CN108727859B - squaric acid dye and application thereof in preparation of colorimetric fluorescence dual-response copper ion probe - Google Patents

Abstract

The invention discloses a squaric acid dye and application thereof in preparation of a colorimetric fluorescence dual-response copper ion probe, wherein the preparation method comprises the following steps: (1) mixing aniline derivative and squaric acid, dissolving in proper solvent, connecting with water separator, and adding water to obtain the final product₂carrying out reduced pressure reflux and water diversion for hours under protection; (2) cooling the reaction mixture obtained in the step (1) to room temperature, and removing the solvent under reduced pressure to obtain a crude product; (3) and purifying the crude product by silica gel column chromatography to obtain a near-infrared squaric acid dye product. The squarylium dye has good stability and excellent optical performance, nitrogen atoms and oxygen atoms on side chains have good binding force with copper ions, and only the copper ions cause fluorescence quenching of the dye and the color of the solution is changed from blue to colorless when various metal ions are added into a system, so that the squarylium dye can be used as a colorimetric/fluorescent probe for detecting the copper ions, can be used for detecting the copper ions in an aqueous medium and shows good detection sensitivity.

Description

Squaric acid dye and application thereof in preparation of colorimetric fluorescence dual-response copper ion probe

Technical Field

The invention belongs to the field of analytical chemistry, and particularly relates to a squaric acid dye and application thereof in preparation of a colorimetric fluorescent dual-response copper ion probe.

background

Copper ions are an essential trace element in living bodies, are widely distributed in biological tissues, have redox activity, and thus participate in the redox process of the body and scavenge free radicals (Uauy, R.; Olivares, M.; Gonzalez, M. availability of cooper in human beings).Am. J. Clin. Nutr. 1998, 67952S-959S.). Copper ions are also important components of some enzymes in the Organism, such as cytochrome C, in maintaining the energy of the cells, while another lysyl oxidase is involved in the crosslinking process of collagen and elastin to form connective tissue, which not only helps to maintain the integrity and elasticity of connective tissue in the heart and blood vessels, but also plays an important role in bone formation (Angelova, m.; Asenova, s.; Nedkova, v.; Koleva-Kolarova, r. Copper in the Human Organism).Trakia J. Sci.2011, 9, 88-98.). Excessive copper ions in humans can cause serious health problems, such as ischemic heart, kidney, anemia, and skeletal diseases (Reddy, S.A.; Reddy, K.J.; Narayan, S.L.; Reddy A.V. Analytical Applications of 2, 6-diacetylpyridinium Bis-4-phenyl-3-thiosemi-carbazone, and Determination of Cu (II) in Food Samples).Food Chem. 2008, 109(3)654 and 659)), and can also cause burden to organs in human body, thereby affecting human health. The maximum value of the copper ion content in the drinking water is 1.30 mg/L, and the concentration range of the copper ion in the food specified by European food safety administration is 1.2-4.2 mg/L. The content of copper ions in drinking water is not higher than 1.0 mg/L according to the provisions of sanitary Standard for Drinking Water in China. Therefore, it is of great interest to develop methods that can be used to accurately detect copper ions in water, environment and organisms.

the current methods for Copper ion detection are reported in the literature mainly as Atomic Absorption spectroscopy (Kabil, M.A.; ElKourasy, A.G.; ElHagrasy, M.A. Flame Atomic Absorption Spectrometry inversion and Determination of Cobalt and Copper Using ethanol and minerals as Chemical Modifiers.J. Anal. Atom. Spectrom.1996, 11, 379-387. Ζemberyová, M.; Barteková, J.; Závadská, M.; Šišoláková, M.Determination of Bioavailable Fractions of Zn, Cu, Ni, Pb and Cd in Soils andSludges by Atomic Absorption Spectrometry.Talanta2007, 711661-1668.), Inductively Coupled Plasma atomic emission Spectrometry (Batista, B.L.; Rodrigues, J.L.; Nunes, J.A.; Tormen, L.; Curtius, A.J.; Barbosa, F. Simultaneous Determination of Cd, Cu, Mn, Ni, Pb and Zn in Nail Samples by inductive coupling of composite Plasma Mass Spectrometry (ICP-MS) after catalytic hydrogenation reaction: composite with ETAAS. Talanta 2008,76575-579), Electrochemical process (Jena, B. K.; Raj, C. R. Gold Nanoelectrode Ensembles for the Simultaneous Electrochemical Detection of Ultratrace Arsenic, Mercury, and Copper. Anal. Chem.2008, 80, 4836-4844.), etc., but these methods have low sensitivity, require expensive instruments and are not suitable for outdoor real-time detection, so it is necessary to develop a rapid and simple detection method, and the fluorescence method has high sensitivity, rapidness, good selectivity and simple operation and is widely used for detecting copper ions.

Squaric acid Dyes are 1, 3-disubstituted derivatives formed by condensation of squaric acid with electron-rich aryl compounds or amine compounds, and are a class of Dyes with resonance-stable zwitterionic structures (Ajayaghosh, A. chemistry of Square-Derived Materials: New-IR Dyes, Low Band Gap Systems, and cation Sensors.Acc. Chem. Res. 2005, 38, 449-459.). The compounds are characterized by narrow and strong absorption band and higher quantum yield from visible region to near infrared region (620-670 nm), and molar absorption coefficient is more than 10⁵L•mol^-1•cm^-1. This photoelectric property is mainly due to the strong intra-molecular charge transfer between donor-acceptor-donor (donor-acceptor). The probe has the characteristics of excellent fluorescence emission performance and electrochemical performance, good optical stability, easy modification and the like, and has wide application prospect in the design of a new generation of chemical small molecular probe.

At present, most of copper ion Fluorescent probes realize identification of copper ions based on fluorescence change signals obtained after copper ions are coordinated with the probes (Wang, W.; Fu, A.; You, J.; Gao, G.; Lan, J.; Chen, L.Squarine-based colorimetry and fluorescence Sensors for Cu)²⁺-specific Detection and Fluorescence Imaging in Living Cells. Tetrahedron 2010, 66, 3695-3701.). The invention modifies the group containing N, O heteroatom on the side chain of squaric acid to synthesize the squaric acid dye colorimetric/fluorescent probe capable of identifying copper ions, and only the copper ions cause the fluorescence quenching of the probe and the color of the solution is changed from blue to colorless when various metal ions are added into the system.

disclosure of Invention

the squaric acid dye has good stability, excellent optical performance, specific selectivity and higher sensitivity to copper ions, realizes colorimetric/fluorescent recognition of the copper ions, can be used for detecting the copper ions in an aqueous medium, and has wide application prospect for detecting the copper ions in other organisms and environments.

In order to achieve the purpose, the invention adopts the following technical scheme:

A near-infrared squarylium dye has a structural formula as follows:

。

The preparation method of the near-infrared squaraine dye comprises the following steps:

(1) Aniline derivativeand squaric acidmixing, dissolving in solvent, connecting water separator, N₂Carrying out pressure reduction and reflux under protection;

(2) cooling the reaction mixture obtained in the step (1) to room temperature, and removing the solvent under reduced pressure to obtain a crude product;

(3) And purifying the crude product by silica gel column chromatography to obtain the near-infrared squaric acid dye.

in the step (1), the solvent is n-heptanol, the pressure is reduced to 76 mmHg, the reaction temperature is 135 ℃, and the reflux time is 9 hours; and (3) the eluent used for the silica gel column chromatography in the step (3) is a mixed solution of chloroform and methanol in a volume ratio of 70: 1.

The aniline derivativeThe synthesis method comprises the following steps:

(A) Will be provided withDissolving in a solvent, adding triethylamine, dropwise adding acetyl chloride in an ice water bath, and stirring at room temperature after dropwise adding;

(B) After the reaction is finished, filtering to remove triethylamine hydrochloride, washing the filtrate with 10wt% of sodium carbonate and water, drying the filtrate with anhydrous sodium carbonate, and concentrating to remove the solvent after filtering to obtain a crude product;

(C) Purifying the crude product by a silica gel column to obtain the aniline derivative。

In the step (A), the solvent is dichloromethane, acetyl chloride is dripped in an ice water bath, the temperature is controlled to be 25-35 ℃, and the stirring time at room temperature is 2 hours; and (C) the eluent used for the silica gel column chromatography in the step (C) is a mixed solution of petroleum ether and ethyl acetate with the volume ratio of 6: 1.

The invention has the following remarkable advantages: the near-infrared squarylium dye has the advantages of easily available raw materials and simple synthetic route, and can be used as a colorimetric/fluorescent probe for detecting copper ions. The side chain of the squaric acid is modified with a group containing N and O heteroatoms with good binding force on copper ions, so that the probe has good anti-interference capability on other various metal ions and anions, the copper ion recognition capability is obviously specific, the detection limit on the copper ions is low, and a good linear relation is achieved.

drawings

FIG. 1 is a graph of the absorption spectra of squarylium dye at a concentration of 2.5. mu.M in different organic solvents;

FIG. 2 is a graph of the change in absorption spectra of squaraine dye at a concentration of 2.5. mu.M in different ratios of PB-MeCN;

FIG. 3 is a graph showing the change in absorption spectra of squaric acid dye at a concentration of 2.5. mu.M in a 5mM SDS solution of PB-MeCN (9:1, v/v) at pH 7.5 with different metal ions added;

FIG. 4 is a graph showing the color change of a solution of squarylium dye at a concentration of 2.5. mu.M in a 5mM SDS pH 7.5 PB-MeCN (9:1, v/v) solution before and after addition of copper ions;

FIG. 5 is richa squaric acid dye with a degree of 2.5. mu.M in a PB-MeCN (9:1, v/v) solution of 5mM SDS pH 7.5 was added the fluorescence spectrum profiles (lambda_ex = 637 nm, slit: 5 nm/5 nm, PMT Volts: 500）；

FIG. 6 is a graph of fluorescence spectrum of cation interference experiment (. lamda.1, v/v) of squaric acid dye at a concentration of 2.5. mu.M in a solution of PB-MeCN (9:1, v/v) in 5mM SDS pH 7.5_ex = 637 nm, slit: 5 nm/5 nm, PMT Volts: 500）；

FIG. 7 is a graph of the fluorescence spectrum of anion interference experiment (. lamda.1, v/v) of squaric acid dye at a concentration of 2.5. mu.M in a solution of PB-MeCN (9:1, v/v) in 5mM SDS pH 7.5_ex = 637 nm, slit: 5 nm/5 nm, PMT Volts: 500）；

FIG. 8 is a graph of the absorbance spectrum titration of copper ions by squaraine dye at a concentration of 2.5. mu.M in a 5mM SDS pH 7.5 PB-MeCN (9:1, v/v) solution;

FIG. 9 is a graph showing the titration of the fluorescence spectrum of squaric acid dye at a concentration of 2.5. mu.M in a PB-MeCN (9:1, v/v) solution of 5mM SDS pH 7.5 against copper ions (. lamda._ex = 637 nm, slit: 5 nm/5 nm, PMT Volts: 500）；

FIG. 10 is a graph showing the trend of 2.5. mu.M squarylium dye change at 650 nm in the concentration of copper ions in a PB-MeCN (9:1, v/v) solution of 5mM SDS pH 7.5 in the range of 25-55. mu.M (. lamda.) (λ. lambda._ex = 637 nm, slit: 5 nm/5 nm, PMT Volts: 500）。

Detailed Description

The present invention will be further described with reference to the following examples, but the present invention is not limited to these examples.

example 1

Aniline derivativesThe preparation of (1):

In a 100 mL two-necked flask, an aniline derivative was added(2.71 g, 15 mmol) and 30 mL of dichloromethane, triethylamine (4.54 g, 45 mmol) was added, and acetyl chloride (3) was added dropwise to the mixture in an ice-water bath53 g, 45 mmol), controlling the temperature at 25 ℃, stirring at room temperature for 2 hours after the dropwise addition, monitoring by TLC, filtering to remove Et after the reaction is complete₃N.hcl, the filtrate was washed with 10% sodium carbonate, once with water, dried with anhydrous sodium carbonate, filtered and the solvent removed to give a dark brown liquid. The crude product was chromatographed on silica gel column with petroleum ether: ethyl acetate (6: 1, v/v) gave 3.82 g of a yellow oil in 96% yield. FTIR (KBr): v_max2961, 1740, 1600, 1506, 1381, 1232, 1196, 1038, 750 cm^-1。

The near-infrared squarylium dyeThe preparation of (1):

In a 100 mL round-bottom flask, an aniline derivative was added(96 mg, 0.36 mmol), squaric acid (20.7 mg, 0.18 mmol), dissolving with 40 mL n-heptanol, reducing pressure to 76 mmHg, removing water by a water separator, heating to 135 ℃, refluxing for reaction for 9 hours, removing n-heptanol under reduced pressure, performing silica gel column chromatography on the residue, eluting with chloroform-methanol (70: 1, v/v), removing the solvent to obtain 58.8 mg of a blue-purple solid with a yield of 54%, a melting point of 207 ~ 209 ℃; FTIR (KBr): 209 ℃_max2960, 1732, 1624, 1590, 1415, 1383, 1347, 1244, 1227, 1192, 1125, 1035, 982, 950, 845, 791, 531 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 8.43 (d, J=8.4 Hz, 4H), 6.89 (d,J=8.2 Hz, 4H), 4.32 (t, J=5.1 Hz, 8H), 3.81 (d, J=5.1 Hz, 8H), 2.04 (s, 12H);¹³C NMR (100 MHz, CDCl₃) δ 191.65, 182.87, 170.69, 153.92, 133.60, 120.82, 112.71, 60.91, 49.68, 20.78; ESI-MS: m/z 609.2 ([M+H]⁺)。

FIG. 1 is the absorption spectrum of squaric acid dye with concentration of 2.5 μ M in different organic solvents, the maximum absorption wavelength of the dye in various solvents is within 619-631 nm, and the molar absorption coefficient ε is more than 10⁵ L.mol^-1.cm^-1Unimodal and narrow peak form, increasing with solvent polarity, dyeingThe maximum absorption wavelength of the material is red-shifted, and the molar absorption coefficient is reduced.

The squarylium dye is easy to self-aggregate in aqueous solution, and the polarity of the solvent and the components of the solvent have great influence on the spectral properties of the dye. FIG. 2 is a graph of the change in absorption spectra of squaraine dye at a concentration of 2.5. mu.M in different ratios of PB-MeCN; as can be seen from the graph, as the water content in the system increases, the maximum absorption peak is red-shifted from 625 nm to 633 nm, and the absorption intensity decreases. When the water content reaches 95%, the absorption value of the dye at 633 nm is significantly reduced, and a broader absorption peak at about 550 nm appears, indicating that the dye begins to aggregate in the system.

Selectivity is an index for evaluating whether a chemical sensor is excellent or not. To a PB-MeCN (9:1, v/v) buffer solution of 5mM SDS at pH 7.5 containing 2.5. mu.M of a dye, 25. mu.M of each metal ion (Li) was added⁺, Na⁺, K⁺, Ag⁺, Mg²⁺, Ca²⁺, Ba²⁺, Pb²⁺, Cd²⁺, Co²⁺, Al³⁺, Cu²⁺, Zn²⁺, Ni²⁺, Hg²⁺) As can be seen from FIG. 3, the absorption intensity decreased only after the addition of copper ions, and the solution faded from blue to colorless (FIG. 4). From the fluorescence spectrum of fig. 5, it can be observed that the system fluorescence is quenched only after the addition of copper ions, while other ions do not respond, and the experimental result shows that the dye can realize double responses to single colorimetric/fluorescence of copper ions.

We further examined the response of the dye to copper ions when other metal ions and copper ions were present, and FIG. 6 is a fluorescence spectrum of a cation interference experiment with squarylium dye at a concentration of 2.5. mu.M in a 5mM SDS solution of PB-MeCN (9:1, v/v) at pH 7.5. Separately adding 50. mu.M Ni to the test system²⁺, Ba²⁺, Ag⁺, Hg²⁺, Ca²⁺, Al³⁺, Pb²⁺, Zn²⁺, Na⁺, K⁺, Co²⁺, Mn²⁺Plasma, the fluorescence intensity of the system is basically not changed, and 25 mu M Cu is added²⁺Later, it is seen from FIG. 6 that the fluorescence of the system is almost quenched, indicating that other metal ionsThe coexistence of the dye and the copper ions does not interfere with the detection of the copper ions, which further indicates that the dye has good selectivity for the copper ions. Coexisting anions generally interfere with metal ion recognition, 50 μ M copper ions are added into a PB-MeCN (9:1, v/v) system of 5mM SDS, the fluorescence of the system is quenched, and then 50 μ M of various anions are added respectively, as can be seen from FIG. 7, the detection of the system on the copper ions is not affected by the presence of the anions, which indicates that the binding force of the dye on the copper ions is strong.

To further investigate the sensitivity of the squarylium dye to copper ion detection, the effect of different concentrations of copper ions on the dye absorption and fluorescence spectra were examined in a PB-MeCN (9:1, v/v) system of 5mM SDS. FIGS. 8 and 9 are the absorbance spectrum and the fluorescence spectrum titration graph of copper ion in PB-MeCN (9:1, v/v) solution at pH 7.5 of 5 mMSDS for squaric acid dye with concentration of 2.5. mu.M, respectively, and as shown in FIG. 8, the absorbance peak at 645 nm gradually decreases with the increase of the copper ion concentration in the system, indicating that the dye forms a complex with the copper ion. Meanwhile, the fluorescence intensity of the system is gradually reduced along with the increase of the concentration of copper ions in the solution (figure 9), the fluorescence intensity has a good linear relation with the concentration of copper ions (25-55 mu M) (figure 10), and the linear equation is F = -6.29[ Cu = -6.29 ]²⁺]+426.46, linear correlation coefficient R²=0.99, wherein F is the fluorescence intensity of the dye after addition of copper ions, according to 3 sigma-kthe detection limit of the dye to the copper ions is calculated to be 5.87 multiplied by 10^-7 M。

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims (1)

1. the application of the near-infrared squarylium dye is characterized in that: the method is used for preparing a copper ion probe with double responses of fluorescence and colorimetry, and the copper ion probe is applied to the detection of copper ions in an aqueous medium;

the structural formula of the near-infrared squaraine dye is as follows:

；

the preparation method of the near-infrared squarylium dye comprises the following steps:

(1) Aniline derivativeAnd squaric acidMixing, dissolving in solvent, connecting water separator, N₂Carrying out pressure reduction and reflux under protection;

(2) Cooling the reaction mixture obtained in the step (1) to room temperature, and removing the solvent under reduced pressure to obtain a crude product;

(3) Purifying the crude product by silica gel column chromatography to obtain the near-infrared squaric acid dye;

In the step (1), the solvent is n-heptanol, the pressure is reduced to 76 mmHg, the reaction temperature is 135 ℃, and the reflux time is 9 hours; the eluent used for silica gel column chromatography in the step (3) is a mixed solution of chloroform and methanol with the volume ratio of 70: 1;

The aniline derivativeThe synthesis method comprises the following steps:

(A) Will be provided withDissolving in a solvent, adding triethylamine, dropwise adding acetyl chloride in an ice water bath, and stirring at room temperature after dropwise adding;

(B) After the reaction is finished, filtering to remove triethylamine hydrochloride, washing the filtrate with 10wt% of sodium carbonate and water, drying the filtrate with anhydrous sodium carbonate, and concentrating to remove the solvent after filtering to obtain a crude product;

(C) Purifying the crude product by a silica gel column to obtain the aniline derivative；

In the step (A), the solvent is dichloromethane, acetyl chloride is dripped in an ice water bath, the temperature is controlled to be 25-35 ℃, and the stirring time at room temperature is 2 hours; and (C) the eluent used for the silica gel column chromatography in the step (C) is a mixed solution of petroleum ether and ethyl acetate with the volume ratio of 6: 1.