Fluorescent carbon dots as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a fluorescent carbon dot based on p-phenylenediamine and triphenylamine as well as a preparation method and application of the fluorescent carbon dot. The fluorescent carbon dots are obtained by taking triphenylamine and p-phenylenediamine as raw materials through a solvothermal method. The invention provides a functionalized carbon dot based on p-phenylenediamine and triphenylamine. The functionalized carbon dot can achieve 599 nm emission under excitation of the wavelength of 365 nm. NADH and active sites on the surfaces of the carbon dots form a non-fluorescent ground state compound through a static quenching mechanism, 599 nm fluorescence quenching and 444 nm emission enhancement are caused, and ratio fluorescence detection of NADH is achieved. The method is good in linear relation, high in selectivity, rapid in response and high in sensitivity, and the detection limit is as low as 108 nM.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to fluorescent carbon dots based on p-phenylenediamine and triphenylamine, and a preparation method and application thereof. Background Art

[0002] Nicotinamide adenine dinucleotide (NADH) is a core coenzyme of cellular redox reactions and a key carrier of mitochondrial energy metabolism and electron transport chain. Its concentration dynamics directly reflects the body's redox homeostasis and mitochondrial functional status. Clinical studies have shown that abnormal fluctuations in serum NADH levels are closely related to a variety of major diseases. For example, in the process of diabetes, the NADH / NAD + An imbalance in the ratio can exacerbate the accumulation of reactive oxygen species, leading to oxidative stress damage in pancreatic beta cells. Malignant tumor cells, through the Warburg effect, overaccumulate NADH, providing metabolic support for their rapid proliferation. However, current quality control of NADH products is severely lagging. Due to the lack of simple and reliable detection methods, the true content and biological activity of NADH in products are difficult to accurately assess. Therefore, the development of highly sensitive and specific NADH quantitative analysis methods is a critical technical need for clinical diagnostics.

[0003] At present, the quantitative analysis of NADH mainly relies on three types of technologies: enzyme cycle method, high performance liquid chromatography (HPLC) and electrochemical sensor. + The NADH cycle amplifies the signal, but its specificity is heavily dependent on enzyme activity, susceptible to interferences, and has high reagent costs. While HPLC can achieve high-precision separations, it requires complex sample pretreatment and is bulky, making it difficult to meet the demands of point-of-care (POCT) testing. Electrochemical sensors, while portable, suffer from the instability of electrode-modified materials, and electroactive substances such as uric acid in serum can easily trigger false-positive signals.

[0004] Fluorescence and colorimetric sensing technologies have attracted significant attention in the field of bioanalysis in recent years due to their rapid response, ease of operation, and signal visualization. However, traditional single-wavelength fluorescent probes are susceptible to interference from ambient temperature, light source fluctuations, and probe concentration differences, resulting in unreliable detection results. In contrast, ratiometric fluorescence sensing, by simultaneously monitoring the signal ratio of two emission channels, enables built-in self-calibration, significantly improving detection stability and accuracy. Furthermore, the inherent color gradient characteristics of dual-emission systems enable semi-quantitative visualization without the need for sophisticated instrumentation, offering a unique advantage for rapid on-site screening. Carbon quantum dots (CDs), as novel fluorescent nanomaterials, have become ideal vehicles for constructing ratiometric fluorescent probes due to their excellent biocompatibility, tunable luminescence properties, and strong resistance to photobleaching. However, existing carbon dot systems are mostly limited to blue-green emission. The few synthesized orange and red carbon dots often require excitation light around 550 nm. The small Stokes shift poses significant challenges to the design of ratiometric fluorescence sensors, making it difficult to distinguish color changes, significantly limiting their potential for application in NADH detection.

[0005] For example, the prior art CN 117887460 A discloses a ratiometric fluorescent carbon dot for tracing nicotinamide adenine dinucleotide in liver cancer glycolysis. The ratiometric fluorescent carbon dot uses aniline derivatives and aromatic disulfide as carbon and nitrogen sources and is obtained by a solvent thermal method. + As the concentration increases, the carbon dots migrate to red fluorescence, and the ratio fluorescence intensity F640 / F520 is related to NAD + The linear relationship is good, and after the action of the glycolysis inhibitor 2-DG, the uptake of carbon dots in HepG2 cells is significantly reduced. However, although the invention mentions that carbon dots are basically non-cytotoxic, experiments have shown that at high concentrations, high-concentration carbon dots will have a certain toxic effect on cells. For example, in Example 1, when the concentration of carbon dots reached 500 μg / mL, the survival rate of HepG2 cells was only 80%, which was significantly lower than that of the low-concentration group, indicating that it is toxic at this concentration. At high concentrations, carbon dots will have a certain negative impact on cells. In addition, the biocompatibility of the carbon dots of this invention is worrying. Secondly, its linear range is too wide and it is difficult to apply to different tissues and cells. For example, in the blood, NAD + The concentration is relatively low, approximately at the micromolar level, and the sensitivity of the test of the invention is difficult to meet.

[0006] Prior art CN 115404074 B discloses a method for preparing a fluorescence detection nanoprobe, comprising the following steps: preparing blue fluorescent carbon dots (BCD) by hydrothermal synthesis; preparing red fluorescent carbon dots (RCD) by solvothermal synthesis; mixing the prepared BCD and RCD in a certain ratio, incubating them with Whatman filter paper, and oven-drying to obtain the fluorescence detection nanoprobe. However, the preparation of this probe involves steps such as incubating the blue and red fluorescent carbon dots with Whatman filter paper. This preparation method can result in insufficient stability of the probe during long-term storage or use, thereby affecting the consistency and reliability of detection. Summary of the Invention

[0007] The purpose of the present invention is to provide a highly sensitive fluorescent carbon dot based on p-phenylenediamine and triphenylamine, and a preparation method and application thereof.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] A fluorescent carbon dot is obtained by using triphenylamine and p-phenylenediamine as raw materials through a solvent thermal method.

[0010] The planar benzene ring of p-phenylenediamine is used as the basic conjugated unit, and the polycyclic structure of triphenylamine undergoes dehydration condensation and carbonization recombination under solvent thermal conditions to form a carbon core structure with a specific multi-level conjugated structure, thereby increasing the π-π * The transition efficiency is improved, which drives the red shift of the fluorescence emission peak of carbon dots, thereby enhancing light absorption and fluorescence stability.

[0011] In a preferred embodiment, the mass ratio of p-phenylenediamine (PDA) to triphenylamine (TPA) is 1-2:2-1.

[0012] In a preferred embodiment, the particle size of the fluorescent carbon dots is 1.80-5.00 nm, and the average particle size is 3.00-3.20 nm.

[0013] A method for preparing fluorescent carbon dots comprises the following steps:

[0014] S1, dissolving triphenylamine and p-phenylenediamine in a reaction solvent, and ultrasonically mixing to obtain a mixed solution;

[0015] S2. The mixed solution is transferred to a polytetrafluoroethylene autoclave for thermal reaction. The obtained product is filtered, dialyzed and freeze-dried to obtain a finished product.

[0016] In one preferred embodiment, in step S1, in the mixed solution, the concentration of triphenylamine is 0.005-0.01 g / mL; the concentration of p-phenylenediamine is 0.005-0.02 g / mL.

[0017] In one preferred embodiment, in step S1, the reaction solvent is anhydrous ethanol.

[0018] In one preferred embodiment, in step S1, the pH of the mixed solution is 8-10.

[0019] In one preferred embodiment, in step S2, the reaction temperature of the thermal reaction is 200-240° C., and the reaction time is 10-15 h.

[0020] This temperature range is conducive to the condensation and conjugation expansion of aromatic rings, enhancing red light emission. High temperature will promote the large size sp 2 The formation of conjugated domains (dominated by π→π* transitions) leads to a red shift in the emission of carbon dots (e.g., from blue to orange). If the temperature is too low, the orange fluorescence will not be produced, while if the temperature is too high (e.g., >250°C), the emission peak will be broadened or even quenched due to excessive graphitization.

[0021] In one preferred embodiment, in step S2, the molecular weight cut-off of the dialysis bag used for dialysis is 1000-1500 Da, and the dialysis time is 24-28 h.

[0022] The molecular weight cut-off of the dialysis bag determines the size range of molecules that can pass through the dialysis bag.

[0023] In one preferred embodiment, in step S2, the size of the filter membrane used for filtration is 0.22-0.24 μm.

[0024] Based on the same inventive concept, the present invention also claims protection for the use of the fluorescent carbon dots in detecting nicotinamide adenine dinucleotide (NADH).

[0025] Based on the same inventive concept, the present invention also claims protection for a method for detecting nicotinamide adenine dinucleotide (NADH), which comprises mixing the fluorescent carbon dots and nicotinamide adenine dinucleotide and testing the fluorescence spectrum of the mixed solution.

[0026] Based on the same inventive concept, the present invention also claims protection for a fluorescent probe, which includes the fluorescent carbon dots.

[0027] In one preferred embodiment, the fluorescent probe is obtained by incubating the fluorescent carbon dots with Whatman filter paper and drying in an oven.

[0028] Based on the same inventive concept, the present invention also claims protection for the application of the fluorescent probe, which is used to prepare early diagnosis reagents for liver cancer, preparations for diabetes detection reagents, and preparations for phenylketonuria detection reagents.

[0029] Based on the same inventive concept, the present invention also claims protection for the application of the fluorescent probe, which is applied to the quality control reagent of NADH capsules.

[0030] Based on the same inventive concept, the present invention also claims protection for a test paper comprising the fluorescent carbon dots.

[0031] In one preferred embodiment, the test paper is prepared by loading the fluorescent carbon dots onto the test paper and freeze-drying the test paper, and comparing the color change to perform quantitative analysis of NADH.

[0032] The present invention is further explained below:

[0033] The present invention provides a functionalized carbon dot (PDA-TPA-CDs) based on p-phenylenediamine (PDA) and triphenylamine (TPA), wherein orange fluorescent carbon dots are synthesized by controlling the pH value through a one-step solvothermal method, and the amino groups on the surface of the carbon dots are closely connected with the carbon core sp 2 The synergistic effect of the conjugated structure imparts a dual-channel excited-state energy level structure to the material, enabling emission at 599 nm under 365 nm excitation. NADH forms a non-fluorescent ground-state complex with the carbon dot surface active sites via a static quenching mechanism, resulting in fluorescence quenching at 599 nm and enhanced emission at 444 nm, enabling ratiometric fluorescence detection of NADH. This method demonstrates excellent linearity, high selectivity, rapid response, and high sensitivity, with a detection limit as low as 108 nM.

[0034] In the present invention, a ratiometric sensing system was successfully constructed based on the static quenching effect of NADH on the 599nm emission peak and the enhanced response of 444nm emission. The system shows an excellent linear relationship in the range of 0-550μM, with a detection limit as low as 108nM, and is highly selective for NADH. The fluorescent probe of the present invention responds rapidly and can respond within seconds, and the color of the sensing solution changes from orange-red to indigo with the NADH concentration gradient, which provides a feasibility basis for subsequent combination with smartphone image analysis programs to complete on-site semi-quantitative detection. Therefore, the present invention provides a new nano-tool for the highly sensitive detection of NADH, and provides a new method for early screening of metabolic diseases (diabetes detection, phenylketonuria), quality monitoring of health products (such as NADH capsules), and personalized health management. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the identification spectrum of PDA-TPA-CDs; wherein, Figure 1 a is the TEM image of PDA-TPA-CDs; Figure 1 b is the size distribution of PDA-TPA-CDs; Figure 1 c is the XRD pattern of PDA-TPA-CDs;

[0036] Figure 2 is the FT-IR spectrum of PDA-TPA-CDs;

[0037] Figure 3 The XPS technology is used to deeply analyze the surface chemical composition and bonding configuration of PDA-TPA-CDs, among which, Figure 3 a is the full spectrum of PDA-TPA-CDs; Figure 3 b is the C1s spectrum of PDA-TPA-CDs; Figure 3 c is the N1s spectrum of PDA-TPA-CDs; Figure 3 d is the O1s spectrum of PDA-TPA-CDs;

[0038] Figure 4 The optimized diagram of the synthesis conditions of PDA-TPA-CDs is shown in FIG. Figure 4 a is the fluorescence curve of PDA-TPA-CDs caused by the change of feed ratio; Figure 4 b is the fluorescence curve of PDA-TPA-CDs caused by solvent change; Figure 4 c is the fluorescence curve of PDA-TPA-CDs caused by pH change; Figure 4 d is the fluorescence curve of PDA-TPA-CDs caused by the constant changes during synthesis;

[0039] Figure 5 Figure 2 is the fluorescence characteristics and detection mechanism of PDA-TPA-CDs; Figure 5 a is the emission spectra of PDA-TPA-CDs at different excitation wavelengths; Figure 5 b is the absorption spectrum (green), excitation spectrum (red) and emission spectrum (blue) of PDA-TPA-CDs; Figure 5 c is the fluorescence lifetime of PDA-TPA-CDs and PDA-TPA-CDs+NADH; Figure 5 d is the UV-visible spectra of PDA-TPA-CDs, NADH, and PDA-TPA-CDs+NADH;

[0040] Figure 6 The fluorescence spectrum and linear relationship of PDA-TPA-CDs in the presence of NADH; Figure 6 a is the fluorescence spectrum of PDA-TPA-CDs in the presence of 0-275 μM NADH; Figure 6 b shows the linear relationship of PDA-TPA-CDs in the presence of 0-275 μM NADH; Figure 6 c is the fluorescence spectrum of PDA-TPA-CDs in the presence of 275-550 μM NADH; Figure 6d is the linear relationship of PDA-TPA-CDs in the presence of 275-550 μM NADH;

[0041] Figure 7 is the detection performance of PDA-TPA-CDs, where Figure 7 a is the emission spectra of PDA-TPA-CDs under NADH (200 μm) and other ten analytes under 365 nm excitation, PBS was used as a control; Figure 7 b is the fluorescence ratio of PDA-TPA-CDs to the other ten analytes under NADH (200 μ m ) excitation at 365 nm; Figure 7 c is the rapid response performance of PDA-TPA-CDs after rapid injection of NADH (1 mM); Figure 7 d is the photostability of PDA-TPA-CDs. DETAILED DESCRIPTION

[0042] The present invention is not limited to the following specific embodiments. Based on the disclosure of the present invention, a person skilled in the art may adopt a variety of other specific embodiments to implement the present invention. Any simple changes or modifications made to the design structure and concept of the present invention fall within the scope of protection of the present invention. It should be noted that the embodiments and features of the embodiments of the present invention may be combined with each other unless they conflict.

[0043] Instruments and reagents

[0044] Triphenylamine (TPA), citric acid (CA) and p-phenylenediamine (PDA) were purchased from Shanghai Adamas Reagent Co., Ltd.; nicotinamide adenine dinucleotide reduced form (NADH), sodium hydroxide (NaOH), phenylalanine (Phe), glutamic acid (Glu), zinc chloride (ZnO) and nicotinamide adenine dinucleotide oxidized form (NAD + ) was supplied by Tokyo Institute of Technology (Shanghai) Chemical Industry Development Co., Ltd.; glucose (Glc), magnesium chloride (MgCl2), calcium chloride (Ca MgCl2), sodium chloride (NaCl) and phosphate buffered saline (PBS) were purchased from Beijing Bailingwei Technology Co., Ltd.; anhydrous ethanol was purchased from Shanghai Titan Technology Co., Ltd.; hydrochloric acid (HCl) was purchased from Nanjing Chemical Reagent Co., Ltd.

[0045] Example 1

[0046] Synthesis of PDA-TPA-CDs

[0047] PDA-TPA-CDs were synthesized using a one-step solvothermal method. 0.3 g of p-phenylenediamine (PDA) and 0.15 g of triphenylamine (TPA) were dissolved in 30 mL of anhydrous ethanol and thoroughly mixed by ultrasonication for 5 minutes. The pH of the solution was then 9. The above solution was placed in a 100 mL polytetrafluoroethylene-lined reactor and heated at 200°C for 12 hours. After the reaction, the mixture was naturally cooled to room temperature, centrifuged at 10,000 rpm for 10 minutes, filtered through a 0.22 μm filter membrane, and dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 1,000 Da. Finally, the product was vacuum dried to obtain PDA-TPA-CDs powder, which was dispersed in anhydrous ethanol to prepare a 150 μg / mL solution for subsequent experiments.

[0048] The obtained PDA-TPA-CDs were subjected to performance tests. The fluorescence spectrum was measured using a Hitachi F-4700 fluorescence spectrometer from Japan, with the following test parameters: 365 nm excitation, slit 10 20, and voltage 400 V. The UV spectrum was measured using a Shimadzu UV-2600i from Japan. Fourier transform infrared spectroscopy (FT-IR) was measured using a Shimadzu IRTracer 100 from Japan. X-ray photoelectron spectroscopy (XPS) was measured using the Thermo Fisher NEXSA from the United States. X-ray polycrystal diffraction (XRD) was obtained using a Bruker D8Advance from Germany. Field emission transmission electron microscopy (TEM) was obtained using a JEOL F200 thermal field emission transmission electron microscope.

[0049] Quantum yield is an important indicator for measuring the optical properties of luminescent materials. It is measured along with fluorescence lifetime using the Edinburgh-FLS1000. Quantum yield can be expressed by the following formula:

[0050]

[0051] Where QY is the absolute quantum yield, L emission is the number of photons emitted by the sample fluorescence, E solvent and E sample are the number of photons excited by the excitation light on the solvent and the sample, respectively. The fluorescence lifetime is calculated using the following formula:

[0052]

[0053] where A1 and A2 are pre-exponential factors and t1 and t2 are decay times.

[0054] The results of morphological analysis using field emission transmission electron microscopy are shown in Figure 2. Figure 1 As shown, Figure 1 a is the TEM image of PDA-TPA-CDs; Figure 1 b is the size distribution of PDA-TPA-CDs; Figure 1 c is the XRD pattern of PDA-TPA-CDs. Figure 1 As shown in a and b, the synthesized PDA-TPA-CDs are spherical and have good monodispersity. The particle size ranges from 1.91 to 4.95 nm, with an average particle size of 3.20 nm. As shown in the high-resolution transmission electron microscopy image, PDA-TPA-CDs have a clear lattice structure with a lattice spacing of 0.21 nm, which can be attributed to the (100) in-plane lattice graphene carbon. The XRD spectrum of PDA-TPA-CDs ( Figure 1 In c), a broad diffraction peak appears at 26.67 2θ, which can be attributed to the typical characteristics of the (002) crystal plane of graphitized carbon.

[0055] The FT-IR spectrum of PDA-TPA-CDs is shown in Figure 2 As shown. 3302cm -1 The broad peak near 3004cm is attributed to the stretching vibration of amino group NH. -1 The peak corresponds to the aromatic ring sp 2 Hybrid CH vibration. 1503cm -1 The characteristic peak at 1259cm is attributed to the C=C vibration of the benzene ring skeleton. -1 The peak at 820cm may be related to the CN stretching vibration of the amino group or amide bond. -1 The peak at 684cm is related to the out-of-plane bending vibration of the para-substituted benzene ring. -1 and 502cm -1 The low-frequency signal can be traced back to the in-plane deformation vibration of the aromatic CH system. Comprehensive analysis confirms that the carbon quantum dots have successfully introduced abundant nitrogen and oxygen functional groups.

[0056] XPS technology was used to deeply analyze the surface chemical composition and bonding configuration of PDA-TPA-CDs. The results are as follows Figure 3 As shown. The results of wide spectrum scanning show that ( Figure 3 a), the synthesized PDA-TPA-CDs are composed of three elements: C, N, and O. The C / N / O atomic percentages are 74.97 / 14.72 / 7.31 respectively after quantitative analysis. High-resolution spectrum analysis shows that: C 1s spectrum ( Figure 3 b) Gaussian peak analysis shows three characteristic peaks (284.13eV, 284.81eV, 289.34eV), corresponding to sp 2 Typical binding energies of hybrid carbon skeletons (CC / C=C), amino / imine bonds (CN / C=N), and carbonyl groups (C=O). N 1s spectrum ( Figure 3 c) shows that the double peaks at 398.76eV and 399.88eV represent the electronic state characteristics of pyridinic (CN=C) and pyrrolic (CNC) nitrogen, respectively. It is worth noting that the O 1s spectrum ( Figure 3d) Characteristic vibrational modes at 531.93 eV and 535.35 eV are clearly identified as chemical fingerprints of carboxyl (C=O) and ether (CO) bonds. These patterns confirm the presence of a rich network of nitrogen and oxygen functional groups on the surface of the material, a property that significantly enhances the colloidal stability and surface active site density of the quantum dots. Furthermore, as measured by a reference method, the carbon quantum dots exhibit an absolute fluorescence quantum yield of 16.34%, exceeding that of conventional carbon-based fluorescent materials.

[0057] Comparative Example 1

[0058] The experimental conditions of Example 1 were followed, except that the reaction raw materials were changed to 0.3 g p-phenylenediamine and 0.15 g triphenylamine dissolved in 30 mL water, and then reacted at 200° C. for 12 h. Other conditions were the same as in Example 1.

[0059] The results showed that the fluorescence intensity of the solution was weak and insufficient for specific detection of NADH.

[0060] Comparative Example 2

[0061] The experimental conditions were the same as those in Example 1, except that the reaction raw materials were changed to 0.3 g p-phenylenediamine and 0.15 g urea dissolved in 30 mL anhydrous ethanol, and the reaction was carried out at 200° C. for 12 h; other conditions were the same as those in Example 1.

[0062] The results showed that no orange-red carbon dots were obtained.

[0063] Comparative Example 3

[0064] The experimental conditions of Example 1 were followed, except that the reaction raw material was changed to 0.3 g p-phenylenediamine dissolved in 30 mL anhydrous ethanol and reacted at 200° C. for 12 h; other conditions were the same as in Example 1.

[0065] The results showed that weak orange-red carbon dots were obtained, but they could not specifically respond to NADH.

[0066] Comparative Example 4

[0067] The experimental conditions of Example 1 were followed, except that the reaction raw materials were changed to 0.3 g p-phenylenediamine and 0.9 g 2,2'-dithiosalicylic acid dissolved in 30 mL anhydrous ethanol and reacted at 200° C. for 12 h. Other conditions were the same as in Example 1.

[0068] The results showed that no orange-red carbon dots were obtained.

[0069] Comparative Example 5

[0070] Based on Example 1, the solvent polarity was changed, and a gradient solvent system was constructed using tetrahydrofuran (ε = 7.6), anhydrous ethanol (ε = 24.3), and deionized water (ε = 80.1) according to the dielectric constant of the solvent system. The volume ratios of tetrahydrofuran: anhydrous ethanol: water were 4:1:0, 5:3:2, 0:1:0, 0:3:2, and 0:1:4, respectively, to prepare four types of carbon quantum dots. The fluorescence spectrum was then tested using a Hitachi F-4700 fluorescence spectrometer in the same manner as in Example 1.

[0071] The results are as follows Figure 4 As shown in b, the results show that the polarity of the reaction solvent has a significant effect on the emission wavelength and fluorescence intensity of CDs. When the solvent is anhydrous ethanol, orange light CDs with better fluorescence properties are obtained.

[0072] Example 2

[0073] On the basis of Example 1, the mass ratio of PDA:TPA was adjusted to 1:2, 1:1, 2:1, 4:1 and 8:1 respectively to obtain carbon quantum dots. The fluorescence spectrum was measured by a Japan Hitachi F-4700 fluorescence spectrometer, and the test method was the same as that of Example 1. The results are shown in Figure 1. Figure 4 As shown in Figure a, the fluorescence performance of carbon quantum dots is good at 1:2, 1:1, and 2:1. However, the R0 (i.e., 599nm emission peak value / 444nm emission peak value) is the smallest at the 2:1 ratio, indicating that the fluorescence performance of carbon quantum dots at the 2:1 ratio is the best.

[0074] Example 3

[0075] Based on Example 1, the pH of the reaction system was precisely controlled using HCl / NaOH solutions to investigate the performance differences of PDA-TPA-CDs synthesized under different pH conditions (pH 3, 5, 7, 9, and 11). Fluorescence spectra were measured using a Hitachi F-4700 fluorescence spectrometer using the same testing method as in Example 1.

[0076] The results are as follows Figure 4 As shown in Figure c, pH has a significant effect on the fluorescence intensity and emission peak position of carbon quantum dots. Under acidic and neutral conditions, the emission peak of PDA-TPA-CDs is around 450nm, emitting bright blue light. As the pH value increases, the emission wavelength red-shifts, emitting orange light at pH 9 and yellow light at pH 11.

[0077] However, to build a ratiometric fluorescence sensing system, two well-separated peaks are required. Materials that emit blue and yellow light too close to the NADH peak itself can affect the peak shape and make it difficult to distinguish, making it difficult to build a good ratiometric fluorescence sensing system. Therefore, the present invention uses orange light, which corresponds to a pH of 8-10.

[0078] Example 4

[0079] Based on Example 1, the thermal reaction time was adjusted to 10h, 11h, 12h, 13h, and 14h to obtain carbon quantum dots. Figure 4 As shown in Figure d, the results show that the synthesis time has little effect on the fluorescence properties of PDA-TPA-CDs.

[0080] Through the coordinated optimization of the above parameters, PDA-TPA-CDs with stable fluorescence properties were successfully prepared, laying the material foundation for the construction of a highly selective ratiometric fluorescence sensing platform.

[0081] Example 5

[0082] Fluorescence characteristics and detection mechanism

[0083] The excitation-emission characteristics and electronic transition mechanism of the PDA-TPA-CDs prepared in Example 1 were analyzed using a fluorescence spectrometer and UV-visible spectrophotometer. Fluorescence spectra were measured using a Hitachi F-4700 fluorescence spectrometer (Japan) with the following test parameters: 365 nm excitation, 10-20 slit, and 400 V. UV spectra were measured using a Shimadzu UV-2600i (Japan).

[0084] From the fluorescence spectrum, we know that the intensity and peak position of fluorescence emission are determined by the excitation wavelength, so the effect of excitation wavelength on fluorescence intensity was investigated. Figure 5 a and b show that PDA-TPA-CDs has double excitation peaks at 365nm and 510nm, indicating that it has a multi-channel excited state energy level structure. It is worth noting that although the excitation wavelength varies in the range of 340-580nm, the emission peak is always stable at 599nm, showing a significant excitation wavelength-independent characteristic, which suggests that a unified radiation recombination center is formed inside the carbon dots. In order to facilitate colorimetric analysis, 365nm was selected as the excitation wavelength for subsequent experiments. Under natural light, the synthesized PDA-TPA-CDs is a clear and transparent light orange solution. Under ultraviolet light with a wavelength of 365nm, PDA-TPA-CDs appear bright orange. As shown Figure 5 As shown in b, PDA-TPA-CDs has two absorption peaks at 242nm and 295nm, which correspond to the transition of lone pair electrons (n orbital) of nitrogen-containing polar groups (such as C-NH2) on the surface to π* antibonding orbital and the transition of sp orbital to carbon nucleus. 2The π-π* electronic transition of the hybrid conjugated aromatic structure. This dual-mode absorption feature confirms the cooperative luminescence mechanism of the surface state and carbon core state of PDA-TPA-CDs, providing theoretical support for its excitation wavelength-independent emission behavior.

[0085] The study of the fluorescence quenching mechanism of carbon dots requires systematically eliminating multiple potential mechanisms, including static quenching, dynamic quenching, energy transfer, photoinduced electron transfer, and internal filtering. Therefore, the fluorescence quenching mechanism of the PDA-TPA-CDs prepared in Example 1 was analyzed using a fluorescence spectrometer and a UV-visible spectrophotometer system.

[0086] The results showed that: since the absorption spectrum of NADH did not overlap effectively with the excitation spectrum of PDA-TPA-CDs ( Figure 4 b, d), so the inner filtration effect is excluded. TPA is a typical electron donor, and its structural characteristics suggest that photoinduced electron transfer may occur. However, after the addition of NADH, a new absorption peak appeared in the PDA-TPA-CDs solution, indicating the formation of a non-fluorescent ground state complex. Therefore, it is inferred that the quenching mechanism is static quenching. The core criterion of static quenching is the constancy of fluorescence lifetime. Therefore, the present invention records the fluorescence decay curves of PDA-TPA-CDs and PDA-TPA-CDs~NADH system. Figure 5 As shown in Figure c, the fluorescence lifetime of the PDA-TPA-CDs solution was approximately 8.75 ns before and after the addition of NADH, with no significant change. Therefore, the fluorescence quenching mechanism was static quenching.

[0087] Example 6

[0088] A dual-emission ratiometric fluorescence sensing platform was constructed based on the characteristic emission peaks of NADH (blue emission at 444 nm) and PDA-TPA-CDs (orange emission at 599 nm). A 0.1 M NADH stock solution was prepared and added incrementally to the PDA-TPA-CDs solution to create a concentration gradient of 0 to 550 μM. After pipetting and homogenization, the fluorescence spectrum was measured using a fluorescence spectrophotometer under 365 nm excitation. Each data set was replicated three times.

[0089] By continuously measuring the fluorescence response signal of the same PDA-TPA-CDs (Example 1) and NADH complex system 6 times to investigate the precision, the relative standard deviation (RSD) of the fluorescence intensity ratio (F444 / F599) was calculated to be 1.31% (n=6), highlighting the high repeatability and operational stability of this method in continuous detection. Under optimal conditions, the dynamic response relationship between the dual emission fluorescence intensity and NADH concentration under 365nm excitation was systematically investigated. It was found that the fluorescence intensity of the system showed a good linear relationship with the NADH concentration. Figure 6As shown in a, as the NADH concentration increases (0-275 μM), the fluorescence intensity at 599 nm gradually decreases, while the fluorescence intensity at 444 nm increases. The concentration of NADH and F444 / F599 show a good linear relationship ( Figure 6 b). When the NADH concentration is higher (275-550 μM), the fluorescence intensity of F444 gradually decreases due to the aggregation quenching effect ( Figure 6 c, d). In addition, according to the formula LOD = 3σ / K (σ is the standard deviation of eleven blank samples, K is the slope of the linear equation, n = 11), the detection limit of NADH is calculated to be 108 nM, which meets the requirements for trace detection of NADH in biological fluids.

[0090] Based on the same method, the detection sensitivity of several other NADH detection methods was analyzed. The detection methods are listed as follows:

[0091] Method 1: The preparation method in the second paragraph of page 14994 of reference [1] was used for testing.

[0092] Method 2: The “Experimental Section” on pages 7119 and 7120 of reference [2] was used for testing.

[0093] Method 3: Detection was performed using the method in Sections 2.3 to 2.5 of Reference [3].

[0094] Method 4: Detection was performed using the method in Sections 2.3 to 2.4 of Reference [4].

[0095] The summary data is as follows:

[0096] Table 1 Detection sensitivity of different NADH detection methods

[0097]

[0098] As can be seen, the detection method of the present invention has a sensitivity nearly 10 times higher than that of Method 1. Furthermore, there is no need for complex photodetector arrays and microcavity integration; the fluorescence spectrum can be directly measured after mixing the carbon dots with the sample, resulting in a simple operation process.

[0099] Method 2 requires engineering nanopore proteins and highly sensitive current recording equipment, which carries high technical barriers and is expensive. Compared to Method 2, this method utilizes a one-step solvothermal method to synthesize carbon dots, which reduces raw material costs and requires only a 30-second mixing reaction. It eliminates the need for specialized equipment and can subsequently develop a mobile phone camera for colorimetric analysis, enabling semi-quantitative detection through smartphone image analysis. This makes it more practical for rapid on-site testing.

[0100] Compared to Method 3, this method requires no electrode modification or potential control, and the detection process only requires a 365nm excitation light source. Furthermore, the ECL signal of Method 3 is susceptible to interference from electroactive substances, while fluorescence detection achieves specific binding through a static quenching mechanism, resulting in higher accuracy.

[0101] Compared with method 4, the linear range is wider and can be applied to a wider range of samples for detection.

[0102] References:

[0103] [1]Z.Xiong,G.Fang,RKMondal,Y.Liao,N.Nie,Y.-C.Chen,M.Kim,On-ChipNADH Detection in Multicellular Models Using an AlGaN / GaN Photodetector Arraywith Enhanced Sensitivity,Nano Letters 24(2024)14993-15000.

[0104] [2]X.Liu, W.Feng, F.Yao, J.Zhang, R.Ayesha, T.Chen, X.Shi, X.Qiao, L.Ma, S.Yu,

[0105] [3]H.Chen,

[0106] [4] N.Wang, X.Cao, D.Sun,

[0107] Example 7

[0108] In order to better apply the developed fluorescent probe in actual samples and verify the reliability of the sensing system in complex matrices, other common analytes (Mg 2+ 、Cl - 、Zn 2+ , Ca 2+ 、Na + , NAD+, Glc, CA, Phe, Glu) were used for selectivity study. The concentration of analyte was 200 μM, and PBS was added as a control. The results are shown in Figure 7 As shown in a and b, the fluorescence signal ratios of other analytes did not change significantly compared with the blank group, while the fluorescence signal ratio of the NADH system increased significantly, fully demonstrating that the fluorescent probe has a highly specific recognition ability for NADH. When NADH is quickly injected into the solution, the PDA-TPA-CDs fluorescent probe responds quickly within a few seconds ( Figure 7 c), indicating that the sensing system meets the needs of point-of-care testing (POCT). In addition, after irradiating the PDA-TPA-CDs solution with a xenon lamp for 30 minutes, the fluorescence intensity hardly changed. Therefore, PDA-TPA-CDs has good resistance to photobleaching ( Figure 7 d), suitable for long-term monitoring scenarios.

[0109] In summary, the present invention innovatively uses p-phenylenediamine and triphenylamine as precursors, and adopts a one-step solvothermal method to synthesize blue, yellow and orange tri-color fluorescent carbon quantum dots under different pH conditions. Among them, the PDA-TPA-CDs prepared at pH = 9 exhibit unique dual excitation center characteristics (λex = 365 / 510nm) and emission characteristics independent of the excitation wavelength. Based on the cooperative luminescence mechanism of its surface state and carbon core state, a self-calibrated ratio fluorescence sensing system was successfully constructed. NADH suppresses the 599nm emission peak through static quenching and enhances the 444nm fluorescence emission. The dual signal ratio (F444 / F599) shows a high-precision linear response to the NADH concentration in the range of 0-550μM, and the detection limit is as low as 108nM. Compared with traditional single-signal carbon dot probes, environmental interference is effectively eliminated and reliability is significantly improved. The feasibility of the method was evaluated, and the results showed that the method has good stability, high sensitivity, and high specificity for NADH. Furthermore, the fluorescence color changes from orange-red to indigo as the NADH concentration gradient increases, providing a technical basis for the development of smartphone-assisted visual instant detection. Therefore, the fluorescence sensing analysis method established in this study provides a new strategy for NADH detection.

[0110] It should be noted that the above embodiments are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. A person skilled in the art would be able to make other variations or modifications based on the above description. It is not possible to enumerate all embodiments here. Any obvious variations or modifications arising from the technical solution of the present invention remain within the scope of protection of the present invention.

Claims

1. A fluorescent carbon dot, characterized in that: It is obtained by solvent thermal method using triphenylamine and p-phenylenediamine as raw materials.

2. The fluorescent carbon dots according to claim 1, wherein The mass ratio of the p-phenylenediamine to triphenylamine is 1-2:2-1.

3. The fluorescent carbon dots according to claim 1, wherein The particle size of the fluorescent carbon dots is 1.80-5.00 nm, and the average particle size is 3.00-3.20 nm.

4. The method for preparing fluorescent carbon dots according to any one of claims 1 to 3, wherein The following steps are involved: S1, dissolving triphenylamine and p-phenylenediamine in a reaction solvent, and ultrasonically mixing to obtain a mixed solution; S2. The mixed solution is transferred to a polytetrafluoroethylene autoclave for thermal reaction. The obtained product is filtered, dialyzed and freeze-dried to obtain a finished product.

5. The preparation method according to claim 4, characterized in that In step S1, in the mixed solution, the concentration of triphenylamine is 0.005-0.01 g / mL; the concentration of p-phenylenediamine is 0.005-0.02 g / mL; the reaction solvent is anhydrous ethanol; and the pH of the mixed solution is 8-10.

6. The preparation method according to claim 4, characterized in that In step S2, the reaction temperature of the thermal reaction is 200-240°C, and the reaction time is 10-15 hours; the molecular weight cutoff of the dialysis bag used for dialysis is 1000-1500 Da, and the dialysis time is 24-28 hours; in step S2, the size of the filter membrane used for filtration is 0.22-0.24 μm.

7. Use of the fluorescent carbon dots according to any one of claims 1 to 3 in detecting nicotinamide adenine dinucleotide.

8. A fluorescent probe, characterized in that The fluorescent probe comprises the fluorescent carbon dots according to any one of claims 1 to 3.

9. The use of the fluorescent probe according to claim 8, characterized in that: The fluorescent probe is used in preparing a liver cancer early diagnosis reagent, a diabetes detection reagent, a phenylketonuria detection reagent, or a quality control reagent for NADH capsules.

10. A test paper, characterized in that The test paper comprises the fluorescent carbon dots according to any one of claims 1 to 3.

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