A method for simultaneous detection of copper and chromium ions based on dual-emission center carbon dots

Abstract

The present invention discloses a method for simultaneously detecting copper ions and chromium ions based on dual-emission center carbon dots. The method comprises the following steps: (a) pre-treating a sample to obtain a copper and chromium ion test solution; (b) synthesizing anemarrhena fluorescent carbon dots (AB-CDs); (c) synthesizing Pu'er tea fluorescent carbon dots (PT-CDs); (d) taking the AB-CDs and PT-CDs solutions and mixing them evenly to prepare dual-emission center carbon dots (AP-CDs), i.e., a ratiometric fluorescent probe; (e) mixing the copper ion test solution, AP-CDs solution, and ascorbic acid (AA) solution, adjusting the pH to 4.78, incubating for 40 minutes, and then heating at 36 The detection was performed at an excitation wavelength of 5 nm, and the fluorescence emission spectrum in the range of 385 to 710 nm was recorded to obtain the fluorescence intensity values ​​at 440 nm and 670 nm. The copper ion content was calculated based on the standard curve. The chromium ion test solution was mixed with the AP-CDs solution, the pH was adjusted to 4.78, and the detection was performed at an excitation wavelength of 365 nm. The fluorescence emission spectrum in the range of 385 to 710 nm was recorded to obtain the fluorescence intensity values ​​at 440 nm and 670 nm. The chromium ion content was calculated based on the standard curve. The detection method of the present invention has the advantages of high selectivity and high sensitivity, and is simple to operate, has obvious phenomena, and is reproducible.

Description

Technical Field

[0001] The present invention relates to the field of fluorescence sensor detection technology, and in particular to a method for simultaneously detecting copper ions and chromium ions based on dual-emission center carbon dots. Background Art

[0002] With the advancement of industrialization, heavy metal pollution has become an increasingly serious global environmental problem. Copper is widely present in the natural environment and is an indispensable trace element for organisms. 2+ Excessive use can cause toxicity to animals, plants and microorganisms. 2+ Excessive accumulation in the environment will destroy the ecological balance and cause food safety risks. The World Health Organization (WHO) stipulates that the amount of Cu in drinking water should be 2+ The permissible concentration for industrial wastewater is 31.5 μM, while the threshold set by the U.S. Environmental Protection Agency (EPA) is even lower, at 20 μM. Chromium is one of the most common heavy metal pollutants in industrial wastewater, primarily existing in two oxidation states: trivalent chromium and hexavalent chromium. The U.S. Environmental Protection Agency recommends that the concentration of hexavalent chromium in drinking water should not exceed 100 ppb (μg / L). Therefore, in order to ensure water safety, it is urgent to take corresponding measures to conduct efficient, accurate, and comprehensive monitoring of heavy metal ions in aquatic environments such as surface water and groundwater.

[0003] At present, a variety of methods for detecting Cu have been successfully developed. 2+ and Cr 6+ There are many techniques for the detection of Cu, including colorimetry, electrochemical methods, atomic absorption spectroscopy, inductively coupled plasma mass spectrometry, chromatography and spectrophotometry. However, these methods require expensive instruments and equipment, complex sample pretreatment procedures and professional technicians, which limits their widespread application. In contrast, the fluorescent probe method is an innovative detection method with the advantages of simple sample preparation, fast response time, wide linear dynamic range, low interference and high sensitivity. It is gradually becoming the most popular method for detecting Cu. 2+ and Cr 6+ A promising analytical technique for the detection of copper and chromium ions in water. Furthermore, sensors constructed with single-emission probes can be affected by various factors, such as changes in probe concentration and light source intensity, which can lead to inaccurate or erroneous results. Sensors constructed with dual-emission probes, on the other hand, can effectively avoid background absorption and reduce errors, significantly improving detection sensitivity. Dual-emission probes have become a research focus in the field of ratiometric fluorescence sensors. Therefore, it is of great significance to construct a ratiometric fluorescence probe with high sensitivity, selectivity, and repeatability for the simultaneous detection of copper and chromium ions in water. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for simultaneously detecting copper ions and chromium ions based on dual-emission central carbon dots, so as to solve the problems of high cost, low sensitivity, poor selectivity and repeatability, and complex operation of existing methods for detecting copper ions and chromium ions in water.

[0005] The object of the present invention is achieved by providing a method for simultaneously detecting copper ions and chromium ions based on dual-emission center carbon dots, comprising the following steps:

[0006] (a) pre-treating the sample to be tested to obtain a copper ion test solution and a chromium ion test solution;

[0007] (b) Synthesis of fluorescent carbon dots from Anemarrhena asphodeloides (denoted as AB-CDs);

[0008] (c) Synthesis of Pu'er tea fluorescent carbon dots (denoted as PT-CDs);

[0009] (d) The above-mentioned Anemarrhena asphodeloides fluorescent carbon dot solution and Pu'er tea fluorescent carbon dot solution were mixed evenly to obtain dual-emission center carbon dots (denoted as AP-CDs) as a ratiometric fluorescent probe;

[0010] (e) detecting the copper ion test solution and the chromium ion test solution; wherein the copper ion detection method comprises: mixing the copper ion test solution with the dual-emission center carbon dot solution in step (d) and a reducing agent, adjusting the pH with a buffer, incubating, detecting at an excitation wavelength of 365 nm, recording the fluorescence emission spectrum in the range of 385 to 710 nm, obtaining fluorescence intensity values ​​at 440 nm and 670 nm, and calculating the copper ion content in combination with a copper ion standard curve; and the chromium ion detection method comprises: mixing the chromium ion test solution with the dual-emission center carbon dot solution in step (d), adjusting the pH with a buffer, detecting at an excitation wavelength of 365 nm, recording the fluorescence emission spectrum in the range of 385 to 710 nm, obtaining fluorescence intensity values ​​at 440 nm and 670 nm, and calculating the chromium ion content in combination with a chromium ion standard curve.

[0011] Furthermore, the Anemarrhena fluorescent carbon dots in step (b) are prepared by using the traditional Chinese medicine Anemarrhena and water through a hydrothermal method.

[0012] Furthermore, the mass-volume ratio of Anemarrhena asphodeloides and water is 0.5 g:20 mL.

[0013] Furthermore, the Pu'er tea fluorescent carbon dots in step (c) are prepared by using Pu'er tea and anhydrous ethanol through ultrasonic extraction.

[0014] Furthermore, the mass-volume ratio of Pu'er tea to anhydrous ethanol is 0.8 g:10 mL.

[0015] Furthermore, in step (d), the volume ratio of the Anemarrhena asphodeloides fluorescent carbon dot solution to the Pu'er tea fluorescent carbon dot solution is 5 μL:100 μL.

[0016] Furthermore, the buffer in step (e) is a BR buffer with a pH of 4.78.

[0017] Furthermore, when the copper ion test solution is detected in step (e), the reducing agent is ascorbic acid, and the incubation time is 40 minutes.

[0018] Furthermore, the copper ion standard curve in step (e) is

[0019] Furthermore, the chromium ion standard curve in step (e) is

[0020] When testing the copper ion test solution, adding ascorbic acid to the test solution can reduce the Cr 6+ , to eliminate its effect on Cu 2+ In addition, ascorbic acid has a strong reducing property, although it may 2+ Reduction to Cu + , but due to Cu + It is extremely unstable and cannot exist stably in solution, so ascorbic acid will not affect Cu 2+ Detection.

[0021] Beneficial effects of the present invention:

[0022] (1) The ratiometric fluorescent probe constructed based on the dual-emission center carbon dots of the present invention can simultaneously detect copper ions and chromium ions and has been applied to the detection of actual water samples. The probe system has good selectivity, sensitivity, and repeatability. The fluorescence ratio F of the ratiometric fluorescent probe is 440 / F 670 It is linearly related to the concentration of copper ions in the range of 0.1-25μM and 50-250μM, and the linear relationship equations are: The minimum detection limit is 0.04μM; the fluorescence ratio F 670 / F 440 It has a linear relationship with the concentration of chromium ions in the range of 1.0 to 300 μM, and the linear relationship equation is: The minimum detection limit is 0.34 μM, which has good practical application value.

[0023] (2) The present invention also studied the interference of 16 metal ions and 8 anions that may be present in water. The results showed that the response signals of the interfering substances were comparable to those of the blank group, indicating that these interfering substances that may be present in water did not affect the detection of copper and chromium ions. The constructed ratiometric fluorescence probe detection system has excellent anti-interference ability and can accurately determine the concentrations of copper and chromium ions in water.

[0024] (3) When Cu 2+ and Cr 6+ When they exist at the same time, due to the interaction between AP-CDs probe and Cu 2+ and Cr 6+ The optimal reaction time of the probe is different. 2+ It takes 40 minutes to incubate, so Cu 2+ The presence of Cr 6+ The influence of Cu can be ignored; 2+ When ascorbic acid is added to the system, the effect of Cr 6+ Reduction can eliminate Cr 6+ impact. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the preparation of fluorescent carbon dots AB-CDs, PT-CDs and dual-emission center carbon dots AP-CDs.

[0026] Figure 2 These are high-resolution transmission electron micrographs and particle size distribution diagrams of the fluorescent carbon dots AB-CDs, PT-CDs, and dual-emission center carbon dots AP-CDs in Example 1.

[0027] Figure 3 A is the ultraviolet absorption spectra of the fluorescent carbon dots AB-CDs, PT-CDs and AP-CDs with dual emission centers in Example 1; 3B is the fluorescence spectra of the three carbon dots, in which the inset Figure 3 BA, 3B-B, and 3B-C are photos of the three carbon dots under sunlight (left) and 365nm ultraviolet light (right), respectively; 3C is the Fourier transform infrared spectra of the three carbon dots; 3D is the Zeta potential diagram of the three carbon dots.

[0028] Figure 4 A is the effect of the placement time on the fluorescence intensity ratio of the dual-emission center carbon dots AP-CDs in Example 1; 4B is the effect of the ion intensity on the fluorescence intensity ratio of the dual-emission center carbon dots AP-CDs.

[0029] Figure 5 A is the AP-CDs, AP-CDs+Cu in Example 2 2+ 、AP-CDs+Cr 6+ Fluorescence emission spectra of the three systems, among which Figure 5 AA, 5A-B, and 5A-C are photos of three systems under 365nm UV light; 5B and 5C are AP-CDs+Cu 2+ 、AP-CDs+Cr 6+ High-resolution transmission electron microscopy images of the system; 5D is AP-CDs, AP-CDs+Cu 2+ 、AP-CDs+Cr 6+ 、Cu 2+ and Cr 6+ UV absorption spectra of five systems; 5E is AP-CDs, AP-CDs+Cu 2+ 、AP-CDs+Cr 6+ Fluorescence lifetime diagrams of three systems; 5F is AP-CDs+Cr 6+ Sterne-Volmer curve of the system.

[0030] Figure 6 The AP-CDs prepared by different ratios of AB-CDs and PT-CDs in Example 3 and Cu 2+ Fluorescence spectrum of the reaction system, in which Figure 6 AA, 6B-B, and 6C-C are photos of the reaction system under 365 nm UV light.

[0031] Figure 7 A is the concentration of AP-CDs and Cu at different pH values ​​in Example 3 2+ The fluorescence intensity ratio of the reaction system (F 440 / F 670 ); 7B is the AP-CDs and Cr at different pH values 6+ The fluorescence intensity ratio of the reaction system (F 670 / F 440 ).

[0032] Figure 8 A is the concentration of AP-CDs and Cu at different reaction times in Example 3 2+ The fluorescence intensity ratio of the reaction system (F 440 / F 670 ); 8B is AP-CDs and Cu 2+ The fluorescence intensity of the reaction system at 440nm; 8C is the fluorescence intensity of AP-CDs and Cu 2+ Fluorescence intensity of the reaction system at 670 nm.

[0033] Figure 9 The results of the reaction between AP-CDs and Cr at different reaction times in Example 3 are as follows: 6+ The fluorescence intensity ratio of the reaction system (F 670 / F 440 ).

[0034] Figure 10 A is the concentration of AP-CDs and Cu in Example 4 2+ Fluorescence spectrum of the reaction system, in which Figure 10 AA is a photo of the solution under 365nm UV light; 10B and 10C are Cu 2+ The linear relationship between concentration and fluorescence intensity ratio; 10D is the linear relationship between AP-CDs and different concentrations of Cr 6+ Fluorescence spectrum of the reaction system, in which Figure 10 DD is a photo of the solution under 365nm UV light; (E) is Cr 6+ The linear relationship between the concentration and the fluorescence intensity ratio.

[0035] Figure 11 A is the AP-CDs in Example 5 and Cu 2+ , and other ion reaction systems; 11B is the fluorescence intensity ratio of AP-CDs and Cr 6+ , the fluorescence intensity difference of other ion reaction systems; Figure 11 AA and 11B-B are photos of AP-CDs incubated with different ions under 365 nm UV light. DETAILED DESCRIPTION

[0036] The embodiments of the present invention are further analyzed and described below in conjunction with the accompanying drawings. Reagents and operations not mentioned in the examples are all carried out according to conventional operations in the art.

[0037] Reagents

[0038] Copper nitrate (Cu(NO3)2·3H2O), cobalt nitrate (Co(NO3)2·6H2O), chromium nitrate (Cr(NO3)3·9H2O), cadmium nitrate (Cd(NO3)2·4H2O), manganese nitrate (Mn(NO3)2), iron nitrate (Fe(NO3)3·9H2O), cerium (III) nitrate (Ce(NO3)3·6H2O) and sodium sulfate (Na2SO4) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Sodium nitrate (NaNO3), potassium nitrate (KNO3), silver nitrate (AgNO3), magnesium nitrate (Mg(NO3)2·6H2O), calcium nitrate (Ca(NO3)2·4H2O), zinc nitrate (Zn(NO3)2·6H2O), nickel nitrate (Ni(NO3)2·6H2O), barium nitrate (Ba(NO3)2), lead nitrate (Pb(NO3)2), and glacial acetic acid (HAc) were purchased from Tianjin Damao Chemical Reagent Factory (Tianjin, China). Ferrous sulfate (FeSO4·7H2O), potassium dichromate (K2Cr2O7), sodium fluoride (NaF), sodium chloride (NaCl), and anhydrous ethanol were purchased from Fuchen (Tianjin) Chemical Reagent Co., Ltd. (Tianjin, China). Aluminum nitrate (Al(NO3)3·9H2O) and sodium oxalate (Na2C2O4) were purchased from Tianjin Guangfu Technology Development Co., Ltd. (Tianjin, China). Anhydrous sodium carbonate (Na2CO3) and anhydrous sodium acetate (CH3COONa) were purchased from Tianjin Beichen Fangzheng Reagent Factory (Tianjin, China). Sodium phosphate (Na3PO4·12H2O) was purchased from Tianjin Standard Technology Co., Ltd. (Tianjin, China). Ultrapure water was used in all experiments.

[0039] instrument

[0040] F-7000 fluorescence spectrophotometer (Japan), TU-1901 double-beam UV-visible spectrophotometer (China), Edinburgh fls 1000 fluorescence spectrophotometer (UK), FEI-Themis G2300 high-resolution transmission electron microscope (USA), Nicolet iS10 Fourier transform infrared spectrometer (USA), FE-28 pH meter (Switzerland), 19-00BS electric blast drying oven (China), KQ-800KDB high-power CNC ultrasonic cleaner (China), H1750R high-speed desktop refrigerated centrifuge (China).

[0041] Example 1

[0042] The preparation diagram of blue fluorescent carbon dots AB-CDs, red fluorescent carbon dots PT-CDs, and dual-emission center carbon dots AP-CDs is shown in the figure. Figure 1 shown.

[0043] (1) Synthesis of blue fluorescent carbon dots AB-CDs by hydrothermal method:

[0044] The traditional Chinese medicine Anemarrhena asphodeloides Bunge was cleaned, dried at 60°C to constant weight, and then ground into a fine powder. Accurately weigh 0.5 g of Anemarrhena powder was dispersed in 20 mL of ultrapure water and ultrasonicated in a high-power, digitally controlled ultrasonic cleaner for 30 minutes. After mixing, the suspension was transferred to a polytetrafluoroethylene-lined reactor and heated at 200°C for 8 hours. After cooling to room temperature, the resulting solution was centrifuged at 10,000 rpm for 10 minutes to remove large particles. The solution was then purified by filtration through a 0.22 μm microporous filter membrane to obtain a blue Anemarrhena fluorescent carbon dot (AB-CDs) solution, which was stored at 4°C until ready for use.

[0045] (2) Synthesis of red fluorescent carbon dots PT-CDs by ultrasonic extraction method:

[0046] Pu-erh tea was ground into powder, and 0.8 g of the powder was precisely weighed and placed in a conical flask. 10 mL of anhydrous ethanol was added, and ultrasonic extraction was performed in a high-power, digitally controlled ultrasonic cleaner for 1 hour. Subsequently, the mixture was centrifuged at 10,000 rpm for 10 minutes to remove insoluble residues. The supernatant was further filtered and purified through a 0.22 μm microporous filter membrane to obtain a red Pu-erh tea fluorescent carbon dot (PT-CDs) solution, which was stored at 4°C until further use.

[0047] (3) Preparation of dual-emission center carbon dots AP-CDs:

[0048] Accurately measure 5 μL of AB-CDs solution and 100 μL of PT-CDs solution, add 400 μL of anhydrous ethanol and 495 μL of ultrapure water, mix evenly with a vortex mixer to obtain a dual-emission central carbon dot (AP-CDs) solution, and store at 4°C for future use.

[0049] The morphology, structure, surface properties and fluorescence properties of blue fluorescent carbon dots AB-CDs, red fluorescent carbon dots PT-CDs and dual-emission center carbon dots AP-CDs were characterized by high-resolution transmission electron microscopy (HRTEM), ultraviolet-visible absorption spectroscopy (UV-Vis), fluorescence spectroscopy, Fourier transform infrared spectroscopy (FT-IR) and zeta potential analysis. Figure 2 and Figure 3 .

[0050] Figure 2 A and 2C are high-resolution transmission electron microscopy (HRTEM) images of blue fluorescent carbon dots AB-CDs and red fluorescent carbon dots PT-CDs, respectively. Figure 2 As shown in Figures A and 2C, both the blue fluorescent carbon dots AB-CDs and the red fluorescent carbon dots PT-CDs are approximately spherical, with uniform size distribution and good dispersion. Figure 2 There are no clear lattice fringes in A and 2C, indicating that the crystallinity of AB-CDs and PT-CDs is low, confirming that their main component is amorphous carbon. Figure 2 As shown in B and 2D, the average particle size of PT-CDs (4.40 nm) is larger than that of AB-CDs (2.36 nm). Figure 2 E is the HRTEM image of dual-emission center carbon dots AP-CDs. Figure 2 As shown in E, the dual-emission center carbon dots AP-CDs are spherical, with excellent dispersion and no obvious agglomeration. The average particle size is 4.54 nm (see Figure 2 F), the particle size range is between 1 and 8 nm.

[0051] The UV-visible absorption spectra of blue fluorescent carbon dots AB-CDs, red fluorescent carbon dots PT-CDs and dual-emission center carbon dots AP-CDs are shown in Figure 2. Figure 3 As shown in A, all three fluorescent carbon dots exhibit an absorption peak at 273 nm, which is attributed to the π-π* transition of the C=C / C=N bond. The absorbance of the dual-emission center carbon dot AP-CDs can be considered as the sum of the absorbances of the blue fluorescent carbon dot AB-CDs and the red fluorescent carbon dot PT-CDs. To evaluate the feasibility of dual-emission center carbon dot AP-CDs in ratiometric fluorescence sensing, the fluorescence spectra of blue fluorescent carbon dot AB-CDs, red fluorescent carbon dot PT-CDs, and dual-emission center carbon dot AP-CDs were measured (see Figure 3 B). Figure 3 As shown in Figure B, the fluorescence spectrum of AP-CDs has two separate emission peaks at 440nm and 670nm, corresponding to the emission peaks of carbon dots AB-CDs and PT-CDs, respectively; Figure 3 BA, 3B-B, and 3B-C are photos of carbon dot AB-CDs, PT-CDs, and AP-CDs solutions under sunlight (left) and 365nm UV light (right). Figure 3 As shown in Figure BA, the AB-CDs solution is almost colorless under sunlight and turns blue under 365nm ultraviolet light. Figure 3 As shown in BB, the PT-CDs solution is light yellow in sunlight and red under 365nm ultraviolet light. Figure 3 As shown in BC, the AP-CDs solution appears light yellow under sunlight and light purple under 365 nm UV light.

[0052] In Fourier transform infrared (FTIR) spectroscopy (see Figure 3 In C), the blue fluorescent carbon dots AB-CDs and the red fluorescent carbon dots PT-CDs have a 3370 cm -1The broad absorption peak at 2850-2932 cm-1 confirmed the OH stretching vibration, indicating the presence of -OH groups on the CDs surface; -1 The absorption peaks between 1706 and 1698 cm are attributed to the asymmetric stretching vibration of the C-H bond; -1 The absorption peak at 1518-1633 cm corresponds to C=O stretching vibration; -1 The absorption peaks in the range 1036-1454 cm show the stretching vibrations associated with the C=N bond; -1 The absorption between them is due to the bending vibration of CO / CC / CN bonds. The infrared spectrum of the dual-emission center carbon dots AP-CDs does not show any new characteristic peaks, indicating that there may be no covalent bond interaction between AB-CDs and PT-CDs. Figure 3 As shown in Figure 2, the Zeta potential of the blue fluorescent carbon dots AB-CDs was measured to be 11.42 mV, indicating the presence of positive charge on the surface. In contrast, the Zeta potential of the red fluorescent carbon dots PT-CDs was -14.15 mV, indicating the presence of negative charge on its surface. The Zeta potential of the dual-emission center carbon dots AP-CDs was -14.79 mV. The Zeta potential of the mixture of AB-CDs and PT-CDs changed less than that of PT-CDs, which may be attributed to their relative proportions, and the mixed system remained in a relatively stable state.

[0053] In addition, the present invention also studied the stability of dual emission center carbon dots AP-CDs, and the results are shown in Figure 4 The changes in fluorescence intensity were detected at room temperature within 3 hours. Figure 4 As shown in A, the fluorescence intensity of AP-CDs remained basically unchanged within 3 hours, indicating that it has good stability at room temperature. After the dual-emission center carbon dot AP-CDs were mixed with different concentrations of NaCl, the fluorescence intensity changes of the system were detected. The results are shown in Figure 4 As shown in Figure (B), the change in fluorescence intensity is negligible with the increase of NaCl concentration, indicating that AP-CDs also have good stability in high-concentration salt ion solutions.

[0054] Example 2

[0055] Principle of ratiometric fluorescence probe AP-CDs for detecting copper and chromium ions

[0056] This example explores the principle of ratiometric fluorescent probe AP-CDs in detecting copper and chromium ions. Figure 5 A is dual emission center carbon dots AP-CDs and their respective 2+ Cr 6+ After mixing (respectively recorded as AP-CDs, AP-CDs+Cu 2+ 、AP-CDs+Cr 6+) fluorescence emission spectrum, Figure 5 AA, 5A-B, and 5A-C are the systems AP-CDs, AP-CDs+Cu under UV light, respectively. 2+ and AP-CDs+Cr 6+ Photos. Figure 5 As shown in A, when Cu 2+ When introduced into the AP-CDs probe system, the fluorescence intensity at 440 nm remained almost unchanged, while that at 670 nm decreased significantly. Under ultraviolet light, the color of the solution changed from light purple to blue (see insets, respectively). Figure 5 AA, 5A-B); when adding Cr 6+ When the fluorescence intensity at 440 nm and 670 nm decreased, the solution became obviously darker under UV irradiation (see insets Figure 5 AA, 5A-C). Figure 5 B and 5C are Cu 2+ and Cr 6+ AP-CDs solution (i.e. AP-CDs+Cu 2+ 、AP-CDs+Cr 6+ ) is a high-resolution transmission electron microscopy (HRTEM) image. Cu is added to the AP-CDs solution. 2+ This causes CDs to aggregate, resulting in unclear boundaries (see Figure 5 B), the presence of various active surface groups on AP-CDs, such as hydroxyl and carboxyl groups, may lead to interactions with analytes, which can cause CDs aggregation and thus cause fluorescence quenching; in contrast, the addition of Cr 6+ Afterwards, larger spherical particles were observed with diameters ranging from 20 to 80 nm (see Figure 5 C) Subsequently, AP-CDs+Cu 2+ 、AP-CDs+Cr 6+ The UV-visible spectrum of the system was analyzed and the results were as follows Figure 5 As shown in D, after adding Cu 2+ After quenching, the UV absorption wavelength of AP-CDs did not change, indicating that no new substances were generated. In order to further understand the quenching mechanism, fluorescence lifetime measurements were also performed (see Figure 5 E). Figure 5 As shown in E, the photoluminescence (PL) lifetime of AP-CDs is 6.34 ns, while that of Cu 2+ In the presence of , the PL lifetime is 6.40 ns, which remains almost unchanged, indicating that it is mainly a static fluorescence quenching phenomenon. In order to gain a deeper understanding of the quenching mechanism, the Sterne-Volmer (SV) equation was used for analysis.

[0057] F0 / F=1+K SV[Q]=1+K q τ0[Q]

[0058] Where F0 and F represent the fluorescence intensity of the fluorophore in the presence and absence of the quencher, respectively; K SV represents the SV quenching constant; [Q] represents the concentration of the quencher; and K q and τ0 represent the quenching rate and fluorescence lifetime in the absence of quencher, respectively. K obtained from the SV equation SV The value is 0.3985×10 6 M -1 , correlation coefficient R 2 is 0.9907. Using τ0 and K SV K q The value is 6.2855×10 13 M -1 s -1 Observed K q The value is significantly higher than the maximum collision quenching constant (2.0×10 10 M -1 s -1 ), thus confirming the static quenching mechanism.

[0059] like Figure 5 D shows, Cr 6+ The UV-visible absorption spectrum of the AP-CDs exhibits two different absorption bands at 255 nm and 365 nm, and the excitation peak of the AP-CDs at 365 nm is consistent with the above-mentioned Cr 6+ The absorption spectra of Cr 6+ The presence of induced AP-CDs and Cr 6+ The internal filter effect (IFE) between the two causes the fluorescence emission to switch from the "on" state to the "off" state. Figure 5 As shown in E, Cr 6+ After quenching, the fluorescence lifetime of AP-CDs decreased from 6.34 ns to 4.77 ns, indicating that the observed fluorescence change may be attributed to the dynamic quenching effect. 6+ The quenching constant for AP-CDs is 0.0018×10 6 M -1 , correlation coefficient R 2 is 0.9993, K q The value is 2.8391×10 11 M -1 s -1 For dynamic quenching, as the temperature increases, the quenching constant also increases, that is, the slope of the SV equation increases. Therefore, the relationship between temperature and quenching constant is further studied. Figure 5As shown in F, with the increase of temperature, the slope of the equation gradually increases. The slopes are 0.0013×10 6 , 0.0018×10 6 and 0.0025×10 6 M -1 The results show that with the increase of temperature, K SV It shows an upward trend, which further proves the existence of the dynamic quenching process.

[0060] In summary, Cu 2+ The quenching mechanism of AP-CDs involves static quenching (SQ) and aggregation-induced quenching (ACQ), while Cr 6+ The quenching mechanism of AP-CDs is attributed to dynamic quenching (DQ) and inner filter effect (IFE).

[0061] Example 3

[0062] Optimization of detection conditions

[0063] (1) Effect of the mixing ratio of AB-CDs and PT-CDs on detection sensitivity

[0064] In order to achieve the best sensing performance, the present invention studied the mixing ratio of blue fluorescent carbon dots AB-CDs and red fluorescent carbon dots PT-CDs in the preparation process of dual-emission center carbon dots AP-CDs. The ratio of AB-CDs and PT-CDs in dual-emission center carbon dots AP-CDs directly determines the ratio of the emission peak intensities of the system at 440nm and 670nm. 2+ The sensitivity of the color change during the detection process has a significant impact, so Cu 2+ The ratio of AB-CDs and PT-CDs was investigated for detection. AB-CDs and PT-CDs carbon dot solutions of different ratios (5μL:100μL, 5μL:200μL, 10μL:100μL) were mixed with anhydrous ethanol (400μL, 300μL, 400μL), and then 150μL of buffer solution (BR, 0.04M, pH 4.78) and different concentrations of Cu were added. 2+ Standard solutions (0, 1.0, 1.5, 2.0, 2.5, 3.0, 5.0, 8.0, 10.0, 15.0, 20.0, 25.0, 50.0, 100.0, and 200.0 μM) were prepared and then added with ultrapure water to a volume of 1 mL. After thorough mixing, the solution was incubated at room temperature for 40 min, and the fluorescence spectra were recorded at an excitation wavelength of 365 nm. The results are shown in Table 1. Figure 6 .

[0065] Figure 6The mixing ratios of AB-CDs and PT-CDs carbon dot solutions in A to C are 5 μL: 100 μL, 5 μL: 200 μL, and 10 μL: 100 μL, respectively. Figure 6 Numbers 1 to 15 in AA, 6B-B, and 6C-C correspond to different Cu 2+ Concentration: 0, 1.0, 1.5, 2.0, 2.5, 3.0, 5.0, 8.0, 10.0, 15.0, 20.0, 25.0, 50.0, 100.0, 200.0 μM.

[0066] like Figure 6 As shown in A, when the mixing ratio of AB-CDs and PT-CDs carbon dot solutions is 5 μL:100 μL, 2.5 μM Cu 2+ After the standard solution was added, the solution turned blue under 365nm UV light (see insert Figure 6 Number 5 in AA corresponds to Cu 2+ concentration 2.5 μM). Figure 6 As shown in B and 6C, when the mixing ratio of AB-CDs and PT-CDs carbon dot solutions is 5 μL: 200 μL ( Figure 6 B) and 10 μL:100 μL ( Figure 6 C) When 5 μM Cu was added to the reaction system 2+ After the standard solution was added, the solution turned blue when irradiated under 365nm UV light (see insert). Figure 6 Number 7 in BB and 6C-C corresponds to Cu 2+ The results showed that when the mixing ratio of AB-CDs and PT-CDs carbon dot solutions was 5 μL:100 μL, the obtained dual-emission carbon dot AP-CDs (i.e., AP-CDs ratio fluorescence probe) could be used as Cu 2+ The sensor has higher sensitivity and is the best detection system.

[0067] (2) Effect of pH value of buffer solution on Cu 2+ and Cr 6+ Effect of fluorescence changes on the detection system

[0068] 5 μL of AB-CDs solution, 100 μL of PT-CDs solution and 400 μL of anhydrous ethanol were mixed, and then 150 μL of buffer solution (BR, 0.04 M, pH = 1.81-11.92) and 5 μM of Cu 2+ Standard solution or 50 μM Cr 6+ The standard solution was finally diluted to 1 mL with ultrapure water, mixed well, and incubated at room temperature for 40 min. Under the excitation of 365 nm, the fluorescence intensity at 440 and 670 nm was recorded. 2+Test results are shown in Figure 7 A, Cr 6+ Test results are shown in Figure 7 B. Such as Figure 7 As shown in A, when the pH is 4.78, Cu 2+ The fluorescence intensity ratio of the AP-CDs probe (F 440 / F 670 ) reaches its maximum; Figure 7 As shown in B, the ratiometric fluorescent probe is sensitive to Cr 6+ The response of AP-CDs as a ratiometric fluorescent probe of Cu 2+ and Cr 6+ The optimal pH value for the sensor is 4.78.

[0069] (3) Effect of reaction time on Cu 2+ and Cr 6+ Effect of fluorescence changes on the detection system

[0070] 5 μL of AB-CDs solution, 100 μL of PT-CDs solution and 400 μL of anhydrous ethanol were mixed, and then 150 μL of buffer solution (BR, 0.04 M, pH 4.78), 5 μM of Cu 2+ Standard solution or 50 μM Cr 6+ The standard solution was then diluted to 1 mL with ultrapure water and mixed evenly. The fluorescence intensity at 440 and 670 nm was measured at different incubation times. 2+ Test results are shown in Figure 8 , Cr 6+ Test results are shown in Figure 9 .like Figure 8 As shown, as Cu 2+ The fluorescence intensity ratio of the sensor, AP-CDs ratio fluorescence probe (F 440 / F 670 ) gradually increases with reaction time (see Figure 8 A), the fluorescence intensity at 440 nm remained relatively stable (see Figure 8 B), the fluorescence intensity at 670 nm gradually decreased and the intensity decrease rate slowed down after 40 min (see Figure 8 C), so 40 min is the optimal reaction time. Figure 9 As shown, as Cr 6+ As the reaction time increases, the fluorescence intensity ratio of the ratiometric fluorescence probe AP-CDs (F 670 / F 440 ) has almost no change and the measurement can be carried out after the solution is fully mixed.

[0071] Example 4

[0072] (1) Detection of copper ions using ratiometric fluorescent probe AP-CDs

[0073] 5 μL of AB-CDs solution, 100 μL of PT-CDs solution and 400 μL of anhydrous ethanol were mixed, and then 150 μL of buffer solution (BR, 0.04 M, pH 4.78) and different concentrations (0.1 to 250 μM) of Cu were added. 2+ The standard solution was finally diluted to 1 mL with ultrapure water, mixed thoroughly, and incubated at room temperature for 40 min. The fluorescence spectrum was recorded at an excitation wavelength of 365 nm, and the fluorescence intensity at 440 and 670 nm was measured.

[0074] like Figure 10 As shown in A, with Cu 2+ With the increase of concentration, the fluorescence intensity of AP-CDs ratio fluorescence probe at 440nm remained relatively stable, while the fluorescence intensity at 670nm gradually decreased; Figure 10 As shown in AA, under 365nm ultraviolet light, the color of the solution changes significantly, gradually quenching from light purple to blue. Figure 10 B, 10C, Cu 2+ The fluorescence intensity ratio F of the standard solution in the range of 0.1~25.0μM and 50.0~250.0μM 440 / F 670 With Cu 2+ The concentration linear relationship is good, and the linear relationship equations are and The minimum detection limit was 0.04 μM.

[0075] (2) Detection of chromium ions using ratiometric fluorescent probe AP-CDs

[0076] 5 μL of AB-CDs solution, 100 μL of PT-CDs solution and 400 μL of anhydrous ethanol were mixed, and then 150 μL of buffer solution (BR, 0.04 M, pH = 4.78) and different concentrations (1.0 to 300 μM) of Cr were added. 6+ The standard solution was finally diluted to 1 mL with ultrapure water and mixed evenly. The fluorescence spectrum was recorded at an excitation wavelength of 365 nm, and the fluorescence intensity at 440 and 670 nm was measured.

[0077] like Figure 10 As shown in D, when Cr 6+ When the concentration increased from 1.0 μM to 300.0 μM, the fluorescence intensity of the ratiometric fluorescent probe AP-CDs at 440 nm and 670 nm decreased to varying degrees; Figure 10 As shown in DD, under 365nm ultraviolet light, the color of the solution gradually changes from light purple to dark purple. Figure 10E shows, Cr 6+ The standard solution is in the range of 1.0~300μM, and the fluorescence intensity ratio F 670 / F 440 With Cr 6+ The concentration linear relationship is good, and the linear relationship equation is The minimum detection limit was 0.34 μM.

[0078] Example 5

[0079] Selectivity of ratiometric fluorescent probe AP-CDs

[0080] This example studies the selectivity of the ratiometric fluorescent probe AP-CDs. Under optimal conditions, the AP-CDs probe was studied with 16 metal ions (Na + , K + 、Ag + , Ca 2+ Mg 2+ 、Ba 2+ , Pb 2+ 、Zn 2+ 、Ni 2+ 、Co 2+ 、Cd 2+ 、Al 3+ 、Fe 2+ 、Fe 3+ Cr 3 + 、Ce 3+ ) and 8 anions (CO3 2- 、C2O4 2- PO4 3- 、F - 、Ac - 、SO4 2- 、Cl - 、NO3 - )The fluorescence intensity of the system changes after the reaction, and the Cu 2+ and Cr 6+ The fluorescence intensity changes of AP-CDs probes were investigated when they were present at the same time. Figure 11 .like Figure 11 As shown in A, Cu 2+ The fluorescence intensity ratio of the system after incubation with AP-CDs probe alone (F 670 / F 440 ) was significantly reduced, and the fluorescence intensity ratios of the other 24 ions after incubation with the AP-CDs probe (F 670 / F 440 ) were comparable to those in the control group. Figure 11 As shown in B, Cr 6+The decrease in fluorescence intensity (ΔF) at 440 nm and 670 nm of the system after incubation with AP-CDs probe alone 440 , ΔF 670 )maximum.

[0081] When Cu 2+ and Cr 6+ When both exist, detect Cu 2+ When Cr 6+ Reduction to eliminate Cr 6+ The impact of Figure 11 As shown in A, there is Cr in the system. 6+ 、Cu 2+ 、AA (ie system Cr 6 + +AA+Cu 2+ ), fluorescence intensity ratio (F 670 / F 440 ) and only Cu 2+ Existing equivalent.

[0082] Since the AP-CDs probe is 2+ and Cr 6+ The optimal reaction time of Cu is different, so 2+ The presence of Cr 6+ The influence of can be ignored. Figure 11 As shown in B, the ratiometric fluorescent probe is combined with Cu 2+ Cr 6+ Fluorescence detection was performed immediately after the solutions were mixed, and the decrease in fluorescence intensity at 440nm and 670nm was ΔF. 440 , ΔF 670 With only Cr 6+ There is no significant difference when it exists. Figure 11 AA and 11B-B are photos of the solution after incubation of AP-CDs probe with different ions (the above 16 metal ions and 8 anions in turn) under 365nm UV light. The above results show that the AP-CDs probe has a strong affinity for Cu 2+ and Cr 6+ The selectivity of the detector is higher than that of other ions, and copper ions and chromium ions can be detected simultaneously.

[0083] Example 6

[0084] Detection of copper and chromium ions in real water samples using ratiometric fluorescence probe AP-CDs

[0085] Based on fluorescence spectroscopy and a spiked method, the ratiometric fluorescence probe AP-CDs was used to detect copper and chromium ions in real water samples. The samples were tap water obtained from the laboratory and spring water from Laiyuan County, Baoding City, Hebei Province. The samples were centrifuged at 10,000 rpm for 10 minutes and then filtered twice through a 0.22 μm microporous membrane to remove insoluble impurities.

[0086] Copper ion detection: Mix 5 μL of AB-CDs solution, 100 μL of PT-CDs solution, and 400 μL of anhydrous ethanol, add 100 μL of sample, then add 150 μL of buffer solution (BR, 0.04 M, pH 4.78) and 175 μM ascorbic acid (AA) solution, and finally add ultrapure water to 1 mL. After thorough mixing, incubate at room temperature for 40 min, record the fluorescence intensity at 365 nm excitation wavelengths of 440 and 670 nm, and calculate the copper ion content in the actual sample according to the linear relationship equation for copper ions in Example 4.

[0087] Chromium ion detection: Mix 5 μL of AB-CDs solution, 100 μL of PT-CDs solution, and 400 μL of anhydrous ethanol, add 100 μL of sample, then add 150 μL of buffer solution (BR, 0.04 M, pH 4.78), and finally add ultrapure water to 1 mL. After mixing, measure the fluorescence intensity at 440 and 670 nm under an excitation wavelength of 365 nm. Calculate the chromium ion content in the actual sample according to the linear relationship equation for chromium ions in Example 4.

[0088] For spike recovery experiments, standard solutions of copper or chromium ions at varying concentrations were added to tap water and spring water samples for analysis and detection. The results, shown in Table 1, showed copper ion recoveries ranging from 95.24% to 102.58%, with relative standard deviations (RSDs) of 1.00% to 3.53%. Chromium ion recoveries ranged from 97.49% to 105.68%, with RSDs of 0.46% to 3.95%. These results demonstrate that the ratiometric fluorescence probe AP-CDs can effectively detect copper and chromium ions in real samples.

[0089] Table 1 Detection of copper and chromium ion contents in water samples using ratiometric fluorescent probe AP-CDs

[0090]

[0091] This study fabricated dual-emission carbon dots (AP-CDs) using blue-fluorescent carbon dots (AB-CDs) and red-fluorescent carbon dots (PT-CDs). These carbon dots serve as ratiometric fluorescent probes for the simultaneous detection of copper and chromium ions in water and have been successfully applied to real-world water samples. The method demonstrates high selectivity, sensitivity, and repeatability for simultaneous copper and chromium ion detection, while also being simple to operate and cost-effective.

Claims

1. A method for simultaneous detection of copper ions and chromium ions based on dual-emission center carbon dots, characterized in that: The following steps are involved: (a) Pre-treating the sample to be tested to obtain a copper ion test solution and a chromium ion test solution; (b) Synthesis of fluorescent carbon dots from Anemarrhena asphodeloides; (c) Synthesis of Pu'er tea fluorescent carbon dots; (d) The above-mentioned Anemarrhena asphodeloides fluorescent carbon dot solution and Pu'er tea fluorescent carbon dot solution were mixed evenly to obtain dual-emission center carbon dots as a ratiometric fluorescent probe; (e) detecting the copper ion test solution and the chromium ion test solution; wherein the copper ion detection method comprises: mixing the copper ion test solution with the dual-emission center carbon dot solution in step (d) and a reducing agent, adjusting the pH with a buffer, incubating, detecting at an excitation wavelength of 365 nm, recording the fluorescence emission spectrum in the range of 385-710 nm, obtaining fluorescence intensity values ​​at 440 nm and 670 nm, and calculating the copper ion content in combination with a copper ion standard curve; the chromium ion detection method comprises: mixing the chromium ion test solution with the dual-emission center carbon dot solution in step (d), adjusting the pH with a buffer, detecting at an excitation wavelength of 365 nm, recording the fluorescence emission spectrum in the range of 385-710 nm, obtaining fluorescence intensity values ​​at 440 nm and 670 nm, and calculating the chromium ion content in combination with a chromium ion standard curve; The Anemarrhena fluorescent carbon dots in step (b) are prepared by a hydrothermal method using the traditional Chinese medicine Anemarrhena and water; The Pu'er tea fluorescent carbon dots in step (c) are prepared by using Pu'er tea and anhydrous ethanol through ultrasonic extraction; When the copper ion test solution is detected in step (e), the reducing agent is ascorbic acid, and the incubation time is 40 minutes.

2. The method for simultaneous detection of copper ions and chromium ions based on dual-emission center carbon dots according to claim 1, characterized in that: The mass-volume ratio of Anemarrhena asphodeloides and water is 0.5 g:20 mL.

3. The method for simultaneous detection of copper ions and chromium ions based on dual-emission center carbon dots according to claim 1, characterized in that: The mass-volume ratio of Pu'er tea to anhydrous ethanol is 0.8 g:10 mL.

4. The method for simultaneous detection of copper ions and chromium ions based on dual-emission center carbon dots according to claim 1, characterized in that: In step (d), the volume ratio of the Anemarrhena asphodeloides fluorescent carbon dot solution to the Pu'er tea fluorescent carbon dot solution is 5 mL:100 mL.

5. The method for simultaneous detection of copper ions and chromium ions in water based on dual-emission center carbon dots according to claim 1, characterized in that: The buffer solution in step (e) is a BR buffer solution with a pH of 4.

78.

6. The method for simultaneous detection of copper ions and chromium ions based on dual-emission center carbon dots according to claim 1, characterized in that: The copper ion standard curve in step (e) is F 440 / F 670 = 1.4994 + 3.3484, : 0.1~25mM; F 440 / F 670 = 0.0654 + 34.6176, :50~250 mM.

7. The method for simultaneous detection of copper ions and chromium ions based on dual-emission center carbon dots according to claim 1, characterized in that: The chromium ion standard curve in step (e) is F 670 / F 440 = 0.0006 + 0.3083, :1.0~300mM.

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