CN108949156B

CN108949156B - Fluorescent composite material and preparation method and application thereof

Info

Publication number: CN108949156B
Application number: CN201811009990.XA
Authority: CN
Inventors: 仉华; 刘俊维; 牛慧玉; 王亚甫; 冯北斗; 王鑫
Original assignee: Henan Normal University
Current assignee: Henan Normal University
Priority date: 2018-08-31
Filing date: 2018-08-31
Publication date: 2021-04-20
Anticipated expiration: 2038-08-31
Also published as: CN108949156A

Abstract

Fluorescent composite material and its preparation process and application. The fluorescent composite material is obtained by compounding organic dye molecules and phosphorus-containing inorganic salt, wherein the organic dye molecules have a structure shown in a general formula I. The fluorescent composite material containing the nitrogenous organic naphthalene heterocyclic derivative and the phosphorus inorganic salt can identify and detect DNA in cell nucleuses in living cells. The material has a certain level of water solubility, simultaneously has good cell membrane permeability, and simultaneously has lower phototoxicity, photobleaching property and proper biotoxicity. The spectral range of which is sufficiently different from the spectral range of the biological sample.

Description

Fluorescent composite material and preparation method and application thereof

Technical Field

The invention relates to a design and synthesis of an organic dye molecule for detecting DNA in a cell nucleus and application thereof in biological analysis, in particular to a fluorescent composite material of the organic dye molecule and phosphorus-containing inorganic salt.

Background

With the increasing concern of people on life and health, people pay more and more attention to the prevention of diseases. Various diseases occur as a result of changes in subcellular organelles. The development of science and technology field makes human research on health field more and more microscopic, even microscopic to fine substances such as amino acid and base. More and more human diseases are being identified as being associated with mutations at the genetic level. Many diseases are caused by gene mutations, but not every gene mutation causes a disease. Mutation of a gene is damage to DNA (deoxyribonucleic acid). DNA is a major component of chromosomes in the nucleus, and functions to store genetic information. DNA is an organic compound with a double-helix structure and consists of nucleotides. The nitrogenous base of the nucleotide is adenine, guanine, cytosine and thymine. The DNA participates in the inheritance of genes in the propagation process and is copied to the next generation in a semi-reserved copying mode to complete the inheritance of the genes. Research shows that there are ten thousand genes in human body, and healthy human body has many variant genes, which will not cause diseases although the genes are mutated. Some genetic variations can lead to disease and even death. More than half of the gene mutations were found to be associated with disease. It has been reported that DNA damage can cause the development of some common diseases, such as lung cancer, breast cancer, etc. Therefore, the method has good application prospect by the method for early diagnosis of DNA damage.

DNA detection has always played an important role in the fields of pathogen analysis, genetic disease diagnosis and forensic testing. Existing detection methods, such as biosensors based on hybridization of capture DNA to target DNA, can detect DNA directly, sensitively, and rapidly without the need for target amplification; based on fluorescence, colorimetry, Surface Plasmon Resonance (SPR), electrochemistry, reflectance spectroscopy, infrared absorption spectroscopy, Surface Enhanced Raman Spectroscopy (SERS), and the like. These methods, while highly sensitive and efficient for routine detection, are expensive and require complex instrumentation and operation. In addition, some methods do not allow for real-time dynamic monitoring of organisms and cells. Therefore, it is of great interest to develop a tool and method that can target organelles and perform DNA detection. In recent years, fluorescent dyes combined with the developing microscopic imaging technology provide a feasible important microscopic monitoring tool for eliminating the defects, and the optical molecular imaging technology taking the fluorescent dyes as the core provides a potential visualization tool for the relevant basic research of DNA. In recent years, nanocomposites have received much attention from many researchers as a new functional material. Researchers have conducted systematic and intensive research and exploration on the preparation method and the application field of the nano composite material. The organic fluorescent composite material is a functional nano composite material prepared by combining organic fluorescent dye and other inorganic materials. The composite material has double functions, not only has excellent performance of a nano-grade material, but also has the fluorescence characteristic of the organic fluorescent dye. Therefore, organic and inorganic composite materials with low/no toxicity, high membrane permeability, high selective recognition and biological imaging are a novel development direction of biological materials, and the design, synthesis and preparation of the organic and inorganic composite materials are still a blank field.

Disclosure of Invention

The invention aims to provide a fluorescent composite material, which is a compound of a nitrogen-containing organic naphthalene heterocyclic derivative and a phosphorus-containing inorganic salt, wherein the fluorescent composite material is obtained by compounding organic dye molecules and a phosphorus-containing inorganic salt (M), wherein the organic dye molecules have a structure shown in a general formula I:

in the general formula I:

k is selected from K₁，K₂And K₃Wherein the dotted bond represents an unsaturated double bond;

l is selected from- (CH)₂)_pN(CH₃)₂、-(CH₂)_pNHCOOH、-(CH₂)_pNHCH₃、-(CH₂)_pCH₃、-(CH₂)_pNH(CH₂)_qCH₃、-(CH₂)_pNH₂、-(CH₂)_pNHOH and- (CH)₂)_pN(COOH)₂Wherein p and q are each independently selected from integers of 1 to 8;

said K₁，K₂And K₃R in (1)₁，R₂，R₃，R₄And R₅Each independently selected from-H, -CN, -NH₂、-OH、-(CH₂)_xCH₃、-(CH₂)_xCN、-(CH₂)_xCOOH、-(CH₂)_xOH、-(CH₂)_xCOCH₃、-(CH₂OCH₂)_xCOOH、-(CH₂OCH₂)_xOH、-COO(CH₂)_xCH₃、-CH[(CH₂)_xCOOH]₂and-CH [ (CH)₂)_xOH]₂Wherein x is independently selected from an integer from 1 to 8.

On the other hand, the invention also provides a preparation method of the fluorescent composite material, which comprises the following steps:

1) respectively reacting 4-bromoacenaphthenequinone with the compounds a, b and c according to the molar ratio of 1:1-1:7 to prepare a compound i, a compound ii and a compound iii;

the reaction temperature is 0-200 ℃, the reaction time is 1-32 hours, and the reaction solvent is selected from ethanol, glacial acetic acid, ethylene glycol monomethyl ether, methanol, dichloromethane, acetone, glycerol, ethyl acetate or the mixture thereof;

2) compounds i, ii, iii are each independently of the compound L-NH₂Reacting in a molar ratio of 1:1 to 1:10 to prepare a compound iv, a compound v and a compound vi;

the reaction temperature is 0-180 ℃, the reaction time is 1-18 hours, and the reaction solvent is selected from ethanol, glacial acetic acid, ethylene glycol monomethyl ether, methanol, dichloromethane, acetone, glycerol, ethyl acetate or the mixture thereof;

3) compounds iv, v, vi are each Ca-exchanged with phosphorus-containing inorganic salts²⁺Participating in reaction complexation, organic dye molecules, Ca² ⁺The reaction molar ratio of the phosphorus-containing inorganic salt to the phosphorus-containing inorganic salt is 1:10:10 to 1:100: 80.

Describing step (3) in the above preparation method more specifically, the organic dye molecule and the phosphorus-containing inorganic salt in the step are compounded through the following reaction steps:

(1) organic dye molecules with Ca (NO)₃)₂.4H₂O in molar ratioMixing and stirring the mixture in a solvent at a ratio of 1:10-1:100 for reaction for 3-12 h; the solvent is selected from ethanol, glacial acetic acid, ethylene glycol monomethyl ether, methanol, dichloromethane, acetone, glycerol, ethyl acetate or a mixture thereof;

(2) adding a phosphorus-containing inorganic salt aqueous solution with the concentration of 0.01mol/L-3.0mol/L into the system obtained in the step (1) under stirring, and then reacting at the temperature of 0-100 ℃ for 36-120h, wherein the PH is kept at 8.0-10 during the reaction.

The fluorescent composite material provided by the invention has double functions, not only has excellent performance of a nano-grade material, but also has the fluorescence characteristic of an organic fluorescent dye. Can be combined with DNA macromolecules through the adsorption effect of the material; the action mechanism of the dye composite material and DNA molecules can be reproduced in real time through the fluorescence characteristic of the organic fluorescent dye.

In addition, the fluorescent composite material has the characteristics of fluorescent dyes such as high sensitivity and resolution, low/no toxicity, proper water solubility, high selective recognition, special optical properties and the like. The spectral range of the biological sample has enough difference with the spectral range of the biological sample, and the generation of biological autofluorescence can be avoided.

Therefore, another object of the present invention is to provide an application of the fluorescent composite material of the present invention in the preparation of DNA targeting detection reagents or anti-tumor drugs. In particular to the application in preparing a nuclear DNA targeting detection reagent.

Drawings

The invention is illustrated in figure 13:

FIG. 1 shows a general structural formula I of the fluorescent composite material of the present invention.

FIG. 2 shows Material A₁Transmission electron micrograph (c).

FIG. 3 shows Material A₁The absorption spectrum of (a).

FIG. 4 shows Material A₂Transmission electron micrograph (c).

FIG. 5 shows Material A₂The emission spectrum of (1).

FIG. 6 shows Material A₃Transmission electron microscope photograph ofAnd (3) slicing.

FIG. 7 shows Material A₃The water solubility detection test result chart of (1).

FIG. 8 shows Material A₄Transmission electron micrograph (c).

FIG. 9 is dye A₄-2 water solubility test result graph.

FIG. 10 is dye molecule A₁-2 image test result chart for intracellular DNA.

FIG. 11 is dye molecule A₂-2 specific recognition and detection result diagram for intracellular DNA.

FIG. 12 is dye molecule A ₃2, a result of the detection of the specific recognition of the intracellular DNA, wherein a in FIG. 12 is a graph of the effect of an undigested control composition image of the cell; fig. 12 b is a graph showing the effect of the experimental composition image after the cell digestion treatment.

FIG. 13 is dye molecule A₄-2 specific recognition and detection result diagram for intracellular DNA. FIG. 13 a is a graph showing the effect of control images of undigested cells; FIG. 13 b is a graph showing the effect of the experimental composition image after the cell digestion treatment.

Detailed Description

The invention mainly aims to provide a fluorescent composite material obtained by compounding organic dye molecules and phosphorus-containing inorganic salt (M). Wherein the organic dye molecule has the structure of formula I:

in one embodiment, K is K₁Wherein R is₁And R₂Each independently selected from-H, -CN, -NH₂、-OH、-(CH₂)_xCH₃、-(CH₂)_xCN、-(CH₂)_xCOOH、-(CH₂)_xOH、-(CH₂)_xCOCH₃、-(CH₂OCH₂)_xCOOH、-(CH₂OCH₂)_xOH、-COO(CH₂)_xCH₃、-CH[(CH₂)_xCOOH]₂and-CH [ (CH)₂)_xOH]₂(ii) a Preferably, R is₁And R₂Independently selected from-H, -CN, -OH, - (CH)₂)_xCN and- (CH)₂)_xAnd (5) OH. Wherein x is an integer from 1 to 8, preferably an integer from 1 to 4.

In a second embodiment, K is K₂Wherein R is₃At K₂Optionally substituted on the phenyl ring, the meaning of said optionally substituted being: any number of substitutions at any position. R₃Selected from-H, -CN, -NH₂、-OH、-(CH₂)_xCH₃、-(CH₂)_xCN、-(CH₂)_xCOOH、-(CH₂)_xOH、-(CH₂)_xCOCH₃、-(CH₂OCH₂)_xCOOH、-(CH₂OCH₂)_xOH、-COO(CH₂)_xCH₃、-CH[(CH₂)_xCOOH]₂and-CH [ (CH)₂)_xOH]₂(ii) a preferably-H, -CN, -OH, - (CH)₂)_xCN and- (CH)₂)_xAnd (5) OH. Wherein x is an integer from 1 to 8, preferably an integer from 1 to 4.

In a third embodiment, K is K₃Wherein R is₄And R₅Each independently selected from-H, -CN, -NH₂、-OH、-(CH₂)_xCH₃、-(CH₂)_xCN、-(CH₂)_xCOOH、-(CH₂)_xOH、-(CH₂)_xCOCH₃、-(CH₂OCH₂)_xCOOH、-(CH₂OCH₂)_xOH、-COO(CH₂)_xCH₃、-CH[(CH₂)_xCOOH]₂and-CH [ (CH)₂)_xOH]₂(ii) a Preferably, R is₁And R₂Independently selected from-H, -CN, -OH, - (CH)₂)_xCN and- (CH)₂)_xAnd (5) OH. Wherein x is an integer from 1 to 8, preferably an integer from 1 to 4.

In another embodiment, L is selected from- (CH)₂)_pN(CH₃)₂、-(CH₂)_pNHCOOH、-(CH₂)_pNHCH₃、-(CH₂)_pCH₃、-(CH₂)_pNH(CH₂)_qCH₃、-(CH₂)_pNH₂、-(CH₂)_pNHOH and- (CH)₂)_pN(COOH)₂(ii) a Preferably, L is selected from- (CH)₂)_pN(CH₃)₂、-(CH₂)_pNHCOOH、-(CH₂)_pNHCH₃And- (CH)₂)_pNH(CH₂)_qCH₃(ii) a Most preferably, L is selected from- (CH)₂)_pN(CH₃)₂And- (CH)₂)_pNHCOOH. Wherein p and q are each independently selected from integers of 1 to 8; preferably an integer of 1 to 4.

In the fluorescent composite material, the organic dye molecules and the phosphorus-containing inorganic salt pass through Ca²⁺Participating in reaction complexation, organic dye molecules, Ca²⁺The reaction molar ratio of the phosphorus-containing inorganic salt to the phosphorus-containing inorganic salt is 1:10:10 to 1:100: 80. More specifically, the organic dye molecule and the phosphorus-containing inorganic salt are compounded through the following reaction steps:

(1) organic dye molecules with Ca (NO)₃)₂.4H₂Mixing and stirring O in a solvent according to a molar ratio of 1:10-1:100 for reaction for 3-12 h; the solvent is selected from ethanol, glacial acetic acid, ethylene glycol monomethyl ether, methanol, dichloromethane and acetoneGlycerol, ethyl acetate or mixtures thereof;

The phosphorus-containing inorganic salt (M) mentioned in the above-mentioned technical means of the present invention is selected from the group consisting of diammonium hydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, Hydroxyapatite (HAP), octacalcium phosphate (Ocp), calcium hydrogen phosphate (DCP), monocalcium phosphate (MCP) and tricalcium phosphate (TCP). Dipotassium hydrogen phosphate, potassium dihydrogen phosphate, Hydroxyapatite (HAP), or calcium dihydrogen phosphate (MCP) is preferred.

The combination of the preferred technical features, leading to the most preferred embodiment of the invention with respect to the fluorescent composite material, can be represented as 1-1, 1-2, 1-3 and 1-4:

in another aspect, the present invention provides a method for preparing the above optical composite material, comprising the steps of:

in a specific embodiment, the reaction temperature of step 1) is preferably 30 to 180 ℃, more preferably 50 to 180 ℃, and most preferably 60 to 160 ℃; the reaction time is preferably 1 to 24 hours, more preferably 1 to 20 hours, and most preferably 1 to 18 hours; the reaction solvent is preferably ethanol, glacial acetic acid, ethylene glycol monomethyl ether, methanol, dichloromethane, acetone, glycerol, ethyl acetate or a mixture thereof, more preferably ethanol, glacial acetic acid, ethylene glycol monomethyl ether, methanol, dichloromethane, acetone, ethyl acetate or a mixture thereof, and most preferably ethanol, glacial acetic acid, ethylene glycol monomethyl ether, methanol, ethyl acetate or a mixture thereof; the reaction of 4-bromoacenaphthenequinone with compounds a, b, c is preferably carried out in a molar ratio of 1:1 to 1:7, more preferably 1:1 to 1:6.5, most preferably 1:1 to 1:6.

in a particular embodiment, the reaction temperature of step 2) is preferably 0 to 150 ℃, more preferably 25 to 140 ℃, most preferably 60 to 140 ℃; the reaction time is preferably 1 to 15 hours, more preferably 1 to 13 hours, and most preferably 1 to 10 hours; the reaction solvent is preferably ethanol, glacial acetic acid, methanol, ethylene glycol monomethyl ether, acetone, ethyl acetate or a mixture thereof, more preferably ethanol, glacial acetic acid, methanol, ethylene glycol monomethyl ether, ethyl acetate or a mixture thereof, and most preferably glacial acetic acid, methanol, ethylene glycol monomethyl ether, ethyl acetate or a mixture thereof; the compounds i, ii, iii are reacted with the compounds L-H, respectively, in a preferred molar ratio of 1:1 to 1:10, more preferably 1:1 to 1:8, most preferably 1:1 to 1: 7.

This step can be further described specifically as comprising a 2-step reaction process:

(1) organic dye molecules with Ca (NO)₃)₂.4H₂Mixing and stirring O in a solvent according to a molar ratio of 1:10-1:100 for reaction for 3-12 h; the solvent is selected from ethanol, glacial acetic acid, ethylene glycol monomethyl ether, methanol, dichloromethane, acetone, glycerol, ethyl acetate or a mixture thereof; it will be readily appreciated that the organic dye molecules referred to therein are compounds iv, v and vi.

Wherein the organic dye molecule is in contact with Ca (NO)₃)₂.4H₂O is preferably present in a molar ratio of 1:10 to 1:100, more preferably 1:15 to 1:90, most preferably 1:20 to 1: 85; the stirring time is preferably 3 to 10 hours, more preferably 3 to 9 hours, and most preferably 3 to 8 hours; the reaction solvent is preferably ethanol, glacial acetic acid, methanol, dichloromethane, acetone, glycerol or a mixture thereof, and most preferably ethanol, methanol, dichloromethane, glycerol, acetone, ethyl acetate or a mixture thereof.

Wherein, the reaction temperature is preferably 10-100 ℃, more preferably 15-90 ℃, and most preferably 20-80 ℃; the reaction time is preferably 36 to 110h, more preferably 40 to 100h, most preferably 50 to 90 h.

K₂HPO₄The mol ratio of the total dropping amount to the organic dye molecules is preferably 1:10-1: 80, more preferably 1:15-1:70, most preferably 1:20-1: 60.

The structure of the fluorescent composite material prepared by the synthesis method is confirmed by a nuclear magnetic resonance spectrogram, a transmission electron microscope and the like.

The fluorescent composite material provided by the invention has the following advantages:

the fluorescent composite material introduces a recognition group capable of being specifically combined with DNA, so that the specificity and specificity of the fluorescent composite material of organic dye molecules and phosphorus-containing inorganic salt on DNA detection in cell nuclei are improved. The organic dye has excellent optical performance, and has low biological photobleaching and photodamage when being applied to biological sample imaging.

The dye compound has the advantages of small toxicity and side effects, easily obtained raw materials, simple structure, easy preparation and easy industrialization.

The fluorescent composite material combines the organic dye and the inorganic salt, so that the fluorescent composite material not only has the excellent performance of a nano-grade material, but also has the self-fluorescent characteristic of the organic fluorescent dye.

In view of the above, the fluorescent composite material of the present invention can be used for the identification and detection of DNA in cell nucleus. In addition to being used directly in the form described herein for the recognition and detection of DNA in the nucleus, compositions containing the fluorescent composites of the present invention can also be used for the recognition and detection of DNA in the nucleus. The composition should contain an effective amount of one of the fluorescent composite materials of the organic dye molecules and the phosphorus-containing inorganic salt provided by the invention. In addition, other components required for incubation of the biological sample may also be included, such as solvents, pH adjusting agents, and the like. These components are all known in the art. The compositions described above may be presented as aqueous solutions or may be presented in other suitable forms for constitution with water as a solution prior to use.

The present invention also provides a method for identifying and detecting DNA in cell nucleus using the above fluorescent composite material of the present invention, which comprises the step of contacting the organic dye molecule and the fluorescent composite material containing inorganic salt of phosphorus type with a biological sample. The term "contacting" as used herein may include contacting in solution or in a solid phase.

The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way.

Example 1

Fluorescent composite material A₁The synthetic route of (2) is as follows:

(1) compound A₁Synthesis of (E) -1

20mmol of 4-bromoacenaphthenequinone and 80mmol of diaminomaleonitrile were added to a solution containing 10mL of glacial acetic acidA round bottom flask in acid solution. The reaction was heated to 110 ℃ and refluxed for 8h before stopping. The solvent was distilled off under reduced pressure and the residue was subjected to column chromatography (dichloromethane: petroleum ether, 3:1, v/v) to give a yellow compound A₁-1，

The yield thereof was found to be 80%.

(2) Compound A₁Synthesis of (E) -2

20mmol of compound A₁-1 and 120mmol of N, N-dimethylethylenediamine were added to a round-bottomed flask containing 20mL of a solution of ethylene glycol monomethyl ether, and the reaction was heated to 125 ℃ under reflux for 6 hours and then stopped. Pouring the mixture into ice water, precipitating, vacuum filtering to obtain brick red crude product, and treating with column chromatography (dichloromethane: methanol, 50:1-50:3, v/v) to obtain brick red solid powder compound A₁-2, yield 60%.

(3) Material A₁Synthesis of (2)

A flask containing 3mL of water was charged with 0.0004mol of Ca (NO)₃)₂.4H₂Stirring and dissolving O to obtain a calcium ion solution for later use; then 0.002g A was added to the flask₁Preparing a dye solution by using-2 and 2mL of absolute ethyl alcohol, adding the dye solution into the prepared calcium ion solution under stirring, and placing the calcium ion solution on a magnetic stirrer to stir for 6 hours for later use.

0.00027mol of K₂HPO₄(i.e., dipotassium hydrogenphosphate) was dissolved in 5mL of water, and the solution was slowly added dropwise to the above mixed solution. And under the condition of constant-temperature condensation and reflux, heating the mixed solution in an oil bath at the temperature of 30 ℃ for reaction for 72 hours. In the reaction process, dilute ammonia water is dripped to maintain the pH of the solution>7.2. After the reaction is finished, the mixture is kept stand and cooled, and light pink solid precipitate is generated. Removing the solution, adding deionized water to repeatedly wash the precipitate at the bottom until the solution is clear and transparent, removing the excessive solution, standing, and naturally drying the precipitate for about 15 days. Material A giving a light pink powder product with a loose structure₁。

(4) Material A₁Is characterized by

To A₁In A₁Characterization of section-2:¹H NMR(600MHz，CD₃Cl)δ8.45(d,J＝6Hz,1H)8.28(d,d J＝6,24Hz,2H),7.84(t,J＝18Hz,1H),6.84(s,1H),6.77(d,J＝12Hz,1H),3.52(s,2H),2.81(s,2H),2.39(s,6H).

to A₁The characterization of (1):¹H NMR(600MHz，CD₃Cl)δ8.45(d,J＝6Hz,1H)8.28(d,d J＝6,24Hz,2H),7.84(t,J＝18Hz,1H),6.92(s,1H),6.77(d,J＝12Hz,1H),3.52(s,2H),2.81(s,2H),2.39(s,6H).

from A₁And A₁-2 of¹And (4) comparing and analyzing the H NMR test result, wherein the analysis shows that: a. the₁NH peak in-2 is still shown, but chemical shift is changed due to A₁-2 is connected with the inorganic phosphate composite nano material through hydrogen bond to form A₁。

The resulting Material A₁The transmission electron micrograph of (A) is shown in FIG. 2. As can be seen, material A₁Is a nano material.

Example 2: absorption spectrum detection test of Material A1

Weighing the synthesized material A₁Adding 0.0002g of the complex into a four-way cuvette containing 3mL of high-purity water, measuring the absorption spectrum of the complex, collecting the absorbance value of the complex at the wavelength of 200 and 800nm, wherein the wavelength corresponding to the maximum absorbance value is the absorption wavelength of the complex. The results are shown in FIG. 3.

The instrument used in this example was an Agilent 8453 uv spectrophotometer, respectively.

Example 3

Fluorescent composite material A₂The synthetic route of (2) is as follows:

(1) compound A₂Synthesis of (E) -1

20mmol of 4-bromoacenaphthenequinone and 80mmol of 3, 4-diaminophenylacetonitrile were added to a round-bottomed flask containing 10mL of glacial acetic acid solution. The reaction was heated to 110 ℃ and refluxed for 8h before stopping. The solvent was distilled off under reduced pressure and the residue was subjected to column chromatography (dichloromethane: petroleum ether, 3:1, v/v) to give a yellow compound A₂-1, yield 80%.

(2) Compound A₂Synthesis of (E) -2

20mmol of compound A₂-1 and 120mmol of N, N-dimethylethylenediamine were added to a round-bottomed flask containing 20mL of a solution of ethylene glycol monomethyl ether, and the reaction was heated to 125 ℃ under reflux for 6 hours and then stopped. Pouring the mixture into ice water, precipitating, vacuum filtering to obtain brick red crude product, and treating with column chromatography (dichloromethane: methanol, 50:1-50:3, v/v) to obtain brick red solid powder compound A₂-2, yield 60%.

(3) Material A₂Synthesis of (2)

A flask containing 3mL of water was charged with 0.0004mol of Ca (NO)₃)₂.4H₂Stirring and dissolving O to prepare a calcium ion solution for later use; then 0.002g A was added to the flask₂Preparing a dye solution by using-2 and 2mL of absolute ethyl alcohol, adding the dye solution into the prepared calcium ion solution under stirring, and placing the calcium ion solution on a magnetic stirrer to stir for 6 hours for later use.

0.00027mol of K₂HPO₄(i.e., dipotassium hydrogenphosphate) was dissolved in 5mL of water, and the solution was slowly added dropwise to the above mixed solution. And under the condition of constant-temperature condensation and reflux, heating the mixed solution in an oil bath at the temperature of 30 ℃ for reaction for 72 hours. In the reaction process, dilute ammonia water is dripped to maintain the pH of the solution>7.2. After the reaction is finished, the mixture is kept stand and cooled, and light pink solid precipitate is generated. Removing the solution, adding deionized water to repeatedly wash the precipitate at the bottom until the solution is clear and transparent, removing the excessive solution, standing, and naturally drying the precipitate for about 15 days. Material A giving a light pink powder product with a loose structure₂。

(4) Material A₂Is characterized by

To A₂In A₂Characterization of section-2:¹H NMR(600MHz，CD₃Cl)δ8.45(d,J＝6Hz,1H)8.28(d,d J＝6,24Hz,2H),7.84(t,J＝18Hz,1H),7.55-7.57(m,2H),6.90(d,J＝6.9Hz,1H)，6.84(s,1H),6.77(d,J＝12Hz,1H),4.33(s,2H),3.52(s,2H),2.81(s,2H),2.39(s,6H).

to A₂The characterization of (1):¹H NMR(600MHz，CD₃Cl)δ8.45(d,J＝6Hz,1H)8.28(d,d J＝6,24Hz,2H),7.84(t,J＝18Hz,1H),7.55-7.57(m,2H),6.92(s,1H),6.90(d,J＝6.9Hz,1H),6.77(d,J＝12Hz,1H),4.33(s,2H),3.52(s,2H),2.81(s,2H),2.39(s,6H).

from A₂And A₂-2, and comparative analysis of 1H NMR test results, analysis showing: a. the₂NH peak in-2 is still shown, but chemical shift is changed due to A₂-2 is connected with the inorganic phosphate composite nano material through hydrogen bond to form A₂。

The resulting Material A₂The transmission electron micrograph of (A) is shown in FIG. 4. As can be seen, material A₂Is a nano material.

Example 4: material A₂Emission spectrum detection test of

Weighing the synthesized material A₂0.0002g of the fluorescent substance is added into a four-way cuvette containing 3mL of high-purity water, the emission spectrum is measured, the excitation wavelength is 455nm, the fluorescence intensity of the wavelength at 480-650nm is collected, and the wavelength corresponding to the position with the strongest fluorescence intensity is the emission wavelength, and the result is shown in FIG. 5.

Example 5

Fluorescent composite material A₃The synthetic route of (2) is as follows:

(1) compound A₃Synthesis of (E) -1

20mmol of 4-bromoacenaphthenequinone and 80mmol of 3, 4-diaminophenylacetonitrile were added to a round-bottomed flask containing 10mL of glacial acetic acid solution. The reaction was heated to 110 ℃ and refluxed for 8h before stopping. The solvent was distilled off under reduced pressure and the residue was subjected to column chromatography (dichloromethane: petroleum ether, 3:1, v/v) to give a yellow compound A₃-1, yield 80%.

(2) Compound A₃Synthesis of (E) -2

20mmol of compound A₃-1 and 120mmol of N, N-dimethylethylenediamine were added to a round-bottomed flask containing 20mL of a solution of ethylene glycol monomethyl ether, and the reaction was heated to 125 ℃ under reflux for 6 hours and then stopped. Pouring the mixture into ice water, precipitating, vacuum filtering to obtain brick red crude product, and treating with column chromatography (dichlorine chloride)Methane and methanol at a ratio of 50:1-50:3, v/v) to obtain brick red solid powder compound A₃-2, yield 60%.

(3) Material A₃Synthesis of (2)

A flask containing 3mL of water was charged with 0.0004mol Ca (NO)₃)₂.4H₂Stirring and dissolving O to prepare a calcium ion solution for later use; then 0.002g A was added to the flask₃-2 and 2mL absolute ethanol to prepare a dye solution; the dye solution is added into the calcium ion solution prepared above under stirring, and the mixture is placed on a magnetic stirrer to be stirred for 6 hours for standby.

0.00027mol of K₂HPO₄(i.e., dipotassium hydrogenphosphate) was dissolved in 5mL of water, and the solution was slowly added dropwise to the above mixed solution. And under the condition of constant-temperature condensation and reflux, heating the mixed solution in an oil bath at the temperature of 30 ℃ for reaction for 72 hours. In the reaction process, dilute ammonia water is dripped to maintain the pH of the solution>7.2. After the reaction is finished, the mixture is kept stand and cooled, and light pink solid precipitate is generated. Removing the solution, adding deionized water to repeatedly wash the precipitate at the bottom until the solution is clear and transparent, removing the excessive solution, standing, and naturally drying the precipitate for about 15 days. Material A giving a light pink powder product with a loose structure₃。

(4) Material A₃Is characterized by

To A₃In A₃Characterization of section-2:¹H NMR(600MHz，CD₃Cl)δ11.09(s,1H),8.45(d,J＝6Hz,1H)8.28(d,d J＝6,24Hz,2H),8.03(s,1H),7.84(t,J＝18Hz,1H),6.84(s,1H),6.77(d,J＝12Hz,1H),3.35(s,2H),3.18(s,2H),1.52-1.49(m,4H).

to A₃The characterization of (1):¹H NMR(600MHz，CD₃Cl)δ11.09(s,1H),8.45(d,J＝6Hz,1H)8.28(d,d J＝6,24Hz,2H),8.03(s,1H),7.84(t,J＝18Hz,1H),6.92(s,1H),6.77(d,J＝12Hz,1H),3.35(s,2H),3.18(s,2H),1.52-1.49(m,4H)

from A₃And A₃-2 of¹And (4) comparing and analyzing the H NMR test result, wherein the analysis shows that: a. the₃NH peak in-2 is still shown, but chemical shift is changed due to A₃-2 is connected with the inorganic phosphate composite nano material through hydrogen bond to form A₃。

The resulting Material A₃The transmission electron micrograph of (A) is shown in FIG. 6. As can be seen, material A₃Is a nano material.

Example 6: material A₃Water solubility detection test of

0.2mg of the above-synthesized material A was weighed out₃Adding into 3mL of high-purity water, dissolving completely, measuring absorbance, adding 0.2mg of material each time to ensure gradient concentration, and measuring A under different addition₃Absorbance at the maximum absorption wavelength of the aqueous solution. The test results are shown in fig. 7, and the results show that: when the material A is₃When the concentration of (A) is more than 0.6mg/mL, the absorbance value deviates from the linear relation, i.e., the material A₃The solubility in water was 0.6 mg/mL.

Example 7

Fluorescent composite material A₄The synthetic route of (2) is as follows:

(1) compound A₄Synthesis of (E) -1

20mmol of 4-bromoacenaphthenequinone and 80mmol of Compound A were added to a round-bottomed flask containing 10mL of glacial acetic acid solution. The reaction was heated to 110 ℃ and refluxed for 8h before stopping. The solvent was distilled off under reduced pressure and the residue was subjected to column chromatography (dichloromethane: petroleum ether, 3:1, v/v) to give a yellow compound A₄-1, yield 80%.

(2) Compound A₄Synthesis of (E) -2

20mmol of compound A₄-1 and 120mmol of compound B are added into a round bottom flask containing 20mL of ethylene glycol monomethyl ether solution, and the reaction is heated to 125 ℃ and refluxed for 6h, and then stopped. The mixture was poured into ice-water and,precipitating, vacuum filtering to obtain brick red crude product, and treating with column chromatography (dichloromethane: methanol, 50:1-50:3, v/v) to obtain brick red solid powder compound A₄-2, yield 60%.

(3) Material A₄Synthesis of (2)

A flask containing 3mL of water was charged with 0.0004mol of Ca (NO)₃)₂.4H₂Stirring and dissolving O to prepare a calcium ion solution for later use; then 0.002g A was added to the flask₄Preparing a dye solution by using 2 and 2mL of absolute ethyl alcohol, adding the dye solution into the calcium ion solution prepared by the above method under stirring, and placing the calcium ion solution on a magnetic stirrer to stir for 6 hours for later use.

0.00027mol of K₂HPO₄(i.e., dipotassium hydrogenphosphate) was dissolved in 5mL of water, and the solution was slowly added dropwise to the above mixed solution. And under the condition of constant-temperature condensation and reflux, heating the mixed solution in an oil bath at the temperature of 30 ℃ for reaction for 72 hours. In the reaction process, dilute ammonia water is dripped to maintain the pH of the solution>7.2. After the reaction is finished, the mixture is kept stand and cooled, and light pink solid precipitate is generated. Removing the solution, adding deionized water to repeatedly wash the precipitate at the bottom until the solution is clear and transparent, removing the excessive solution, standing, and naturally drying the precipitate for about 15 days. Material A giving a light pink powder product with a loose structure₄。

(4) Material A₄Is characterized by

To A₄In A₄Characterization of section-2:¹H NMR(600MHz，CD₃Cl)δ11.09(s,1H),8.45(d,J＝6Hz,1H)8.28(d,d J＝6,24Hz,2H),8.03(s,1H),7.84(t,J＝18Hz,1H),7.58(s,2H),6.84(s,1H),6.77(d,J＝12Hz,1H),3.66(t,4H),3.65(s,2H),3.35(s,2H),3.18(s,2H),2.77(t,4H)，1.52-1.49(m,4H).

to A₄The characterization of (1):¹H NMR(600MHz，CD₃Cl)δ11.09(s,1H),8.45(d,J＝6Hz,1H)8.28(d,d J＝6,24Hz,2H),8.03(s,1H),7.84(t,J＝18Hz,1H),7.58(s,2H),6.92(s,1H),6.77(d,J＝12Hz,1H),3.66(t,4H),3.65(s,2H),3.35(s,2H),3.18(s,2H),2.77(t,4H)，1.52-1.49(m,4H).

from A₄And A₄-2 of¹And (4) comparing and analyzing the H NMR test result, wherein the analysis shows that: a. the₄NH peak in-2 is still shown, but chemical shift is changed due to A₄-2 is connected with the inorganic phosphate composite nano material through hydrogen bond to form A₄。

The resulting Material A₄The transmission electron micrograph of (A) is shown in FIG. 8. As can be seen, material A₄Is a nano material.

Example 8: dye A₄-2 Water solubility detection test

Weighing a certain amount of the synthesized dye A₄-2 preparing a 3mM DMSO solution, pipetting 1. mu.L of the above stock solution into 3mL water using a pipette, and adding each time in a gradient, stirring and dissolving sufficiently while ensuring that the final DMSO addition volume is less than one thousandth of the total volume. The absorbance values were measured, and the results are shown in FIG. 9. The results show that: when dye A₄At concentrations of-2 greater than 10. mu.M, the absorbance values deviate from a linear relationship, i.e.dye A₄Solubility in water of-2 is 10. mu.M.

Example 9: dye A₁-2 test for detecting cancer cell toxicity

HeLa cells were selected as the study subjects. The magnitude of the cytotoxicity of the dye was characterized by the cell viability. At 5 ﹡ 10⁴Cell density per mL was seeded in 96-well plates in a volume of 100. mu.L per well at 37 ℃ with 5% CO₂Culturing for 24h under the condition. Then adding the dye molecule A in gradient concentration₁-2 setting 5 multiple wells per gradient concentration in the culture medium, setting a blank control, and detecting the survival rate of the cells after culture. When in detection, 20 mu L of 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazolium bromide (MTT) solution is added into each hole, and the temperature is 37 ℃ and the CO content is 5 percent₂Culturing for 4h under the condition. The original culture was then removed and DMSO (150. mu.L/well) was added, and the OD was then measured three times using a microplate reader, and the results are shown in FIG. 10. The results show that: the above synthesized Material A₁-2 has a greater toxicity for cancer cells.

Example 10: dye molecule A₂-2 in intracellular imaging assays

HeLa cells were selected as the study subjects. Incubate in a confocal dish at 37 ℃ and 5% CO2 for 24 h. The medium was then decanted, rinsed three times with ice methanol, added with a volume of ice methanol and incubated at 4 ℃ for 30min, the ice methanol removed and washed 2-3 times with PBS. Then 2mL of PBS was added, followed by dye A at a final concentration of 30. mu.M₂And (5) 2, incubating for 1h, and carrying out laser confocal imaging. Representative areas were selected and observed with an oil-glass (60 ×) for three replicates, the results are shown in fig. 11. The results show that: fluorescent dye Compound A₂-2 has a strong fluorescence signal in the HeLa cell nucleus, the site where DNA is mainly concentrated.

Example 11: dye molecule A₃-2 specific recognition detection assay for intracellular DNA

HeLa cells were selected as the study subjects. Incubate in a confocal dish at 37 ℃ and 5% CO2 for 24 h. The medium was then decanted, rinsed three times with ice methanol, added with a volume of ice methanol and incubated at 4 ℃ for 30min, the ice methanol removed and washed 2-3 times with PBS. Then 2mL of PBS was added, followed by dye A at a final concentration of 30. mu.M₃After 1h of incubation, DNase digestive enzyme was added to the experimental group (b) and not to the control group (a), and laser confocal imaging was performed after a certain period of digestion. Representative areas were selected and observed with an oil-glass (60 ×), which was repeated three times. The results are shown in FIG. 12. The results show that: the imaging effect of the control group (a) in which the cells were not digested is shown in a of fig. 12, and the fluorescence signal is mainly strong in the nuclei of the cells, while the imaging effect of the experimental group (b) in which the cells were digested for a certain period of time is shown in b of fig. 12, and the fluorescence signal is reduced. This result shows that the above-synthesized dye molecule A₃-2 is an example of a dye that specifically recognizes DNA.

Example 12: dye molecule A₄-2 specific recognition detection assay for intracellular DNA

HeLa cells were selected as the study subjects. Incubate in a confocal dish at 37 ℃ and 5% CO2 for 24 h. Then pouring out the culture medium, washing with ice methanol for three times, and adding a certain amount of waterAfter incubation for 30min at 4 ℃ with a volume of ice methanol, the ice methanol was removed and washed 2-3 times with PBS. Then 2mL of PBS was added, followed by dye A at a final concentration of 30. mu.M₄After 1h of incubation, DNase digestive enzyme was added to the experimental group (b) and not to the control group (a), and laser confocal imaging was performed after a certain period of digestion. Representative areas were selected and observed with an oil-glass (60 ×), which was repeated three times. The results are shown in FIG. 13. The results show that: the imaging effect of the control group (a) in which the cells were not digested is shown in a of fig. 13, and the fluorescence signal is mainly strong in the nuclei of the cells, while the imaging effect of the experimental group (b) in which the cells were digested for a certain period of time is shown in b of fig. 13, and the fluorescence signal is reduced. This result shows that the above-synthesized dye molecule A₄-2 is an example of a dye that specifically recognizes DNA.

Claims

1. A fluorescent composite material is prepared by compounding organic dye molecules and phosphorus-containing inorganic salt, wherein the organic dye molecules have a structure shown in a general formula I:

in the general formula I:

k is selected from K₂And K₃Wherein the dotted bond represents an unsaturated double bond;

the L is selected from: - (CH)₂)_pN(CH₃)₂、-(CH₂)_pNHCOOH、-(CH₂)_pNHCH₃And- (CH)₂)_pNH(CH₂)_qCH₃P and q are each independently selected from integers of 1 to 4;

said K₂And K₃R in (1)₃，R₄And R₅Each independently selected from: -H, -CN, -NH₂、-OH、-(CH₂)_xCH₃、-(CH₂)_xCN、-(CH₂)_xCOOH、-(CH₂)_xOH、-(CH₂)_xCOCH₃、-(CH₂OCH₂)_xCOOH、-(CH₂OCH₂)_xOH、-COO(CH₂)_xCH₃、-CH[(CH₂)_xCOOH]₂and-CH [ (CH)₂)_xOH]₂Wherein x is independently selected from an integer from 1 to 8.

2. The fluorescent composite of claim 1, wherein L is selected from the group consisting of- (CH)₂)_pN(CH₃)₂And- (CH)₂)_pNHCOOH。

3. The fluorescent composite of claim 1, wherein R is₃，R₄And R₅Each independently selected from-H, -CN, -OH, - (CH)₂)_xCN and- (CH)₂)_xOH, wherein x is selected from an integer of 1 to 4.

4. The fluorescent composite of claim 1, wherein the inorganic salt comprising phosphorus is selected from the group consisting of diammonium hydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, hydroxyapatite, octacalcium phosphate, calcium hydrogen phosphate, calcium dihydrogen phosphate, and tricalcium phosphate.

5. The fluorescent composite of claim 4, wherein the inorganic salt of phosphorus is selected from the group consisting of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, hydroxyapatite and calcium dihydrogen phosphate.

6. The fluorescent composite of claim 1, wherein the organic dye molecules and the phosphorus-containing inorganic salt are Ca-doped²⁺Participating in reaction complexation, organic dye molecules, Ca²⁺The reaction molar ratio of the phosphorus-containing inorganic salt to the phosphorus-containing inorganic salt is 1:10:10 to 1:100: 80.

7. The fluorescent composite of claim 1, being a compound 1-2, or 1-4:

wherein M is a phosphorus-containing inorganic salt.

8. The method of preparing the fluorescent composite of claim 1, comprising the steps of:

1) respectively reacting 4-bromoacenaphthenequinone with the compounds b and c according to the molar ratio of 1:1-1:7 to prepare a compound ii and a compound iii;

2) compounds ii, iii are each independently of the compound L-NH₂Reacting in a molar ratio of 1:1-1:10 to prepare a compound v and a compound vi;

3) the compounds v, vi are each reacted with phosphorus-containing inorganic salts via Ca²⁺Participating in reaction complexation, organic dye molecules, Ca²⁺The reaction molar ratio of the phosphorus-containing inorganic salt to the phosphorus-containing inorganic salt is 1:10:10 to 1:100: 80.

9. The fluorescent composite material of claim 1, in the preparation of a DNA targeting detection reagent or an anti-tumor drug.