A ratio phosphorescent probe material for detecting 2,6-pyridinedicarboxylic acid and a preparation method and application thereof

Abstract

The application discloses a kind of ratio phosphorescent probe materials for detecting 2,6-pyridine dicarboxylic acid and its preparation method and application.The material is europium ion modified nitrogen / chlorine co-doped carbon dot@AlPO-5 molecular sieve composite material, wherein carbon dot is in situ confined in molecular sieve channel, and europium ion is loaded on the surface or in the channel of molecular sieve.In the presence of 2,6-pyridine dicarboxylic acid, due to the confinement protection effect of molecular sieve, the phosphorescent lifetime of sensitized europium ion is 0.135 s, which is much higher than the microsecond-level lifetime of traditional europium complex.During detection, the phosphorescence of carbon dot is quenched while the phosphorescence of europium ion is enhanced, forming a dual-channel ratio response.Combined with the time-resolved detection mode of delay time 0.1 s, high sensitivity and high selectivity quantitative detection of 2,6-pyridine dicarboxylic acid can be realized.The material of the application is simple to prepare, has high detection sensitivity, good selectivity, strong anti-interference ability, and has wide application prospect in the field of public health monitoring.

Description

Technical Field

[0001] This invention belongs to the field of luminescent materials and biological detection technology, specifically relating to a ratiometric phosphorescent probe material for detecting 2,6-pyridinedicarboxylic acid, its preparation method, and its application. Background Technology

[0002] 2,6-Pyridinedicarboxylic acid (DPA, also known as pyridine-2,6-dicarboxylic acid) is a unique chemical component of the cortex of Bacillus spores, accounting for 5% to 15% of the dry weight of the spores. It is an important biomarker for detecting Bacillus spores (such as Bacillus anthracis). Bacillus anthracis spores possess extremely strong resistance and pathogenicity, posing a serious threat to public health and safety. Therefore, developing rapid, sensitive, and selective methods for detecting DPA is of great significance for food safety monitoring, clinical diagnosis, environmental monitoring, and bioterrorism.

[0003] Currently, common methods for detecting DPA include high-performance liquid chromatography (HPLC), polymerase chain reaction (PCR), and colorimetry. However, these methods generally suffer from drawbacks such as cumbersome operation, long detection cycles, expensive equipment, and difficulty in achieving rapid on-site detection. Optical detection methods, due to their advantages of simple operation, rapid response, and high sensitivity, have gradually become a research hotspot in the field of DPA detection. Among them, fluorescent probe methods and phosphorescent probe methods have attracted much attention because they can achieve real-time, in-situ detection.

[0004] Ratio-response optical probes, by measuring the ratio of luminescence intensity at two different wavelengths, can effectively avoid interference from probe concentration, excitation light intensity fluctuations, and environmental factors, enabling precise quantitative detection. They have been widely used in the field of biosensing in recent years. Time-resolved detection technology, by setting an appropriate delay time, can eliminate interference from short-lived background fluorescence (such as the autofluorescence of proteins and amino acids in biological samples), further improving the signal-to-noise ratio and accuracy. Therefore, developing probe materials that simultaneously possess ratio-response characteristics and long-lived luminescence signals has significant application value.

[0005] rare earth ion Eu 3+ It possesses a unique line emission spectrum, a large Stokes shift, and a long luminescence lifetime, and its luminescence is easily sensitized and enhanced by DPA molecules through the "antenna effect," thus it is widely used for DPA detection. However, traditional Eu... 3+ The luminescence lifetime of complexes in solution is typically only in the microsecond range. Although this differs from the lifetime of background fluorescence in biological samples (in the nanosecond range), it is still difficult to completely eliminate interference from long-lived backgrounds such as phosphorescence and scattered light in complex biological matrices. To achieve more accurate time-resolved detection, the lifetime of the response signal needs to be further extended to the millisecond or even hundreds of millisecond range.

[0006] Carbon dots, as a novel zero-dimensional carbon-based nanomaterial, possess excellent optical properties, good biocompatibility, low toxicity, and ease of functionalization. Studies have shown that confining carbon dots within a molecular sieve matrix can effectively suppress their non-radiative transitions, resulting in stable, long-lifetime room-temperature phosphorescence emission. However, how to integrate the stable phosphorescence of carbon dots with Eu... 3+ By organically combining responsive luminescence with the structure of DPA, a DPA probe material with both long lifetime and ratiometric response capabilities was constructed, and Eu after DPA sensitization was achieved. 3+ No significant improvement in luminescence lifetime has been reported in the literature to date.

[0007] In the existing technology, there are already literature reports on Eu 3+ Co-loading of luminescent guests with porous materials for DPA detection. For example, Chinese patent application CN110903826A discloses a fluorescent probe based on a zeolite-based imidazolium ester (ZIF) framework. This probe is constructed by co-loading CdS quantum dots, rhodamine 6G dye, and europium ions onto a ZIF framework, utilizing DPA and Europium ions... 3+ The principle of post-recovery fluorescence of CdS quantum dots is used to achieve ratiometric fluorescence detection of DPA. However, this technique has the following drawbacks: First, the organic framework structure of ZIF materials has relatively poor stability and may degrade in complex detection environments; second, CdS quantum dots contain the heavy metal cadmium, which has potential biotoxicity, limiting its application in biological samples; third, this probe is still based on steady-state fluorescence detection and does not involve the regulation and utilization of luminescence lifetime, making it difficult to completely eliminate long-lifetime background interference.

[0008] In view of the above-mentioned shortcomings of the existing technology, developing a novel DPA probe material with good biocompatibility, high stability, ultra-long response signal and time-resolved ratio detection remains an urgent technical problem to be solved in this field. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention provides a ratiometric phosphorescent probe material for detecting 2,6-pyridinedicarboxylic acid, its preparation method, and its application. The material utilizes the molecular sieve confinement effect to sensitize Eu with DPA. 3+ The luminescence lifetime is significantly extended to over 0.1s, and combined with carbon dot phosphorescence and Eu 3+ The synergistic response of phosphorescence through a dual-channel system of "one decrease and one increase" enables the detection of 2,6-pyridinedicarboxylic acid with high sensitivity and high selectivity at a time resolution ratio.

[0010] This invention is achieved through the following technical solution:

[0011] A ratiometric phosphorescent probe material for detecting 2,6-pyridinedicarboxylic acid, wherein the material is a europium ion-modified nitrogen / chlorine co-doped carbon dot@AlPO-5 molecular sieve composite material, wherein:

[0012] The nitrogen / chlorine co-doped carbon dots were synthesized in one step by solid-phase grinding combined with hydrothermal crystallization using an ethylenediamine-hydrochloric acid system as a precursor and confined in situ within the pore structure of AlPO-5 molecular sieve.

[0013] The europium ions are loaded on the surface or within the pores of the molecular sieve;

[0014] In the presence of 2,6-pyridinedicarboxylic acid, the phosphorescence lifetime of the europium ions after sensitization is greater than 0.1 s;

[0015] The material emits both room-temperature phosphorescence of carbon dots and characteristic phosphorescence of europium ions under excitation light irradiation.

[0016] Preferably, in the presence of 2,6-pyridinedicarboxylic acid, the phosphorescence lifetime of the europium ion after sensitization is 0.15 s; and when detecting 2,6-pyridinedicarboxylic acid, the characteristic phosphorescence intensity of the europium ion is enhanced, while the room temperature phosphorescence intensity of the carbon dots undergoes concentration-dependent quenching, forming a dual-channel ratio response.

[0017] The preparation method of the above-mentioned ratio phosphorescent probe material includes the following steps:

[0018] Step 1) Preparation of nitrogen / chlorine co-doped carbon dot precursor solution: Mix ethylenediamine with hydrochloric acid to react and obtain nitrogen / chlorine co-doped carbon dot precursor solution;

[0019] Step 2) Preparation of phosphorus source precursor: Di-n-propylamine is reacted with phosphoric acid to obtain phosphoric acid-di-n-propylamine precursor;

[0020] Step 3) Preparation of carbon dot @AlPO-5 composite material: The aluminum source, the phosphate-di-n-propylamine precursor obtained in step 2) and the template agent are mixed and ground, and the nitrogen / chlorine co-doped carbon dot precursor solution obtained in step 1) is added. After further grinding, the mixture is subjected to hydrothermal crystallization to obtain the nitrogen / chlorine co-doped carbon dot @AlPO-5 composite material.

[0021] Step 4) Europium ion post-modification: The nitrogen / chlorine co-doped carbon dot @AlPO-5 composite material obtained in step 3) is immersed in europium ion solution and subjected to low-temperature hydrothermal reaction to obtain europium ion modified nitrogen / chlorine co-doped carbon dot @AlPO-5 composite material.

[0022] Preferably, the volume ratio of ethylenediamine to hydrochloric acid in step 1) is 0.5:1; the volume ratio of di-n-propylamine to phosphoric acid in step 2) is 14:5; and the mass ratio of aluminum source, phosphate-di-n-propylamine precursor and template agent in step 3) is 0.36:0.80:0.42.

[0023] Preferably, the aluminum source in step 3) is boehmite, the template agent is tetraethylammonium bromide, and the hydrothermal crystallization temperature is 220°C for 24 hours.

[0024] Preferably, the europium ion solution in step 4) is europium nitrate or europium chloride solution with a concentration of 100 μM; the temperature of the low-temperature hydrothermal reaction is 60°C and the time is 2 h.

[0025] A method for detecting 2,6-pyridinedicarboxylic acid, comprising quantitative detection and selective detection;

[0026] The quantitative detection steps are as follows: the ratio phosphorescent probe material described above is mixed with the sample to be tested containing 2,6-pyridinedicarboxylic acid, and the intensity ratio of europium ion characteristic phosphorescence to carbon point room temperature phosphorescence is determined by time-resolved phosphorescence spectroscopy. Based on the functional relationship between this ratio and the concentration of 2,6-pyridinedicarboxylic acid, the quantitative detection of 2,6-pyridinedicarboxylic acid is achieved.

[0027] The selective detection steps are as follows: the above-mentioned ratio phosphorescent probe material is mixed with 2,6-pyridinedicarboxylic acid and potential interfering substances respectively, and the intensity ratio of europium ion characteristic phosphorescence to carbon point room temperature phosphorescence is determined by time-resolved phosphorescence spectroscopy to evaluate the material's selective response to 2,6-pyridinedicarboxylic acid and its anti-interference ability.

[0028] Preferably, the time-resolved phosphorescence spectroscopy method is set with a delay time of 0.1 s; the characteristic phosphorescence of europium ions is phosphorescence at 616 nm, and the room-temperature phosphorescence of carbon dots is phosphorescence at 515 nm; the potential interfering substances include amino acids, glucose, citric acid, urea, nicotinic acid, phthalic acid, isophthalic acid, terephthalic acid, boric acid, 2,5-pyridinedicarboxylic acid, and inorganic ions.

[0029] The above-mentioned ratiometric phosphorescent probe material is used in the detection of 2,6-pyridinedicarboxylic acid.

[0030] The application of the aforementioned ratiometric phosphorescent probe material in the preparation of a kit for detecting Bacillus spores.

[0031] The beneficial effects of this invention are as follows:

[0032] (1) This invention is the first to combine the stable room-temperature phosphorescence of carbon dots with the responsive phosphorescence of rare-earth europium ions in the same AlPO-5 molecular sieve matrix, constructing a novel ratiometric phosphorescent probe, which is then applied to the detection of 2,6-pyridinedicarboxylic acid. This material can simultaneously emit room-temperature phosphorescence (~515 nm) of carbon dots and characteristic phosphorescence (~616 nm) of europium ions under a single excitation wavelength, providing a new technical solution for the ratiometric detection of DPA and expanding the application scope of carbon dot@molecular sieve composite materials in the field of biosensing.

[0033] (2) This invention uses AlPO-5 type aluminum phosphate molecular sieve as the confinement matrix, which has an electrically neutral framework and a one-dimensional straight-channel structure (pore size of about 0.73 nm), providing an ideal confinement environment for carbon dots. This confinement effect effectively suppresses the nonradiative transitions of carbon dots, enabling them to obtain stable long-lifetime room-temperature phosphorescence emission. At the same time, the regular channel structure of AlPO-5 molecular sieve is beneficial to Eu 3+ The coordination loading and diffusion enrichment of DPA molecules, along with the good thermal and chemical stability of its inorganic framework, enable the composite material to maintain structural integrity and stable optical properties in complex testing environments.

[0034] (3) In the nitrogen / chlorine co-doped carbon dot @AlPO-5 confined system of the present invention, the DPA molecule acts as an "antenna ligand" and interacts with Eu. 3+ Coordination effectively sensitizes its luminescence. Due to the confinement protection effect of AlPO-5 molecular sieve, Eu... 3+ The nonradiative transition pathway of the -DPA complex is significantly suppressed, making the DPA-sensitized Eu... 3+ The luminescence lifetime reaches 0.135 s, which is significantly longer than that of traditional Eu. 3+ The complex exhibits a microsecond-level lifetime improvement of approximately 2 to 3 orders of magnitude in solution. This characteristic enables time-resolved detection of the material with a delay time of 0.1 s, effectively eliminating interference from short-lived background fluorescence (nanosecond to microsecond level) in biological samples, and significantly improving the detection signal-to-noise ratio and accuracy.

[0035] (4) The material of the present invention has unique dual-channel response characteristics: on the one hand, DPA acts as an "antenna ligand" and Eu 3+ Coordination sensitizes its luminescence, resulting in a phosphorescence intensity (P616) at 616 nm. 616 The phosphorescence of the carbon dots at 515 nm is significantly enhanced; on the other hand, there is an interaction between DPA and carbon dots, resulting in room temperature phosphorescence (P) of the carbon dots at 515 nm. 515 Concentration-dependent quenching occurs. This coordinated signal change of "one increase and one decrease" causes P to... 616 / P 515 The ratio changes more significantly with DPA concentration, achieving synergistic signal amplification and effectively improving detection sensitivity. Simultaneously, the ratio detection mode can automatically correct for interference from probe concentration, excitation light intensity fluctuations, and environmental factors, ensuring the accuracy of quantitative results.

[0036] (5) The material of the present invention exhibits excellent analytical performance for DPA detection: within the concentration range of 1~100 μM, P 616 / P 515The ratio showed a good linear relationship with DPA concentration, with a detection limit as low as 100 nM. Selectivity experiments demonstrated that the probe had a specific response to DPA; common amino acids, sugars, organic acids, aromatic acid analogs (such as phthalic acid, isophthalic acid, terephthalic acid, and 2,5-pyridinedicarboxylic acid), and inorganic ions did not cause significant changes in the ratio, indicating strong anti-interference ability. In actual water sample (tap water and lake water) spiked recovery experiments, the recovery rate reached 95.9%–103.2%, with a relative standard deviation of less than 7%, verifying its reliability and practicality in complex matrices.

[0037] (6) This invention employs a solid-state grinding combined with hydrothermal crystallization method to achieve the synthesis of nitrogen / chlorine co-doped carbon dots and their in-situ confinement in the pores of AlPO-5 molecular sieves in one step. The process is simple, convenient, and reproducible. The preparation process does not require complex equipment, the reaction conditions are mild, the raw materials are widely available and inexpensive, making it suitable for large-scale production and widespread application.

[0038] (7) The carbon dots used in this invention have good biocompatibility and low toxicity. AlPO-5 molecular sieve is an inorganic material with high biological safety. Compared with quantum dot probes containing heavy metals (such as cadmium), it is more suitable for biological sample detection and potential in vivo applications. Attached Figure Description

[0039] Figure 1 The X-ray diffraction (XRD) patterns of the AlPO-5, N,Cl-CDs@AlPO-5 and Eu&N,Cl-CDs@AlPO-5 composite materials in Example 1 are shown.

[0040] Figure 2 Characterization of the Eu&N,Cl-CDs@AlPO-5 composite material in Example 1: A is a transmission electron microscope image (scale 500 nm), B is a transmission electron microscope image (scale 50 nm), C is a transmission electron microscope image (scale 20 nm), and D is an elemental mapping diagram.

[0041] Figure 3 The following are X-ray photoelectron spectroscopy (XPS) images of the N,Cl-CDs@AlPO-5 and Eu&N,Cl-CDs@AlPO-5 composite materials in Example 1: A is the full XPS spectrum, and B is the high-resolution XPS image of Eu.

[0042] Figure 4 The images show the room temperature phosphorescence emission spectra of the Eu&N,Cl-CDs@AlPO-5 composite material in Example 2 before and after the addition of 2,6-pyridinedicarboxylic acid.

[0043] Figure 5 The phosphorescence lifetime decay curve of the Eu&N,Cl-CDs@AlPO-5 composite material in Example 2 is shown.

[0044] Figure 6 For N,Cl-CDs / Eu in Example 2 3+ Phosphorescence spectra of the system after adding different concentrations of 2,6-pyridinedicarboxylic acid;

[0045] Figure 7 Quantitative analysis of 2,6-pyridinedicarboxylic acid in the Eu&N,Cl-CDs@AlPO-5 composite material in Example 3: A shows the phosphorescence spectra of the material after adding different concentrations of 2,6-pyridinedicarboxylic acid; B shows the phosphorescence intensity ratio (P0.05). 616 / P 515 The linear relationship between the concentration of 2,6-pyridinedicarboxylic acid and its concentration;

[0046] Figure 8 The selective response and anti-interference analysis of the Eu&N,Cl-CDs@AlPO-5 composite material in Example 4 to different interfering substances: A represents amino acids and other organic substances; B represents inorganic ions.

[0047] Figure 9 This is a bar chart showing the change in DPA release over time during the germination of Bacillus subtilis spores in Example 6. Detailed Implementation

[0048] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0049] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the experimental methods without specific conditions are all conventional methods in the art.

[0050] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0051] Example 1: Synthesis and Characterization of Eu&N,Cl-CDs@AlPO-5 Composite Material

[0052] 1. Synthesis of Eu&N,Cl-CDs@AlPO-5 composite material

[0053] (1) Preparation of N,Cl-CDs precursor solution

[0054] Mix 0.5 mL of ethylenediamine with 1 mL of deionized water thoroughly. While stirring continuously, slowly add 1 mL of concentrated hydrochloric acid (36%~38%) to the mixture and stir until the reaction is complete to obtain a yellow nitrogen / chlorine co-doped carbon dot (N,Cl-CDs) precursor solution.

[0055] (2) Preparation of phosphate-di-n-propylamine precursor

[0056] 14 mL of di-n-propylamine was dissolved in 30 mL of ethanol, and 5 mL of phosphoric acid was slowly added dropwise while stirring continuously. The reaction was continued for 4 h. The resulting mixture was filtered under reduced pressure, and the precipitate was washed several times with anhydrous ethanol and dried at 60 °C for 8 h to obtain a white di-n-propylamine phosphate powder.

[0057] (3) Preparation of N,Cl-CDs@AlPO-5 composite material

[0058] Weigh 0.36 g of boehmite (AlOOH), 0.80 g of di-n-propylamine phosphate powder, and 0.42 g of tetraethylammonium bromide (TEABr) and place them in a mortar, then grind and mix thoroughly. Next, add 100 μL of the N,Cl-CDs precursor solution prepared in step (1) and continue grinding for 10 min to ensure homogeneity. Transfer the mixture to a reaction vessel and hydrothermally crystallize at 220 °C for 24 h. The resulting product is washed several times with anhydrous ethanol, centrifuged at 8000 rpm for 15 min, and finally dried at 60 °C for 12 h to obtain N,Cl-CDs@AlPO-5 powder.

[0059] (4) Preparation of Eu&N,Cl-CDs@AlPO-5 composite material

[0060] Europium ion post-modification was performed using a low-temperature hydrothermal method. The N,Cl-CDs@AlPO-5 powder prepared in step (3) was impregnated in a 100 μM EuCl3 solution and stirred for 20 min. It was then transferred to a reaction vessel and crystallized at 60 °C for 2 h. The product was washed multiple times with deionized water, centrifuged at 8000 rpm for 15 min, and finally dried at 60 °C for 12 h to obtain the europium ion-modified nitrogen / chlorine co-doped carbon dots@AlPO-5 composite material (Eu&N,Cl-CDs@AlPO-5).

[0061] 2. Material Characterization

[0062] The crystal structures of N,Cl-CDs@AlPO-5 and Eu&N,Cl-CDs@AlPO-5 composites were characterized by X-ray diffraction (XRD) analysis, such as... Figure 1 As shown, AlPO-5, N,Cl-CDs@AlPO-5 and Eu&N,Cl-CDs@AlPO-5 composites all formed AFI topologies, confirming the successful synthesis of AlPO-5 molecular sieves and demonstrating that the introduction of N,Cl-CDs and europium ion modification did not affect the AlPO-5 framework structure.

[0063] The obtained Eu&N,Cl-CDs@AlPO-5 composite material was characterized by transmission electron microscopy (TEM), such as... Figure 2As shown in AC, carbon dots are uniformly distributed within the AlPO-5 molecular sieve framework, with uniform particle size and no obvious agglomeration. Elemental mapping analysis ( Figure 2 The results showed that nitrogen (N), chlorine (Cl), carbon (C), oxygen (O), phosphorus (P), aluminum (Al), europium (Eu) and other elements were uniformly distributed in the composite material, confirming the successful synthesis of nitrogen / chlorine co-doped carbon dots and their confinement in the molecular sieve channels, as well as the successful loading of europium ions.

[0064] Furthermore, X-ray photoelectron spectroscopy (XPS) was used to investigate the elemental composition and chemical state of N,Cl-CDs@AlPO-5 and Eu&N,Cl-CDs@AlPO-5, and the results are as follows: Figure 3 As shown. XPS full spectrum of N,Cl-CDs@AlPO-5 ( Figure 3 The spectrum of Eu&N,Cl-CDs@AlPO-5 shows characteristic peaks corresponding to C 1s, N 1s, O 1s, Cl 2p, P 2p, and Al 2p, while Eu&N,Cl-CDs@AlPO-5 exhibits additional Eu 3d binding peaks in the full spectrum. This confirms the successful doping of nitrogen / chlorine in N,Cl-CDs and the introduction of Eu into the composite material. (High-resolution Eu 3d spectrum of Eu&N,Cl-CDs@AlPO-5) Figure 3 In (B), the characteristic peaks at 1135.1 eV and 1165.0 eV are attributed to Eu, respectively. 3+ 3D 5 / 2 and 3D 3 / 2 The absence of Eu2O3 diffraction peaks in the XRD pattern of Eu&N,Cl-CDs@AlPO-5 indicates that Eu exists in the composite material in an ionic state and can coordinate with the molecular sieve framework or electronegative functional groups on the surface of N,Cl-CDs.

[0065] Example 2: Phosphorescence lifetime determination of Eu&N,Cl-CDs@AlPO-5 composite material

[0066] 1. Experimental Methods

[0067] The Eu&N,Cl-CDs@AlPO-5 composite material prepared in Example 1 was dispersed in 10 mM phosphate buffer (pH=4) to prepare a 1 g / L suspension. 50 μM of 2,6-pyridinedicarboxylic acid (DPA) was added, and the mixture was thoroughly mixed and allowed to stand at room temperature for 5 min.

[0068] The phosphorescence decay curve of the sample was determined using a time-resolved detection mode of a fluorescence spectrometer, with an excitation wavelength of 275 nm and a monitoring wavelength of 616 nm (Eu). 3+ Characteristic emission). Emission lifetime was calculated using a single exponential fit.

[0069] 2. Experimental Results and Analysis

[0070] like Figure 4 As shown, the carbon dot emission peak (515 nm) in the room temperature phosphorescence emission spectrum of the Eu&N,Cl-CDs@AlPO-5 composite material decreased slightly before and after the addition of DPA, while the Eu&N,Cl-CDs@AlPO-5 composite material showed a slight decrease. 3+ The emission peak (616 nm) was significantly enhanced.

[0071] like Figure 5 As shown, in the presence of DPA, Eu in Eu&N,Cl-CDs@AlPO-5 composite material 3+ Its phosphorescence lifetime is 0.135 s, which is much higher than that of traditional Eu. 3+ The microsecond-level lifetime of the complex.

[0072] In addition, the N,Cl-CDs / Eu ratio was compared and investigated. 3+ The system's time-resolved detection performance for DPA under a delay time of 0.1 s is shown in the following figures. Figure 6 As shown, N,Cl-CDs / Eu 3+ In aqueous solution, it is easily quenched by the -OH bonds of water molecules and dissolved oxygen, causing its excited triplet state to dissipate in large quantities through non-radiative transitions, resulting in an extremely weak phosphorescent signal when DPA is detected.

[0073] The experimental results of this embodiment show that, in the confined system of the present invention, the DPA molecule acts as an "antenna ligand" and interacts with Eu. 3+ Coordination sensitizes its luminescence, and due to the confinement protection effect of AlPO-5 molecular sieve, Eu 3+ - The nonradiative transitions of the DPA complex are effectively suppressed, making the DPA-sensitized Eu 3+ The luminescence lifetime reaches 0.135 s, which significantly extends its luminescence lifetime and lays the foundation for the time-resolved detection application of this material.

[0074] Example 3 Quantitative detection and standard curve establishment of 2,6-pyridinedicarboxylic acid

[0075] 1. Experimental Methods

[0076] Prepare a series of test tubes and add 1 mL of the Eu&N,Cl-CDs@AlPO-5 suspension prepared in Example 1 (1 g / L, dispersed in 10 mM phosphate buffer, pH=4). Add different volumes of DPA standard solution to each tube sequentially to achieve final DPA concentrations of 0, 1, 2, 5, 10, 20, 30, 60, and 100 μM. Adjust the volume of all samples to 2 mL with buffer solution, mix thoroughly, and let stand at room temperature for 5 min.

[0077] Phosphorescence spectra of all samples were determined using a time-resolved fluorescence spectrometer under the following conditions: excitation wavelength 275 nm, excitation slit 10 nm, emission slit 20 nm, delay time 0.1 s, integration time 1 ms, and scan range 500–700 nm.

[0078] 2. Experimental Results and Analysis

[0079] The measurement results are as follows Figure 7 As shown in Figure A, with the increase of DPA concentration, Eu at 616 nm... 3+ Characteristic phosphorescence intensity (P) 616 The phosphorescence intensity of the carbon dots at 515 nm gradually increases, while the room temperature phosphorescence intensity (P) gradually increases. 515 The phosphorescence intensity gradually decreases. Calculate the phosphorescence intensity ratio P at each concentration. 616 / P 515 and with P 616 / P 515 The ratio is on the ordinate, and the DPA concentration (C) is on the ordinate. DPA A standard curve was plotted with the x-axis as the horizontal axis. The result is as follows: Figure 7 As shown in Figure B, within the concentration range of 1–100 μM, P 616 / P 515 The ratio showed a good linear relationship with DPA concentration, and the linear regression equation was P. 616 / P 515 =0.0487C DPA +0.01006, linear correlation coefficient R 2 =0.9979. The detection limit for DPA calculated using the 3σ / slope method is approximately 100 nM.

[0080] The experimental results of this embodiment show that the Eu&N,Cl-CDs@AlPO-5 composite material prepared in Example 1 can effectively eliminate the interference of short-lived background fluorescence (nanosecond to microsecond level) in the sample under time-resolved detection mode (the delay time in this embodiment is 0.1 s), while ensuring the long-lived Eu sensitized by DPA. 3+ The signal was detected stably. P 616 / P 515 The ratio detection further eliminates the influence of environmental factor fluctuations, realizing high signal-to-noise ratio and high accuracy in DPA quantitative analysis.

[0081] Example 4: Selectivity Evaluation of the Detection System

[0082] 1. Experimental Methods

[0083] (1) Selection of interfering substances

[0084] To evaluate the selectivity of Eu&N,Cl-CDs@AlPO-5 composites for DPA, the following potential interfering substances were selected for testing:

[0085] Inorganic ions: K + Ca 2+ Na + Mg 2+ Zn 2+ Fe 2+ Fe 3+ Cr 3+ Cu 2+ CO3 2- SO4 2- NO3 - CH3COO - PO4 3- ,Br - Cl - I - ;

[0086] Amino acids: L-leucine, L-glutamic acid, L-cysteine, L-proline, L-serine, L-lysine, L-histidine, L-glycine, L-alanine;

[0087] Other organic compounds: glucose, citric acid, urea, nicotinic acid, phthalic acid, isophthalic acid, terephthalic acid, boric acid, 2,5-pyridinedicarboxylic acid.

[0088] (2) Detection steps

[0089] Take a series of test tubes and add 1 mL of the Eu&N,Cl-CDs@AlPO-5 suspension prepared in Example 1 (1 g / L, dispersed in 10 mM phosphate buffer, pH=4). Add DPA or the above-mentioned interfering substance to each tube to make the final concentration 100 μM (a 30 μM concentration group was set up for the DPA group). Make up to 2 mL with buffer, mix thoroughly, and let stand at room temperature for 5 min. Measure the phosphorescence spectrum of each sample according to the test conditions of Example 3 and calculate P. 616 / P 515 ratio.

[0090] (3) Anti-interference capability test

[0091] To investigate the anti-interference ability in complex matrices, the following experimental group was set up: 30 μM DPA was added to a suspension containing 100 μM of interfering agent Eu&N,Cl-CDs@AlPO-5, mixed well, and allowed to stand for 5 min. The phosphorescence spectrum was then measured, and P was calculated. 616 / P 515 The ratio was compared with that of the pure DPA (30 μM) group.

[0092] 2. Experimental Results and Analysis

[0093] like Figure 8 As shown, the selective test results (blue bars in the figure) indicate that the sample group with only DPA added caused P 616 / P 515 The ratio increased significantly, while the P-value of the sample group with added other interfering substances was significantly higher. 616 / P 515 The ratio showed no significant change compared to the blank group, indicating that the probe has excellent selectivity for DPA. The anti-interference test results (red bars in the figure) show that, in the presence of various potential interfering substances at a concentration of 100 μM, the response signal (P) of the probe of this invention to 30 μM DPA is... 616 / P 515 The ratio (total deviation) fluctuates within ±17% compared to the pure DPA system. Considering that the concentration of interfering substances is much higher than that of DPA (more than 3 times), and the types of interfering species are diverse (covering inorganic ions, amino acids, other organic substances, etc.), this result indicates that the probe of this invention has excellent stability and superior anti-interference ability in complex biological matrices, which provides support for its practical application in selectively and reliably detecting DPA in real samples.

[0094] Example 5: Spiked Recovery Experiment of DPA in Actual Water Samples

[0095] 1. Experimental Methods

[0096] (1) Sample collection and pretreatment

[0097] Water samples were collected from the laboratory tap water and the lake on the Nantong University campus. The lake water samples were first centrifuged (4000 rpm, 10 min) to remove large suspended particles, and the supernatant was filtered through a 0.45 μm filter membrane. Tap water was used directly without pretreatment.

[0098] (2) Spiked recovery experiment

[0099] Pretreated tap water and lake water samples were taken and DPA standard solution was added to them to make spiking concentrations of 5, 20, and 60 μM, respectively. The DPA concentration in each spiked sample was determined according to the detection procedure in Example 3. Each concentration was measured in triplicate, and the spike recovery rate and relative standard deviation (RSD) were calculated.

[0100] 2. Experimental Results and Analysis

[0101] Table 1. Results of DPA spike recovery experiments in actual water samples

[0102]

[0103] As shown in Table 1, the recoveries of the three spiked concentrations in the tap water samples ranged from 95.9% to 102.4%, with RSDs ranging from 1.17% to 3.63%; while in the lake water samples, the recoveries ranged from 98.0% to 103.2%, with RSDs ranging from 2.62% to 6.01%. The recoveries were all between 95.9% and 103.2%, and the RSDs were all less than 7%.

[0104] The experimental results of this embodiment show that the material of the present invention has high accuracy and good repeatability in actual water sample testing, and can meet the requirements for quantitative detection of DPA in complex matrices.

[0105] Example 6 Monitoring of DPA release during Bacillus subtilis spore germination

[0106] 1. Experimental Methods

[0107] Non-infectious Bacillus subtilis rich in DPA was selected as the research subject to evaluate the application of this probe in bacterial spore detection. Bacillus subtilis was cultured in nutrient agar medium, and third-generation Bacillus subtilis was cultured at 37°C for 72 h to promote spore formation. Spores were collected and centrifuged at 10,000 rpm for 15 min at 4°C, repeated three times to remove the culture medium. Subsequently, the purified spores (approximately 1.0 × 10⁻⁶) were... 9 (1 spore) was redispersed in 1 mL of phosphate buffer (10 mM, pH=4) containing Eu&N,Cl-CDs@AlPO-5 (1 g / L) and placed in a 5 mL test tube. To promote spore germination, 1 mL of 10 mM L-alanine was added as a nutrient source. The sample solution was heated at 75 °C for different time intervals (5, 10, 20, 30, 45, 60, 75, 90 min) to promote the release of DPA from the spores. The phosphorescence spectra of each sample were measured according to the test conditions in Example 3, and P was calculated. 616 / P 515 The ratio was used to quantitatively analyze the DPA released by bacterial spores based on the standard curve established in Example 3.

[0108] 2. Experimental Results and Analysis

[0109] The probe was applied to measure the DPA released by Bacillus subtilis spores, and the results were as follows: Figure 9As shown, the release of DPA gradually increased with the extension of spore incubation time. After 45 min of incubation, the DPA concentration was measured to be 50 μM, accounting for approximately 70% of the total spore release. Sufficient L-alanine (a germination promoter for Bacillus subtilis spores) can promote rapid spore growth and DPA release. However, as spore growth and L-alanine are gradually consumed, the spore growth rate slows down, leading to a decrease in the DPA release rate and a stabilization of the total release. After 60 min of incubation, the measured DPA concentration no longer increased significantly, indicating that the spore germination process was basically completed.

[0110] The experimental results of this embodiment show that the ratio phosphorescent probe prepared by the present invention can be used to rapidly assess the growth cycle and germination stage of bacterial spores, providing a practical and convenient tool for real-time monitoring of bacterial reproduction.

[0111] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. The scope of protection of the present invention is determined by the scope claimed in the claims. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A ratiometric phosphorescent probe material for detecting 2,6-pyridinedicarboxylic acid, characterized in that, The material is a europium ion-modified nitrogen / chlorine co-doped carbon dot@AlPO-5 molecular sieve composite material, wherein: The nitrogen / chlorine co-doped carbon dots were synthesized in one step by solid-phase grinding combined with hydrothermal crystallization using an ethylenediamine-hydrochloric acid system as a precursor and confined in situ within the pore structure of AlPO-5 molecular sieve. The europium ions are loaded on the surface or within the pores of the molecular sieve; In the presence of 2,6-pyridinedicarboxylic acid, the phosphorescence lifetime of the europium ions after sensitization is greater than 0.1 s; The material emits both room-temperature phosphorescence of carbon dots and characteristic phosphorescence of europium ions under excitation light irradiation.

2. The ratiometric phosphorescent probe material for detecting 2,6-pyridinedicarboxylic acid according to claim 1, characterized in that, In the presence of 2,6-pyridinedicarboxylic acid, the phosphorescence lifetime of the europium ion after sensitization is 0.15 s; and when detecting 2,6-pyridinedicarboxylic acid, the characteristic phosphorescence intensity of the europium ion is enhanced, while the room temperature phosphorescence intensity of the carbon dots undergoes concentration-dependent quenching, forming a dual-channel ratio response.

3. A method for preparing a ratiometric phosphorescent probe material as described in claim 1 or 2, characterized in that, Includes the following steps: Step 1) Preparation of nitrogen / chlorine co-doped carbon dot precursor solution: Mix ethylenediamine with hydrochloric acid to react and obtain nitrogen / chlorine co-doped carbon dot precursor solution; Step 2) Preparation of phosphorus source precursor: Di-n-propylamine is reacted with phosphoric acid to obtain phosphoric acid-di-n-propylamine precursor; Step 3) Preparation of carbon dot @AlPO-5 composite material: The aluminum source, the phosphate-di-n-propylamine precursor obtained in step 2) and the template agent are mixed and ground, and the nitrogen / chlorine co-doped carbon dot precursor solution obtained in step 1) is added. After further grinding, the mixture is subjected to hydrothermal crystallization to obtain the nitrogen / chlorine co-doped carbon dot @AlPO-5 composite material. Step 4) Europium ion post-modification: The nitrogen / chlorine co-doped carbon dot @AlPO-5 composite material obtained in step 3) is immersed in europium ion solution and subjected to low-temperature hydrothermal reaction to obtain europium ion modified nitrogen / chlorine co-doped carbon dot @AlPO-5 composite material.

4. The preparation method according to claim 3, characterized in that, Step 1) The volume ratio of ethylenediamine to hydrochloric acid is 0.5:1; Step 2) The volume ratio of di-n-propylamine to phosphoric acid is 14:5; Step 3) The mass ratio of aluminum source, phosphate-di-n-propylamine precursor and template agent is 0.36:0.80:0.

42.

5. The preparation method according to claim 3, characterized in that, Step 3) The aluminum source is boehmite, and the template agent is tetraethylammonium bromide; the hydrothermal crystallization temperature is 220℃ and the time is 24 h.

6. The preparation method according to claim 3, characterized in that, Step 4) The europium ion solution is europium nitrate or europium chloride solution with a concentration of 100 μM; the temperature of the low-temperature hydrothermal reaction is 60℃ and the time is 2 h.

7. A method for detecting 2,6-pyridinedicarboxylic acid, characterized in that, Including quantitative detection and selective detection; The quantitative detection step is as follows: the ratio phosphorescent probe material as described in claim 1 or 2 is mixed with the test sample containing 2,6-pyridinedicarboxylic acid, and the intensity ratio of europium ion characteristic phosphorescence to carbon point room temperature phosphorescence is determined by time-resolved phosphorescence spectroscopy. Based on the functional relationship between this ratio and the concentration of 2,6-pyridinedicarboxylic acid, the quantitative detection of 2,6-pyridinedicarboxylic acid is achieved. The selective detection step is as follows: the ratio phosphorescent probe material as described in claim 1 or 2 is mixed with 2,6-pyridinedicarboxylic acid and potential interfering substances respectively, and the intensity ratio of europium ion characteristic phosphorescence to carbon point room temperature phosphorescence is determined by time-resolved phosphorescence spectroscopy to evaluate the selective response and anti-interference ability of the material to 2,6-pyridinedicarboxylic acid.

8. The method for detecting 2,6-pyridinedicarboxylic acid according to claim 7, characterized in that, The time-resolved phosphorescence spectroscopy method is set with a delay time of 0.1 s; the characteristic phosphorescence of europium ions is phosphorescence at 616 nm, and the room-temperature phosphorescence of carbon dots is phosphorescence at 515 nm; the potential interfering substances include amino acids, glucose, citric acid, urea, nicotinic acid, phthalic acid, isophthalic acid, terephthalic acid, boric acid, 2,5-pyridinedicarboxylic acid, and inorganic ions.

9. The application of the ratiometric phosphorescent probe material as described in claim 1 or 2 in the detection of 2,6-pyridinedicarboxylic acid.

10. The use of the ratiometric phosphorescent probe material as described in claim 1 or 2 in the preparation of a kit for detecting Bacillus spores.

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