CN107686479B

CN107686479B - Near-infrared fluorescent probe compound and preparation method and application thereof

Info

Publication number: CN107686479B
Application number: CN201710913917.4A
Authority: CN
Inventors: 李海涛; 谷标; 苏伟; 张友玉; 姚守拙
Original assignee: Hunan Normal University
Current assignee: Hunan Normal University
Priority date: 2017-09-30
Filing date: 2017-09-30
Publication date: 2020-11-13
Anticipated expiration: 2037-09-30
Also published as: CN107686479A

Abstract

The invention provides near-infrared fluorescenceA probe compound and a preparation method and application thereof. The structural formula of the fluorescent probe compound provided by the invention is shown as a formula (1). The probe compound has good selectivity and sensitivity to copper ions and is not interfered by other metal ions, and the quantitative and qualitative detection of the copper ions in an environmental water sample and living cells is successfully realized.

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Description

Near-infrared fluorescent probe compound and preparation method and application thereof

Technical Field

The invention belongs to the technical field of analysis and detection, and particularly relates to a near-infrared fluorescent probe compound, a preparation method thereof and application thereof in copper ion detection.

Background

Copper (Cu)²⁺) Is considered to be the third important trace element in life after iron and zinc, which plays an important role in various physiopathological processes. However, high concentrations of copper ions can be toxic and harmful to the natural environment and human health. Excessive copper ions in cells have been reported to cause a serious health problem including gastrointestinal disorders, permanent damage to kidneys and nerves, and the like. Therefore, it would be of great interest to develop efficient and reliable methods for detecting copper ions in environments and biological systems.

Trace copper ions in environmental samples can be detected by traditional methods such as inductively coupled plasma mass spectrometry, atomic absorption spectrometry and stripping voltammetry. However, these methods have the disadvantages of expensive instruments, complicated processing, and destructive detection, and are difficult to satisfy in situ detection in biological samples. In contrast, fluorescence methods have received great attention because of their advantages such as high sensitivity, good selectivity, easy and simple operation, and non-destructive detection.

To date, many fluorescent probes have been reported for copper ion detection. Depending on the reaction mechanism, the copper ion fluorescent probes can be divided into two categories: the specific reaction induced by (I) copper ion and receptor complexation and (II) copper ion. The first copper ion fluorescent probe has an earlier development time, and usually utilizes the paramagnetic effect of copper ions to cause fluorescence quenching, which is mostly represented by an on-off type (quenching type) fluorescence change. Such fluorescent probes are less sensitive and are susceptible to interference from competing metal ions of similar ionic radius and chemical nature, such as iron, cobalt, nickel and mercury ions. The second kind of copper ion fluorescent probe is also called as active probe, and utilizes the specific chemical reaction of copper ion to induce the recognition group on the probe to hydrolyze and release fluorescent signal group to cause the fluorescent recovery. The probe has higher selection for copper ions, can effectively avoid the paramagnetic effect of the copper ions in the identification process, and has great attention in the design and preparation of probe molecules because the output signal is fluorescence enhancement type (recovery type). Unfortunately, most of the reported copper ion-active fluorescent probe emissions are in the uv-vis region and are susceptible to interference from background fluorescence in biological applications. The near-infrared (650-900nm) fluorescent probe provides possibility for people to research the existence of copper ions in living cells due to the advantages of low background interference, low light damage, deep tissue penetrability and the like. Therefore, the enhanced near-infrared fluorescent probe is used for detecting copper ions in environmental water samples and living cells with high sensitivity and high selectivity, and is very meaningful for environmental protection and human disease diagnosis.

Disclosure of Invention

The invention aims to provide a near-infrared fluorescent probe compound and a preparation method and application thereof. The fluorescent probe provided by the invention has higher sensitivity and selectivity to copper ions and is not interfered by other metal ions, so that the quantitative and qualitative detection of the copper ions in an environmental water sample and living cells is successfully realized, and a very effective detection method is provided for environmental protection and copper ion-induced disease diagnosis.

The invention provides a near-infrared fluorescent probe compound, which has a chemical structural formula shown as a formula (1) and is referred to as DCM-P for short.

The method for preparing the infrared fluorescent probe compound comprises the following steps:

(1) taking 4- (dicyanomethylene) -2- (4-hydroxystyrene) -4H-1-benzopyran as a raw material, adding picolinic acid, 4-dimethylaminopyridine, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and dichloromethane into the raw material, and uniformly mixing to obtain a mixed solution; the 4- (dicyanomethylene) -2- (4-hydroxystyryl) -4H-1-benzopyran is abbreviated as DCM-OH, the 4-dimethylaminopyridine is abbreviated as DMAP, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is abbreviated as EDC, and the formula proportion of the mixed solution is as follows:

(2) stirring the mixed solution at 20-40 ℃ for 5-24 hours;

(3) after the reaction is finished, a yellow precipitate is separated out, and the yellow precipitate is filtered and separated to obtain the pure probe DCM-P.

The formula proportion of the mixed solution in the step (1) is preferably as follows:

the formula proportion of the mixed solution in the step (1) is further preferably as follows:

step (2) is preferably stirred at 20-40 ℃ for 12-24 hours.

Structural identification of the compound:

the probe DCM-P NMR spectrum results are as follows:

¹HNMR(500MHz,DMSO-d₆)：8.84(d,J＝3.2Hz，1H)，8.73(d，J＝8.3Hz，1H)，8.26(d，J＝7.7Hz，1H)，8.10(t，J＝7.5Hz,1H)，7.98–7.86(m，3H)，7.85–7.71(m，3H)，7.62(t，J＝7.5Hz，1H)，7.54(d，J＝16.0Hz，1H)，7.44(d，J＝8.2Hz，2H)，7.05(s，1H)。

the probe DCM-P NMR carbon spectrum results are as follows:

¹³CNMR(126MHz,DMSO-d₆)：163.23，157.89，152.88，150.00，146.60，137.74，135.46，132.97，129.47，128.03，126.19,125.76，124.63，122.54，119.97，119.06，117.05，115.72，106.91，60.56。

the probe DCM-P high resolution mass spectrum result is as follows:

ESI-MSm/z：[M+Na]⁺440.1032 (experimentally determined values), 440.1006 (theoretically calculated values).

The invention also provides application of the near-infrared fluorescent probe compound in copper ion detection.

Preferably, the copper ions include copper ions in an environmental water sample and exogenous copper ions in living cells.

In a further preferred scheme, the method for detecting the copper ions in the environmental water sample by using the near-infrared fluorescent probe compound comprises the following steps:

(1) accurately weighing a certain amount of 4- (dicyanomethylene) -2- (4-pyridine formyl ester styryl) -4H-1-benzopyran compound (DCM-P) and dissolving in chromatographic pure dimethyl sulfoxide (DMSO) to prepare the compound with the concentration of 1.0X 10^-3Storing the DCM-P stock solution of mol/L at the temperature of 2-5 ℃;

(2) accurately weighing a certain amount of copper nitrate, respectively dissolving in deionized water to prepare the copper nitrate with the concentration of 1.0 multiplied by 10^-2Storing the copper ion stock solution in mol/L at the temperature of 2-5 ℃; diluting copper ion stock solutions with different volumes by using deionized water to obtain copper ion standard solutions with different concentrations;

(3) respectively adding 1mL of dimethyl sulfoxide and 0.5mL of phosphate buffer solution into a plurality of 2mL test tubes, respectively adding 0.02mL of DCM-P stock solution in the step (1), respectively adding copper ion standard solutions with different concentrations in the step (2), and fixing the volume to 2mL by using deionized water, wherein the volume of DMSO: H in the system at the moment is controlled₂O＝4:6v/v-6:4v/vAfter the mixed solution reacts for at least 30 minutes, measuring the fluorescence intensity of each solution by using a fluorescence spectrophotometer, determining the quantitative relation between the fluorescence intensity and the concentration of copper ions, and making a standard curve;

(4) adding 1mL of DMSO and 0.5mL of phosphate buffer solution into a 2mL test tube, adding 0.02mL of DCM-P stock solution in the step (1), adding the solution to be tested, diluting the volume to 2mL with deionized water, measuring the fluorescence intensity of the mixed solution after the mixed solution reacts for a period of time, and determining the concentration of copper ions in the solution to be tested according to a standard curve.

Wherein the phosphate buffer is preferably 10mM Na with pH 7.4₂HPO₄-NaH₂PO₄And (4) preparing the system.

Wherein the volume ratio of the DMSO to the water phase in the detection system is preferably 5:5 v/v.

Wherein the fluorescence intensity of the solution is measured after the mixing reaction is preferably 30 minutes to 40 minutes.

The fluorescence spectrophotometer parameters are set as an excitation slit 10nm, an emission slit 10nm, an excitation wavelength 560nm, an emission wavelength 677nm and a scanning range 695-850 nm.

In a further preferred scheme, the method for detecting the exogenous copper ions in the living cells by using the near-infrared fluorescent probe compound comprises the following steps:

(1) placing the living cells in a culture medium containing 10% fetal bovine serum and 1.0X 10^-5Culturing the probe DCM-P in a DMEM medium at the temperature of 37 ℃ for 30 minutes under the condition of 5% carbon dioxide in mol/L;

(2) sucking out the culture medium by using a suction pipe, washing the cells for 3 times by using phosphate buffer solution at 37 ℃, and then imaging by using a fluorescence inverted microscope;

(3) adding 1.0X 10 to the cells of step (2)^-5mol/L copper ion, incubation for 30 min, washing the cells 3 times with 37 ℃ phosphate buffer, and imaging with a fluorescence inverted microscope.

According to the invention, through experimental study on the recognition effect of the compound DCM-P and various metal ions, the DCM-P is found to have specific response to copper ions. The method can be used in the near infrared region (lambda)_em>650nm) implementationHigh sensitivity and high selectivity fluorescence detection of copper ions, and has larger Stokes shift (116 nm). In the aspect of practical application, the compound DCM-P can not only quantitatively detect copper ions in an environmental water sample, but also qualitatively detect exogenous copper ions in living cells. The preparation method of the near-infrared fluorescent probe has the advantages of mild reaction conditions, high yield, simple separation and the like.

Drawings

FIG. 1 is a hydrogen spectrum of probe DCM-P provided by the invention, and it can be seen from FIG. 1 that probe DCM-P is relatively pure and has no other impurities;

FIG. 2 is a carbon spectrum of probe DCM-P provided by the invention;

FIG. 3 is a high resolution mass spectrum of probe DCM-P provided by the invention; the peak at 440.1032 is [ DCM-P + Na]⁺A molecular ion peak;

FIG. 4 is a graph showing the change of fluorescence intensity of a mixed system after copper ions are added into probe DCM-P with different concentrations in example 14 of the present invention;

FIG. 5 is a histogram of fluorescence intensity of probe DCM-P in example 14 after adding copper ions in different buffer systems;

FIG. 6 is a graph showing the change of fluorescence intensity of probe DCM-P in example 14 after adding copper ions in different aqueous phases compared with organic phases;

FIG. 7 is a graph showing the change of the fluorescence intensity of the mixed system after adding copper ions into the probe DCM-P in example 14 of the present invention with pH

FIG. 8 is a graph showing the change of the fluorescence intensity of the mixed system with reaction time after adding copper ions into the probe DCM-P in example 14 of the present invention;

FIG. 9 is a fluorescence emission spectrum of a mixed system after adding copper ions with different concentrations into probe DCM-P in example 15 of the present invention;

FIG. 10A is a graph showing the change of fluorescence intensity of a mixed system after adding copper ions of different concentrations to the probe DCM-P in example 15 of the present invention, and FIG. 10B is a linear regression curve showing the fluorescence emission intensity of copper ions of different concentrations;

FIG. 11 is a fluorescence emission spectrum of a mixed system after different metal ions are added into probe DCM-P in example 16 of the present invention;

FIG. 12 is a histogram of fluorescence intensity of a mixed system in which copper ions are added to the probe DCM-P in the presence of other metal ions in example 17 of the present invention;

FIG. 13 is a graph showing the change of hydrogen spectrum of probe DCM-P before and after addition of copper ions in example 18 of the present invention;

FIG. 14 is a high resolution mass spectrum of probe DCM-P of example 18 after reacting with copper ion;

FIG. 15 is a photomicrograph of Hela cells incubated with probe DCM-P of example 20 of the present invention before and after addition of copper ions.

Detailed Description

The present invention is described in more detail below with reference to examples, but it should not be construed that the scope of the present invention is limited to the examples below, and any technologies implemented based on the above contents of the present invention are within the scope of the present invention.

Example 1

187.4 mg of DCM-OH, 49.2 mg of picolinic acid, 7.3 mg of DMAP and 138.0 mg of EDC were weighed out accurately in a dry round bottom flask, then 20 ml of dichloromethane was added and stirred at 30 ℃ for 12 hours; after the reaction was complete, a yellow solid precipitated and was monitored by TLC spot plate, and it was found that some of the starting material DCM-OH was not reacted completely. The reaction was filtered to give the pure probe DCM-P as a solid (42% yield).

Example 2

187.4 mg of DCM-OH, 81.3 mg of picolinic acid, 7.3 mg of DMAP and 138.0 mg of EDC were weighed out accurately in a dry round bottom flask, then 20 ml of dichloromethane was added and stirred at 30 ℃ for 12 hours; after the reaction was complete, a large amount of yellow solid precipitated and was directly filtered to give the pure probe DCM-P (85% yield).

Example 3

187.4 mg of DCM-OH, 147.7 mg of picolinic acid, 7.3 mg of DMAP and 138.0 mg of EDC were weighed out accurately in a dry round-bottomed flask, then 20 ml of dichloromethane were added and stirred at 30 ℃ for 12 hours; after the reaction was complete, a large amount of yellow solid precipitated and was directly filtered to give the crude product, which was recrystallized from pure dichloromethane to give the pure probe DCM-P (76% yield).

Example 4

187.4 mg of DCM-OH, 81.3 mg of picolinic acid, 2.4 mg of DMAP and 138.0 mg of EDC were weighed out accurately in a dry round bottom flask, then 20 ml of dichloromethane was added and stirred at 30 ℃ for 12 hours; after the reaction was complete, a yellow solid precipitated and was directly filtered to give the pure probe DCM-P (51% yield).

Example 5

187.4 mg of DCM-OH, 81.3 mg of picolinic acid, 22.0 mg of DMAP and 138.0 mg of EDC were weighed out accurately in a dry round bottom flask, then 20 ml of dichloromethane was added and stirred at 30 ℃ for 12 hours; after the reaction was complete, a large amount of yellow solid precipitated and was directly filtered to give the pure probe DCM-P (84% yield).

Example 6

187.4 mg of DCM-OH, 81.3 mg of picolinic acid, 7.3 mg of DMAP and 57.5 mg of EDC were weighed out accurately in a dry round bottom flask, then 20 ml of dichloromethane was added and stirred at 30 ℃ for 12 hours; after the reaction was complete, a yellow solid precipitated and was directly filtered to give the pure probe DCM-P (35% yield).

Example 7

A dry round bottom flask was accurately weighed with 187.4 mg DCM-OH, 81.3 mg picolinic acid, 7.3 mg DMAP and 287.6 mg EDC, then 20 mL dichloromethane was added and stirred at 30 deg.C for 12 hours; after the reaction was complete, a large amount of yellow solid precipitated and was directly filtered to give the pure probe DCM-P (82% yield).

Example 8

187.4 mg of DCM-OH, 81.3 mg of picolinic acid, 7.3 mg of DMAP and 138.0 mg of EDC were weighed out accurately in a dry round bottom flask, 10 ml of dichloromethane was added and stirred at 30 ℃ for 12 hours; after the reaction was complete, a large amount of yellow solid precipitated and was directly filtered to give the pure probe DCM-P (83% yield).

Example 9

187.4 mg of DCM-OH, 81.3 mg of picolinic acid, 7.3 mg of DMAP and 138.0 mg of EDC were weighed out accurately in a dry round bottom flask, then 50 ml of dichloromethane was added and stirred at 30 ℃ for 12 hours; after the reaction was complete, a yellow solid precipitated and was directly filtered to give the pure probe DCM-P (62% yield).

Example 10

187.4 mg of DCM-OH, 81.3 mg of picolinic acid, 7.3 mg of DMAP and 138.0 mg of EDC were weighed out accurately in a dry round bottom flask, 10 ml of dichloromethane was added and stirred at 20 ℃ for 12 hours; after the reaction was complete, a yellow solid precipitated and was directly filtered to give the pure probe DCM-P (61% yield).

Example 11

187.4 mg of DCM-OH, 81.3 mg of picolinic acid, 7.3 mg of DMAP and 138.0 mg of EDC were weighed out accurately in a dry round bottom flask, 10 ml of dichloromethane was added and stirred at 40 ℃ for 12 hours; after the reaction was complete, a large amount of yellow solid precipitated and was directly filtered to give the pure probe DCM-P (88% yield).

Example 12

187.4 mg of DCM-OH, 81.3 mg of picolinic acid, 7.3 mg of DMAP and 138.0 mg of EDC were weighed out accurately in a dry round bottom flask, then 20 ml of dichloromethane was added and stirred at 30 ℃ for 5 hours; after the reaction was complete, a yellow solid precipitated and was directly filtered to give the pure probe DCM-P (53% yield).

Example 13

187.4 mg of DCM-OH, 81.3 mg of picolinic acid, 7.3 mg of DMAP and 138.0 mg of EDC were weighed out accurately in a dry round bottom flask, then 20 ml of dichloromethane was added and stirred at 30 ℃ for 24 hours; after the reaction was complete, a large amount of yellow solid precipitated and was directly filtered to give the pure probe DCM-P (87% yield).

Example 14

An application of near infrared fluorescence in copper ion detection comprises the following specific steps:

(1) accurately weighing a certain amount of DCM-P, dissolving in chromatographic pure DMSO to prepare the mixture with the concentration of 1.0X 10^-3Storing the DCM-P stock solution in mol/L in a refrigerator at 2-5 ℃;

(2)accurately weighing a certain amount of metal nitrate (Cu)²⁺,Li⁺,Na⁺,K⁺,Ag⁺,Cd²⁺,Co²⁺,Hg²⁺,Ni²⁺,Zn² ⁺,Pb²⁺,Mn²⁺,Mg²⁺,Al³⁺,Cr³⁺) Respectively dissolved in deionized water to prepare the solution with the concentration of 1.0 multiplied by 10^-2Storing the metal ion stock solution in mol/L in a refrigerator at 2-5 ℃;

(3) 1mL of DMSO,0.5mL of phosphate buffer, and 0.02mL of DCM-P stock solutions (1.0X 10) were added to each of 2mL test tubes^-3mol/L), then adding copper ion standard solutions with different concentrations respectively, and using deionized water to fix the volume to 2mL (at the moment, organic phase DMSO: H in the system is₂O ═ 5:5v/v), after the mixed solution reacted for a while, the fluorescence intensity of each solution was measured with a fluorescence spectrophotometer (HitachiF-4500, Japan), the quantitative relationship between the fluorescence intensity and the copper ion concentration was determined, and a standard curve was prepared;

(4) to a 2mL test tube, 1mL DMSO,0.5mL phosphate buffer, and 0.02mL DCM-P stock solution (1.0X 10)^-3mol/L), adding the solution to be detected, diluting the solution to 2mL with deionized water, measuring the fluorescence intensity of the mixed solution after the mixed solution reacts for a period of time, and determining the concentration of copper ions in the solution to be detected according to a standard curve.

Optimization and selection of test conditions

1. Selection of probe DCM-P concentration

The fluorescence method is used to analyze the substance to be detected, most of which is to prepare a solution and then detect the solution. The level of probe concentration directly affects the sensitivity and linear range of detection. If the concentration of the probe is too low, the signal generated by the interaction of the substance to be detected is weaker, and the sensitivity is reduced. If the concentration of the probe is too high, a molecular aggregation quenching effect occurs, and the linearity of the standard curve is reduced. In a specific study, we fixed the concentration of copper ions (1.0X 10)^-5) And other experimental conditions, the concentration of the probe DCM-P was changed, and then the fluorescence intensity of each solution was measured. As shown in FIG. 4, when the probe is DCM-P concentration>0.8×10^-5DCM-P has the best effect on copper ions at mol/LShould be used. The optimal concentration of the probe is chosen to be 1.0X 10, taking into account the sensitivity and linear range of the method^-5mol/L。(1.0×10^-5mol/L-3.0×10^-5mol/L all can)

2. Selection of buffer solution

In a specific study, we fixed the concentration of copper ions (1.0X 10)^-5mol/L), concentration of probe DCM-P (1.0X 10)^-5mol/L) and other experimental conditions, the types of buffer systems such as Tris (Tris buffer), 4-hydroxyethylpiperazineethanesulfonic acid (HEPES buffer), phosphate (PBS buffer) were changed, and the fluorescence intensity of the corresponding solutions was measured. As shown in FIG. 5, the probe reacts with copper ions in a phosphate buffer system to generate an optimal fluorescence signal.

3. Selection of the ratio of aqueous phase to organic phase

The content of the organic phase in the test system directly influences the reaction condition between the probe and the object to be tested. The content of the organic phase in the test system is too low, so that the solubility of the probe in the test solution is reduced, and the reaction between the probe and the object to be tested is not facilitated. The content of organic phase in the test system is too high, which limits the biological application of the probe. In a specific study, the concentration of copper ions (1.0X 10) was determined by fixing^- ⁵mol/L), concentration of probe DCM-P (1.0X 10)^-5mol/L), buffer solution (Na)₂HPO₄-NaH₂PO₄System), and other experimental conditions, the ratio of organic phase to aqueous phase in the test system was varied, and the fluorescence intensity of each solution was then measured. Shown by FIG. 6, in DMSO/H₂In the system of 0-4/6-6/4 (v/v), probe DCM-P reacts with copper ions to generate optimal fluorescence signals. Therefore, the ratio of the organic phase to the aqueous phase in the analytical test is preferably 4/6 to 6/4, and more preferably 5/5.

4. Selection of the pH of the solution

In a specific study, we fixed the concentration of copper ions (1.0X 10)^-5mol/L) and the concentration of probe DCM-P (1.0X 10)^-5mol/L), buffer solution (Na)₂HPO₄-NaH₂PO₄System), ratio of organic to aqueous phases (5/5, v/v), and other experimental conditions, the pH was varied in the test system and then measuredThe fluorescence intensity of each solution was determined. As shown in fig. 7, probe DCM-P reacted with copper ions to generate optimal fluorescence signals when the pH of the solution was in the range of 7.0 to 10.0, indicating that probe DCM-P is suitable for physiological pH range (pH 7.4). Since this study is intended to realize the recognition effect of a probe on copper ions in living cells, phosphate buffer at pH 7.4 was selected to simulate the intracellular environment.

5. Selection of reaction time.

In a specific study, we fixed the concentration of copper ions (1.0X 10)^-5mol/L) and the concentration of probe DCM-P (1.0X 10)^-5mol/L), buffer solution (Na)₂HPO₄-NaH₂PO₄System), ratio of organic phase to aqueous phase (5/5, v/v), pH 7.4 and other experimental conditions, and then the fluorescence intensity of the solution at different reaction times was measured. As shown in FIG. 8, the fluorescence intensity of the solution increases with the increase of the reaction time, reaches a maximum after 30 minutes, and then becomes stable, so that the optimum test time is preferably 30 to 40 minutes.

Example 15

To each of 2mL test tubes, 1mL of DMSO,0.5mL of phosphate buffer (pH 7.4) and 0.02mL of DCM-P stock solution (1.0 × 10) were added^-3mol/L) and then adding copper ion solutions ([ Cu ] with different concentrations respectively²⁺](×10^-6mol/L): 0. 0.1, 0.2, 0.4, 0.8, 1, 2, 4, 6, 8, 10, 14, 18, 20, 25, 30 μ M), and a volume of 2mL with deionized water, and after the mixture solution reacted for 30 minutes, the fluorescence intensity of each solution was measured with a fluorescence spectrophotometer (HitachiF-4500, Japan). The fluorescence test parameters are 10nm of excitation slit, 10nm of emission slit, 560nm of excitation wavelength, 677nm of emission wavelength and 695-850nm of scanning range. FIG. 9 is the fluorescence emission spectrum of probe DCM-P after the action of copper ions with different concentrations. It can be seen that the fluorescence intensity at 676nm increases with the increase of the concentration of copper ions, when the concentration of copper ions reaches 1.0X 10^-5And the fluorescence intensity of the detection system tends to be stable at mol/L. The fluorescent emission of the probe DCM-P is positioned in a near infrared region after the probe DCM-P reacts with copper ions, and the interference of background fluorescence can be effectively avoided, so that the probe DCM-P has bright application in biological applicationThe method has obvious advantages. The relationship between the copper ion concentration and the emission peak intensity is shown in FIG. 10. It can be seen that the concentration of copper ions is 0 to 8X 10^-6In the mol/L range, the fluorescence intensity of the detection system and the concentration of copper ions present a good linear relationship. The linear equation is that Y is 0.0799X +0.1617, R²0.9985, where Y is the normalized fluorescence intensity, X is the copper ion concentration, and the detection limit is 3.6 × 10^-8mol/L。

Example 16

To each of 2mL test tubes, 1mL of DMSO,0.5mL of phosphate buffer (pH 7.4) and 0.02mL of DCM-P stock solution (1.0 × 10) were added^-3mol/L) and then 0.02mL (1.0X 10) was added^-2mol/L) of stock solutions of different metal ions (Li)⁺,Na⁺,K⁺,Ag⁺,Cd²⁺,Co²⁺,Hg²⁺,Ni²⁺,Zn²⁺,Pb²⁺,Mn²⁺,Mg²⁺,Al³⁺,Cr³⁺) The volume of the solution was adjusted to 2mL with deionized water, and after the mixture was reacted for 30 minutes, the fluorescence intensity of each solution was measured with a fluorescence spectrophotometer (HitachiF-4500, Japan). As shown in FIG. 11, when various metal ions with the same concentration are added into the solution containing the probe DCM-P respectively for a period of time, only copper ions cause the fluorescence enhancement of the detection system, and other metal ions do not cause obvious fluorescence change. The probe DCM-P is shown to have good selectivity for identifying copper ions.

Example 17

In order to further investigate the selectivity and anti-interference performance of the probe DCM-P on copper ions, the influence of various metal ions possibly existing in an environmental water sample and the coexistence of the copper ions on the fluorescence intensity of a detection system is tested. In FIG. 12, none is DCM-P (1.0X 10) containing only probe^-5mol/L), white rectangles represent different metal ions (1.0X 10)^-4mol/L) and DCM-P (1.0X 10^-5mol/L) fluorescence intensity after mixing, black rectangles indicate the presence of different metal ions (1.0X 10)^-4mol/L) and DCM-P (1.0X 10^-5mol/L) of copper ions (1.0X 10)^-4Fluorescence after mol/L)Strength. As can be seen from FIG. 12, the probe DCM-P is hardly interfered by other metal ions for copper ion detection, and has good anti-interference performance.

Example 18

The probe DCM-P molecule consists of two fragments of 4- (dicyanomethylene) -2- (2-hydroxystyryl) -4H-1-benzopyran and pyridine formyl. Wherein 4- (dicyanomethylene) -2- (2-hydroxy styryl) -4H-1-benzopyran is used as a fluorescence signal unit, and pyridine formyl is used as a recognition unit. Before the reaction with copper ions, the intramolecular charge transfer process of the probe DCM-P is blocked, so that the fluorescence is quenched. After the copper ions are added, nitrogen atoms on the pyridine formyl groups are easy to coordinate with the copper ions, so that ester bonds are catalyzed to hydrolyze, and a fluorescent signal unit is released. At the moment, an obvious intramolecular electron transfer process exists in the fluorescent signal unit, and near infrared fluorescence emission is generated under the excitation of light.

To demonstrate the above reaction mechanism, we monitored the entire reaction process by nuclear magnetic resonance spectroscopy. As shown in FIGS. 13-14, the proton signal peaks at the picolinoyl group appeared at 8.8,8.3,8.1 and 7.4ppm before the probe DCM-P acted on the copper ion. After the addition of copper ions, the proton signal peak at the picolinoyl group disappeared while the proton signal peak at the phenolic hydroxyl group appeared at 10.1ppm (FIG. 13). In addition, we characterized the probe DCM-P and the product of copper ion by high resolution mass spectrometry, and obtained an ion fragment peak corresponding to the fluorescence signal unit (FIG. 14).

Example 19

The probe DCM-P was used for detection of copper ions in an environmental water sample in this example.

(1) The water sample is taken from tap water in Changsha city, Hunan river water, in Hunan province, and is filtered by a 0.22 micron membrane before analysis and test, and the pH is adjusted to 7.4.

(2) Considering that the concentration of copper ions in a water sample is too low, the concentration of the copper ions is measured by adopting a standard addition method. The method specifically comprises the following steps: to the pretreated water sample, copper ions (0, 0.2. mu.M, 2. mu.M) of known concentration were added and the fluorescence intensity was measured under optimum conditions, and the recovery amount and recovery rate were calculated from the standard curve.

In order to verify the practicability of the probe in the environmental field, the concentration of copper ions in tap water in Changsha city and water samples in Hunan river is measured by adopting a standard addition method. When the probe is directly added into a water sample, the fluorescence is not obviously changed, when copper ions with different known concentrations are added into the water sample, the fluorescence is changed to a certain extent, and the measured fluorescence intensity change value is substituted into a standard curve to calculate the concentration of the copper ions. The detection results are shown in table 1, and it can be seen that the recovery rate obtained by the method is between 98.5% and 103%, which indicates that the probe DCM-P can meet the requirement of detecting copper ions in a water sample. Therefore, the probe DCM-P can be used as a powerful tool for quantitatively detecting the concentration of copper ions in a water sample.

TABLE 1 analysis results of detection of copper ions in environmental water sample with probe DCM-P

Example 20

The probe DCM-P was used for detection of copper ions in HeLa cells in this example.

(1) Placing Heila cells in a culture medium containing 10% fetal bovine serum and 1.0 × 10^-5mol/L probe DCM-P in DMEM medium, at 37 ℃ and 5% carbon dioxide conditions were cultured for 30 minutes.

(2) The medium was aspirated with a pipette, and the cells were washed 3 times with warm (37 ℃) phosphate buffer and imaged with a fluorescence inverted microscope (Nikon, eclipseTi-S).

(3) Adding 1.0X 10 to the cells of step (2)^-5mol/L copper ions, incubation for a further 30 minutes, washing the cells 3 times with warm (37 ℃) phosphate buffer, and then using a fluorescence inverted microscope (Nikon, Ecli)pseTi-S) was imaged.

FIG. 15 is a photograph of fluorescence micrographs of HeLa cells, in which A represents the bright field of cells cultured in the medium containing probe DCM-P for 30 minutes, and B represents the bright field of cells cultured in the medium containing probe DCM-P for 30 minutes before adding copper ions and continuing the culture for 30 minutes. C and D are fluorescence imaging images of A and B respectively. It can be seen that the probe DCM-P provided by the embodiment can accurately determine the distribution of copper ions in cells.

Claims

1. A preparation method of a near-infrared fluorescent probe compound is characterized by comprising the following steps:

DCM-OH 0.60 mmol

0.66-0.9 mmol of picolinic acid

DMAP 0.06-0.18 mmol

0.72 to 1.50 mmole of EDC

The volume of the dichloromethane is 10-20 ml;

(2) stirring the mixed solution at 30-40 ℃ for 12-24 hours;

(3) after the reaction is finished, a yellow precipitate is separated out, and the yellow precipitate is filtered and separated to obtain a pure probe DCM-P, wherein the chemical structural formula of the pure probe DCM-P is shown as a formula (1):

formula (1).

2. The method for preparing a near-infrared fluorescent probe compound according to claim 1, wherein the formula ratio of the mixed solution in the step (1) is as follows:

DCM-OH 0.60 mmol

0.66-0.9 mmol of picolinic acid

DMAP 0.06 mmol

EDC 0.72 mmol

The volume of dichloromethane is 10-20 ml.