CN115477761B - Detection Fe (CN) 63- Fluorescent probe of ion and preparation method and application thereof - Google Patents

Abstract

The invention discloses a method for detecting Fe (CN) ₆ ^3‑ An ionic fluorescent probe, a preparation method and application thereof. The fluorescent probe is prepared by hinging a quaternary melon ring and 4,4' - (nitrilotris (benzene-4, 1-diyl)) tris (1-butylpyridine-1-onium). The fluorescent probe of the invention can be used for detecting Fe (CN) in aqueous solution and human renal cortex proximal tubular epithelial cells ₆ ^3‑ The ion detection method has the characteristics of low detection limit, high sensitivity, good biocompatibility, simple sample treatment, convenient operation and quick measurement.

Description

Detection Fe (CN) 63- Fluorescent probe of ion and preparation method and application thereof

Technical Field

The invention relates to a fluorescent probe, a preparation method and application thereof, in particular to a method for detecting Fe (CN) ₆ ^3- An ionic fluorescent probe, a preparation method and application thereof.

Background

Potassium ferricyanide (K) ₃ [Fe{CN} ₆ ]) In the solid state, it is a red crystal that is easily decomposed into highly toxic cyanide (KCN, HCN) by alkaline or light in aqueous solution. Cyanide is extremely toxic to organisms and can be inhaled through the mouth, respiratory tract or skin. After inhalation, the mitochondria are induced to generate active oxygen (hydroxyl free radicals); leading to mitochondrial dysfunction; and causes convulsions, vomiting, loss of consciousness, and even death.

According to the regulations of the world health organization, a minimum cyanide concentration of 1.9 μm in drinking water is permissible, but for organisms cyanide concentrations in blood exceeding 20 μm are determined to be toxic. Cyanide is often accidentally released into aqueous solutions in industry, posing a significant risk to human life and health, as well as environmental pollution.

Therefore, developing a rapid and effective method for detecting potassium ferricyanide is very important for environmental monitoring and life health. Among the many different analytical methods, the fluorescent probe method has received great attention because of its simple technology, high sensitivity, non-invasiveness, fast reaction time, and availability for biological environments.

Disclosure of Invention

The invention aims to provide a method for detecting Fe (CN) ₆ ^3- An ionic fluorescent probe, a preparation method and application thereof. The fluorescent probe of the invention can be used for detecting Fe (CN) in aqueous solution and human renal cortex proximal tubular epithelial cells ₆ ^3- The ion detection method has the advantages of low detection limit, high sensitivity, good biocompatibility, simple sample treatment, convenient operation and detectionQuick speed.

The technical scheme of the invention is as follows: detection Fe (CN) ₆ ^3- Fluorescent probe of ion with molecular formula of 3C ₈₄ H ₈₄ N ₅₆ O ₂₈ @C ₄₅ H ₅₁ N ₄ Br ₃ The structural formula is as follows:

wherein,,

preparation of the aforementioned detection Fe (CN) ₆ ^3- The method of the ionic fluorescent probe is prepared by hinging a quaternary melon ring and 4,4' - (nitrilotris (benzene-4, 1-diyl)) tris (1-butylpyridin-1-ium).

Preparation and detection of Fe (CN) as described above ₆ ^3- A method of fluorescent probe of an ion, the method comprising in particular:

(1) Taking a hinged ten-quaternary melon ring, then adding a mixed solvent, and uniformly mixing to obtain a solution A;

(2) Taking 4,4' - (nitrilotris (benzene-4, 1-diyl)) tris (1-butylpyridine-1-onium), and then adding a mixed solvent to mix uniformly to obtain a solution B;

(3) And mixing the solution A and the solution B, and then reacting at normal temperature to obtain the fluorescent probe.

Preparation and detection of Fe (CN) as described above ₆ ^3- And the mixed solvent is secondary water.

Preparation and detection of Fe (CN) as described above ₆ ^3- Method for the fluorescent probe of ions, the concentration of the hinged ten-four melon rings in the solution A being 0.1X10 ^-3 -10×10 ^-3 mol/L; the concentration of 4,4' - (nitrilotris (benzene-4, 1-diyl)) tris (1-butylpyridin-1-ium) in the solution B was 0.1X10 ^-3 -10×10 ^-3 mol/L。

Preparation and detection of Fe (CN) as described above ₆ ^3- A method of ionic fluorescent probes, wherein the solution a and the solution B are mixed, and the molar ratio of the hinged quaternary melon ring to the 4,4',4"- (nitrilotris (benzene-4, 1-diyl)) tris (1-butylpyridin-1-ium) is greater than 3:1.

The fluorescent probe is used for detecting Fe (CN) in aqueous solution ₆ ^3- Use of an ionic probe.

The fluorescent probe as described above was used for detecting Fe (CN) ₆ ^3- The application method of the ion probe comprises the following steps:

(1) Taking the fluorescent probe, diluting with secondary water to obtain a concentration of 2×10 ^-5 A mol/L probe standard solution;

(2) Adding an aqueous solution of a sample to be detected into the probe standard solution prepared in the step (1), standing for 3-10min, then carrying out fluorescence emission spectrometry at a fixed excitation wavelength of 455nm, and drawing a change curve of fluorescence intensity at the laser wavelength;

(3) Adding Fe (CN) into the standard solution of the probe according to the curve calculation in the step (2) ₆ ^3- The Fe (CN) in the aqueous solution of the sample to be detected can be obtained by corresponding the change value delta I of the fluorescence emission spectrum intensity at 575nm before and after the aqueous solution of the sample to be detected ₆ ^3- And detecting ions.

The fluorescent probe is used for detecting Fe (CN) in human renal cortex proximal tubular epithelial cells ₆ ^3- Use of an ionic probe.

The fluorescent probe as described above was used for detecting Fe (CN) ₆ ^3- The application of the ion probe is characterized in that the application method comprises the following steps:

treating cells with fluorescent probe under serum-free DMEM culture conditions for 20-40min, adding 5 μM and 20 μ M K respectively ₃ Fe(CN) ₆ After the cells were treated for 1 hour, the presence or absence of a significant change in fluorescence of the intracellular probe was observed after the cells were fixed, and if the presence of fluorescence was reduced or quenched, it was revealed that Fe (CN) was contained ₆ ^3- If not, it indicates that Fe (CN) is not contained ₆ ^3- 。

The beneficial effects of the invention are that

The invention utilizes a hinged ten-quaternary melon ring (tQ 14 for short]) And 4,4' - (nitrilotris (benzene-4, 1-diyl)) tris (1-butylpyridin-1-ium) (TPAPy for short) as raw materials, and constructing an AIE effect supermolecule fluorescent probe (TPAPy@tQ14) by interaction of host and guest])。 TPAPy@tQ[14]Can sensitively detect potential cyanide (K) ₃ [Fe{CN} ₆ ]) The detection limit is as low as 3.28X10 ^-7 M。TPAPy@tQ[14]The unique property of positive charge of (a) can rapidly cross the glomerular filtration barrier into human renal cortex proximal tubular epithelial cells (HK-2 cells). In addition, the probe was successfully applied to distinguish excess Fe (CN) in HK-2 cells ₆ ^3- Cell imaging was performed.

The fluorescent probe has the advantages of higher sensitivity, good biocompatibility, simple sample treatment, convenient operation and quick measurement.

Drawings

FIG. 1 is a schematic representation of the structural formulae of a hinged ten-membered melon ring (tQ 14) and 4,4' - (nitrilotris (benzene-4, 1-diyl)) tris (1-butylpyridin-1-ium) (TPAPy);

FIG. 2 is a fluorescence spectrum of a hinged ten-membered melon ring (tQ 14) and 4,4' - (nitrilotris (benzene-4, 1-diyl)) tris (1-butylpyridin-1-ium) (TPAPy) data processed using origin software; wherein: (a) Is the AIE effect of TPAPy in water and tetrahydrofuran solvents; (b) is the solvent effect of TPAPy in solvents of different polarity; (c) 0.0.1. to 6 equivalents of tQ [14] to the fluorescence spectrum of TPAPy; (d) Job's diagram of TPAPy@tQ [14 ];

FIG. 3 is a diagram of tQ 14 and TPAPy nuclear magnetic titration and inclusion patterns; wherein (i) TPAPy; (ii) tpapy:tq [14] =1: 1, a step of; (iii) tpapy:tq [14] =1: 2; (iv) tpapy:tq [14] =1: 3.

FIG. 4A probe contains 15 potassium salt anions vs. Fe (CN) ₆ ^3- Is a histogram of the immunity against interference;

FIG. 5 is a fluorescence spectrum obtained by detecting the probe and anion and performing data processing by using origin software; wherein: (a) a specific selective fluorescence spectrum for 16 anions; (b) For Fe (CN) ₆ ^3- Is specifically recognized by (a)The mechanism is as follows; (c) Adding Fe (CN) with different concentrations into the probe standard solution ₆ ^3- Fluorescence titration spectrum curve of ion in solution; (d) Adding Fe (CN) to the probe standard solution ₆ ^3- Fluorescence titration detection limit for ionic solutions;

FIG. 6 is a fluorescence contrast plot of the probe against 16 potassium salt anion specific selection cuvettes;

FIG. 7 is a bar graph of the biocompatibility of the probe in HK-2 cells;

FIG. 8 is a cell imaging of HK-2 cells with probes; wherein (a) is a staining pattern of the probe pair HK-2 cells; (b) The probe pair HK-2 cells contained 5. Mu. Mol/LFe (CN) ₆ ^3- Is a cell staining pattern of (2); (c) The probe pair HK-2 cells contained 20. Mu. Mol/LFe (CN) ₆ ^3- Is shown in the figure.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to be limiting.

Embodiments of the invention

Example 1

Preparation of fluorescent probes:

(1) Accurately weighing a proper amount of hinged ten-quaternary melon rings, dissolving with secondary water, and fixing volume to 100mL volumetric flask to obtain 1.00×10 ^-3 A ten-four-membered melon ring solution is hinged in mol/L;

(2) Accurately weighing appropriate amount of 4,4' - (nitrilotris (benzene-4, 1-diyl)) tris (1-butylpyridin-1-ium), dissolving with secondary water, and metering to 100mL volumetric flask to obtain 1.00X10-concentration ^-3 mol/L of 4,4' - (nitrilotris (benzene-4, 1-diyl)) tris (1-butylpyridin-1-ium) solution.

(3) The two solutions are mixed according to the volume ratio V _tQ[14] :V _TPAPy =3: 1, and standing at normal temperature for a while to obtain a concentration of 2.00×10 ^-5 mol/L fluorescent probe standard solution.

Example 2

The method for detecting potassium ferricyanide in water by using the fluorescent probe in example 1 comprises the following steps:

(1) The probe is taken and diluted by secondary water to prepare the probe with the concentration of 2.00 multiplied by 10 ^-5 A mol/L fluorescent probe standard solution;

(2) Adding a solution to be detected into the probe standard solution prepared in the step 1), standing for 5min, performing fluorescence emission spectrometry at a fixed excitation wavelength of 455nm, and drawing a change curve of fluorescence intensity at an emission wavelength of 574 nm;

(3) Calculating a change value delta I of fluorescence emission spectrum intensity at 574nm corresponding to water to be detected in the fluorescent probe solution according to the curve of the step 2), and indicating that the water to be detected contains Fe (CN) if the intensity is obviously weakened when the fluorescence emission spectrum at 574nm corresponding to water to be detected is added ₆ ^3- And the concentration is TPAPy@tQ [14]]0-2 times (excluding 0 and 2), when the fluorescence emission spectrum at 572nm before and after adding the water to be measured does not change significantly in intensity, it indicates that the water does not contain Fe (CN) ₆ ^3- 。

Example 3

The method for detecting HK-2 cells using the fluorescent probe described in example 1 was as follows:

(1) Accurately weighing appropriate amount of hinged ten-quaternary melon ring, dissolving with secondary water, and fixing volume to 100mL volumetric flask to obtain 1×10 ^-3 A ten-four-membered melon ring solution is hinged in mol/L;

(2) Accurately weighing appropriate amount of 4,4' - (nitrilotris (benzene-4, 1-diyl)) tris (1-butylpyridin-1-ium), dissolving with secondary water, and metering to 100mL volumetric flask to obtain 1×10 concentration ^-3 mol/L of a solution of 4,4' - (nitrilotris (benzene-4, 1-diyl)) tris (1-butylpyridin-1-ium);

(3) The two solutions are mixed according to the volume ratio V _tQ[14] :V _TPAPy =3: 1, and standing at normal temperature for a while to obtain a concentration of 1×10 ^-4 A mol/L fluorescent probe standard solution;

(4) Under the culture condition of serum-free DMEM, the cells are treated with 100 mu M probe for 30min, and 5 mu mol/L and 20 mu mol/LK are added respectively ₃ Fe(CN) ₆ The HK-2 cells are treated and co-cultured for 1h, after the cells are fixed, the fluorescence of the intracellular probe is observed to have no obvious change, if the fluorescence is reduced or quenched, the fluorescence is reduced or quenchedDescription of Fe (CN) contained therein ₆ ^3- The method comprises the steps of carrying out a first treatment on the surface of the If not, it indicates that Fe (CN) is not contained ₆ ^3- 。

Experimental example:

1. preparing a standard solution of iron cyanide ions:

accurately weighing proper amount of potassium ferricyanide, dissolving with secondary water with pH=6.75, and fixing volume to 10mL to obtain 1.00×10 ^-1 And (3) mol/L of standard solution of ferricyanide ions.

2. Drawing a standard curve:

taking a quartz fluorescent cuvette, adding 2.00×10 ^-5 After 3000. Mu.L of the mol/L fluorescent probe solution, 2.00×10 was accurately added ^-1 mol/L Fe (CN) ₆ ^3- 1.0. Mu.L of the standard solution was stirred in a cuvette and fluorescence emission spectrometry was performed at a fixed excitation wavelength of 455 nm.

According to the above procedure, a quantitative amount of 0.5. Mu.L Fe (CN) was continuously added to the 3000. Mu.L probe solution ₆ ^3- And (3) standard solution, and measuring a series of fluorescence curves under the excitation wavelength of 455nm until the ordinate value of the fluorescence curves changes slowly, so that the titration operation can be stopped.

Then with Fe (CN) ₆ ^3- The concentration is on the abscissa, and the fluorescence emission intensity of the probe at 574nm emission wavelength is equal to that of the probe added with Fe (CN) with different concentrations ₆ ^3- The difference in fluorescence emission intensities of (2) is the ordinate to obtain a standard curve.

3. Biocompatibility testing of probes:

HK-2 cells were seeded in 96-well U-shaped bottom plates at 5% CO ₂ And culturing at 37deg.C for 24 hr, and then culturing with different concentrations of TPAPy and TPAPy@tQ [14]]Treatment is carried out for 12h and 24h. Subsequently, after removal of the supernatant, MTT (5 mg/mL medium, 20. Mu.L/well) was added to the well, followed by incubation at 37℃for 4 hours. The supernatant was removed, 150 μl DMSO was added per well to dissolve, and the absorbance values were tested after shaking the plate for 4 minutes.

4. Cell imaging of the probe:

HK-2 cells were spread in DMEM containing 10% FBS (fetal bovine serum) and 2% antibiotic solution on 35mm diameter round glassOn a glass culture dish, 5% CO ₂ And incubation in incubator at 37 ℃ for 24 hours. Cells were incubated with DMSO (ph=6.75) for 5min at 37 ℃. The cells were then washed with pre-warmed PBS (ph=6.75) for 5 minutes. Subsequently, the cells were incubated with the probe (100 μm) dissolved in water at 37 ℃ for 5 minutes, and then washed 3 times with pre-warmed PBS (ph=6.75). For the detection of Fe (CN) in living cells ₆ ^3- Fe (CN) was used in the petri dishes ₆ ^3- Cells were stimulated (5. Mu.M and 20. Mu.M) and fluorescence microscopy images were obtained within 30 seconds.

5. Fe (CN) of HK-2 cells ₆ ^3- And (3) detection:

cells were treated with 100. Mu.M probe for 30mins under serum-free DMEM culture conditions, 5. Mu.M and 20. Mu. M K, respectively ₃ Fe(CN) ₆ When HK-2 cells were treated for 1h, no significant change in fluorescence of the intracellular probe was observed after immobilization of the cells, and if fluorescence was reduced or quenched, it was revealed that Fe (CN) was contained therein ₆ ^3- The method comprises the steps of carrying out a first treatment on the surface of the If not, it indicates that Fe (CN) is not contained ₆ ^3- 。

While the invention has been described with reference to the preferred embodiments, it should be understood that the invention is not limited to the embodiments described above, but is intended to cover modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

Claims (9)

1. Detection Fe (CN) ₆ ^3- An ionic fluorescent probe characterized by: molecular formula is 3C ₈₄ H ₈₄ N ₅₆ O ₂₈ @C ₄₅ H ₅₁ N ₄ Br ₃ The structural formula is as follows:

wherein,,

2. a method for preparing the Fe (CN) detector of claim 1 ₆ ^3- A method of fluorescent probe for ions, characterized by: is prepared from hinged quaternary melon rings and a compound I shown in the structural formula of the following figure;

3. preparation and detection of Fe (CN) according to claim 2 ₆ ^3- A method for fluorescence probe of ions, characterized in that it comprises the following specific steps:

(1) Taking a hinged ten-quaternary melon ring, then adding secondary water, and uniformly mixing to obtain a solution A;

(2) Taking a compound I, then adding secondary water, and uniformly mixing to obtain a solution B;

(3) And mixing the solution A and the solution B, and then reacting at normal temperature to obtain the fluorescent probe.

4. The preparation and detection Fe (CN) according to claim 3 ₆ ^3- A method of fluorescent probe for ions, characterized by: the concentration of the hinged ten-quaternary melon rings in the solution A is 0.1 multiplied by 10 ^-3 -10×10 ^-3 mol/L; the concentration of compound I in the solution B was 0.1X10 ^-3 -10×10 ^-3 mol/L。

5. The preparation and detection Fe (CN) according to claim 3 ₆ ^3- A method of fluorescent probe for ions, characterized by: when the solution A and the solution B are mixed, the molar ratio of the hinged quaternary melon ring to the compound I is more than 3:1.

6. A fluorescent probe according to claim 1 as a probe for detecting Fe (CN) in an aqueous solution ₆ ^3- Use of an ionic probe.

7. The fluorescent probe according to claim 6 as a probe for detecting Fe (CN) in an aqueous solution ₆ ^3- The application of the ion probe is characterized in that the application method comprises the following steps:

(1) Taking the fluorescent probe, diluting with secondary water to obtain a concentration of 2×10 ^-5 A mol/L probe standard solution;

(2) Adding an aqueous solution of a sample to be detected into the probe standard solution prepared in the step (1), standing for 3-10min, then carrying out fluorescence emission spectrometry at a fixed excitation wavelength of 455nm, and drawing a change curve of fluorescence intensity at the laser wavelength;

(3) Adding Fe (CN) into the standard solution of the probe according to the curve calculation in the step (2) ₆ ^3- The Fe (CN) in the aqueous solution of the sample to be detected can be obtained by corresponding the change value delta I of the fluorescence emission spectrum intensity at 575nm before and after the aqueous solution of the sample to be detected ₆ ^3- And detecting ions.

8. A fluorescent probe according to claim 1 for use as a detection of Fe (CN) in human renal cortex proximal tubular epithelial cells ₆ ^3- Use of an ionic probe.

9. Fluorescent probe according to claim 8 as a method for detecting Fe (CN) in human renal cortex proximal tubular epithelial cells ₆ ^3- The application of the ion probe is characterized in that the application method comprises the following steps:

treating cells with fluorescent probe under serum-free DMEM culture conditions for 20-40min, adding 5 μM and 20 μ M K respectively ₃ Fe(CN) ₆ After the cells were treated for 1 hour, the presence or absence of a significant change in fluorescence of the intracellular probe was observed after the cells were fixed, and if the presence of fluorescence was reduced or quenched, it was revealed that Fe (CN) was contained ₆ ^3- If not, it indicates that Fe (CN) is not contained ₆ ^3- 。

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