Fluorescent carbon dots and preparation method and application thereof

Abstract

The application belongs to the technical field of pharmaceutical preparations, and particularly relates to fluorescent carbon dots based on p-phenylenediamine and triphenylamine as well as a preparation method and application thereof. The fluorescent carbon dots are obtained by taking triphenylamine and p-phenylenediamine as raw materials through a solvothermal method. The application provides functionalized carbon dots based on p-phenylenediamine and triphenylamine, so that the carbon dots can realize 599 nm emission under 365 nm wavelength excitation. NADH forms a non-fluorescent ground-state complex with active sites on the surface of the carbon dots through a static quenching mechanism, resulting in 599 nm fluorescence quenching and 444 nm emission enhancement, and realizing ratio fluorescence detection of NADH. The method has good linear relationship, high selectivity, rapid response, high sensitivity, and a detection limit as low as 108 nM.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, specifically relating to a fluorescent carbon dot based on p-phenylenediamine and triphenylamine, its preparation method and application. Background Technology

[0002] Nicotinamide adenine dinucleotide (NADH), a core coenzyme in cellular redox reactions, is a key carrier of mitochondrial energy metabolism and the electron transport chain. Its concentration dynamics directly reflect the body's redox homeostasis and mitochondrial functional status. Clinical studies have shown that abnormal fluctuations in serum NADH levels are closely related to various major diseases, such as the progression of diabetes, where the NADH / NAD ratio is significantly affected. + An imbalance in the ratio can exacerbate the accumulation of reactive oxygen species, leading to oxidative stress damage to pancreatic β-cells; malignant tumor cells can excessively accumulate NADH through the Warburg effect, 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, developing highly sensitive and specific quantitative analytical methods for NADH is a crucial technical requirement for clinical diagnosis.

[0003] Currently, the quantitative analysis of NADH mainly relies on three types of techniques: enzymatic cycling, high-performance liquid chromatography (HPLC), and electrochemical sensors. Enzymatic methods utilize NAD+... + While NADH cyclic reactions amplify the signal, their specificity is heavily dependent on enzyme activity, easily affected by interfering substances, and reagent costs are high. HPLC, although capable of high-precision separation, requires complex sample pretreatment and has a large instrument size, making it difficult to meet the needs of point-of-care testing (POCT). Electrochemical sensors offer portability, but the stability of electrode modification materials is insufficient, and electroactive substances such as uric acid in serum can easily induce false positive signals.

[0004] Fluorescence and colorimetric sensing technologies have garnered significant attention in the field of bioanalysis in recent years due to their rapid response, ease of operation, and signal visualization capabilities. However, traditional single-wavelength fluorescent probes are susceptible to interference from ambient temperature, light source fluctuations, and probe concentration variations, leading to insufficient reliability of detection results. In contrast, ratiometric fluorescence sensing, by simultaneously monitoring the signal ratio of two emission channels, achieves built-in self-calibration, significantly improving detection stability and accuracy. Furthermore, the inherent color gradient characteristics of the dual-emission system enable semi-quantitative visual analysis without the need for sophisticated instruments, providing a unique advantage for rapid on-site screening. Carbon quantum dots (CDs), as novel fluorescent nanomaterials, have become an ideal carrier 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 light emission, and the few synthesized orange and red carbon dots often require excitation at around 550 nm. The small Stokes shift poses a significant challenge to the design of ratiometric fluorescence sensors and makes it difficult to distinguish color changes, greatly limiting their application potential in NADH detection.

[0005] For example, prior art CN 117887460 A discloses a ratiometric fluorescent carbon dot for tracing nicotinamide adenine dinucleotide in liver cancer glycolysis. This ratiometric fluorescent carbon dot is obtained via a solvothermal method using aniline derivatives and aromatic disulfides as carbon and nitrogen sources, respectively. The carbon dots prepared by this invention show a significant increase in NAD+ concentration. + As concentration increases, carbon dots migrate towards red fluorescence, and the ratio of fluorescence intensity F640 / F520 to NAD... + The linear relationship was good, and the uptake of carbon dots in HepG2 cells was significantly reduced after treatment with the glycolysis inhibitor 2-DG. However, although the invention states that the carbon dots are essentially non-cytotoxic, experiments have shown that high concentrations of carbon dots can have a certain toxic effect on cells. For example, in Example 1, when the carbon dot concentration 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 can have a certain negative impact on cells. In addition, the biocompatibility of the carbon dots in this invention is questionable. Secondly, its linear range is too broad, making it difficult to apply to different tissues and cells. For example, in blood, NAD... + At relatively low concentrations, around the micromolar level, the sensitivity of the invention's tests is insufficient.

[0006] Existing technology CN 115404074 B discloses a method for preparing a fluorescent detection nanoprobe, which includes the following steps: synthesizing blue fluorescent carbon dots (BCD) via a hydrothermal method; synthesizing red fluorescent carbon dots (RCD) via a solvothermal method; mixing the prepared BCD and RCD in a certain proportion, co-incubating with Waterman filter paper, and drying in an oven to obtain the fluorescent detection nanoprobe. However, it is evident that the preparation of this probe involves steps such as co-incubating blue and red fluorescent carbon dots with Waterman filter paper. This preparation method can lead to insufficient stability of the probe during long-term storage or use, thus affecting the consistency and reliability of detection. Summary of the Invention

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

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

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

[0010] The planar benzene ring of p-phenylenediamine serves as the basic conjugated unit. Under solvothermal conditions, it undergoes dehydration condensation and carbonization recombination with the polycyclic structure of triphenylamine to form a carbon core structure containing specific multi-level conjugated structures, thereby enhancing the π-π structure of the carbon dots. * The transition efficiency drives the red shift of the fluorescence emission peak of carbon dots, thereby enhancing light absorption and fluorescence stability.

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

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

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

[0014] S1. Dissolve triphenylamine and p-phenylenediamine in a reaction solvent and mix them by ultrasonication to obtain a mixed solution;

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

[0016] In one preferred embodiment, in step S1, the concentration of triphenylamine in the mixed solution is 0.005–0.01 g / mL; and 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 thermal reaction temperature is 200-240°C and the reaction time is 10-15 hours.

[0020] This temperature range favors the condensation and conjugated expansion of aromatic rings, enhancing red light emission. Higher temperatures promote the growth of larger splines. 2 The formation of conjugated domains (dominated by π→π* transitions) leads to a redshift in carbon point emission (e.g., from blue to orange). Too low a temperature will prevent the production of orange fluorescence, while too high a temperature (e.g., >250℃) will cause excessive graphitization, resulting in broadening or even quenching of the emission peak.

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

[0022] The molecular weight cutoff of a dialysis bag determines the range of molecular sizes of substances that can pass through the dialysis bag.

[0023] In one preferred embodiment, in step S2, the size of the filtered membrane 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 the detection of nicotinamide adenine dinucleotide (NADH).

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

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

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

[0028] Based on the same inventive concept, this invention also claims protection for the application of the fluorescent probe, specifically its use in the preparation of reagents for early diagnosis of liver cancer, reagents for diabetes detection, and reagents for phenylketonuria detection.

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

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

[0031] In one preferred embodiment, the test strip is prepared by loading the fluorescent carbon dots onto the test strip and lyophilizing it, and comparing the color changes to perform quantitative analysis of NADH.

[0032] The present invention will be further explained below:

[0033] This invention provides a functionalized carbon dot (PDA-TPA-CDs) based on p-phenylenediamine (PDA) and triphenylamine (TPA). Orange fluorescent carbon dots were synthesized by controlling the pH value through a one-step solvothermal method, and the amino groups on the surface of the carbon dots and the sp groups on the carbon core were highly reactive. 2 The synergistic effect of the conjugated structure endows the material with a dual-channel excited-state energy level structure, enabling it to achieve 599nm emission under 365nm excitation. NADH forms a non-fluorescent ground-state complex with the active sites on the carbon dot surface through a static quenching mechanism, resulting in 599nm fluorescence quenching and 444nm emission enhancement, thus achieving ratiometric fluorescence detection of NADH. This method exhibits good linearity, high selectivity, rapid response, high sensitivity, and a detection limit as low as 108 nm.

[0034] In this invention, a ratio sensing system was successfully constructed based on the static quenching effect of NADH on the 599 nm emission peak and the enhanced response to the 444 nm emission peak. This system exhibits excellent linearity in the 0-550 μM range, with a detection limit as low as 108 nM, and demonstrates high selectivity for NADH. Furthermore, the fluorescent probe of this invention responds rapidly, within seconds, and the color of the sensing solution changes from orange-red to indigo with the NADH concentration gradient, providing a feasible basis for subsequent integration with smartphone image analysis programs to complete on-site semi-quantitative detection. Therefore, this invention provides a novel nanotool for the highly sensitive detection of NADH, offering new methods for early screening of metabolic diseases (diabetes detection, phenylketonuria), quality monitoring of health supplements (such as NADH capsules), and personalized health management. Attached Figure Description

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

[0036] Figure 2 The FT-IR spectra of PDA-TPA-CDs;

[0037] Figure 3 To provide an in-depth analysis of the surface chemical composition and bonding configuration of PDA-TPA-CDs using XPS technology, among other things, 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 diagram shows the optimized synthesis conditions for PDA-TPA-CDs; where, Figure 4 a is the fluorescence curve of PDA-TPA-CDs caused by changes in the 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 shows the fluorescence curves of PDA-TPA-CDs caused by frequent changes in synthesis time;

[0039] Figure 5 The diagram shows the fluorescence characteristics and detection mechanism of PDA-TPA-CDs; among them, Figure 5 a represents the emission spectra of PDA-TPA-CDs at different excitation wavelengths; Figure 5 b shows the absorption spectrum (green), excitation spectrum (red), and emission spectrum (blue) of PDA-TPA-CDs; Figure 5 c represents the fluorescence lifetime of PDA-TPA-CDs and PDA-TPA-CDs+NADH; Figure 5 d represents the UV-Vis spectra of PDA-TPA-CDs, NADH, and PDA-TPA-CDs+NADH;

[0040] Figure 6 The fluorescence spectra and linear relationships of PDA-TPA-CDs in the presence of NADH are shown; among them, Figure 6 a is the fluorescence spectrum of PDA-TPA-CDs in the presence of 0-275 μM NADH; Figure 6 b represents the linear relationship between 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 represents the linear relationship between PDA-TPA-CDs in the presence of 275-550 μM NADH;

[0041] Figure 7 For the detection performance of PDA-TPA-CDs, among which, Figure 7 a shows the emission spectra of PDA-TPA-CDs under NADH (200 Mm) and ten other analytes under 365 nm excitation, with PBS used as a control; Figure 7 b represents the fluorescence ratio of PDA-TPA-CDs under NADH (200 Mm) excitation to ten other analytes under 365 nm excitation; Figure 7 c represents the rapid response performance of PDA-TPA-CDs after rapid injection of NADH (1mM); Figure 7 d represents the photostability of PDA-TPA-CDs. Detailed Implementation

[0042] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[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 (NADH) were also present. + The following were supplied by TCI (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.; and 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 sonication for 5 min, resulting in a solution pH of 9. The solution was then placed in a 100 mL polytetrafluoroethylene-lined reactor and heated at 200 °C for 12 h. After the reaction, the mixture was allowed to cool naturally to room temperature, centrifuged at 10,000 rpm for 10 min, filtered through a 0.22 μm filter, and dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 1000 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 performance of the obtained PDA-TPA-CDs was tested. Fluorescence spectra were obtained using a Hitachi F-4700 fluorescence spectrometer (Japan), with the following parameters: 365 nm excitation, slit width 10-20 mm, and voltage 400 V. Ultraviolet spectra were obtained using a Shimadzu UV-2600i (Japan). Fourier transform infrared spectroscopy (FT-IR) was performed using a Shimadzu IRTracer 100 (Japan). X-ray photoelectron spectroscopy (XPS) was performed using a Thermo Fisher Scientific NEXSA (USA). X-ray polycrystalline diffraction (XRD) was performed using a Bruker D8 Advance (Germany). Field emission transmission electron microscopy (TEM) was performed using a JEOL F200 thermal field emission transmission electron microscope.

[0049] Quantum yield is an important indicator for evaluating the optical properties of luminescent materials, and both it and fluorescence lifetime were measured using the Edinburgh-FLS1000. Quantum yield can be expressed by the following formula:

[0050]

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

[0052]

[0053] Where A1 and A2 are the exponential factors, and t1 and t2 are the decay times.

[0054] The results of morphological analysis by field emission transmission electron microscopy are as follows: Figure 1 As shown, where Figure 1 a is a TEM image of PDA-TPA-CDs; Figure 1 b represents the size distribution of PDA-TPA-CDs; Figure 1 c is the XRD image of the PDA-TPA-CDs. For example... Figure 1 As shown in a and b, the synthesized PDA-TPA-CDs are spherical and exhibit good monodispersity, with particle sizes ranging from 1.91 to 4.95 nm and an average particle size of 3.20 nm. The high-resolution transmission electron microscopy (TEM) images are shown in the figures. PDA-TPA-CDs possess a distinct lattice structure with a lattice spacing of 0.21 nm, which can be attributed to in-plane (100) lattice graphene carbon. The XRD pattern of PDA-TPA-CDs (…) Figure 1 In c), a broad diffraction peak appears at 2θ = 26.67, which can be attributed to the typical characteristics of the (002) crystal plane of graphitized carbon.

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

[0056] XPS technology was used to analyze the surface chemical composition and bonding configuration of PDA-TPA-CDs in depth, and the results are as follows: Figure 3 As shown. Broadband scanning results indicate that ( Figure 3 a) The synthesized PDA-TPA-CDs are composed of three elements: C, N, and O. Quantitative analysis showed that their C / N / O atomic percentages were 74.97 / 14.72 / 7.31, respectively. High-resolution spectral analysis showed that the C 1s spectrum ( Figure 3 b) Gaussian peak analysis revealed three characteristic peaks (284.13 eV, 284.81 eV, 289.34 eV), 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 The spectroscopic deconvolution results in c) show that the doublets at 398.76 eV and 399.88 eV characterize the electronic states of pyridine (CN=C) and pyrrole (CNC) nitrogen, respectively. Notably, the O 1s spectrum ( Figure 3d) Characteristic vibrational modes are exhibited at 531.93 eV and 535.35 eV, which can be clearly identified as chemical fingerprints of carboxyl groups (C=O) and ether bonds (CO). These collectively confirm the presence of a rich network of nitrogen and oxygen functional groups on the material surface, a characteristic that significantly enhances the colloidal stability and surface active site density of the quantum dots. Furthermore, measurements using a reference method show that these carbon quantum dots exhibit an absolute fluorescence quantum yield of 16.34%, superior to conventional carbon-based fluorescent materials.

[0057] Comparative Example 1

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

[0062] The results showed that orange-red carbon dots could not be obtained.

[0063] Comparative Example 3

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

[0065] The results showed that weak orange-red carbon dots were obtained, but they did not have a specific response to NADH.

[0066] Comparative Example 4

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

[0068] The results showed that orange-red carbon dots could not be obtained.

[0069] Comparative Example 5

[0070] Based on Example 1, by changing the solvent polarity, 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 spectra were then measured using a Hitachi F-4700 fluorescence spectrometer, following the same testing method 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 impact on the emission wavelength and fluorescence intensity of CDs. When the solvent is anhydrous ethanol, orange CDs with better fluorescence performance are obtained.

[0072] Example 2

[0073] Based on Example 1, the PDA:TPA mass ratio was adjusted to five ratios: 1:2, 1:1, 2:1, 4:1, and 8:1, respectively, to obtain carbon quantum dots. Fluorescence spectra were then measured using a Hitachi F-4700 fluorescence spectrometer, following the same testing method as in Example 1. The results are as follows: Figure 4 As shown in Figure a, carbon quantum dots exhibit good fluorescence performance at ratios of 1:2, 1:1, and 2:1. However, the lowest R0 (i.e., the ratio of emission peak value at 599 nm to emission peak value at 444 nm) is obtained at a 2:1 ratio, indicating that carbon quantum dots with the best fluorescence performance are obtained at the 2:1 ratio.

[0074] Example 3

[0075] Based on Example 1, the pH value of the reaction system was precisely controlled using HCl / NaOH solution to investigate the performance differences of synthesized PDA-TPA-CDs under different pH conditions (pH values ​​of 3, 5, 7, 9, and 11). Fluorescence spectra were measured using a Hitachi F-4700 fluorescence spectrometer, following 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 impact on the fluorescence intensity and emission peak position of carbon quantum dots. Under acidic and neutral conditions, PDA-TPA-CDs emit bright blue light around 450 nm. As the pH value increases, the emission wavelength redshifts, emitting orange light at pH 9 and yellow light at pH 11.

[0077] However, to construct a ratiometric fluorescence sensing system, two peaks with good separation are required. If the material using blue and yellow light is too close to the peak of NADH itself, the peak shape will be affected and indistinguishable, making it impossible to construct a good ratiometric fluorescence sensing system. Therefore, this invention uses orange light, i.e., at a pH of 8-10.

[0078] Example 4

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

[0080] Through synergistic optimization of the above parameters, PDA-TPA-CDs with stable fluorescence performance were successfully prepared, laying a material foundation for building a high-selectivity ratio fluorescence sensing platform.

[0081] Example 5

[0082] Fluorescence properties 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 a UV-Vis spectrophotometer system. Fluorescence spectra were obtained using a Hitachi F-4700 fluorescence spectrometer (Japan), with the following parameters: 365 nm excitation, slit width 10 20, and voltage 400 V. UV spectra were obtained using a Shimadzu UV-2600i (Japan).

[0084] As shown in the fluorescence spectrum, the intensity and peak position of fluorescence emission are determined by the excitation wavelength; therefore, the effect of the excitation wavelength on the fluorescence intensity was investigated. Figure 5 As shown in a and b, PDA-TPA-CDs exhibit dual excitation peaks at 365 nm and 510 nm, indicating a multi-channel excited-state energy level structure. Notably, despite the excitation wavelength varying within the 340-580 nm range, the emission peak remains stable at 599 nm, demonstrating significant excitation wavelength independence, suggesting the formation of a unified radiative recombination center within the carbon dots. For ease of colorimetric analysis, 365 nm was chosen as the excitation wavelength for subsequent experiments. Under natural light, the synthesized PDA-TPA-CDs are a clear, transparent, light orange solution. Under a 365 nm UV lamp, PDA-TPA-CDs exhibit a bright orange color. Figure 5 As shown in b, PDA-TPA-CDs exhibit two absorption peaks at 242 nm and 295 nm, corresponding to the transition of lone pair electrons (n ​​orbitals) from nitrogen-containing polar groups (such as C-NH2) on the surface to π* antibonding orbitals and the transition of sp electrons in the carbon core, respectively. 2The π-π* electronic transitions in the hybrid conjugated aromatic structure. This dual-mode absorption characteristic confirms the co-luminescence mechanism of the surface states and carbon core states of PDA-TPA-CDs, providing theoretical support for their wavelength-independent emission behavior.

[0085] Studies on the fluorescence quenching mechanism based on carbon dots require the systematic exclusion of multiple potential mechanisms, including static quenching, dynamic quenching, energy transfer, photoinduced electron transfer, and internal filtering effects. Therefore, the fluorescence quenching mechanism of the PDA-TPA-CDs prepared in Example 1 was analyzed using a fluorescence spectrometer and a UV-Vis spectrophotometer system.

[0086] The results show that the absorption spectrum of NADH does not effectively overlap with the excitation spectrum of PDA-TPA-CDs. Figure 4 b) and d), thus ruling out the internal filtration effect. TPA, as a typical electron donor, suggests the possibility of photoinduced electron transfer due to its structural characteristics. However, the addition of NADH resulted in a new absorption peak in the PDA-TPA-CDs solution, indicating the formation of a non-fluorescent ground-state complex. Therefore, the quenching mechanism is inferred to be static quenching. The core criterion for static quenching lies in the constancy of fluorescence lifetime; therefore, this invention recorded the fluorescence decay curves of PDA-TPA-CDs and the PDA-TPA-CDs~NADH system. Figure 5 As shown in 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] Based on the characteristic emission peaks of NADH (blue emission at 444 nm) and PDA-TPA-CDs (orange emission at 599 nm), a dual-emission ratio fluorescence sensing platform was constructed. A 0.1 M NADH solution was prepared as a stock solution. The NADH stock solution was then sequentially added to the PDA-TPA-CDs solution to prepare a mixed analyte solution with a concentration gradient of 0–550 μM. After homogenization, the fluorescence spectra under 365 nm excitation were measured using a fluorescence spectrophotometer, with each data point repeated three times.

[0089] Precision was assessed by continuously measuring the fluorescence response signal of the same PDA-TPA-CDs (Example 1) and NADH composite system six times. 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 dual-emission fluorescence intensity and NADH concentration under 365nm excitation was systematically investigated. A good linear relationship was found between the fluorescence intensity and NADH concentration. Figure 6As shown in Figure a, with increasing NADH concentration (0–275 μM), the fluorescence intensity at 599 nm gradually decreased, while the fluorescence intensity at 444 nm increased. The concentration of NADH showed a good linear relationship with the F444 / F599 ratio. Figure 6 b). At higher NADH concentrations (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 below:

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

[0092] Method 2: The test was performed on the “Experimental Section” on pages 7119 and 7120 of reference [2].

[0093] Method 3: The detection is performed using the methods described in sections 2.3 to 2.5 of reference [3].

[0094] Method 4: The detection is performed using the methods described in sections 2.3 to 2.4 of reference [4].

[0095] The summarized data is as follows:

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

[0097]

[0098] As can be seen, compared with Method 1, the detection method of the present invention has a sensitivity that is nearly 10 times higher. Furthermore, it eliminates the need for complex photodetector arrays and microcavity integration; the fluorescence spectrum can be directly measured simply by mixing carbon dots with the sample, making the operation process simple.

[0099] Method 2 requires engineered nanoporous proteins and highly sensitive current recording equipment, making it technically challenging and expensive. In contrast, Method 2 employs a one-step solvothermal method to synthesize carbon dots, resulting in lower raw material costs. The detection process requires only 30 seconds of mixing and reaction, eliminates the need for specialized equipment, and allows for subsequent development of smartphone image analysis for semi-quantitative detection. This makes it more practical for rapid on-site detection.

[0100] Compared to Method 3, this method requires no electrode modification or potential control, and the detection process only needs a 365nm excitation 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 to method 4, it has a wider linear range 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] To better apply the developed fluorescent probe to real samples and verify the reliability of the sensing system in complex matrices, other common analytes (Mg) were selected. 2+ Cl - Zn 2+ Ca 2+ Na + Selectivity studies were conducted on NAD+, Glc, CA, Phe, and Glu. The analyte concentration was 200 μM, with PBS added as a control. Results are as follows: Figure 7 As shown in a and b, the fluorescence signal ratios of other analyte systems did not change significantly compared to the blank group, while the fluorescence signal ratio of the NADH system increased significantly, fully demonstrating that the fluorescent probe has a high specific recognition ability for NADH. When NADH is rapidly injected into the solution, the PDA-TPA-CDs fluorescent probe responds rapidly within a few seconds. Figure 7 c) This indicates that the sensing system meets the requirements of point-of-care testing (POCT). Furthermore, after irradiating the PDA-TPA-CDs solution with a xenon lamp for 30 minutes, the fluorescence intensity showed almost no change; therefore, PDA-TPA-CDs exhibit good resistance to photobleaching. Figure 7 d) Suitable for long-term monitoring scenarios.

[0109] In summary, this invention innovatively synthesizes carbon quantum dots with blue, yellow, and orange fluorescence under different pH conditions using p-phenylenediamine and triphenylamine as precursors via a one-step solvothermal method. Among them, PDA-TPA-CDs prepared at pH=9 exhibit unique dual-excitation-center characteristics (λex=365 / 510nm) and wavelength-independent emission features. Based on the synergistic luminescence mechanism of their surface and carbon core states, a self-calibrating ratiometric fluorescence sensing system was successfully constructed. NADH suppresses the 599nm emission peak through static quenching while enhancing the 444nm fluorescence emission. The dual-signal ratio (F444 / F599) shows a high-precision linear response to NADH concentration in the 0-550μM range, with a detection limit as low as 108nM. Compared to traditional single-signal carbon dot probes, this method effectively eliminates environmental interference and significantly improves reliability. The feasibility of the method is evaluated, and the results show that it has good stability, high sensitivity, and high specificity for NADH. Furthermore, the fluorescence color gradually changes from orange-red to indigo with the NADH concentration gradient, providing a technological basis for developing smartphone-assisted, visualized, real-time 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 clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A fluorescent carbon dot, characterized in that, It is obtained by using triphenylamine and p-phenylenediamine as raw materials through a solvothermal method; during the reaction, the solvent is anhydrous ethanol, and the pH of the reaction solution is 8-10.

2. The fluorescent carbon dot according to claim 1, characterized in that, The mass ratio of p-phenylenediamine to triphenylamine is 1-2:2-1.

3. The fluorescent carbon dot according to claim 1, characterized in that, The fluorescent carbon dots have a particle size of 1.80-5.00 nm and an average particle size of 3.00-3.20 nm.

4. The method for preparing fluorescent carbon dots according to any one of claims 1-3, characterized in that, Includes the following steps: S1. Dissolve triphenylamine and p-phenylenediamine in a reaction solvent and mix them by ultrasonication to obtain a mixed solution; S2. The mixed solution is transferred to a polytetrafluoroethylene autoclave for thermal reaction. The resulting product is then filtered, dialyzed, and freeze-dried to obtain the final product.

5. The preparation method according to claim 4, characterized in that, In step S1, the concentration of triphenylamine in the mixed solution 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℃, and the reaction time is 10-15h; the molecular weight cutoff of the dialysis bag used for dialysis is 1000-1500Da, and the dialysis time is 24-28h; in step S2, the size of the filter membrane used for filtration is 0.22-0.24μm.

7. The use of the fluorescent carbon dots according to any one of claims 1-3 in the detection of nicotinamide adenine dinucleotide.

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

9. The application of the fluorescent probe according to claim 8, characterized in that, The fluorescent probe can be used to prepare reagents for early diagnosis of liver cancer, reagents for diabetes detection, reagents for phenylketonuria detection, or as a quality control reagent for NADH capsules.

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

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