reason for the relatively high reaction rate of Cys may be that the Cys molecular structure is smaller and the reaction steric hindrance is smaller. The initial rate constants of Hcy and GSH are relatively close, being 0.0138 and 0.0133 respectively. Although there are differences among the three, the overall differences are not significant. Especially in the later stage of the reaction, the rate curves of the three are close to parallel.

[0087] 5. Correlation study on the concentration of three kinds of biothiols and fluorescence recovery

[0088] (1) Prepare stock solutions of Cys, Hcy, and GSH with a concentration of 1 mmol / L respectively for later use.

[0089] (2) Take multiple portions of 2 mL of the above PBS solution with pH = 7.4. After adding 20 μL of the 1 mg / mL CDs-CNPMD stock solution to each, then add a series of 0 - 150 μL of the stock solutions of Cys, Hcy, or GSH with a concentration of 1 mmol / L respectively. After mixing and standing for 120 min, record the changes in their fluorescence spectra.

[0090] The results are as Figure 8 shown. With the continuous increase in the concentration of the three kinds of biothiols, under the same reaction conditions, the fluorescence intensity of the probe solution gradually increases and finally tends to be flat. The linear ranges for the detection of the three kinds of biothiols by the probe are similar, all being 1 - 30 μM, and the detection limits are 0.98 μM, 1.14 μM, and 0.87 μM respectively.

[0091] 6. Selectivity and interference experiments

[0092] Prepare stock solutions of cysteine, homocysteine, glutathione, histidine, serine, threonine, valine, tryptophan, lysine, glycine, tyrosine, phenylalanine, arginine, aspartic acid, glutamic acid, glucose, NaBr, KI, NaHPO4, FeCl3, MgSO4, NaHCO3, NaClO, Na2CO3, NaNO2, CH3COONa, CaCl2, NaHS, ZnSO4, and H2O2 with a concentration of 1 mmol / L respectively for later use.

[0093] (1) Selectivity experiment

[0094] Take multiple portions of 2 mL of the same PBS solution with pH = 7.4 respectively. After adding 20 μL of the 1 mg / mL CDs-CNPMD stock solution to each, then add 20 μL of the stock solutions of Cys, Hcy, GSH, and the above-mentioned amino acids and ions respectively. After mixing and standing for 10 min, record the changes in their fluorescence spectra to observe whether CDs-CNPMD has high selectivity for Cys.

[0095] (2) Interference experiment

[0096] Take three groups of the same PBS solutions with pH = 7.4, with 31 samples in each group (KI solution is used as K + and I - for the investigation of two kinds of ions), 2 mL for each, add 20 μL of 1 mg / mL CDs-CNPMD stock solution, then add 20 μL of Cys, Hcy, GSH and the stock solutions of the above amino acids and ions to each group respectively. After mixing evenly, add 20 μL of 1 mmol / L Cys, Hcy or GSH stock solution to each group of solutions respectively. After mixing evenly and standing for 120 min, record the changes in their fluorescence spectra to observe whether the presence of these amino acids or ions affects the fluorescence recovery of Cys to CDs-CNPMD.

[0097] The results are as Figure 9 shown. It can be seen that the probe has high selectivity for the three biothiols. Only after reacting with the three biothiols, the fluorescence intensity will increase significantly. The presence of other amino acids or ions will not interfere with the fluorescence recovery of the three thiols, indicating that CDs-CNPMD has strong anti-interference ability in detecting the three biothiols.

[0098] 7. Determination method for total biothiols in fruits and vegetables

[0099] Thoroughly wash cucumbers, cherry tomatoes, tomatoes, grapes, oranges and apples with detergent and dry the surface moisture. After weighing, put them into a homogenizer for homogenization respectively, then transfer them to an ultrasonic cleaner and ultrasonicate for 20 minutes. Then take out 10 g of the paste and put it into a centrifuge for centrifugation for 10 minutes (9000 rpm). Take the supernatant obtained after centrifugation and filter it with a 0.22 μm filter membrane, and accurately measure 1 mL of each of the obtained liquids as the stock solution. If necessary, the obtained stock solution can be diluted to further obtain a sample solution for testing. Incubate the obtained stock solution or sample solution with 10 μg / mL CDs-CNPMD solution for 120 min, and at the same time measure the biothiol content by the standard Ellman method and make a comparison.

[0100] The results are shown in Table 1. The results show that the test results of the probe are close to those of the standard Ellman method, proving the reliability of the method.

[0101] Table 1 Comparison of the results of measuring total biothiols by the CDs-CNPMD probe method and the standard Ellman method

[0102]

[0103] In summary, the present invention uses catechol as a raw material to synthesize CDs with uniform particle size and a graphite-like structure. After modification of the CDs, XPS and IR indicate that the p-nitropyrimidine group has been successfully linked to the carbon quantum dots. The conditional experiments show that CDs-CNPMD can detect biological thiols with high selectivity, high sensitivity, and strong anti-interference ability. The linear ranges of the probe for detecting three biological thiols are similar, all being 0 - 30 μM, and the detection limits are 0.98 μM, 1.14 μM, and 0.87 μM, respectively. Therefore, the probe has good detection ability for biological thiols. The total biological thiols in cucumber, cherry tomato, tomato, grape, orange, and apple were detected respectively, and the results are close to those of the standard Ellman method, which proves the reliability of the method.

[0104] The above are only the preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present application should be covered by the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A preparation method of a 5-nitropyrimidine-functionalized carbon dot fluorescent probe, characterized in that, It includes the following steps: First, prepare CDs by the high-temperature reflux method, and then sequentially add 2-chloro-5-nitropyrimidine and triethylamine to the DMF solution of the obtained CDs for reaction. The temperature of the reaction is 40 °C and the time is 24 h. After the reaction, remove the solvent under reduced pressure, and then purify by column chromatography to obtain the 5-nitropyrimidine-functionalized carbon dots; The preparation of CDs by the high-temperature reflux method includes the following steps: Add concentrated phosphoric acid to the ethylene glycol solution of resorcinol, carry out a reflux reaction in air. After the reaction, remove the solvent, then add water and adjust the pH to precipitate. After purifying the obtained precipitate, the CDs are obtained.

2. The preparation method of a 5-nitropyrimidine-functionalized carbon dot fluorescent probe according to claim 1, characterized in that, The addition ratio of resorcinol, ethylene glycol and concentrated phosphoric acid is 500 mg: 50 mL: 150 μL.

3. The preparation method of a 5-nitropyrimidine-functionalized carbon dot fluorescent probe according to claim 1, characterized in that, The temperature of the reflux reaction is 180 °C and the time is 5 h.

4. The preparation method of a 5-nitropyrimidine-functionalized carbon dot fluorescent probe according to claim 1, characterized in that, Adjusting the pH means adjusting to pH = 13 and then adjusting to pH = 6.

5. The preparation method of a 5-nitropyrimidine-functionalized carbon dot fluorescent probe according to claim 1, characterized in that, The addition ratio of CDs, DMF, 2-chloro-5-nitropyrimidine and triethylamine is 100 mg: 5 mL: 50 mg: 50 μL.

6. A 5-nitropyrimidine-functionalized carbon dot fluorescent probe prepared by the preparation method of a 5-nitropyrimidine-functionalized carbon dot fluorescent probe according to any one of claims 1-5.

7. An application of the 5-nitropyrimidine-functionalized carbon dot fluorescent probe according to claim 6 in thiol detection.

Images