阅读说明：本技术 一种基于丹磺酸结构的亚铁离子荧光探针分子、制备方法及应用 (Ferrous ion fluorescent probe molecule based on dansyl acid structure, preparation method and application ) 是由 高光芹 谢普会 王志敏 周晶晶 申丽婕 党玉丽 苏惠 于 2019-10-09 设计创作，主要内容包括：本发明提供了一种基于丹磺酸结构的亚铁离子荧光探针分子、制备方法及应用,其分子式为C<Sub>12</Sub>H<Sub>14</Sub>NO<Sub>4</Sub>S,简称为：FeP1。该探针通过商品化试剂丹磺酰氯一步反应制得,合成产率高,制备成本低。通过荧光光谱仪研究了探针FeP1在Tris-DMSO(4:1,pH=7.4,v/v)溶液中与金属离子、常见阴离子、活性硫、活性氧等小分子物质的识别特性。结果表明：探针FeP1对亚铁离子(Fe<Sup>2+</Sup>)具有高效专一的选择性,具有较强的抗干扰能力,识别速度快,酸碱度适用范围广,其最低检出限为18 nM,可以广泛应用于生物体系和环境样品中亚铁离子的检测。(The invention provides a dansyl acid structure-based ferrous ion fluorescent probe molecule, a preparation method and application thereof, wherein the molecular formula of the probe molecule is C 12 H 14 NO 4 S, abbreviated as: FeP 1. The probe is prepared by one-step reaction of a commercialized reagent dansyl chloride, and has high synthesis yield and low preparation cost. The recognition characteristics of the probe FeP1 with metal ions, common anions, active sulfur, active oxygen and other small molecular substances in a Tris-DMSO (4:1, pH =7.4, v/v) solution were studied by a fluorescence spectrometer. The results show that: probe FeP1 for ferrous ion (Fe) 2+ ) Has high-efficiency and specific selectivity, strong anti-interference capability, high identification speed and pH value application rangeThe kit has wide range, the lowest detection limit is 18 nM, and the kit can be widely applied to detection of ferrous ions in biological systems and environmental samples.)

1. A ferrous ion fluorescent probe molecule based on a dansyl acid structure is characterized in that the structural formula of the fluorescent probe is as follows:。

2. The preparation method of the dansyl acid structure-based ferrous ion fluorescent probe molecule according to claim 1, characterized by comprising the following steps:

(1) Dissolving m-chloroperoxybenzoic acid in anhydrous dichloromethane to obtain a solution A;

(2) dissolving dansyl chloride in anhydrous dichloromethane to obtain a solution B, slowly dropwise adding the solution A into the solution B under the condition of ice bath stirring, heating to room temperature for reaction for a period of time, adding a certain amount of alkaline alumina, stirring for a period of time, removing the solvent under reduced pressure, and separating by silica gel column chromatography to obtain a product FeP1, namely the fluorescent probe molecule.

3. The method for preparing the dansyl acid structure-based ferrous ion fluorescent probe molecule according to claim 1, wherein: the molar ratio of the intermediate chloroperoxybenzoic acid in the step (1) to the dansyl chloride in the step (2) is (1-5): 1, the room-temperature reaction time in the step (2) is 2-12 h.

4. The method for preparing the dansyl acid structure-based ferrous ion fluorescent probe molecule according to claim 1, wherein: the molar ratio of dansyl chloride to basic alumina in the step (2) is 1: (2-8) stirring for 1-4 h.

5. The method for preparing the dansyl acid structure-based ferrous ion fluorescent probe molecule according to claim 1, wherein: the silica gel column chromatography separation in the step (2) adopts the eluent of methanol and dichloromethane with the volume ratio of 1 (4-15), and the yield is 40-90%.

6. The application of the fluorescent probe molecule prepared by the preparation method of any one of claims 2 to 5 in the field of high-sensitivity and specific detection of ferrous ions.

Technical Field

The invention relates to the field of metal ion fluorescent probe detection, in particular to a dansyl acid structure-based ferrous ion fluorescent probe molecule, a preparation method and application.

Background

Iron is an indispensable metal element in the living body, and is involved in many physiological activities of the living body, such as oxygen transport in cells, maintenance of hematopoietic function, synthesis of many coenzymes, and DNA synthesis. Iron is an important component of hemoglobin, myoglobin and cytochrome, participates in the metabolic process of maintaining a living body, and directly participates in the transportation and storage of oxygen in the living body. Iron must be maintained at a certain concentration in the organism, otherwise it will have a very adverse effect on the health of the organism.

The major regulatory role in the organism is active iron, which is considered to be an important factor in maintaining the intracellular balance of iron metabolism. In most cases, the active iron is predominantly ferrous iron (Fe)²⁺) Is present in the form of (1). Therefore, for studying physiological processes related to iron element in organisms, and some diseases caused by the change of iron concentration, active Fe can be studied²⁺The concentration of (b) is determined. By detecting the content of ferrous ions, the content of iron elements in cells can be reflected in real time, and abnormal conditions of organisms can be found in time.

The common ferrous ion detection methods at present mainly comprise inductively coupled plasma emission spectroscopy, atomic absorption and spectrophotometry. However, these methods require expensive instruments, are complicated to operate, require complicated pretreatment when measuring a sample, and cannot be applied to real-time in-situ detection of metal ions in a living body. Therefore, a novel simple Fe was developed²⁺The detection method has important significance.

compared with the traditional instrument detection methods (such as mass spectrometry, electrochemical method, chromatography and the like), the organic fluorescent probe serving as a novel fluorescence analysis technology has the advantages of simplicity, convenience, low cost, simplicity in instrument operation, high selectivity, high sensitivity and the like. Most of the reports to date relate to the detection of Fe³⁺Quenching and enhancement fluorescent probes of (2), and for Fe²⁺Few fluorescent probes are reported in detection, and the fluorescent probes are used for Fe in terms of the development of the current iron ion probes²⁺The development of efficiently discriminating fluorescent probes is challenging and opportunistic.

disclosure of Invention

The invention provides a ferrous ion fluorescent probe molecule based on a dansyl acid structure, a preparation method and application, wherein the ferrous ion fluorescent probe molecule is prepared by a simple one-step reaction, nitrogen atoms in a commercialized dansyl chloride structure are subjected to nitrogen oxidation reaction through commercialized m-chloroperoxybenzoic acid, and acid generated by the neutralization reaction of alkaline aluminum oxide, so that a dansyl acid-based probe is obtained, and the probe can be used as Fe in a Tris-DMSO solution system²⁺the highly selective fluorescent probe of (1).

The technical scheme for realizing the invention is as follows:

A ferrous ion fluorescent probe molecule based on dansyl acid structure and having a molecular formula of C₁₂H₁₄NO₄S, abbreviated as: FeP1, and the structural formula of the fluorescent probe is as follows:。

The reaction structure formula is as follows:

。

the preparation method of the ferrous ion fluorescent probe molecule based on the dansyl acid structure comprises the following steps:

(1) Dissolving m-chloroperoxybenzoic acid in anhydrous dichloromethane to obtain a solution A;

(2) Dissolving dansyl chloride in anhydrous dichloromethane to obtain a solution B, slowly dropwise adding the solution A into the solution B under the condition of ice bath stirring, heating to room temperature for reaction for a period of time, adding a certain amount of alkaline alumina, stirring for a period of time, removing the solvent under reduced pressure, and separating by silica gel column chromatography to obtain a product FeP1, namely the fluorescent probe molecule.

The molar ratio of the intermediate chloroperoxybenzoic acid in the step (1) to the dansyl chloride in the step (2) is (1-5): 1, the room-temperature reaction time in the step (2) is 2-12 h.

The molar ratio of dansyl chloride to basic alumina in the step (2) is 1: (2-8) stirring for 1-4 h.

The silica gel column chromatography separation in the step (2) adopts the eluent of methanol and dichloromethane with the volume ratio of 1 (4-15), and the yield is 40-90%.

The dansyl acid structure-based fluorescent probe molecule is applied to the field of high-sensitivity and specific detection of ferrous ions.

The invention has the beneficial effects that:

(1) The fluorescent probe is a dansyl acid-based compound, almost no fluorescence exists in a solution due to the existence of nitric oxide in the probe structure, when the probe and ferrous ions are subjected to specific identification, the probe is reduced to be a new product dansyl acid, an Intramolecular Charge Transfer (ICT) effect occurs in the probe structure, and strong fluorescence is released, so that the fluorescent identification of the ferrous ions is realized. The reaction mechanism is verified by means of high-resolution mass spectrometry (as shown in figure 5), and the high-resolution mass spectrometry test result of the solution after the ferrous ions are identified by the fluorescent probe FeP1 is 250.0543 ([ M-H ]⁺]^-) The theoretical calculation value of the negative ion of the final reduction product dansyl acid is 250.0543. This data corroborates the mechanism of action shown in FIG. 4.

(2) The fluorescent probe is prepared by one-step reaction of a commercialized reagent dansyl chloride and a commercialized m-chloroperoxybenzoic acid, and has the advantages of high synthesis yield, low preparation cost and wide practicability.

(3) According to the invention, the identification performance of the probe FeP1 on related analytes in a Tris-DMSO solution is researched through a fluorescence spectrometer, the probe has specific selectivity on ferrous ions, has strong anti-interference performance on metal cations and common anions, and has the lowest detection limit on the ferrous ions of 18 nM. The test result shows that the probe FeP1 has practical value for detecting ferrous ions in environment and biological systems, and has wide application prospect in the field of environment and biological system detection.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of a fluorescent probe FeP 1.

FIG. 2 is a nuclear magnetic resonance carbon spectrum of a fluorescent probe FeP 1.

FIG. 3 is a high-resolution mass spectrum of the fluorescent probe FeP1 of the invention.

FIG. 4 shows that the fluorescent probe FeP1 of the invention recognizes Fe²⁺The reaction mechanism diagram of (1).

FIG. 5 shows that the fluorescent probe FeP1 of the invention recognizes Fe²⁺High resolution verification of the reaction mechanism (2).

FIG. 6 is a graph showing the fluorescence selectivity of the fluorescent probe FeP1 of the invention to common metal ions, and the excitation wavelength is 350 nm.

FIG. 7 is a fluorescence selectivity diagram of the fluorescent probe FeP1 of the invention for common anion, active sulfur and active oxygen small molecules, and the excitation wavelength is 350 nm.

FIG. 8 shows that the fluorescent probe FeP1 of the invention recognizes Fe²⁺The anti-interference graph of common metal ions has an excitation wavelength of 350 nm and an emission wavelength of 489 nm.

FIG. 9 shows that the fluorescent probe FeP1 of the invention recognizes Fe²⁺The anti-interference graph of common anions, active sulfur and active oxygen has an excitation wavelength of 350 nm and an emission wavelength of 489 nm.

FIG. 10 shows that the fluorescent probe FeP1 of the invention recognizes Fe²⁺The pH value of the fluorescent powder is shown in an application range diagram, the excitation wavelength is 350 nm, and the emission wavelength is 489 nm.

FIG. 11 shows that the fluorescent probe FeP1 of the invention recognizes Fe²⁺Fluorescence titration plot of (a), excitation wavelength 350 nm.

FIG. 12 shows that the fluorescent probe FeP1 of the invention recognizes Fe²⁺The excitation wavelength is 350 nm, and the emission wavelength is 489 nm.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.

The chemical reagent, solvent, metal ion and the like used in the process of preparing the fluorescent probe FeP1 are all purchased from Aladdin reagent company. In the characterization and performance test process of the fluorescent probe FeP1, a DTX-400 nuclear magnetic resonance spectrometer of Bruke company is adopted, the solvent is deuterated chloroform, and a nuclear magnetic resonance hydrogen spectrum and a carbon spectrum are recorded by taking TMS as an internal standard. High resolution mass spectral data were recorded using a Q-exact HR-MS mass spectrometer from Thermo.