An up-conversion dual-emission fluorescent carbon dot, a preparation method thereof, and uses thereof

Abstract

The present invention relates to the technical field of nano-luminescent materials, and particularly to an up / down-conversion dual-emission fluorescent carbon dot, a preparation method thereof, and uses thereof. An up / down-conversion dual-emission fluorescent carbon dot, wherein the carbon dot comprises acridine yellow and 2-aminoterephthalic acid. An application of the up / down-conversion dual-emission fluorescent carbon dot, which is to apply the carbon dot in the preparation of a probe for detecting CA. The up / down-conversion dual-emission fluorescent carbon dot, a preparation method thereof, and uses thereof provided by the present invention synthesize fluorescent carbon dots (U / D-CDs) with up / down-conversion dual emission by using acridine yellow and 2-aminoterephthalic acid as raw materials and adopting a one-step mixed solvent thermal method; the U / D-CDs have blue and green fluorescence emission peaks in both up / down-conversion dual excitation channels; and the U / D-CDs also have good photostability, photobleaching resistance, and salt tolerance.

Description

Technical Field

[0001] The present invention relates to the technical field of nano-luminescent materials, and particularly to an up-conversion and down-conversion dual-emission fluorescent carbon dot and its preparation method and use. Background Art

[0002] Caffeic acid (3,4-dihydroxycinnamic acid, CA) is one of the cinnamates and is widely distributed in vegetables, fruits, coffee, and red wine, etc. [1,2] . All along, caffeic acid has excellent pharmacological properties in clinical treatment and healthcare, and thus has been widely used. [3,4] . In modern medicine, caffeic acid is used to treat leukopenia and thrombocytopenia, and it also has multiple functions such as hemostasis, antioxidant property, and anti-inflammatory property. [5,6] . However, reports show that even at low dose levels, caffeic acid may have carcinogenic effects. [7] . Therefore, the quantitative detection of caffeic acid is of great significance to our daily life. So far, the detection of caffeic acid has attracted extensive attention, and many methods have been developed, mainly including gas chromatography, high performance liquid chromatography, electrochemical method, and ultraviolet spectroscopy analysis method. [8-11] . Although the detection limits are low, these methods are costly, complex to process, and time-consuming. Therefore, it is very important to establish and develop a simple and efficient platform.

[0003] Carbon dots (CDs), as a class of novel zero-dimensional nanomaterials with a size less than 10 nm, have been widely applied in various fields such as fluorescence chemical sensing, bioimaging, catalysis, and optoelectronic devices due to their excellent optical properties, stability, good biocompatibility, low toxicity, and easy synthesis, etc. [12-15] . So far, many CDs-based fluorescent probes have been developed, demonstrating the characteristics of rapid, sensitive, and simple fluorescence detection. [16,17] . Currently, most reported CDs-based fluorescent probes are single-emission fluorescence, and the detection system is easily affected by factors such as background noise and probe concentration, thus limiting their application in the field of analytical probes. [18,19] . Therefore, CDs-based ratio fluorescent probes have attracted people's attention due to their self-calibration ability and visualization. [20-22] .

[0004] However, currently most CDs-based ratio fluorescent probes are based on down-conversion fluorescence, which is easily affected by high background signals, and continuous exposure to ultraviolet light may cause optical damage and mutations, which greatly hinders their application in organisms. [23,24] .

[0005] Therefore, upconversion photoluminescence (UCPL) has attracted extensive attention. Upconversion nanoparticles (UCNPs) have anti-Stokes luminescence characteristics with emission wavelengths shorter than the excitation wavelength.

[25] Compared with downconversion fluorescent materials, UCNPs have the advantages of non-invasiveness, large penetration depth, and low background, making them very suitable for sensing and imaging applications in biomedicine. [26,27] .

[0006] Currently, most reported upconversion fluorescent nanoparticles are based on lanthanide-doped upconversion nanoparticles, and the research has tended to be mature. [28-30] However, the raw materials of lanthanide-doped upconversion nanoparticles are expensive, the synthesis process is cumbersome and complex, and it will bring many environmental problems. [31,32] Therefore, up / downconversion dual-emission fluorescent carbon dots (CDs) exhibit unique advantages, such as excellent multiphoton excitation properties, simple synthesis methods, good biocompatibility, and easy large-scale preparation.

[33] There are few research reports on the use of up / downconversion dual-emission fluorescent CDs for detection and analysis. Therefore, it is very necessary to develop a ratiometric fluorescence probe based on up / downconversion dual-emission fluorescent carbon dots;

[0007] For this reason, we designed an up / downconversion dual-emission fluorescent carbon dot and its preparation method and use to provide another technical solution to the above technical problems. Summary of the Invention

[0008] Based on this, it is necessary to provide an up / downconversion dual-emission fluorescent carbon dot and its preparation method and use to solve the technical problems mentioned in the above background technology.

[0009] To solve the above technical problems, the present invention adopts the following technical solutions:

[0010] An up / downconversion dual-emission fluorescent carbon dot, wherein the carbon dot comprises acridine yellow and 2-aminoterephthalic acid.

[0011] A preparation method of an up / downconversion dual-emission fluorescent carbon dot for an up / downconversion dual-emission fluorescent carbon dot, the steps are as follows:

[0012] S1: Dissolve the raw materials in a solvent and react at 120-220 °C for 1-10 h;

[0013] S2: Centrifuge the liquid obtained in step S1 for 10 minutes to remove insoluble large particles;

[0014] S3: Dialyze the supernatant of the material obtained in step S2 through a cellulose ester dialysis membrane;

[0015] S4: Freeze-dry for 48 h to obtain a solid powder, which is the target carbon dot.

[0016] As a preferred embodiment of the preparation method of the up-conversion dual-emission fluorescent carbon dots provided by the present invention, the raw materials are acridine yellow and 2-aminoterephthalic acid. For every 5 mL of solvent, 40-140 mg of raw materials are used, and the mass ratio of acridine yellow to 2-aminoterephthalic acid is 1:1-6.

[0017] As a preferred embodiment of the preparation method of the up-conversion dual-emission fluorescent carbon dots provided by the present invention, the solvent is a mixed solution of pure water and ethanol, and the volume ratio of pure water to ethanol in the solvent is 5-0:0-5.

[0018] As a preferred embodiment of the preparation method of the up-conversion dual-emission fluorescent carbon dots provided by the present invention, in the S1 step, the reaction temperature is 180 °C, the reaction time is 2 h, the volume ratio of pure water to ethanol in the solvent is 1:1, and the mass ratio of acridine yellow to 2-aminoterephthalic acid is 1:4.

[0019] As a preferred embodiment of the preparation method of the up-conversion dual-emission fluorescent carbon dots provided by the present invention, in the S2 step, centrifugation is performed at 10000 rpm for 10 minutes to remove insoluble large particles;

[0020] In the S3 step, the supernatant is dialyzed through a cellulose ester dialysis membrane with 300 MWCO.

[0021] An application of up-conversion dual-emission fluorescent carbon dots, for an up-conversion dual-emission fluorescent carbon dot, applying the carbon dots in the preparation of a probe for detecting CA.

[0022] An application of up-conversion dual-emission fluorescent carbon dots, for an up-conversion dual-emission fluorescent carbon dot, the steps are as follows:

[0023] Use the prepared carbon dots to detect CA;

[0024] Mix the sample to be tested with the prepared carbon dots and incubate, and measure the fluorescence emission spectra under excitation lights of 330 nm and 670 nm;

[0025] Before mixing, add HBO3-Na2B4O7·10H2O buffer solution to the carbon dots.

[0026] As a preferred embodiment of the application of the up-conversion dual-emission fluorescent carbon dots provided by the present invention, the pH of the buffer solution is 7.4, the incubation time is 0-80 min, and the incubation temperature is 5-55 °C.

[0027] It can be seen without doubt that through the above technical solutions of the present application, the technical problems to be solved by the present application can surely be solved.

[0028] Meanwhile, through the above technical solutions, the present invention has at least the following beneficial effects:

[0029] A kind of up / down-conversion dual-emission fluorescent carbon dots provided by the present invention, its preparation method and uses, uses acridine yellow and 2-aminoterephthalic acid as raw materials, and synthesizes fluorescent carbon dots (U / D-CDs) with up / down-conversion dual-emission by a one-step mixed solvent thermal method; U / D-CDs have blue and green fluorescence emission peaks in both up / down-conversion dual-excitation channels; and U / D-CDs also have good photostability, photobleaching resistance and salt tolerance;

[0030] Based on U / D-CDs, an up / down-conversion dual-channel ratio fluorescence probe is established for the quantitative detection of caffeic acid (CA). The linear ranges of up / down-conversion are both 0.5 - 150 μM, and the two detection limits are as low as 0.27 μM and 0.29 μM respectively;

[0031] Based on this carbon dot, an up / down-conversion dual-channel ratio fluorescence probe is established, making the probe have high sensitivity, good selectivity and anti-interference ability; compared with other down-conversion fluorescence sensors for detecting CA, the up-conversion detection channel of the present invention has advantages such as large penetration depth, low background and little harm to organisms;

[0032] The ratio fluorescence probe of the present invention also shows satisfactory performance in the quantitative detection of caffeic acid (CA) in human serum, avoiding potential background interference in actual samples and improving the accuracy and selectivity of CA detection in complex samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0034] Figure 1 It is a schematic diagram of the synthesis of U / D-CDs of the present invention and its application in the ratio fluorescence detection of caffeic acid;

[0035] Figure 2 It is a schematic diagram of the fluorescence emission spectrum of U / D-CDs of the present invention at different reaction temperatures under different synthesis conditions during synthesis;

[0036] Figure 3 It is a schematic diagram of the fluorescence emission spectrum of U / D-CDs of the present invention under different synthesis conditions;

[0037] Figure 4Schematic diagram of the surface morphology and size distribution of U / D-CDs by the transmission electron microscope (TEM) of the present invention;

[0038] Figure 5 Schematic diagram of the high-resolution XPS spectra of C1s (a), N1s (b) and O1s (c) of U / D-CDs of the present invention;

[0039] Figure 6 Schematic diagram of studying the optical properties of U / D-CDs by ultraviolet-visible absorption spectroscopy and fluorescence emission spectroscopy of the present invention;

[0040] Figure 7 Schematic diagram of the fluorescence emission spectra of U / D-CDs of the present invention in different solvents and different pH environments;

[0041] Figure 8 Schematic diagram of continuous irradiation of U / D-CDs of the present invention under ultraviolet lamps at 330 nm and 670 nm respectively;

[0042] Figure 9 Schematic diagram of the influence of U / D-CDs on the response to CA under different detection conditions of the present invention;

[0043] Figure 10 Schematic diagram of different excitation wavelengths of the present invention;

[0044] Figure 11 Schematic diagram of the difference in adding different substances after excitation of the present invention;

[0045] Figure 12 Schematic diagram of fluorescence quenching of the present invention. Detailed implementation manners

[0046] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0047] It should be noted that similar reference numerals and letters denote similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0048] Refer to Figures 1 - 12 , an up / down-conversion dual-emission fluorescent carbon dot and its preparation method and uses.

[0049] I. The dual-emission fluorescent carbon dots are obtained from acridine yellow and 2-aminoterephthalic acid as raw materials;

[0050] Preferably, under excitation light with wavelengths of 330 nm and 670 nm, the carbon dots exhibit two fluorescence emissions located near 420 nm and 500 nm.

[0051] II. Preparation method of dual-emission fluorescent carbon dots, the steps are as follows:

[0052] S1: Dissolve the raw materials in a solvent and react at 120 - 220 °C for 1 - 10 h;

[0053] S2: Centrifuge the liquid obtained in step S1 at 10000 rpm for 10 minutes to remove insoluble large particles;

[0054] S3: Dialyze the supernatant of the material obtained in step S2 through a cellulose ester dialysis membrane with a molecular weight cut-off of 300 MWCO;

[0055] S4: Freeze-dry for 48 h to obtain a solid powder, which is the target carbon dots.

[0056] Preferably, the raw materials are acridine yellow and 2-aminoterephthalic acid. For every 5 mL of solvent, 40 - 140 mg of raw materials are used, and the mass ratio of acridine yellow to 2-aminoterephthalic acid is 1:1 - 6.

[0057] Preferably, the solvent is a mixed solution of pure water and ethanol, and the volume ratio of pure water to ethanol in the solvent is 5 - 0:0 - 5.

[0058] Preferably, in step S1, the reaction temperature is 180 °C, the reaction time is 2 h, the volume ratio of pure water to ethanol in the solvent is 1:1, and the mass ratio of acridine yellow to 2-aminoterephthalic acid is 1:4.

[0059] III. Apply the carbon dots in the preparation of a probe for detecting CA.

[0060] IV. Use the prepared carbon dots to detect CA;

[0061] Mix the sample to be tested with the prepared carbon dots and incubate, then measure the fluorescence emission spectra under excitation lights of 330 nm and 670 nm;

[0062] Before mixing, add HBO3-Na2B4O7·10H2O buffer solution to the carbon dots.

[0063] Preferably, the pH of the buffer solution is 7.4, the incubation time is 0 - 80 min, and the incubation temperature is 5 - 55 °C.

[0064] V. Method for detecting CA in human serum using carbon dots, the steps are as follows:

[0065] Pre-treatment was carried out to collect serum samples. The supernatant of the plasma samples refrigerated overnight was centrifuged at 10000 rpm for 10 minutes. Then, the obtained supernatant was filtered through a microporous membrane (0.22 μm), and further diluted 100-fold with deionized water for further experiments. Finally, 100 μL of U / D-CDs and different volumes of CA standard solution were added to the diluted serum samples to prepare a series of spiked samples, and fluorescence measurements were carried out after mixing.

[0066] VI. Main reagents used: acridine yellow, 2-aminoterephthalic acid, caffeic acid (CA, 99%), CaCO3, MnCl2, MgSO4, ZnSO4, L-cysteine (Cys), glycine (Gly), arginine (Arg), tryptophan (Trp), leucine (Leu), alanine (Ala), bovine serum albumin (BSA) and ethanol. All chemical reagents were of analytical grade and used directly without further purification. Ultra-pure water (18.2 Ω·cm at 25 °C) was used throughout the manufacturing and detection process.

[0067] 1. Screening of synthesis methods

[0068] U / D-CD was prepared using a simple mixed solvent thermal method. The specific steps are as follows: 20 mg of acridine yellow and 80 mg of 2-aminoterephthalic acid were mixed and dispersed in 2.5 mL of deionized water and 2.5 mL of ethanol, transferred to a 25 mL Teflon liner, and the Teflon liner was placed in a Teflon autoclave and heated at 180 °C for 2 h. When the autoclave cooled to room temperature, a brown solution containing U / D-CDs was obtained. Then the product was dissolved in ethanol and centrifuged at 10000 rpm for 10 minutes to remove insoluble large particles. Finally, the supernatant was dialyzed using a cellulose ester dialysis membrane (300 MWCO), and solid U / D-CD powder was obtained by freeze-drying for 48 h for subsequent experiments.

[0069] In order to obtain up / down-conversion dual-emission fluorescent CDs with the maximum fluorescence intensity, the widest double-peak spacing and the best peak shape, the synthesis conditions of U / D-CDs (raw material mass ratio, solvent volume ratio, synthesis time and synthesis temperature) were optimized. The results are as Figure 2 shown in the fluorescence emission spectra of U / D-CDs synthesized at different (a) precursor mass ratios; (b) solvent volume ratios; (c) reaction times and (d) reaction temperatures. To obtain the optimal U / D-CDs, the best raw material ratio (m acridine yellow: m 2-aminoterephthalic acid) was selected as 1:4, the best solvent volume ratio (V water: V ethanol) was 1:1, the best synthesis time was 2 h, and the best synthesis temperature was 180 °C.

[0070] To verify the accuracy of the optimized experimental results, a four-factor and three-level orthogonal experiment (Table 1) was designed with the raw material ratio, solvent ratio, time, and temperature as four different factors. The results are as Figure 3 shown. Under the fifth synthesis conditions (i.e., the conditions selected in the above optimization experiment), the U / D-CDs synthesized have the highest fluorescence intensity, the widest bimodal peak spacing, and the optimal bimodal peak shape. Therefore, the U / D-CDs synthesized under these optimal synthesis conditions will be further used in subsequent experiments.

[0071] Table 1 Four-factor and three-level orthogonal table

[0072]

[0073]

[0074] 2. Characterization of U / D-CDs

[0075] The surface morphology and size distribution of U / D-CDs were characterized using transmission electron microscopy (TEM), as Figure 4 shown. (a) TEM image of U / D-CDs. (b) Particle size distribution diagram of U / D-CDs (inset: HRTEM image of U / D-CDs). (c) Raman spectrum of U / D-CDs. (d) XRD pattern of U / D-CDs. (e) FT-IR spectrum of U / D-CDs. (f) XPS spectrum of U / D-CDs, as Figure 4 shown in a and b. It can be seen that U / D-CDs are well-dispersed, with the particle size distributed between 3.5 - 6 nm, and the average particle size is 4.72 nm. Moreover, obvious and distinguishable lattice fringes are shown in the high-resolution TEM image (HRTEM) ( Figure 4 inset of b) of U / D-CDs, and the lattice spacing is 0.22 nm, which is attributed to the (100) plane of graphene

[34] . The Raman spectrum of U / D-CDs ( Figure 4 c) indicates the presence of graphite properties in U / D-CDs. Two broad peaks appear at 1400 cm -1 and 1580 cm -1 , corresponding to the D band (sp 3 hybridization) and G band (sp 2 hybridization) [35,36] , and the ID / IG value is 0.57, indicating a high degree of graphitization in the prepared U / D-CDs

[37] . Figure 4 d is the X-ray diffraction pattern of U / D-CDs. An obvious peak (2θ = 26.7°) is observed, corresponding to the (002) crystal plane of graphene

[34] and the peak at 15.0° corresponds to the (100) crystal plane of graphene, which is attributed to highly disordered carbon atoms

[38] .

[0076] The chemical composition and functional group distribution of U / D-CDs were investigated by FTIR spectroscopy and XPS spectroscopy. The FTIR spectrum ( Figure 4 e) shows that the broad absorption peak at 3400 - 3150 cm -1 is mainly due to the stretching vibrations of O-H and N-H

[39] . The absorption peaks at 3050 cm -1 and 810 cm -1 are mainly attributed to the stretching and bending vibrations of Ar-H, the absorption peak at 1700 cm -1 can be attributed to the stretching vibration of C=O, and the absorption peaks at 1630 cm -1 and 1590 cm -1 are attributed to the stretching vibrations of C=N and C=C respectively

[40] . The absorption peaks at 1370 cm -1 and 1150 cm -1 are the result of the stretching vibrations of C-N and C-O [41,42] .

[0077] The full-scan XPS spectrum of U / D-CDs ( Figure 4 f) shows three characteristic peaks at 284.77 eV, 399.399 eV, and 532.04 eV, which confirm the presence of C1s, N1s, and O1s respectively, and the contents are 73.23%, 8.51%, and 18.26% respectively. The high-resolution XPS spectrum of C1s ( Figure 5 a) is divided into three peaks at 287.85 eV, 285.43 eV, and 283.95 eV, proving the presence of C=O / C=N, C-N / C-O, and C-C / C=C [43-45] . The high-resolution XPS spectrum of N1s ( Figure 5 b) can be decomposed into two peaks, which are attributed to C-N (399.42 eV) and pyridine nitrogen (398.56 eV) [46,47] . In the high-resolution XPS spectrum of O1s ( Figure 5 c), the two peaks at 532.45 eV and 530.91 eV are attributed to C-OH / C-O-C and C=O respectively [48,49] . This is basically consistent with the results of the above FTIR spectrum. These results prove that U / D-CDs have been successfully prepared, and there are abundant hydrophilic groups (-NH2, -COOH, -OH, etc.) on the surface, with excellent water solubility and biocompatibility.

[0078] 3. Optical Properties of U / D-CDs

[0079] As shown Figure 6 in the figure, (a) UV-Vis absorption spectrum and fluorescence emission spectrum of U / D-CDs (inset: photographs of U / D-CDs solution under daylight and 310 nm ultraviolet light); (b) fluorescence emission spectrum of U / D-CDs at excitation wavelengths from 320 nm to 380 nm; (c) fluorescence emission spectrum of U / D-CDs at excitation wavelengths from 660 nm to 730 nm. The optical properties of U / D-CDs were studied by UV-Vis absorption spectrum and fluorescence emission spectrum. The optical properties of U / D-CDs were studied by UV-Vis absorption spectrum and fluorescence emission spectrum. As can be seen Figure 6 from Fig. a, U / D-CDs have an obvious absorption peak at 250 nm, which can be attributed to the π→π* transition of the C═C bond. At the same time, there is a relatively weak absorption peak at 320 nm, which is attributed to the n→π* transition of the C═O bond [50,51] . In addition, there is a broad absorption peak at 450 nm, which can be attributed to the coupling of π states, indicating that U / D-CDs can absorb light with longer wavelengths, avoiding the damage of ultraviolet light to excitation

[52] . The fluorescence emission spectrum shows that U / D-CDs have unique dual-emission behavior, and the maximum excitation wavelength is 330 nm. And the inset shows that under the 310 nm ultraviolet light, U / D-CDs emit bright blue-green light. As shown Figure 6 in Fig. b, under the excitation of 320 - 380 nm ultraviolet light, U / D-CDs show fluorescence emission with excitation-dependent characteristics. At the same time, as shown Figure 6 in Fig. c, when the excitation wavelength range is 660 - 730 nm, U / D-CDs also show unique upconversion dual-emission characteristics, which are consistent with the downconversion dual-emission behavior

[0080] As shown Figure 7 in the figure, (a) fluorescence emission spectra of U / D-CDs in different solvents; (b) fluorescence emission spectra of U / D-CDs at different pH values; (c) fluorescence decay curves of blue and green emissions in U / D-CDs. Since U / D-CDs show excitation-dependent PL emission behavior, we assume that both of these emission centers are caused by the diversity of surface emission sites

[53] . The fluorescence emission spectra of U / D-CDs in different solvents and different pH environments were measured. The results are as shown Figure 7 in Figs. a and b. In different solvents, both emissions have a certain blue shift or red shift. And with the increase of pH, the blue emission shows a certain blue shift and the fluorescence intensity continuously increases, while the peak position of the green emission almost remains unchanged and the fluorescence intensity slightly increases. To further study the origin of these two emissions, time-resolved fluorescence measurements were carried out. As shown Figure 7As shown in c, the FL lifetime decay curves of the blue and green emission bands of U / D-CDs both conform to the single-exponential function, with fluorescence lifetimes of 13.98 ns and 14.40 ns respectively, indicating that there is only one luminescence center in both emission bands.

[54] This further indicates that the blue and green emission centers of U / D-CDs are both attributed to surface state emission.

[0081] 4. Stability of U / D-CDs

[0082] The stability of U / D-CDs was studied under different conditions (ultraviolet light, ionic strength, and time). Specifically as follows:

[0083] Photobleaching: U / D-CDs were exposed to ultraviolet light with wavelengths of 330 nm and 670 nm for half an hour, and a fluorescence spectrophotometer was used to measure the change in emission intensity during the exposure process.

[0084] Salt tolerance: 100 μL of U / D-CDs solution was transferred to an EP tube, and then 0 - 0.1 M sodium chloride solution was added and diluted to 1 mL with ultrapure water. Then, its fluorescence emission spectrum was measured to observe the change in fluorescence intensity of the emission peak.

[0085] Photostability: After storing U / D-CDs at room temperature for 120 days, its fluorescence emission spectrum was measured to observe the change in fluorescence intensity of the emission peak.

[0086] As Figure 8 shown, (a) Photobleaching of U / D-CDs at λex = 330 nm; (b) Photobleaching of U / D-CDs at λex = 670 nm; Effects of different concentrations of NaCl on the dual-emission fluorescence intensity of U / D-CDs at excitation wavelengths of 330 nm (c) and 670 nm (d); Fluorescence emission spectra of U / D-CDs before and after 120 days at λex = 330 nm (e) and λex = 670 nm (f). The results are as Figure 8 shown in a and b, when U / D-CDs are continuously irradiated under ultraviolet light at 330 nm and 670 nm for 30 min respectively, it can be clearly seen that the fluorescence intensity of upconversion hardly changes, indicating that U / D-CDs have excellent anti-photobleaching properties. And from Figure 8 c and d, it can be seen that even in an ionic environment of 0.1 M NaCl, the fluorescence intensity of U / D-CDs can basically remain constant. Therefore, U / D-CDs have good salt tolerance. In addition, after continuous storage of U / D-CDs for 120 days ( Figure 8e, f), the fluorescence intensity of their up / down conversion can still remain above 90% of the original fluorescence intensity. Therefore, the above results indicate that U / D-CDs can remain stable under different conditions and have excellent photostability, laying a foundation for the practical application of U / D-CDs.

[0087] 5. Up / Down Conversion Dual-Channel Ratio Fluorescence Detection of U / D-CDs for CA

[0088] Due to the excellent optical properties of U / D-CDs, an up / down conversion dual-channel ratio fluorescence probe based on U / D-CDs was developed for the sensitive detection of CA. To obtain better analytical performance, a series of parameters affecting the probe performance were optimized, including pH value, buffer type, incubation time, temperature, and ionic strength, and [(F1 / F2)0 - F1 / F2] (where F1 / F2 represents the ratio of the fluorescence intensity of the blue peak to the green peak of U / D-CDs) was used as the quenching efficiency for discussion.

[0089] The optimization process was as follows: on the basis of sensing CA, the final concentration of CA was fixed at 30 μM, and conditions such as the pH, type, incubation time, temperature, and ionic strength of the buffer were changed. Specific operations:

[0090] 1. pH value: Transfer 100 μL of U / D-CDs solution to an EP tube, then add 100 μL of buffer solution with different pH values, and then add 100 μL of CA standard solution to the mixture and dilute it to 1 mL with ultrapure water. After vortex oscillation, incubate for a period of time. Finally, measure its fluorescence emission spectrum.

[0091] 2. Buffer type: On the basis of the above operations, determine the pH of the buffer solution and change the type of buffer, with other conditions remaining unchanged.

[0092] 3. Incubation time: On the basis of determining the pH value and buffer type, with other conditions remaining unchanged, only change the incubation time to 0 - 80 minutes. And measure the fluorescence emission spectrum every ten minutes.

[0093] After fixing all the above conditions, only change the incubation temperature of the solution, that is, after incubating for the determined incubation time at 5 - 55 °C (every 10 °C), measure the fluorescence emission spectrum.

[0094] As Figure 9 shown, the effects of U / D-CDs on the response to CA under different detection conditions: (a) pH; (b) buffer type (KH2PO4-NaOH, NaH2PO4-C6H8O, Tris-HCl, H3BO3-Na2B4O7·10H2O, and BR buffer); (c) incubation time; (d) temperature; (e) ionic strength, and the results are as Figure 9As shown in a and b, [(F1 / F2)0 - F1 / F2] does not change significantly in the pH range of 7 - 9, indicating that the dual-channel fluorescent probe has better stability in an alkaline environment. The pH of 7.4 and the buffer type of HBO3 - Na2B4O7·10H2O are selected as the optimal pH and the optimal buffer solution. At the same time, 7.4 is also the normal pH value of a healthy human body. From Figure 9 As can be seen from c, once the fluorescent probe is exposed to the CA environment, the probe immediately responds to CA, and [(F1 / F2)0 - F1 / F2] does not change significantly with the increase of the incubation time, indicating that the reaction quickly reaches stability. Therefore, 10 minutes is set as the optimal incubation time. At the same time, the changes in temperature and ionic strength have little effect on [(F1 / F2)0 - F1 / F2] ( Figure 9 d, e), considering the convenience of experimental operation, 25°C and no NaCl are selected as the optimal experimental conditions.

[0095] Under the above optimal conditions, the quantitative determination of CA in solution is successfully achieved. As Figure 10 shown, the fluorescence emission spectra of U / D - CDs when different concentrations of CA are added at the excitation wavelengths of (a) 330 nm and (c) 670 nm; (b) the linear relationship between F 422 / F 500 and the CA concentration in the range of 0.5 - 150 μM; (d) the linear relationship between F 422 / F 500 and the CA concentration in the range of 0.5 - 150 μM. As Figure 10 shown in a and c, with the gradual increase of the CA concentration, the fluorescence intensities of U / D - CDs at 422 nm and 500 nm continuously decrease, indicating that the fluorescent probe is sensitive to various concentrations of CA, and U / D - CDs can effectively interact with CA. The down - conversion channel for detecting CA is as Figure 10 shown in b. By calculating the fluorescence intensity ratio (F 422 / F 500 ), F 422 / F 500 and the CA molecular concentration show a good linear relationship in the range of 0.5 - 150 μM. The linear equation is y = -0.00237x + 1.64980, and the correlation coefficient is 0.9996. The detection limit (LOD) is calculated to be 0.27 μM. At the same time, the fluorescence response of the up - conversion channel to CA molecules is studied ( Figure 10d), Upconversion has results similar to those of downconversion, showing a good linear relationship (y = -0.00221x + 1.5355) in the range of 0.5 - 150 μM, with a correlation coefficient of 0.9963. The limit of detection (LOD) was calculated to be 0.29 μM. Therefore, the ratiometric fluorescence probe based on U / D-CDs achieved the synchronous detection of CA under the dual excitation channels of up / downconversion, greatly improving the reliability of CA determination in complex samples.

[0096] In addition, compared with other reported methods for detecting CA (Table 2), the developed ratiometric fluorescence sensor has a lower limit of detection and a good linear range. At the same time, compared with the downconversion fluorescence sensor for detecting CA, the upconversion detection channel we developed also has other excellent properties, including large penetration depth, low background, and little harm to organisms.

[0097] Table 2 Parameters of different CA probes

[0098]

[0099] To evaluate the practical applicability of the developed fluorescence sensor, the fluorescence responses of the up / downconversion dual-channel fluorescence sensor to common metal ions, anions, and bioactive molecules in serum were studied, such as Figure 11 shown, under excitation at 330 nm (a) and 670 nm (d), the fluorescence emission spectra after adding different interfering substances (K + , Na + , Ca 2+ , Mg 2+ , Zn 2+ , Mn 2+ , Cl - , H2PO4 - , HCO3 - , CO3 2- , BrO3 - , Br - , SO4 2- , NO3 - , Arg, Gly, Cys, BSA, Trp, Leu, and Ala (where the concentration of BSA is 0.1 mg / mL and the concentration of all other interfering substances is 500 μM)); the selectivity of the ratiometric fluorescence probe for CA after adding different metal cations and anions under excitation at 330 nm (b) and 670 nm (e); the interference test of the ratiometric fluorescence probe for CA after adding interfering substances under excitation at 330 nm (e) and 670 nm (f). As Figure 11As shown in b and e, in the selectivity experiment, whether in the down-conversion channel or the up-conversion channel, the response signal of the fluorescence sensor to CA is much greater than that to other ions and molecules, indicating that the up / down-conversion dual-channel fluorescence sensor has excellent selectivity for the detection of CA. Then, an interference experiment was also carried out. The results are as Figure 11 shown in c and 10f. When CA coexists with other ions and molecules, the fluorescence responses of other ions and molecules can be ignored. Therefore, this detection method has good anti-interference ability and potential application for detecting CA in complex environments.

[0100] The mechanism of fluorescence quenching was further studied. As Figure 12 shown, (a) The UV-Vis absorption spectrum of CA and the fluorescence excitation spectrum of U / D-CDs; (b) The UV-Vis absorption spectra of U / D-CDs, CA, and their mixture; (c) The fluorescence decay curve of U / D-CDs at a fluorescence emission of 422 nm before and after adding CA; (d) The fluorescence decay curve of U / D-CDs at a fluorescence emission of 500 nm before and after adding CA. As Figure 12 shown in a, there is partial overlap between the absorption spectrum of CA molecules in the range of 250 nm - 350 nm and the excitation spectra of U / D-CDs at fluorescence emission wavelengths of 422 nm and 500 nm, indicating that CA molecules may cause the fluorescence quenching of U / D-CDs through the inner filter effect (IFE) or fluorescence resonance energy transfer (FRET). [64，65] . At the same time, the UV-Vis absorption spectra of U / D-CDs, CA, and U / D-CDs with added CA ( Figure 12 b) show that no new substance is formed after mixing CA and U / D-CDs, but only the sum of the individual absorptions of these two components. Therefore, the possibility of static quenching is excluded. To further study the quenching mechanism, time-resolved fluorescence decay analysis was carried out on U / D-CDs. As Figure 12 shown in c and d, the fluorescence lifetime of the blue emission in U / D-CDs (emitting at 422 nm when excited at 330 nm) hardly changes before and after adding CA (from 13.98 ns to 13.94 ns); the fluorescence lifetimes of the green emission in U / D-CDs (emitting at 500 nm when excited at 330 nm) are 14.40 ns and 14.10 ns before and after adding CA, respectively, with very little difference. Therefore, this confirms that IFE plays a major role in the fluorescence quenching process of U / D-CDs, rather than the influence of FRET.

[0101] 6. Detection in actual samples

[0102] The practical applicability of the developed up / down-conversion dual-channel ratio fluorescence sensor was evaluated by detecting CA in human serum samples. The standard addition method was used for detection, and the measurement was repeated 5 times. Different concentrations of CA were added to the diluted human serum samples to simulate real samples.

[0103] The specific steps are as follows: A blank plasma sample was collected from a healthy volunteer. First, pretreatment was carried out to collect the serum sample. The supernatant of the plasma sample refrigerated overnight was centrifuged at 10000 rpm / min for 10 minutes. Then, the obtained supernatant was filtered through a microporous membrane (0.22 μm) and diluted 100-fold with deionized water for further experiments. Finally, 100 μL of U / D-CDs and different volumes of CA standard solution were added to the diluted serum sample to prepare a series of spiked samples, and fluorescence measurement was carried out after mixing.

[0104] The results are shown in Table 3 and Table 4. In the down-conversion excitation channel, the recovery rate was as high as 94.94%-112.2%, and the relative standard deviation (RSD) did not exceed 6.68%. And in the up-conversion excitation channel, the recovery rate was in the range of 96.7%-115%, and the RSD did not exceed 6.86%. The above results confirmed the reliability of the up / down-conversion dual-channel ratio fluorescence probe based on U / D-CDs for detecting CA in real samples.

[0105] Table 3. Recovery rate detection of standard addition in serum samples at λex = 330 nm (n = 5)

[0106]

[0107] Table 4. Recovery rate detection of standard addition in serum samples at λex = 680 nm (n = 5)

[0108]

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[0175] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the present invention to the specific embodiments described. Obviously, many modifications and variations can be made according to the content of this specification. These embodiments are selected and specifically described in this specification to better explain the principle and practical application of the present invention, so that those skilled in the art can well understand and utilize the present invention. The present invention is only limited by the claims and their full scope and equivalents.

Claims

1. A preparation method of up-conversion dual-emission fluorescent carbon dots, characterized in that, The steps are as follows: S1: Dissolve the raw materials in a solvent and react at 120 - 220 °C for 1 - 10 h; S2: Centrifuge the liquid obtained in step S1 for 10 minutes to remove insoluble large particles; S3: Dialyze the supernatant of the material obtained in step S2 through a cellulose acetate dialysis membrane; S4: Freeze-dry for 48 h to obtain a solid powder, which is the target carbon dots; The raw materials are acridine yellow and 2-aminoterephthalic acid. 40 - 140 mg of raw materials are used per 5 mL of solvent, and the mass ratio of acridine yellow to 2-aminoterephthalic acid is 1:2 - 6; The solvent is pure water and / or ethanol.

2. The preparation method of an up-conversion and down-conversion dual-emission fluorescent carbon dot according to claim 1, characterized in that, The volume ratio of pure water to ethanol in the solvent is 5 - 0:0 - 5.

3. The preparation method of an up-conversion and down-conversion dual-emission fluorescent carbon dot according to claim 1, wherein, In step S1, the reaction temperature is 180 °C, the reaction time is 2 h, the volume ratio of pure water to ethanol in the solvent is 1:1, and the mass ratio of acridine yellow to 2-aminoterephthalic acid is 1:

4.

4. The preparation method of an up-conversion and down-conversion dual-emission fluorescent carbon dot according to claim 1, characterized in that, In step S2, centrifuge at 10000 rpm / min for 10 minutes to remove insoluble large particles; In step S3, dialyze the supernatant through a cellulose acetate dialysis membrane with 300 MWCO.

5. A carbon dot prepared by the preparation method according to any one of claims 1 - 4.

6. Use of the carbon dots as described in claim 5, characterized in that, Use the carbon dots according to claim 5 to prepare a probe for detecting caffeic acid.

7. An application of the carbon dots as described in claim 5, characterized in that, The application steps are as follows: Use the prepared carbon dots to detect caffeic acid; Mix the sample to be tested with the prepared carbon dots and incubate, and measure the fluorescence emission spectra under excitation lights of 330 nm and 670 nm; Before mixing, add HBO3-Na2B4O7·10H2O buffer solution to the carbon dots.

8. The application according to claim 7, wherein The pH of the buffer solution is 7.4, the incubation time is 0 - 80 min, and the incubation temperature is 5 - 55 °C.

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