CN116642868A - Application of rare earth complex fluorescent probe in detection of 2, 6-pyridine dicarboxylic acid - Google Patents
Application of rare earth complex fluorescent probe in detection of 2, 6-pyridine dicarboxylic acid Download PDFInfo
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Abstract
The invention relates to the technical field of biological sample detection, in particular to application of a rare earth complex fluorescent probe in detection of 2, 6-pyridine dicarboxylic acid, wherein the rare earth complex fluorescent probe is a Pi-regulated Luminol-Tb fluorescent probe, and comprises Luminol, rare earth terbium ions and phosphate radicals in a molar ratio of 1:1:1, the Luminol is taken as a ligand, the terbium ions are taken as a metal center, and the phosphate radicals are taken as regulating and controlling complex ions, and the complex Luminol-Tb-Pi is formed through self-assembly reaction in a room temperature water phase. An analytical method for the detection of 2, 6-pyridinedicarboxylic acid by Pi-regulated luminel-Tb fluorescent probes, comprising: constructing a Pi-regulated Luminol-Tb fluorescent probe; detecting DPA by using a fluorescent probe; the fluorescent probe is applied to the analysis of DPA in a water sample. The invention can realize the ratio fluorescence detection of DPA based on the double emission and opposite fluorescence signal change. Compared with other single-emission fluorescent probes, the fluorescent probe can eliminate the influence of probe concentration, instrument factors and external environment factors, and has better detection accuracy and higher detection sensitivity.
Description
Technical Field
The invention relates to the technical field of biological sample detection, in particular to application of a rare earth complex fluorescent probe in detection of 2, 6-pyridine dicarboxylic acid.
Background
Anthracnose is an acute infectious disease caused by bacillus anthracis and is seriously threatening the survival of human beings and the healthy development of animal husbandry due to high infectivity, high pathogenicity and high death rate. Currently 2, 6-pyridinedicarboxylic acid (DPA) has been identified as the sole biomarker for identifying Bacillus anthracis (DPA structure is shown in formula 1) and Bacillus anthracis spore concentration can be assessed by detecting DPA content. Therefore, the realization of the specific identification and the simple and rapid detection of the concentration of bacillus anthracis spores is of great significance for maintaining public safety.
Various methods have been developed for DPA detection, such as surface enhanced Raman spectroscopy, high performance liquid chromatography and liquid chromatography, but such detection instruments are large in size, long in detection period, expensive in instrument cost and cannot meet the requirements of on-site rapid detection. The new methods such as electrochemical detection, immunoassay, fluorescence analysis technology and the like which are gradually developed have the characteristics of simple operation, high sensitivity and low cost, wherein the fluorescence analysis technology provides a theoretical basis for in-situ detection of DPA by the characteristics of rapid detection, visualization and the like, so that the establishment of a fluorescence detection method capable of realizing sensitive and selective detection of DPA has important significance. However, the photostability of fluorescent probes and the complex construction process will greatly limit their popularization and application. The optical probe based on lanthanide luminescence is widely applied in the fields of sensing analysis, biological imaging, photoelectric devices and the like due to the characteristics of long fluorescence lifetime, larger Stokes displacement, good light stability, high emitted light purity and the like, so that the establishment of the novel fluorescent probe based on lanthanide luminescence provides technical conditions for the simple and rapid detection of DPA.
Disclosure of Invention
The invention aims to solve the defects in the prior art, and provides an application of a rare earth complex fluorescent probe in detecting 2, 6-pyridine dicarboxylic acid, which can realize rapid and simple detection of the 2, 6-pyridine dicarboxylic acid.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the application of a rare earth complex fluorescent probe in detecting 2, 6-pyridine dicarboxylic acid comprises Luminol, rare earth terbium ions and phosphate radicals in a molar ratio of 1:1:1, wherein Luminol is taken as a ligand, terbium ions are taken as a metal center body, phosphate radicals are taken as regulating and controlling complex ions, and the complex Luminol-Tb-Pi is formed through self-assembly reaction in a room temperature water phase.
Preferably, an analytical method for the detection of 2, 6-pyridinedicarboxylic acid based on Pi-regulated luminel-Tb fluorescent probes comprises the steps of:
(1) Construction of Pi-regulated Luminol-Tb fluorescent probes:
in a 5.0mL cuvette, a certain amount of the following solutions were added sequentially: the concentration is 1.0mol.L -1 500. Mu.L Tris-HCl buffer solution with pH=7.5 at a concentration of 1.0 mol.L -1 160. Mu.L of Luminol solution with a concentration of 5 mol.L -1 30. Mu.L of Tb 3+ The solution and the concentration are 10.0mol.L -1 Constructing a fluorescent probe of a Luminol-Tb-Pi complex from 100 mu L of Pi solution;
(2) Detection of DPA by fluorescent probe:
adding 0.2-45 mu M DPA standard solution or pretreated sample into the probe, and finally, using deionized water to fix the volume by 10.0mL; after uniformly mixing the samples, placing the samples at room temperature for 20min, and finally sequentially measuring the fluorescence spectrum of each sample within 300-600nm under the condition that the excitation wavelength is 280nm and the excitation slit and the emission slit are 5nm by using a fluorescence spectrometer; fluorescence intensity values at 545 and 423nm were used for quantitative analysis of DPA as a function of DPA concentration. In the selectivity test, other possible interfering substances are used to replace DPA, the same assay is used for analytical detection, the selectivity of the fluorescent probe for DPA is explored, and the interference of the possible coexisting substances on DPA detection is explored.
(3) The fluorescent probe is applied to the analysis of DPA in a water sample:
and (3) water sample collection: the water samples are respectively collected from lake water and spinning building tap water of university of south China;
pretreatment of a water sample: taking 25mL of a collected water sample, placing the collected water sample in a 50mL beaker, standing for 2h, and taking a supernatant; filtering the supernatant with a 0.22 μm filter head, and diluting 100 times for later use;
and (3) water sample measurement: DPA (5, 10, 40 mu M) with a certain concentration is added into the pretreated water sample, then a labeled sample is added into a fluorescent probe, and the DPA content in the sample is analyzed by adopting the same measuring process as the quantitative analysis.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention can realize the ratio fluorescence detection of DPA based on the double emission and opposite fluorescence signal change. Compared with other single-emission fluorescent probes, the fluorescent probe can eliminate the influence of probe concentration, instrument factors and external environment factors, and has better detection accuracy and higher detection sensitivity.
2. The fluorescent probe can complete the selective response to DPA within 2min, so that compared with detection technologies such as liquid chromatography-mass spectrometry, high performance liquid chromatography, capillary electrophoresis, chemiluminescence and the like, the fluorescent probe can be used for rapid detection of DPA, and the preparation process is simple and easy to implement and the use cost is low.
Drawings
FIG. 1 is a fluorescence spectrum of different coordination systems in the invention; wherein, luminol: 50. Mu.M; tb (Tb) 3+ ：50μM；Pi：50μM；DPA：20μM；Tris-HCl buffer：10mM(pH＝7.5)；λ ex ＝280nm；
In FIG. 2, (A) is a fluorescence spectrum of the Luminol-Tb-Pi complex system after adding DPA at different concentrations; (B) Is F in the probe system of (A) 545 /F 423 A graph of linear relationship with DPA concentration; (C) Fluorescent spectrograms of the Luminol-Tb complex system after DPA with different concentrations is added; (D) Is F in the probe system of (C) 545 /F 423 A graph of linear relationship with DPA concentration;
FIG. 3 is a schematic diagram showing the detection of DPA selectivity and interference resistance of the Luminol-Tb-Pi probe system of the present invention; wherein, DPA: 20. Mu.M; the organic compound and inorganic ion are both 20 muM；λ ex ＝280nm。
Detailed Description
The following technical solutions in the embodiments of the present invention will be clearly and completely described with reference to the accompanying drawings, so that those skilled in the art can better understand the advantages and features of the present invention, and thus the protection scope of the present invention is more clearly defined. The described embodiments of the present invention are intended to be only a few, but not all embodiments of the present invention, and all other embodiments that may be made by one of ordinary skill in the art without inventive faculty are intended to be within the scope of the present invention.
The application of a rare earth complex fluorescent probe in detecting 2, 6-pyridine dicarboxylic acid comprises Luminol, rare earth terbium ions and phosphate radicals in a molar ratio of 1:1:1, wherein Luminol is taken as a ligand, terbium ions are taken as a metal center body, phosphate radicals are taken as regulating and controlling complex ions, and the complex Luminol-Tb-Pi is formed through self-assembly reaction in a room temperature water phase.
Specifically, the analysis method for detecting 2, 6-dipicolinic acid by using a Pi-regulated Luminol-Tb fluorescent probe comprises the following steps:
(1) Construction of Pi-regulated Luminol-Tb fluorescent probes:
in a 5.0mL cuvette, a certain amount of the following solutions were added sequentially: the concentration is 1.0mol.L -1 500. Mu.L Tris-HCl buffer solution with pH=7.5 at a concentration of 1.0 mol.L -1 160. Mu.L of Luminol solution with a concentration of 5 mol.L -1 30. Mu.L of Tb 3+ The solution and the concentration are 10.0mol.L -1 Constructing a fluorescent probe of a Luminol-Tb-Pi complex from 100 mu L of Pi solution;
(2) Detection of DPA by fluorescent probe:
adding 0.2-45 mu M DPA standard solution or pretreated sample into the probe, and finally, using deionized water to fix the volume by 10.0mL; after uniformly mixing the samples, placing the samples at room temperature for 20min, and finally sequentially measuring the fluorescence spectrum of each sample within 300-600nm under the condition that the excitation wavelength is 280nm and the excitation slit and the emission slit are 5nm by using a fluorescence spectrometer; fluorescence intensity values at 545 and 423nm were used for quantitative analysis of DPA as a function of DPA concentration. In the selectivity test, other possible interfering substances are used to replace DPA, the same assay is used for analytical detection, the selectivity of the fluorescent probe for DPA is explored, and the interference of the possible coexisting substances on DPA detection is explored.
(3) The fluorescent probe is applied to the analysis of DPA in a water sample:
and (3) water sample collection: the water samples are respectively collected from lake water and spinning building tap water of university of south China;
pretreatment of a water sample: taking 25mL of a collected water sample, placing the collected water sample in a 50mL beaker, standing for 2h, and taking a supernatant; filtering the supernatant with a 0.22 μm filter head, and diluting 100 times for later use;
and (3) water sample measurement: DPA (5, 10, 40 mu M) with a certain concentration is added into the pretreated water sample, then a labeled sample is added into a fluorescent probe, and the DPA content in the sample is analyzed by adopting the same measuring process as the quantitative analysis.
In this example, a Pi-regulated Luminol-Tb fluorescent complex (Luminol-Tb-Pi) was constructed for the simple and rapid detection of Bacillus anthracis biomarkers (2, 6-pyridinedicarboxylic acid, DPA). As shown in formula 2, taking Luminol as organic ligand, tb 3+ Is a metal central body, under the regulation of Pi, DPA can be added to ensure that Tb 3+ The characteristic fluorescence is significantly enhanced, and the fluorescence quenching of Luminol is caused. DPA through and Tb 3+ Coordination binding to Tb 3+ Energy transfer is carried out to sensitize the luminescence of the light; pi by scaling down DPA and Tb 3+ The distance between the two components enhances the sensitization of DPA; in addition, pi passes through Tb 3+ The coordination of (C) can also reduce the water molecules to Tb 3+ Further enhance Tb 3+ Fluorescence. Due to DPA and Tb 3+ Competitive binding of Luminol to Tb 3+ Part of the lumineol is released into the free state and its aggregation-induced fluorescence-enhanced state is destroyed, resulting in its fluorescence quenching. Thus, based on Tb 3+ The double signal change of characteristic fluorescence enhancement and Luminol fluorescence quenching can realize the ratio detection of DPA, and has good specificity and anti-interference performance. In addition, as the concentration of DPA increases, the fluorescence of the sensing system is gradually changed from blue to green, and the visual detection of DPA can be realized.
Examples:
(1) Sensing mechanism of fluorescent probe pair DPA
The fluorescence spectrum changes before and after the addition of DPA to the Luminol-Tb-Pi system were measured at an excitation wavelength of 280nm, as shown in FIG. 1. Before DPA is added, only the characteristic fluorescence of the Luminol exists in the Luminol-Tb-Pi system; after DPA addition, three fluorescence peaks at 489, 545 and 584nm, respectively derived from Tb 3+ Transition of [ ] 5 D 4 → 7 F J (j=6, 5, 4), indicating DPA as antenna ligand to Tb 3+ Energy transfer is carried out, and luminescence is sensitized; in addition, after DPA addition, there was a slight quenching of the Luminol fluorescence. This is due to DPA and Tb 3+ Competitive binding of Luminol to Tb 3+ Part of the lumineol is released into the free state and its aggregation-induced fluorescence-enhanced state is destroyed, resulting in its fluorescence quenching.
In addition, to demonstrate the Pi-modulated enhanced fluorescence response, the fluorescence spectrum changes before and after DPA addition were measured simultaneously for the Luminol-Tb system at an excitation wavelength of 280 nm. The results indicate that the Pi-modulated Luminol-Tb system has a more pronounced fluorescent response to DPA relative to the Luminol-Tb system, due to the Pi passing through Tb 3+ Coordination is enhanced, coordination effect between coordination systems is enhanced, and DPA and Tb are effectively reduced 3+ Distance between them, thereby enhancing DPA to Tb 3+ Is a sensitization of (a); in addition, pi passes through Tb 3+ The coordination of (C) can also reduce the water molecules to Tb 3+ Further enhance Tb 3+ Fluorescence. Thus, based on Luminol and Tb in the Luminol-Tb-Pi probe system 3+ The dual fluorescent signal response of (2) can realize the ratio fluorescent detection of DPA.
(2) Quantitative analysis of DPA by fluorescent probe
Under optimized conditions (Luminol: 50. Mu.M; tb) 3+ : 50. Mu.M; pi: 50. Mu.M; tris-HCl buffer:10mM, pH=7.5), the present invention explores the detection performance of the Luminol-Tb-Pi fluorescent probe on DPA. As shown in FIG. 2A, tb was found to be present as the DPA concentration in the probe system gradually increased (0.2-45. Mu.M) 3+ The characteristic fluorescence of (2) gradually increases, and the fluorescence of Luminol in the system gradually decreases. And DPA is in the concentration range of 0.2-45 mu M, and the fluorescence intensity ratio F of the probe is found by drawing a dose-response curve 545 /F 423 There was a good linear relationship with DPA concentration, the linear regression equation was y= 0.04893x-0.21202, r 2 = 0.9891 (fig. 2B). Furthermore, based on lod=3σ/k (σ means standard deviation of 11 blank sample signal values, k is slope of linear regression equation, i.e. 0.04893), the detection limit of the fluorescent probe on DPA was calculated to be 0.051 μm, and LOD of the method is far lower than the infection dose of bacillus anthracis spores on human (60 μm), indicating the sensitive detection performance of the sensing method on DPA.
In addition, the response performance of the Luminol-Tb complex system without Pi regulation to DPA is examined in comparison, and the results are shown in figures 2C and D. The response of the Luminol-Tb complex system to DPA is relatively weak, i.e.DPA to Tb, compared to the Luminol-Tb-Pi fluorescent probe 3+ Is significantly weaker and the ratio of fluorescence intensities F 545 /F 423 And the DPA concentration, the comparison result shows that the Luminol-Tb-Pi probe based on Pi regulation has higher detection sensitivity to DPA.
(3) Analysis of selectivity and interference immunity of fluorescent probe to DPA
To evaluate the potential of the fluorescent probe for DPA detection, the specificity of the Luminol-Tb-Pi system for DPA detection was examined, as shown in FIG. 3. DPA alone causes the fluorescence intensity ratio F of the probe system 545 /F 423 Rapidly increases, and when other organic compounds (L-leucine, L-glutamic acid, L-cysteine, D-alanine, guanosine Monophosphate (GMP), adenosine Monophosphate (AMP), bovine Serum Albumin (BSA), glucose are added(glucose), citric acid (citric acid), uric acid (uric acid), nicotinic acid (nicotinic acid), oxytetracycline (oxytetracycline)) or a plurality of inorganic ions (K) + 、Na + 、Ca 2+ 、Mg 2+ 、Ba 2+ 、Fe 3+ 、Fe 2+ 、Cu 2+ 、Cr 3+ 、CO 3 2- 、SO 3 2- 、SO 4 2- 、NO 3 - 、CH 3 COO - 、Cl - 、Br - ) After that, the fluorescence intensity ratio is not changed obviously, which indicates that the fluorescence probe has good selectivity for DPA detection.
In addition, anti-interference is one of important conditions for evaluating the application performance of the fluorescent probe, and the anti-interference performance of the fluorescent probe on the Luminol-Tb-Pi probe is examined under the condition that a plurality of possible coexisting interferents exist. The results show that the organic substances hardly interfere with the DPA measurement, and that other common ions which may coexist hardly interfere with the measurement. For biological organisms such as bacterial spores, the concentration of these metal ions in the organism is very low and therefore the interference with DPA detection is negligible. The result shows that the fluorescent probe has better selectivity for DPA measurement and has certain application potential in actual sample detection.
(4) Analysis application of fluorescent probe to DPA in water sample
To further investigate the applicability of the proposed method, the recovery of DPA in the samples was investigated by standard addition methods and the measurement results are shown in Table 1. The recovery rate of the fluorescent probe for detecting DPA in the sample is 90.98-108.35%, and the standard deviation value is not more than 0.5%, which shows that the method can be used for reliably detecting DPA in the sample. Therefore, the fluorescent probe developed by the work has a certain application prospect in the aspect of evaluating the bacillus anthracis concentration in environmental samples.
TABLE 1 detection of DPA in actual samples
In summary, the present invention can realize the ratio fluorescence detection of DPA based on the double emission and the opposite fluorescence signal change. Compared with other single-emission fluorescent probes, the fluorescent probe can eliminate the influence of probe concentration, instrument factors and external environment factors, and has better detection accuracy and higher detection sensitivity. In addition, the fluorescent probe can complete the selective response to DPA within 2min, so that compared with detection technologies such as liquid chromatography-mass spectrometry, high performance liquid chromatography, capillary electrophoresis, chemiluminescence and the like, the fluorescent probe can be used for rapid detection of DPA, and the preparation process is simple and easy to implement, and the use cost is low.
The description and practice of the invention disclosed herein will be readily apparent to those skilled in the art, and may be modified and adapted in several ways without departing from the principles of the invention. Accordingly, modifications or improvements may be made without departing from the spirit of the invention and are also to be considered within the scope of the invention.
Claims (2)
1. The application of the rare earth complex fluorescent probe in detecting 2, 6-pyridine dicarboxylic acid is characterized in that the rare earth complex fluorescent probe is a Pi-regulated Luminol-Tb fluorescent probe, and comprises Luminol, rare earth terbium ions and phosphate radicals in a molar ratio of 1:1:1, wherein Luminol is used as a ligand, terbium ions are used as a metal center, phosphate radicals are used as regulating and controlling complex ions, and the complex Luminol-Tb-Pi is formed through self-assembly reaction in a room temperature water phase.
2. The use according to claim 1, characterized in that the analysis method based on the detection of 2, 6-pyridinedicarboxylic acid by Pi-regulated luminel-Tb fluorescent probe comprises the following steps:
(1) Construction of Pi-regulated Luminol-Tb fluorescent probes:
in a 5.0mL cuvette, a certain amount of the following solutions were added sequentially: the concentration is 1.0mol.L -1 500. Mu.L Tris-HCl buffer solution with pH=7.5 at a concentration of 1.0 mol.L -1 160. Mu.L of Luminol solution with a concentration of 5 mol.L -1 30. Mu.L of Tb 3+ The solution and the concentration are 10.0mol.L -1 Constructing a fluorescent probe of a Luminol-Tb-Pi complex from 100 mu L of Pi solution;
(2) Detection of DPA by fluorescent probe:
adding 0.2-45 mu M DPA standard solution or pretreated sample into the probe, and finally, using deionized water to fix the volume by 10.0mL; after uniformly mixing the samples, placing the samples at room temperature for 20min, and finally sequentially measuring the fluorescence spectrum of each sample within 300-600nm under the condition that the excitation wavelength is 280nm and the excitation slit and the emission slit are 5nm by using a fluorescence spectrometer; fluorescence intensity values at 545 and 423nm were used for quantitative analysis of DPA as a function of DPA concentration;
(3) The fluorescent probe is applied to the analysis of DPA in a water sample:
collecting a water sample;
pretreatment of a water sample: taking 25mL of a collected water sample, placing the collected water sample in a 50mL beaker, standing for 2h, and taking a supernatant; filtering the supernatant with a 0.22 μm filter head, and diluting 100 times for later use;
and (3) water sample measurement: adding DPA with a certain concentration into the pretreated water sample, adding a labeled sample into a fluorescent probe, and analyzing the DPA content in the sample by adopting the same measurement process as the quantitative analysis.
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| CN116970116B (en) * | 2023-09-22 | 2024-01-02 | 南昌大学 | Polymer copolymer containing coumarin-Tb complex, and preparation method and application thereof |
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