CN109115743B - A method for detecting aluminum ions in cells by long-wave emission fluorescence imaging - Google Patents

Abstract

A method for detecting aluminum ions in cells by long-wave emission fluorescence imaging, using a fluorescent probe L as a fluorescent imaging probe for detecting trace amounts of Al ³⁺ in cells by fluorescence imaging, and detecting trace amounts of Al ³⁺ in cells by long-wave emission fluorescence imaging, The chemical structural formula of the fluorescent probe L is:

During detection, firstly use the fluorescent probe L solution to incubate the cells with the fluorescent probe L to enter the active cells, and then incubate the active cells with the fluorescent probe L with Al ³⁺ to make the probe and Al ³⁺ in the active cells. The reaction generates a compound capable of emitting fluorescence at a characteristic wavelength, and realizes the staining and imaging of Al ³⁺ ions in active cells by the probe. The advantages are: high selectivity and good sensitivity, fluorescence imaging to detect trace Al ³⁺ in cells at a longer emission wavelength, and strong tissue penetration, avoiding light damage and background fluorescence self-interference.

Description

A method for detecting aluminum ions in cells by long-wave emission fluorescence imaging

technical field

The invention relates to a method for detecting aluminum ions in cells by long-wave emission fluorescence imaging.

Background technique

It is well known that the development of chemical sensors for detecting metal ions has received extensive attention due to their important roles in medicine, living systems, and the environment. Aluminum (Al) is the third most ubiquitous and abundant metallic element in the earth's crust, and people are widely exposed to aluminum as it is dispersed in areas such as water treatment, food additives, storage tools or cooking utensils, pharmaceuticals, and light alloys. At the same time, Al ³⁺ is also an essential element in the life system, but excessive intake of Al ³⁺ will affect the absorption of calcium in the intestinal tract, causing softening, atrophy and even deformation of the bones, and affecting the absorption of iron in the blood, causing anemia. In addition, Al ³⁺ toxicity causes damage to the central nervous system and leads to neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, osteomalacia and renal failure. The World Health Organization (WHO) stipulates that the average human intake of Al ³⁺ is about 3-10 mg per day and limits the concentration in drinking water to 7.41 mmol/L. The weekly tolerable intake of Al ³⁺ in humans is estimated to be 7 mg/Kg.

In recent years, there have been many designs based on the recognition of Al ³⁺ fluorescent probe molecules, for example, Biosens Bioelectron (2015), 68, 749-756; Spectrochim Acta A Mol Biomol Spectrosc (2018), 19, 2257-2262; Sensors and Actuators B: Chemical (2017), 247, 451-460; Biosens Bioelectron (2017), 77, 530-536; Sensors and Actuators B: Chemical (2018), 266, 95-105; Anal Chim Acta (2016), 942, 104-111; Tetrahedron Letters (2016) , 57(8), 953-958; these documents are all for the detection of multiple cations, and do not realize the specific recognition of Al ³⁺ , Sensors and Actuators B: Chemical (2016), 229, 138-144; Spectrochim Acta A Mol Biomol Spectrosc (2018), 201, 185-192; Analytical Methods (2012), 4(7), 1906; Sensors and Actuators B: Chemical (2017), 240, 916-925; Journal of Photochemistry and Photobiology A: Chemistry (2017), 332, 101-111; Sensors and Actuators B: Chemical (2017), 238, 128-137; Sensors and Actuators B: Chemical (2018), 264, 304-311; Although these documents can specifically identify Al ³⁺ , the emission wavelength is short and cannot be Detection in the long wavelength range is easy to cause photodamage to cells or living biological samples, causing background fluorescence self-interference to a certain extent, and most cells have poor permeability and cannot be used to identify Al ³⁺ in cells. Therefore, the design and synthesis are better. The Al ³⁺ fluorescent probe is of great significance.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a method for detecting aluminum ions in cells by long-wave emission fluorescence imaging, which can realize the fluorescence imaging detection of Al ³⁺ at a longer emission wavelength, and has strong tissue penetration, avoiding the need for Photodamage and background fluorescence self-interference.

The technical solution of the present invention is:

A method for detecting aluminum ions in cells by long-wave emission fluorescence imaging, which is special in that a fluorescent probe L is used as a fluorescent imaging probe for detecting trace amounts of Al ³⁺ in cells by fluorescence imaging, and cells are detected by long-wave emission fluorescence imaging. Medium and trace Al ³⁺ , the chemical structural formula of the fluorescent probe L is:

Further, when detecting trace amounts of Al ³⁺ in cells by long-wave emission fluorescence imaging, firstly use 5 μmol/L-10 μmol/L solution of fluorescent probe L to incubate with cells to allow the fluorescent probe L to enter the active cells, and then add the fluorescent probe L to the cells. The active cells of L were cultured with Al ³⁺ , and the probe and Al ³⁺ reacted in the active cells to generate compounds that could emit fluorescence at characteristic wavelengths, and the probes were able to image the Al ³⁺ ions in the active cells, using confocal laser fluorescence. Fluorescence images of viable cells after culture were observed by microscope.

Further, the solvent of the fluorescent probe L solution is PBS buffer.

Further, under the excitation of 488nm wavelength, observe with 630nm-650nm channel, if red fluorescence appears, there is Al ³⁺ .

Further, the active cells are HeLa cells, abbreviated as HeLa cells.

Further, the specific synthesis method of the fluorescent probe L is as follows:

和水杨苯甲酰肼

按照摩尔比1:1～1:3进行投料，加热回流反应8h～12h，反应结束后有黄色固体析出，用乙醇进行重结晶，得到荧光探针L

Using ethanol as solvent, raw material 1,4-diethyl-7-hydroxytetrahydroquinoxaline-6-carbaldehyde

and salicyl hydrazide

Feeding is carried out according to the molar ratio of 1:1～1:3, and the reaction is heated and refluxed for 8h～12h. After the reaction is completed, a yellow solid is precipitated, which is recrystallized with ethanol to obtain the fluorescent probe L

Further, the fluorescent probe L can also fluorescently detect Al ³⁺ in an aqueous solvent, and the solvent for identifying Al ³⁺ is the pH=4～10 of the solvent for identifying Al ³⁺ in a volume ratio of DMSO to Tris buffer solution of 7:3. .

Beneficial effects of the present invention:

(1) The separation and purification process of synthetic fluorescent probes is easy; the fluorescent probes can identify Al ³⁺ with long wavelength (642 nm) fluorescence enhancement in aqueous media, with high selectivity and good sensitivity, and can be applied to detect Al in cells ³⁺ .

(2) Using 1,4-diethyl-7-hydroxytetrahydroquinoxaline-6-carbaldehyde as the substrate, by introducing salicylaldehyde hydrazide to extend the conjugation system and increase the chelation site, Al ³ After ⁺ binding, the isomerization of the C=N double bond is inhibited, and the molecule is coplanar, which increases the rigidity of the molecule, so that the fluorescence is enhanced to recognize Al ³⁺ .

Description of drawings

Fig. 1 is the ¹ H NMR spectrum of the fluorescent probe L used for the long-wave emission fluorescence imaging detection of aluminum ions in cells of the present invention;

Fig. 2 is the ¹³ C NMR spectrum of the fluorescent probe L used for detecting aluminum ions in cells by long-wave emission fluorescence imaging of the present invention;

Fig. 3 is the mass spectrogram of the fluorescent probe L used in the long-wave emission fluorescence imaging of the present invention to detect aluminum ions in cells;

Fig. 4 is the fluorescence emission spectrum of Al ³⁺ detected by fluorescent probe L;

Fig. 5 is a graph showing the effect of coexisting metal ions on the fluorescence intensity of Al ³⁺ detected by fluorescent probe L;

Fig. 6 is the fluorescence titration spectrogram of different concentrations of Al ³⁺ to probe;

Fig. 7 is a graph showing the detection limit of Al ³⁺ by the fluorescent probe L used for the detection of aluminum ions in cells by long-wave emission fluorescence imaging;

Fig. 8 is the toxicity of the fluorescent probe L used for the detection of aluminum ions in cells to HeLa cells by long-wave emission fluorescence imaging;

Fig. 9 is the detection photos of the fluorescence imaging of HeLa cells with the fluorescent probe L after being reacted with different concentrations of Al ³⁺ .

Detailed ways

The technical solutions of the present invention will be described in further detail below with reference to specific embodiments.

Example 1-Example 3 is the specific synthesis of the fluorescent probe L used in the long-wave emission fluorescence imaging of the present invention to detect aluminum ions in cells.

The specific synthetic route of fluorescent probe L is as follows:

Example 1

Compound 1,4-diethyl-7-hydroxytetrahydroquinoxaline-6-carbaldehyde (234 mg, 1 mmol) and salicyl hydrazide (152 mg, 1 mmol) were dissolved in ethanol and refluxed for 8 h, cooled to room temperature, and a yellow color was precipitated The solid was purified by recrystallization from ethanol to obtain 237 mg of a yellow solid, which was the acceptor L, and the yield was 64.3%. The ¹ H NMR spectrum, the ¹³ C NMR spectrum and the mass spectrum of the fluorescent probe L are shown in Figures 1-3.

¹ H NMR (400MHz, DMSO-d ₆ ) δ 12.07(s, 1H), 11.77(s, 1H), 10.75(s, 1H), 8.46(s, 1H), 7.90(d, J=7.7Hz, 1H), 7.44(t, J=7.6Hz, 1H), 6.96(t, J=8.1Hz, 2H), 6.54(s, 1H), 6.08(s, 1H), 3.41(s, 2H), 3.34( d, J=7.0Hz, 2H), 3.27–3.17 (m, 2H), 3.11 (s, 2H), 1.09 (td, J=6.6, 2.9Hz, 6H).

¹³ C NMR (101MHz, DMSO-d ₆ )δ164.26,159.74,153.26,151.39,140.10,134.09,128.53,128.32,119.27,117.72,115.77,111.26,97.42,47.11,415.12,10.

HRMS(ESI ⁺ )calcd for C ₂₀ H ₂₄ N ₄ O ₃ [M+H] ⁺ :369.1848,found:369.1910.

Example 2

Compound 1,4-diethyl-7-hydroxytetrahydroquinoxaline-6-carbaldehyde (234 mg, 1 mmol) and salicyl hydrazide (304 mg, 2 mmol) were dissolved in ethanol and refluxed for 10 h, cooled to room temperature, and a yellow color was precipitated The solid was purified by recrystallization from ethanol to obtain 257 mg of red solid, which was the fluorescent probe L, and the yield was 69.8%. The ¹ H NMR spectrum, the ¹³ C NMR spectrum and the mass spectrum of the fluorescent probe L are shown in Figures 1-3.

Example 3

Compound 1,4-diethyl-7-hydroxytetrahydroquinoxaline-6-carbaldehyde (234 mg, 1 mmol) and salicyl hydrazide (456 mg, 3 mmol) were dissolved in ethanol and refluxed for 12 h, cooled to room temperature, and a yellow color was precipitated The solid was purified by recrystallization from ethanol to obtain 267 mg of red solid, which was the acceptor L, and the yield was 72.5%. The ¹ H NMR spectrum, the ¹³ C NMR spectrum and the mass spectrum of the fluorescent probe L are shown in Figures 1-3.

Fluorescent probe L detects Al ³⁺ in cells:

When detecting the trace amount of Al ³⁺ in cells by long-wave emission fluorescence imaging, firstly use 5μmol/L-10μmol/L fluorescent probe L solution (the solvent is PBS buffer) to incubate the cells to make the fluorescent probe L enter the active cells, and then put the fluorescent probe L into the active cells. The active cells with the fluorescent probe L are incubated with Al ³⁺ , so that the probe and Al ³⁺ react in the active cells to generate compounds that can emit fluorescence at characteristic wavelengths, and the probes can be used to stain and image the Al ³⁺ ions in the active cells. Fluorescence images of viable cells after culture were observed by confocal fluorescence microscopy. Under the excitation of 488nm wavelength, observe with 630nm-650nm channel, if red fluorescence appears, there is Al ³⁺ .

We used HeLa cells for live cell imaging experiments. HeLa cells were incubated with 10 μmol/L fluorescent probe L solution (the solvent was PBS buffer) at 37°C for 30 minutes, and then HeLa cells were washed three times with PBS buffer, and then Al ³ was added. ⁺ (10μM, 50μM, 100μM) for 30 minutes, the red fluorescence brightness was observed to increase with the increase of Al ³⁺ concentration (see Figure 9), these results indicate that probe L has good cell permeability and can detect Al in HeLa cells ³⁺ . This can be achieved by using conventional HeLa cell culture medium during the culture process.

The selective detection of Al ³⁺ by fluorescent probe L:

10μmol/L fluorescent probe L in DMSO:Tris=7:3 (v/v, pH=7.4) buffer solution, to which 8μL (50mmol/L) cations (Mg ²⁺ , Fe ²⁺ , Fe ³⁺ were added respectively , Al ³⁺ , Mn ²⁺ , Pb ²⁺ , Na ⁺ , Cu ²⁺ , Cd ²⁺ , Cr ³⁺ , ^{Ca 2+} , K ⁺ , Ba ²⁺ , Co ²⁺ , Ni ²⁺ , Sr ²⁺ , Zn ²⁺ ), and the fluorescence emission spectrum changes of the solution were detected, as shown in Figure 4. It can be seen from Figure 4 that when cations are added, only Al ³⁺ can cause a significant change in the fluorescence intensity, that is, the fluorescence intensity at 608 nm and 642 nm is enhanced after the addition of Al ³⁺ , while the addition of other cations has no obvious effect on the fluorescence intensity. , it can be seen that the fluorescent probe L has a high degree of selectivity for Al ³⁺ .

Anti-interference detection of Al ³⁺ recognition by fluorescent probe L:

10μmol/L fluorescent probe L in DMSO:Tris=7:3 (v/v, pH=7.4) solution, to which 8μL (50mmol/L) 17 kinds of cations (Mg ²⁺ , Fe ²⁺ , Fe ³ were added respectively ⁺ , Al ³⁺ , Mn ²⁺ , Pb ²⁺ , Na ⁺ , Cu ²⁺ , Cd ²⁺ , Cr ³⁺ , Ca ²⁺ , K ⁺ , Ba ²⁺ , Co ²⁺ , Ni ²⁺ , Sr ^{2 +} , Zn ²⁺ ), detect the fluorescence emission spectrum of the solution, then add 8 μL (50 mmol/L) of Al ³⁺ to each of the above solutions containing cations respectively, detect the fluorescence emission spectrum of the solution, take the corresponding maximum emission wavelength As shown in Figure 5, in the presence of other cations, Al ³⁺ can also lead to the fluorescence enhancement of probe L, indicating that the fluorescent probe L recognizes Al ³⁺ and is interfered by some cations.

Titration test of Al ³⁺ by fluorescent probe L:

10μmol/L fluorescent probe L in DMSO:Tris=7:3 (v/v, pH=7.4) buffer solution, add 0-20 times (50mmol/L) Al ³⁺ respectively, and detect the fluorescence emission spectrum of the solution change, as shown in Figure 6. It can be seen from Figure 6 that with the continuous addition of Al ³⁺ , the emission peak at 640 nm gradually increases. When 20 times of Al ³⁺ is added, the emission peak at 640 nm no longer increases, indicating that at this time reached saturation.

Detection limit test of Al ³⁺ by fluorescent probe L:

In the DMSO:Tris=7:3 (v/v, pH=7.4) buffer solution of probe L, the fluorescence intensity of 20 parallel samples was tested, according to the formula ∑(X _i -X) ² =(X ₁ - X) ² +(X ₂ -X) ² +...+(X _n -X) ² Calculate the sum of squared differences (X _i is the fluorescence intensity value of the receptor itself for each measurement, X is the average fluorescence intensity, n is the number of tests, n is greater than or equal to 11), then according to the formula S=[∑(X _i -X) ² /(n-1)] ^0.5 to find S, and then according to the detection limit formula 3S/K, K is the selected straight line Part of the slope (Note: the straight line is based on the titration to make a dot plot, the abscissa is the ion concentration, and the ordinate is the fluorescence intensity), the detection line is 5.73×10 ^-7 mol/L (see Figure 7), which shows that the probe is The needle has a lower detection limit, can detect Al ³⁺ with a lower concentration, has a higher sensitivity, and has a certain practical application value.

Toxicity detection of fluorescent probe L on HeLa cells:

We used the MTT method to detect the toxicity of probe L to HeLa cells. When 10 μM probe L was incubated with HeLa cells in DMEM medium for 24 h, the cell viability was still close to 100%, indicating that the toxicity of probe L to HeLa cells was very low (see Figure 8).

The above are only specific embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (7)

1. A method for detecting aluminum ions in cells by long-wave emission fluorescence imaging is characterized in that: the fluorescent probe L is used for detecting trace Al in the cells by fluorescence imaging³⁺The fluorescent imaging probe detects trace Al in cells through long-wave emission fluorescent imaging^{3 +}The long wave length is 608nm-650nm, and the chemical structural formula of the fluorescent probe L is as follows:

2. the method of claim 1, wherein the long-wave emission fluorescence imaging is used for detecting aluminum ions in the cells, and the method comprises the following steps: long wave emission wavelength is 608nm-650nm, fluorescence imaging detects trace Al in cells³⁺When in use, firstly, 5 mu mol/L-10 mu mol/L of fluorescent probe L solution is used for culturing cells to ensure that the fluorescent probe L enters active cells, and then the active cells with the fluorescent probe L are cultured with Al³⁺Culturing, the probe and Al³⁺Reacting in active cell to generate compound capable of emitting fluorescence with characteristic wavelength to realize probe to active cell Al³⁺Ion dyeingImaging, namely observing a fluorescence image of the cultured active cells by using a laser confocal fluorescence microscope.

3. The method of claim 2, wherein the long-wave emission fluorescence imaging is used for detecting aluminum ions in the cells, and the method comprises the following steps: the solvent of the fluorescent probe L solution is PBS buffer solution, and the long-wave wavelength is 608nm-650 nm.

4. The method of claim 2, wherein the long-wave emission fluorescence imaging is used for detecting aluminum ions in the cells, and the method comprises the following steps: observing the fluorescence image of the cultured active cells by using a laser confocal fluorescence microscope, observing by using a 630nm-650nm channel under the excitation of 488nm wavelength, and if red fluorescence appears, then Al exists³⁺。

5. The method of claim 2, wherein the long-wave emission fluorescence imaging is used for detecting aluminum ions in the cells, and the method comprises the following steps: the active cells are Hela cells, and the long wave length is 608nm-650 nm.

6. The method of claim 1, wherein the long-wave emission fluorescence imaging is used for detecting aluminum ions in the cells, and the method comprises the following steps: the long wave wavelength is 608nm-650nm, and the fluorescent probe L is synthesized in the following specific mode:

taking ethanol as a solvent, feeding 1, 4-diethyl-7-hydroxytetrahydroquinoxaline-6-formaldehyde and salicylbenzoic hydrazide as raw materials according to a molar ratio of 1: 1-1: 3, heating and refluxing for 8-12 h, separating out a yellow solid after the reaction is finished, and recrystallizing with ethanol to obtain the fluorescent probe L.

7. The method of claim 1, wherein the long-wave emission fluorescence imaging is used for detecting aluminum ions in the cells, and the method comprises the following steps: the fluorescent probe L can also detect Al in an aqueous solvent by fluorescence³⁺Identification of Al³⁺The solvent of (1) is DMSO and Tris buffer solution with the volume ratio of 7:3, and Al is identified³⁺The pH of the solvent is 4-10, and the long-wave wavelength is 608nm-650 nm.

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