Red-emitting carbon dots with large stokes shift, preparation method thereof and application in multi-mechanism detection of fluorine and tetracycline

Abstract

The application belongs to the technical field of fluorescent probe, and provides a red-emitting carbon dot with large stokes shift, a preparation method thereof and application in multi-mechanism detection of fluorine and tetracycline. The red-emitting carbon dot R-CDs is synthesized by one-step solvothermal synthesis method with p-phenylenediamine as a precursor and nickel chloride hexahydrate as a catalyst. Based on fluorescence "switching" and competitive binding mechanism, a set R-CDs-Fe 3+ The fluorescence enhancement sensor is used for quantitative detection of fluorine ions, and is successfully applied to analysis of fluorine ions in actual samples. Based on the synergistic effect of inner filter effect and static quenching, R-CDs can also be used as a fluorescence quenching sensor for tetracycline detection, with a linear range of 0.5-100 μmol / L and a detection limit of 0.11 μmol / L. Obviously, R-CDs is a potential sensing material for detecting fluorine ions and tetracycline.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fluorescent probes, and particularly relates to a red-emitting carbon dot with a large stokes shift, a preparation method thereof and application thereof in multi-mechanism detection of fluorine and tetracycline. BACKGROUND

[0002] Fluoride ions are widely present in drinking water, toothpaste and osteoporosis drugs, and have attracted widespread attention due to their potential health risks. Its proper use is essential for dental care and bone health, but excessive intake can cause damage to human organs such as stomach, kidney and bone. In addition, long-term exposure to fluoride ions has a negative impact on the intelligence quotient of children. Notably, there are many areas in the world where groundwater contains a large amount of fluoride ions, which puts many humans at risk. Therefore, it is very worthwhile to develop a simple, sensitive and selective method for detecting fluoride ions.

[0003] Tetracycline, as a broad-spectrum antibiotic in the tetracycline family, can bind to tRNA to achieve antibacterial effect. Antibiotics are usually used to treat serious bacterial infections in humans. However, the World Health Organization prohibits the use of growth-promoting antibiotics because their use is associated with various human health problems. Tetracycline is deposited in teeth, bones and even nails, and can also cause enamel hypoplasia. Therefore, quantitative detection of tetracycline is of great significance in the fields of food and life sciences.

[0004] At present, various detection technologies such as chromatography, colorimetry and electrochemistry have been successfully developed to determine fluoride ions and tetracycline drugs. Although these methods have high accuracy and high sensitivity, they still have the shortcomings of complex instruments, cumbersome operation procedures and unstable electrodes, which to some extent limit their widespread application. Therefore, it is urgent to develop a new method for rapid, efficient and quantitative detection of fluoride ions and tetracycline.

[0005] In order to make up for the above shortcomings, fluorescent analysis technology represented by carbon dots is favored due to its low cost, simple operation, fast response, strong selectivity and extremely high sensitivity. However, most carbon dots emit in the short-wavelength region (blue and green), which limits their further biological analysis applications due to the spontaneous blue fluorescence of biological substrates. Therefore, long-wavelength emitting carbon dots have received more and more attention and become a new darling in the field of nano-sensors and environmental monitoring.

[0006] At present, many carbon dot-based fluorescent sensors have been developed to detect fluoride ions. Zhang et al. designed a hybrid nanosystem composed of yellow-emitting carbon dots and curcumin, which realized the sensitive detection of fluoride ions through the differentiation of internal filter effect (Anal. Chem. 2018, 90, 12573-12579). J. Hazard. Mater., 2021, 411: 125184). In addition, various functional carbon dots have been used for the determination of tetracycline. Li et al. developed a red-emitting carbon dot based on the aggregation-induced emission enhancement mechanism for the detection of tetracycline ( Sens. Actuators B Chem., 2021, 332: 129513 ). Although the emission of these carbon dots is in the long wavelength region, they have the disadvantage of small Stokes shift. Therefore, it is urgent to develop long wavelength emitting carbon dots with large Stokes shift.

[0007] To our knowledge, although many fluorescent sensors have been developed for detection, few published reports show that the integration of multi-mechanism detection into a single carbon dot-based chemical sensor can simultaneously detect two targets. SUMMARY

[0008] In order to solve the problem that the current carbon dot-based fluorescent sensor has the disadvantage of small Stokes shift, and cannot be integrated into a single carbon dot-based chemical sensor for simultaneous detection of two targets, the present application provides a red-emitting carbon dot with large Stokes shift, a preparation method thereof and application thereof in multi-mechanism detection of fluoride and tetracycline.

[0009] The present application is realized by the following technical solutions: a R-CDs with large Stokes shift, which is obtained by the following method: taking p-phenylenediamine as a precursor and NiCl2·6H2O as a catalyst, R-CDs are prepared by one-step solvothermal reaction, and the obtained R-CDs powder is further subjected to dialysis, filtration and freeze-drying.

[0010] The specific preparation method of the R-CDs according to the present application is as follows:

[0011] (1) 0.03 g of p-phenylenediamine and 0.006 g of NiCl2·6H2O are weighed and dissolved in 10 mL of ethanol, and then ultrasonic treatment is carried out at 15 KHz and 25℃ for 5 minutes; the obtained clear solution is transferred to a high-pressure kettle containing a polytetrafluoroethylene liner and heated in an oven; after the reaction is completed, the temperature is naturally cooled to room temperature to obtain a deep purple solution;

[0012] (2) the obtained deep purple solution is transferred to a dialysis bag with a molecular weight cut-off of 1000 Da and placed in water, and dialysis is carried out for 24 h, with water being changed every 6 h; the liquid after dialysis is filtered through a 0.22 μm filter membrane, and the filtrate is freeze-dried to collect black powder, which is R-CDs.

[0013] Further, the heating temperature is 160℃, the time is 6 h, and the heating rate is 10℃ / min; the freezing temperature is-60℃, and the time is 36 h.

[0014] The R-CDs according to the present application or the R-CDs obtained by the method are used to construct a set R-CDs-Fe 3+The specific steps of the fluorescence enhancement sensor are as follows:

[0015] (1) Preparation of R-CDs diluent: accurately weigh R-CDs powder, dissolve in ethanol to prepare R-CDs stock solution with a concentration of 1.0 mg / mL; the R-CDs diluent is diluted with ethanol to obtain;

[0016] (2) Preparation of Fe 3+ stock solution: accurately weigh FeCl3·6H2O powder, dissolve in ultrapure water to prepare Fe 3+ stock solution with a concentration of 0.01 mol / L;

[0017] (3) 500 μL of R-CDs diluent with a concentration of 0.02 mg / mL, 10 μL of Fe 3+ stock solution with a concentration of 0.01 mol / L, and 400 μL of Tris-HCl buffer solution with a concentration of 0.1 mol / L and a pH of 6.8 are sequentially mixed, and incubated at room temperature for 1 minute, and the obtained mixture is a set R-CDs-Fe 3+ fluorescence enhancement sensor system.

[0018] The set R-CDs-Fe 3+ fluorescence enhancement sensor constructed by the application has the application in the detection of fluoride ions, and the specific steps are as follows:

[0019] (1) Preparation of fluoride ion stock solution: accurately weigh sodium fluoride powder, dissolve in ultrapure water to prepare fluoride ion stock solution with a concentration of 0.01 mol / L;

[0020] (2) Establishment of standard curve: a plurality of volume gradients of the fluoride ion stock solution are added to the set R-CDs-Fe 3+ fluorescence enhancement sensor system, diluted with Tris-HCl buffer solution to 1.0 mL as the final volume, and the fluorescence emission intensity at 600 nm is measured and recorded under the excitation wavelength of 285 nm; the linear fitting of the fluoride ion concentration and the fluorescence intensity of the R-CDs-Fe 3+ system is carried out through Origin software, and the linear equation is: ΔF = 1.830c (氟离子) + 5.617, R 2 = 0.998; the linear range is 0.5~120 μmol / L, and the lowest detection limit is 0.25 μmol / L.

[0021] (3) Determination of fluoride ions in actual samples: 500 μL of R-CDs diluent (0.02 mg / mL), 10 μL of Fe 3+The reserve solution (0.01 mol / L) and 490 μL of the sample solution to be detected are mixed, the fluorescence intensity is measured under the excitation wavelength of 285 nm, and the content of fluoride ions in the sample to be detected is calculated by substituting into the linear equation.

[0022] Further, the R-CDs and Fe 3+ have a concentration ratio of 10 μg / mL:100 μmol / L.

[0023] The application of the R-CDs or the R-CDs prepared by the method in tetracycline detection has the characteristics that the specific steps are as follows:

[0024] (1) Preparation of the R-CDs diluent: R-CDs powder is accurately weighed, dissolved in ethanol to prepare R-CDs reserve solution with a concentration of 1.0 mg / mL; and the R-CDs diluent is obtained by diluting with ethanol;

[0025] (2) Preparation of the tetracycline reserve solution: tetracycline hydrochloride powder is accurately weighed, dissolved in ultrapure water to prepare tetracycline reserve solution with a concentration of 0.01 mol / L;

[0026] (3) Establishment of the standard curve: 500 μL of the R-CDs diluent (0.02 mg / mL), 400 μL of the Tris-HCl buffer solution (0.1 mol / L, pH 6.8) and a certain volume of the tetracycline reserve solution are added to a centrifugal tube, diluted with the Tris-HCl buffer solution to 1.0 mL as the final volume, the fluorescence emission intensity at 600 nm is measured and recorded under the excitation wavelength of 285 nm; the linear equation: ΔF = 4.065c (四环素) + 10.555, R 2 = 0.996 is obtained by linear fitting the tetracycline concentration and the R-CDs fluorescence intensity through the Origin software; the linear range is 0.5-100 μmol / L, and the lowest detection limit is 0.11 μmol / L.

[0027] (4) Determination of tetracycline in the actual sample: 500 μL of the R-CDs diluent (0.02 mg / mL) is mixed with 500 μL of the sample solution to be detected, the fluorescence intensity is measured under the excitation wavelength of 285 nm, and the content of tetracycline in the sample to be detected is calculated by substituting into the linear equation.

[0028] The application also provides the application of the R-CDs or the R-CDs prepared by the method in detecting fluoride ions and tetracycline in the actual sample, and the actual sample needs to be treated before detection, and the specific treatment method is as follows:

[0029] The actual sample is milk, 1 mL of 300 g / L trichloroacetic acid is added to 99 mL of milk, ultrasonic treatment is carried out for 20 min to remove protein and lipid, and centrifugation is carried out at 8000 rpm for 5 min; the centrifugal supernatant is filtered by using a 0.22 μm filter membrane, and the pH of the filtered solution is adjusted to 7.0 by using 5 mol / L sodium hydroxide;

[0030] The actual sample is honey and mineral water, the diluted 20 times honey water and mineral water are filtered by using a 0.22 μm filter membrane, and are prepared for use;

[0031] The actual sample is toothpaste or tea, 20 g of toothpaste or tea is dispersed in 1000 mL of ultrapure water, heating is carried out at 70 ℃ for 3 h, after cooling to room temperature, the mixture is centrifuged at 13000 rpm for 10 min, and the centrifugal supernatant is filtered by using a 0.22 μm filter membrane.

[0032] The present application has the following beneficial effects:

[0033] The present application integrates two sensing mechanisms for detecting fluoride ions and tetracycline into a chemical sensor based on R-CDs. First, Fe 3+ is combined with the amino group / carboxyl group on the surface of the R-CDs, resulting in fluorescence quenching of the R-CDs, then fluoride ions are added, which compete with Fe 3+ to combine with the surface of the R-CDs, so that the fluorescence intensity of the R-CDs is restored, and thus a fluorescence enhancement sensor R-CDs-Fe 3+ can be constructed to detect fluoride ions. In addition, the excitation spectrum of the R-CDs overlaps with the absorption spectrum of tetracycline, so that a fluorescence quenching sensor R-CDs can be established by the synergistic effect of the inner filter effect (IFE) and static quenching to detect tetracycline.

[0034] In summary, compared with other methods for detecting fluoride ions and tetracycline, the present application has the advantages of rapidity, high efficiency, high sensitivity and good selectivity, and provides a novel method for detecting fluoride ions and tetracycline. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 It is a transmission electron microscope image of the R-CDs prepared in Example 1;

[0036] Figure 2 It is a fluorescence excitation emission spectrum image of the R-CDs prepared in Example 1;

[0037] Figure 3 It is an ultraviolet absorption and fluorescence spectrum image of the R-CDs prepared in Example 1;

[0038] Figure 4 It is a result graph of the selectivity experiment of the R-CDs to different metal ions in Example 2;

[0039] Figure 5 For Example 3, based on R-CDs-Fe 3+ The anti-interference experiment results of the fluorescence enhancement sensor for detecting fluoride ions;

[0040] Figure 6 For Example 4, based on R-CDs-Fe 3+ The fluorescence curve change diagram of the fluorescence enhancement sensor for detecting fluoride ions;

[0041] Figure 7 For Example 4, based on R-CDs-Fe 3+ The standard curve diagram of the fluorescence enhancement sensor for detecting fluoride ions;

[0042] Figure 8 For Example 6, the anti-interference experiment results of the fluorescence quenching sensor based on R-CDs for detecting tetracycline;

[0043] Figure 9 For Example 7, the fluorescence change curve diagram of the R-CDs fluorescence quenching sensor for detecting tetracycline;

[0044] Figure 10 For Example 7, the standard curve diagram of the R-CDs fluorescence quenching sensor for detecting tetracycline. DETAILED DESCRIPTION

[0045] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, and the materials cited and referred to herein are incorporated by reference.

[0047] The equivalent techniques of the described specific embodiments, which can be realized by those skilled in the art through routine experiments, are included in the present application.

[0048] In the following examples, the experimental methods are all routine methods unless otherwise specified. In the following examples, the instrument equipment used is all routine laboratory instrument equipment unless otherwise specified; in the following examples, the experimental materials used are all purchased from routine biochemical reagent stores unless otherwise specified.

[0049] Example 1: Preparation and characterization of R-CDs

[0050] Step 1: Weigh 0.03 g of p-phenylenediamine and 0.006 g of NiCl2·6H2O, dissolve them in 10 mL of ethanol, and sonicate for 5 minutes. Transfer the resulting clear solution to a polytetrafluoroethylene-lined autoclave and heat in an oven at 160°C for 6 hours. After the reaction is complete, cool naturally to room temperature to obtain a dark purple solution.

[0051] Step 2: Transfer the resulting dark purple solution to a dialysis bag with a molecular weight cutoff of 1000 Da and place it in water for 24 hours, changing the water every 6 hours. The dialyzed liquid is passed through a 0.22 μm filter membrane, and the filtrate is freeze-dried to collect the black powder, which is R-CDs.

[0052] Step 3: Accurately weigh 0.01 g of R-CDs powder and dissolve it in 10 mL of ethanol to obtain a 1.0 mg / mL R-CDs stock solution.

[0053] For morphology and properties, see Figure 1 、 Figure 2 and Figure 3 .from Figure 1 It can be seen that the prepared R-CDs are spherical and the particle size is relatively uniform; Figure 2 The researchers demonstrated that the optimal excitation and emission wavelengths for R-CDs are 285 nm and 600 nm, respectively, with a Stokes shift of 315 nm. This large Stokes shift not only reduces the impact of background excitation light on fluorescence measurements but also avoids self-quenching, which is beneficial to the sensitivity and detection reliability of R-CDs. Figure 3 The UV absorption spectrum of R-CDs has two sharp absorption peaks at 250 nm and 280 nm, which are attributed to the aromatic sp 2 π→π of the domain * In addition, the broad peak at 523 nm is due to the n→π transition of the surface functional groups. * The emission spectra of R-CDs at different excitation wavelengths show excitation wavelength independence, which may be attributed to the uniformity of the surface structure of R-CDs.

[0054] Example 2: Selectivity of R-CDs for metal ions

[0055] Step 1: Weigh different masses of metal salts (NaCl, KCl, BaCl2, CdCl2, CuSO4, ZnNO3, CaCl2, PbCl2, CoCl2, MgCl2, AlCl3, AgNO3, CrCl3, NiCl2, and FeCl3), add 10 mL of ultrapure water, and prepare 0.1 mol / L metal salt stock solutions.

[0056] Step two, 500 μL R-CDs (0.02 mg / mL) was mixed with 500 μL Tris-HCl buffer solution (0.1 mol / L, pH 6.8), and the fluorescence intensity was measured and recorded as F0; 10 μL metal salt stock solution was added to 500 μL R-CDs (0.02 mg / mL), and diluted to 1.0 mL with Tris-HCl buffer solution, at this time the concentration of metal salt was 1.0 mmol / L, and the fluorescence intensity was measured and recorded as F; the column chart was calculated by F / F0, and the experimental results were shown in Figure 4 .

[0057] As shown in Figure 4 , the fluorescence of R-CDs had good selectivity for Fe 3+ , and the R-CDs-Fe 3+ fluorescence-enhanced sensor could be further constructed to detect fluoride ions.

[0058] Example 3: Anti-interference experiment of R-CDs-Fe 3+ fluorescence-enhanced sensor for detecting fluoride ions

[0059] Step one, FeCl3stock solution was diluted to obtain a 0.01 mol / L Fe 3+ standard solution.

[0060] Step two, different amounts of sodium salt (NaNO2, NaNO3, NaSO3, Na2SO4, NaCO3, NaHCO3, NaSCN, Na2S2O3, NaClO4, NaS2, NaCl, NaBr, NaI and NaF) were weighed, 10 mL ultrapure water was added, and a 0.1 mol / L sodium salt stock solution was prepared.

[0061] Step three, 10 μL Fe 3+ standard solution was added to 500 μL R-CDs (0.02 mg / mL), and diluted to 1.0 mL with Tris-HCl buffer solution (0.1 mol / L, pH 6.8), at this time, the concentration ratio of R-CDs to Fe 3+ was 0.01 mg / mL: 100 μmol / L, and the fluorescence intensity was measured and recorded as F0; 10 μL Fe 3+ standard solution and 10 μL sodium salt stock solution were added to 500 μL R-CDs (0.02 mg / mL), and diluted to 1.0 mL with Tris-HCl buffer solution (0.1 mol / L, pH 6.8), at this time the concentration of sodium salt was 1.0 mmol / L, and the fluorescence intensity was measured and recorded as F; the column chart was calculated by (F-F0) / F0, and the experimental results were shown in Figure 5 .

[0062] As Figure 5 shown, the fluorescence of R-CDs-Fe 3+ system was recovered significantly, and the presence of other metal ions did not affect the fluorescence of R-CDs-Fe 3+ system, indicating that the R-CDs-Fe 3+ system had good anti-interference performance in detecting fluoride ions.

[0063] Example 4: R-CDs-Fe 3+ fluorescence-enhanced sensor was used to establish a standard curve for fluoride ion detection

[0064] Step one, dilute the fluoride ion stock solution to obtain a 0.01 mol / L fluoride ion standard solution.

[0065] Step one, add 10 μL of Fe 3+ standard solution to 500 μL of R-CDs (0.02 mg / mL), and dilute to 1.0 mL with Tris-HCl buffer solution (0.1 mol / L, pH 6.8). At this time, the concentration ratio of R-CDs to Fe 3+ is 0.01 mg / mL: 100 μmol / L, measure the fluorescence intensity, and record it as F0.

[0066] Step two, add 10 μL of Fe 3+ standard solution and different volumes of fluoride ion standard solution to 500 μL of R-CDs (0.02 mg / mL), and dilute to 1.0 mL with Tris-HCl buffer solution (0.1 mol / L, pH 6.8). Record the fluorescence intensity, and record it as F. The fluorescence change is shown in Figure 6 .

[0067] Step three, use Origin software to fit the linear equation between the fluorescence intensity change (ΔF = F-F0) and the fluoride ion concentration, and the results are shown in Figure 7 .

[0068] Figure 6 It is shown that with the increase of fluoride ion concentration, the fluorescence of R-CDs-Fe 3+ system is enhanced, indicating that fluoride ions can specifically recover the fluorescence of R-CDs-Fe 3+ system; Figure 7 It is shown that the fluorescence intensity change value of R-CDs-Fe 3+ system and the fluoride ion concentration establishes a linear equation of ΔF = 1.830c (氟离子) + 5.617, R 2 = 0.998; the linear range is 0.5~120 μmol / L, and the lowest detection limit is 0.25 μmol / L.

[0069] Example 5: Spiked recovery experiment of fluoride ion in real samples

[0070] Step one, toothpaste and tea leaves were dispersed in ultrapure water, heated at 70°C for 3 hours, after cooling to room temperature, the mixture was centrifuged at 13000 rpm for 10 minutes, the supernatant was filtered with 0.22 μm filter membrane, ready for use.

[0071] Step two, mineral water was filtered with 0.22 μm filter membrane, ready for use.

[0072] Step three, 10 μL Fe 3+ The standard solution was added to 500 μL R-CDs (0.02 mg / mL), diluted to 1.0 mL with Tris-HCl buffer solution (0.1 mol / L, pH 6.8), at this time, R-CDs and Fe 3+ The concentration ratio was 0.01 mg / mL: 100 μmol / L, the fluorescence intensity was measured, recorded as F0.

[0073] Step four, 10 μL Fe 3+ The standard solution, 500 μL R-CDs (0.02 mg / mL) and 490 μL sample solution to be tested were mixed, followed by the addition of 2 μL fluoride ion standard solution (0.01 mol / L), the fluorescence intensity was measured, recorded as F1.

[0074] Step five, F1-F0 was substituted into the linear equation, the content of fluoride ion in the corresponding sample was calculated, recorded as c1.

[0075] Step six, 10 μL fluoride ion standard solution (0.001 mol / L) was added to the mixture of step four, the fluorescence intensity was measured, recorded as F2.

[0076] Step seven, F2-F0 was substituted into the linear equation, the content of fluoride ion in the spiked sample was calculated, recorded as c2.

[0077] Step eight, according to the spiked recovery formula (c2-c1) / c, the spiked recovery rate was calculated, c was the known concentration of fluoride ion added.

[0078] The results are shown in Table 1, Table 1 shows that the spiked recovery rate of fluoride ion in real samples is between 97.9~102.8%, indicating that the R-CDs-Fe 3+ The fluorescence-enhanced sensor can be used for the detection of fluoride ion in real samples, and the method has good reproducibility.

[0079] Table 1: Content of fluoride ion in real samples and spiked recovery results

[0080]

[0081] Example 6: Anti-interference experiment of R-CDs fluorescence quenching sensor for detecting tetracycline

[0082] Step one, weigh different amounts of amino acids (proline, alanine, leucine, glycine, aspartic acid, glutamic acid, valine, serine, lysine, arginine, cysteine, histidine, methionine, threonine) and antibiotics (penicillin, ornidazole, tinidazole, erythromycin, gentamicin, kanamycin, thiamphenicol, azithromycin, amoxicillin, tetracycline), add 10 mL ultrapure water to prepare 0.01 mol / L drug stock solution.

[0083] Step three, mix 500 μL R-CDs (0.02 mg / mL) with 500 μL Tris-HCl buffer solution (0.1 mol / L, pH 6.8), measure the fluorescence intensity, and record it as F0; add 100 μL drug stock solution to 500 μL R-CDs (0.02 mg / mL), dilute with Tris-HCl buffer solution (0.1 mol / L, pH 6.8) to 1.0 mL, at this time the drug concentration is 1.0 mmol / L, measure the fluorescence intensity, and record it as F; calculate the column chart from F / F0, and the experimental results are shown in Figure 8 .

[0084] As Figure 8 shown, tetracycline has a significant fluorescence quenching effect on R-CDs, and the presence of other drugs does not affect the fluorescence of R-CDs, indicating that R-CDs has good anti-interference property when detecting tetracycline.

[0085] Example 7: Establishment of standard curve for tetracycline detection based on R-CDs fluorescence quenching sensor

[0086] Step one, mix 500 μL R-CDs (0.02 mg / mL) with 500 μL Tris-HCl buffer solution (0.1 mol / L, pH 6.8), measure the fluorescence intensity, and record it as F0.

[0087] Step two, add different volumes of tetracycline stock solution to 500 μL R-CDs (0.02 mg / mL), dilute with Tris-HCl buffer solution (0.1 mol / L, pH 6.8) to 1.0 mL, and measure the fluorescence intensity, and record it as F; the fluorescence change is shown in Figure 9 .

[0088] Step three, use Origin software to fit the linear equation between the fluorescence intensity change (ΔF = F-F0) and the tetracycline concentration, and the results are shown in Figure 10 .

[0089] Figure 9 It is shown that the fluorescence of R-CDs is weakened with the increase of tetracycline concentration, indicating that tetracycline can specifically quench the fluorescence of R-CDs; Figure 10 It is found that the linear equation of the change value of the fluorescence intensity of R-CDs and the tetracycline concentration is ΔF = 4.065c (四环素) + 10.555, R 2 = 0.996; the linear range is 0.5~100 μmol / L, and the lowest detection limit is 0.11 μmol / L.

[0090] Example 8: Detection of tetracycline content in actual samples and standard addition recovery experiment

[0091] Step one, mix milk with 1% (v / v) trichloroacetic acid (300 g / L), and treat with ultrasonic for 20 min to remove protein and lipid, and centrifuge at 8000 rpm for 5 min; filter the supernatant with a 0.22 μm filter membrane, and adjust the pH of the filtered solution to 7.0 with 5 mol / L sodium hydroxide, and reserve for use.

[0092] Step two, dilute honey with ultrapure water by 20 times, and filter with a 0.22 μm filter membrane, and reserve for use.

[0093] Step three, mix 500 μL R-CDs (0.02 mg / mL) with 500 μL Tris-HCl buffer solution (0.1 mol / L, pH 6.8), and measure the fluorescence intensity, and record as F0.

[0094] Step four, mix 500 μL R-CDs (0.02 mg / mL) with 500 μL sample solution to be detected, and measure the fluorescence intensity, and record as F1.

[0095] Step five, substitute F1-F0 into the linear equation, and calculate the tetracycline content in the corresponding sample, and record as c1.

[0096] Step six, add 10 μL tetracycline standard solution (0.001 mol / L) to the mixture in step four, and measure the fluorescence intensity, and record as F2.

[0097] Step seven, substitute F2-F0 into the linear equation, and calculate the tetracycline content in the standard addition sample, and record as c2.

[0098] Step eight, calculate the standard addition recovery rate according to the standard addition recovery formula (c2-c1) / c, and c is the known tetracycline concentration added.

[0099] The results are shown in Table 2, which shows that the standard addition recovery rate of tetracycline in the actual sample is between 94.9 and 103.8%, indicating that the R-CDs fluorescence quenching sensor can be used for the detection of tetracycline in the actual sample, and the method has good reproducibility.

[0100] Table 2: Content and standard addition recovery results of tetracycline in actual samples

[0101]

[0102] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A red-emitting carbon dot (R-CD) with a large Stokes shift, characterized by: R-CDs were synthesized by a one-step solvothermal synthesis using p-phenylenediamine as a precursor and NiCl2·6H2O as a catalyst. The specific steps of the preparation method are as follows: (1) Weigh 0.03 g of p-phenylenediamine and 0.006 g of NiCl2·6H2O, dissolve them in 10 mL of ethanol, and sonicate at 15 kHz and 25°C for 5 min. Transfer the resulting clear solution to a polytetrafluoroethylene-lined autoclave and heat it in an oven. After the reaction is complete, cool the solution to room temperature to obtain a dark purple solution. (2) The obtained dark purple solution was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da and placed in water for 24 h. The water was changed every 6 h. The dialyzed liquid was passed through a 0.22 μm filter membrane. The filtrate was freeze-dried and the black powder was collected, which was R-CDs.

2. The red-emitting carbon dots (R-CDs) with a large Stokes shift according to claim 1, characterized in that: The heating temperature of the oven heating in step (1) is 160°C, the time is 6 hours, and the heating rate is 10°C / minute; the freezing temperature of the filtrate freeze-drying in step (2) is -60°C, and the time is 36 hours.

3. Using the R-CDs of claim 1 to construct a containerized R-CDs-Fe 3+ A fluorescence enhancement sensor, characterized in that: The specific steps are as follows: (1) Preparation of R-CDs diluent: Accurately weigh R-CDs powder and dissolve it in ethanol to prepare a 1.0 mg / mL R-CDs stock solution; dilute the R-CDs stock solution with ethanol to obtain an R-CDs diluent; (2) Fe 3+ Preparation of stock solution: Accurately weigh FeCl3·6H2O powder and dissolve it in ultrapure water to prepare a FeCl3·6H2O stock solution with a concentration of 0.01 mol / L. 3+ stock solution; (3) R-CDs-Fe 3+ Construction of fluorescence enhanced sensor: 500 μL of R-CDs dilution with a concentration of 0.02 mg / mL and 0.01 mol / L Fe 3+ 10 μL of the stock solution and 400 μL of a 0.1 mol / L, pH 6.8 Tris-HCl buffer solution were mixed in sequence and incubated at room temperature for 1 minute. The resulting mixture was the assembled R-CDs-Fe 3+ Fluorescence enhancement sensor system.

4. The assembled R-CDs-Fe according to claim 3 3+ The application of fluorescence enhancement sensor in fluoride ion detection is characterized by: The specific steps are as follows: (1) Preparation of fluoride ion stock solution: Accurately weigh sodium fluoride powder and dissolve it in ultrapure water to prepare a fluoride ion stock solution with a concentration of 0.01 mol / L; (2) Establishment of standard curve: Add several volume gradients of fluoride ion stock solution to the containerized R-CDs-Fe 3+ In the fluorescence enhanced sensor system, the final volume was 1.0 mL diluted with Tris-HCl buffer solution, and the fluorescence emission intensity at 600 nm was measured and recorded under an excitation wavelength of 285 nm. The fluoride ion concentration and R-CDs-Fe were linearly fitted by Origin software. 3+ Fluorescence intensity of the system, the linear equation is: ΔF = 1.830c (氟离子) +5.617, R 2 = 0.998; linear range: 0.5~120 μmol / L, minimum detection limit: 0.25 μmol / L; (3) Determination of fluoride ions in actual samples: 500 μL of R-CDs dilution solution with a concentration of 0.02 mg / mL and 0.01 mol / L Fe 3+ Mix 10 μL of the stock solution and 490 μL of the sample solution to be tested. Measure the fluorescence intensity at an excitation wavelength of 285 nm and substitute it into the linear equation to calculate the fluoride ion content in the sample to be tested.

5. The use according to claim 4, characterized in that: The R-CDs and Fe 3+ The concentration ratio is 10 μg / mL:100 μmol / L.

6. The use of the R-CDs according to claim 1 in tetracycline detection, characterized in that: The specific steps are as follows: (1) Preparation of R-CDs diluent: Accurately weigh R-CDs powder and dissolve it in ethanol to prepare a 1.0 mg / mL R-CDs stock solution; dilute the R-CDs stock solution with ethanol to obtain an R-CDs diluent; (2) Preparation of tetracycline stock solution: Accurately weigh tetracycline hydrochloride powder and dissolve it in ultrapure water to prepare a tetracycline stock solution with a concentration of 0.01 mol / L; (3) Establishment of standard curve: 500 μL of 0.02 mg / mL R-CDs dilution solution, 400 μL of 0.1 mol / L Tris-HCl buffer solution with a pH of 6.8, and several volume gradients of tetracycline stock solution were mixed in sequence, diluted to 1.0 mL with Tris-HCl buffer solution as the final volume, and the fluorescence intensity was measured at 600 nm under an excitation wavelength of 285 nm. The tetracycline concentration and R-CDs fluorescence intensity were linearly fitted by Origin software, and the linear equation was obtained: ΔF = 4.065c (四环素) +10.555, R 2 = 0.996; linear range: 0.5~100 μmol / L, minimum detection limit: 0.11 μmol / L; (4) Determination of tetracycline in actual samples: Mix 500 μL of R-CDs dilution solution with a concentration of 0.02 mg / mL with 500 μL of the sample solution to be tested. Measure the fluorescence intensity at an excitation wavelength of 285 nm and substitute it into the linear equation to calculate the content of tetracycline in the sample to be tested.

7. Use of the R-CDs according to claim 1 for detecting fluoride ions and tetracycline in actual samples, characterized in that: Before testing, the actual sample is pre-treated. The specific treatment method is as follows: The actual sample is milk. 1 mL of 300 g / L trichloroacetic acid was added to 99 mL of milk, and the milk was ultrasonically treated for 20 minutes to remove proteins and lipids. The milk was then centrifuged at 8000 rpm for 5 minutes. The supernatant was filtered through a 0.22 μm filter membrane, and the pH of the filtered solution was adjusted to 7.0 with 5 mol / L sodium hydroxide. The actual samples are honey and mineral water, and the honey water and mineral water diluted 20 times are filtered using a 0.22 μm filter membrane; The actual sample is toothpaste or tea. 20 g of toothpaste or tea is dispersed in 1000 mL of ultrapure water, heated at 70°C for 3 h, cooled to room temperature, and then centrifuged at 13,000 rpm for 10 min. The supernatant is filtered through a 0.22 μm filter membrane.

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