CN103245646A - Application of 1, 3, 4-oxadiazole derivative fluorescent probe in test of cadmium ions - Google Patents

Abstract

The invention discloses an application of a 1, 3, 4-oxadiazole derivative fluorescent probe in a test of cadmium ions. The probe is specifically used for detecting cadmium ions (Cd2<+>) in water bodies and cells. The probe has pmol level response to Cd2<+>, and the low response is not reported. The fluorescent probe has the advantages of high sensitivity and good selectivity to Cd2<+> in an aqueous liquor, simpleness and convenience in operation, excitation of visible light and large Stocks displacement, proportional measurement and the like. As the probe is greater in color change before and after being combined with Cd2<+>, the probe can be conveniently identified and judged by naked eyes. In addition, the probe can be further convenient to be loaded in cells, so that the condition that imaging cells are polluted by Cd2<+> can be better observed in cellular level through a laser scanning confocal microscope.

Description

The application of 1,3,4-oxadiazole analog derivative fluorescence probe in cadmium ion is measured

Technical field:

This invention She is Ji the purposes of oxazole compounds specifically is that Yi Zhong oxadiazole analog derivative is as the application of fluorescence probe cadmium ion in detecting water body and cell.

Background technology:

Cadmium is widely used in the industry as one of important transition metal, as: plating, smelting, alloy, dyestuff etc.Because the widespread use of cadmium, a large amount of dischargings of industrial waste water containing Cd have been caused, caused the severe contamination of soil and water body, make the toxic metals cadmium enter into human body by food chain, in human body, cause slow poisoning by biological concentration, caused the generation of numerous disease, as: pulmonary emphysema, anosmia, the yellow ring of tooth glaze, renal damage, malacosteon and certain cancers etc.Long-term food and the water that is polluted by Trace Cadmium of taking in also can cause Itai-itai diseases, and this disease once occurred in Europe, and basin, magic river, Toyama County, Japan is because the Itai-itai diseases that cadmium pollution causes are known to all especially.Therefore, a kind of method that can measure cadmium ion content in water body and the cell accurately, simply, fast of development has very important significance.

Measure Cd at present ²⁺Method atomic absorption method, inductively coupled plasma emission spectrometry (ICP-AES), electrochemical process, fluorometry etc. are arranged.In these methods, fluorometry with its simple to operate, high sensitivity and selectivity, fast response speed and can cellular level directly, Cd in the observation of cell in real time ²⁺Variation and do not influence in the cell normal physiology, biochemical process and demonstrate special advantages.

The Cd of present bibliographical information ²⁺There is poorly water-soluble in fluorescence probe, major part, need exciting or having a Zn of ultraviolet light ²⁺Problems such as serious interference, what is more important is the Cd of report now ²⁺The detectability of probe be far longer than living body biological patient maximum and by Cd in the water body of U.S. environmental protection general administration and World Trade Organization's defined ²⁺High-load (4-40nM) (a, Liu, people such as W.M., Org.Lett., 2007,9,3829-3832.b.Peng, people such as X.J., J.Am.Chem.Soc., 2007,129,1500-1501.c.Cheng, people such as T., J.Am.Chem.Soc., 2008,130,16160-16161.d.Taki, M., J.Am.Chem.Soc., 2008,130,12564-12565.e.Tian, people such as H., J.Mater.Chem., 2011,21,10298-10303.), therefore develop the Cd with using value ²⁺Probe has very important actual application value.

Summary of the invention:

The purpose of this invention is to provide a kind of 1,3, the new purposes of 4-oxadiazole derivant, specifically as fluorescence probe for detection of water body and intracellular Cd ²⁺This probe is to Cd ²⁺High sensitivity and selectivity are arranged, use excited by visible light, can effectively reduce uv excitation light to the damage of cell and biological sample.

Provided by the invention a kind of 1,3,4-oxadiazole analog derivative is as Cd ²⁺Fluorescence probe Cd in detecting water body and cell ²⁺Application, described 1,3, the structural formula (I) of 4-oxadiazole analog derivative is:

Wherein, R ₁To R ₇Be hydrogen atom, halogen or methyl; R ₈Be methyl, ethyl or propyl group; R ₉Be methyl, ethyl or its lithium salts;

Perhaps structural formula (II) is:

Wherein, R ₁To R ₄, R ₆, R ₇Be hydrogen atom, halogen or methyl; R ₈Be methyl, ethyl or propyl group; R ₉Be methyl, ethyl or its lithium salts.

Said structure formula (I) and (II) 1,3,4-oxadiazole analog derivative specifically can be used for detecting the fluorescence probe of cadmium ion in water body and the cell, for example is used for surface water, tap water Cd ²⁺Detection; Perhaps be used for simulation living things system Cd ²⁺Detection, Cd in biological living cells and the living tissue ²⁺Analyzing and testing and fluorescence imaging detect and Cd in the various types of cells that interrelates with clinical medicine or the tissue ²⁺Mensuration.

Compared with prior art, the present invention synthesize 1,3,4-oxadiazole analog derivative is as Cd ²⁺Fluorescence probe belongs to ratio type probe, can effectively eliminate the error that factors such as, biological sample uneven thickness irregular because of the probe negative staining and equipment cause, thereby obtains than non-ratio type fluorescence probe measurement result more accurately.And, this probe has exciting and big Stokes displacement at visible region, can effectively reduce uv excitation light to the damage of cell and biological sample and the interference of cell or biological sample autofluorescence, improve selectivity and the sensitivity of detection method, more be conducive to the detection of live body.Fluorescence probe of the present invention is based on intramolecular charge transfer (ICT) design.Because probe and Cd ²⁺Combination caused in the molecule electron-donating group the weakening of sub-ability of powering, show to absorb and blue shift and probe and the Cd of emission spectrum ²⁺In conjunction with the front and back change in color, can rely on bore hole to judge probe and Cd like this ²⁺In conjunction with situation.In addition, this probe can be hatched and be entered cell, but by the imaging of laser co-focusing micro-imaging technique by Cd ²⁺Cd in the cell that pollutes ²⁺1,3,4-oxadiazole class Cd ²⁺It is simple that fluorescence probe has synthetic method, characteristics with low cost since have picomole ultralow like this to Cd ²⁺Therefore response has very important actual application value.

Description of drawings

Figure 11, the lithium salt compound of 3,4-oxadiazole and the Cd of variable concentrations ²⁺Emission spectrum figure in HEPES buffer solution.

Fig. 2 becomes the fluorescence intensity F of ratio ₅₃₀/ F ₆₇₀With Cd ²⁺The concentration linear graph

Under Fig. 3 natural light and the uviol lamp 1,3, the lithium salt compound of 4-oxadiazole is in conjunction with Cd ²⁺The change color figure of front and back

Figure 41, the lithium salt compound of 3,4-oxadiazole and the Cd of variable concentrations ²⁺Abosrption spectrogram in HEPES buffer solution

Fig. 5 becomes the absorption intensity A of ratio ₃₅₄/ A ₄₀₅With Cd ²⁺The linear graph of concentration

Under Fig. 6 natural light and the uviol lamp 1,3, the lithium salt compound of 4-oxadiazole is in conjunction with Cd ²⁺The change color figure of front and back

Figure 71, the compound of 3,4-oxadiazole ester is not in conjunction with Cd ²⁺The time cell imaging

Figure 81, the compound of 3,4-oxadiazole ester is in conjunction with Cd ²⁺After cell imaging

Figure 91, the compound of 3,4-oxadiazole ester is not in conjunction with Cd ²⁺The time cell imaging

Figure 101, the compound of 3,4-oxadiazole ester is in conjunction with Cd ²⁺After cell imaging

Embodiment

Embodiment 1

1,3,4-oxadiazole analog derivative is as Cd ²⁺Fluorescence probe Cd in detecting water body and cell ²⁺Application, described 1,3, the structural formula (I) of 4-oxadiazole analog derivative is:

Wherein, R ₁To R ₇Be hydrogen atom, R ₈Be ethyl, R ₉Be ethyl or lithium salts;

Perhaps structural formula (II) is:

Wherein, R ₁To R ₄, R ₆, R ₇Be hydrogen atom, R ₈Be ethyl, R ₉Be ethyl or lithium salts.

Above-claimed cpd is prepared from reference to the document of number of patent application 201210317190.0.

Embodiment 2:

To contain 1,3 of single fluorophore, the lithium salt compound of 4-oxadiazole joins in the HEPES buffer solution as probe and (contains 10mmol/L EGTA, pH7.2), be made into the solution of 1.0 μ mol/L, with the CdCl of 0.2mol/L ₂Solution joins and contains 1,3 of single fluorophore, in the HEPES damping fluid of 4-oxadiazole lithium salt compound as probe, along with Cd free in the solution ²⁺The increase of concentration (people such as Patricia M., J.Biol.Chem.1992,267,25553-25559), fluorescence spectrum is blue shift gradually, and fluorescence intensity significantly strengthens, occur one at the 619nm place and significantly waited launching site, presented the characteristics (Fig. 1) of tangible one-tenth ratio emission.The fluorescence intensity ratio F of emission ₅₃₀/ F ₆₇₀Be strengthened to 8.0 from 2.2, and and Cd ²⁺Concentration presents favorable linearity correlativity (Fig. 2), and the color of solution also becomes faint yellow by original buff; Under uviol lamp, solution colour becomes bright green (Fig. 3) by original redness.

Embodiment 3

To contain 1,3 of two fluorophores, the lithium salt compound of 4-oxadiazole joins in the HEPES buffer solution as probe and (contains 10mmol/L EGTA, pH7.2), be made into the solution of 1.0 μ mol/L, with the CdCl of 0.2mol/L ₂Solution joins and contains 1,3 of two fluorophores, in the HEPES damping fluid of the lithium salt compound of 4-oxadiazole as probe, along with Cd free in the solution ²⁺The increase of concentration (people such as Patricia M., J.Biol.Chem.1992,267,25553-25559), absorption spectrum is blue shift gradually, and absorption intensity strengthens gradually, two clear and definite isobestic points (Fig. 4) occurred at 313nm and 370nm place, and absorption intensity compares A ₃₅₄/ A ₄₀₅With Cd ²⁺Concentration presents favorable linearity correlativity (Fig. 5), the color of solution also by original orange red become faint yellow; Under uviol lamp, solution colour becomes bright green (Fig. 6) by original redness.

Embodiment 4

To contain 1,3 of single fluorophore, the compound of 4-oxadiazole ester is made into the solution that concentration is 5.0 μ mol/L as probe in DMSO, with it at 37 ° of C, 5%CO ₂Join in the incubator in the Human umbilical vein endothelial cells, hatch and it was entered in the cell in 40 minutes.Afterwards, with HEPES buffer solution flushing 3 times, be put under the confocal laser scanning microscope, CLSM, fixedly excitation wavelength is 405nm, and emission wavelength is 570-610nm, can see that cell imaging is kermesinus (Fig. 7).Then with the CdCl of 10.0 μ mol/L ₂Join in the above-mentioned cell, after continuing to hatch 30 minutes, be put in again under the confocal laser scanning microscope, CLSM, emission wavelength is 510-550nm, and at this moment cell presents bright green (Fig. 8).

Embodiment 5

To contain 1,3 of two fluorophores, the compound of 4-oxadiazole ester is made into the solution that concentration is 5.0 μ mol/L as probe in DMSO, with it at 37 ° of C, 5%CO ₂Join in the incubator in the Human umbilical vein endothelial cells, hatch and it was entered in the cell in 40 minutes.Afterwards, with HEPES buffer solution flushing 3 times, be put under the confocal laser scanning microscope, CLSM, fixedly excitation wavelength is 405nm, and emission wavelength is 570-610nm, can see that cell imaging is kermesinus (Fig. 9).Then with the CdCl of 10.0 μ mol/L ₂Join in the above-mentioned cell, after continuing to hatch 30 minutes, be put in again under the confocal laser scanning microscope, CLSM, emission wavelength is 500-550nm, and at this moment cell presents bright green (Figure 10).

Claims (1)

1. one kind 1,3,4-oxadiazole analog derivative is as Cd ²⁺Fluorescence probe Cd in detecting water body and cell ²⁺Application, described 1,3, the structural formula of 4-oxadiazole analog derivative is:

Wherein, R ₁To R ₇Be hydrogen atom, halogen or methyl; R ₈Be methyl, ethyl or propyl group; R ₉Be methyl, ethyl or its lithium salts;

Perhaps be:

Wherein, R ₁To R ₄, R ₆, R ₇Be hydrogen atom, halogen or methyl; R ₈Be methyl, ethyl or propyl group; R ₉Be methyl, ethyl or its lithium salts.

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