CN112939886A

CN112939886A - Near-infrared fluorescent probe capable of detecting content of GSTs (glutathione S-terminal transferase) as well as synthetic method and application thereof

Info

Publication number: CN112939886A
Application number: CN202110155050.7A
Authority: CN
Inventors: 温莹; 龙志清; 阴彩霞; 霍方俊
Original assignee: Shanxi University
Current assignee: Lianyungang Juwen Trading Co ltd
Priority date: 2021-02-04
Filing date: 2021-02-04
Publication date: 2021-06-11
Anticipated expiration: 2041-02-04
Also published as: CN112939886B

Abstract

The invention provides a near-infrared fluorescent probe capable of detecting the content of GSTs, and a synthetic method and application thereof. The name of the probe is 4- (2,4-dinitrophenoxy) benzyl 3,7-bis (diethylamino) -10H-benzoxazine-10-carboxylate (4- (2,4-dinitrophenoxy) benzyl 3,7-bis (diethylamino) -10H-benzoxazine-10-carboxylate), or 4- (2,4-dinitrophenoxy) benzyl 3,7-bis (dimethylamino) -10H-phenothiazine-10-carboxylate (4- (2,4-dinitrophenoxy) benzyl 3,7-bis (dimethylamino) -10H-phenothiazine-10-carboxylate), the invention also provides a method for detecting the content of the GSTs in vivo, namely the probe is in a phosphate buffer solution pH 7: ethanol/9: 1(v/v) system, and (4) quantitatively detecting the content of GSTs by a fluorescence spectrophotometer. The detection method has high sensitivity and is simple and convenient.

Description

Near-infrared fluorescent probe capable of detecting content of GSTs (glutathione S-terminal transferase) as well as synthetic method and application thereof

Technical Field

The invention relates to a near-infrared fluorescent probe based on basic dye, in particular to a near-infrared fluorescent probe based on basic dye, a synthetic method thereof and application thereof in detecting GSTs.

Background

Chemotherapeutic drugs are toxic to the body, and tumor cells protect themselves from chemotherapeutic drugs by overexpressing GSTs. GSTs are a family of multifunctional biphasic metabolic enzymes that are excreted in vitro mainly by coupling thiol groups in activated GSH (reduced glutathione) to electrophilic groups, which increases the hydrophobicity of the conjugate. It is also an important detoxification system for resisting cell injury and cancer. Near infrared fluorescent probes (650- & ltSUB- & gt 900nm) are regarded by researchers due to their higher signal-to-noise ratio, higher penetration depth and higher imaging resolution. Currently, common near-infrared fluorescent chromophores include: cyanine, rhodamine, squaric acid, porphyrin, BODIPY, basic dye (basic blue, methylene blue), etc. Among them, basic dye fluorescence chromophore is a commonly used tissue dye in the field of bioanalysis. The absorption peak in the near infrared region is strong and sharp, and the molar extinction coefficient is high. Therefore, the basic dye and the derivative thereof can be used as a good skeleton for constructing the probe.

Based on the above, the near-infrared fluorescent probe with high stability, high selectivity, high sensitivity and low cytotoxicity is particularly important for detecting the level change of intracellular GSTs, and a powerful scientific tool is provided for the current biomedical development.

In the invention, two near-infrared fluorescent probes for detecting GSTs are synthesized based on basic dye fluorophores, and the GSTs can activate sulfydryl in GSH to react with the probes to release the basic dye fluorophores which emit red fluorescence. Thereby enabling detection of GSTs.

Disclosure of Invention

The invention aims to provide a near-infrared fluorescent probe capable of detecting the content of GSTs, a synthetic method thereof and application of the probe in detecting the GSTs. The detection process is simple and convenient, the sensitivity is high, the light stability is good, and the detection limit is low.

The two near-infrared fluorescent probes capable of detecting the content of GSTs are named as 4- (2,4-dinitrophenoxy) benzyl 3,7-bis (diethylamino) -10H-benzoxazine-10-carboxylate (4- (2,4-dinitrophenoxy) benzyl 3,7-bis (dimethylamino) -10H-phenoxazine-10-carboxylate) and 4- (2,4-dinitrophenoxy) benzyl 3,7-bis (dimethylamino) -10H-phenothiazine-10-carboxylate (4- (2,4-dinitrophenoxy) benzyl 3,7-bis (dimethylamino) -10H-phenothiazine-10-carboxylate), which are respectively named as L1 and L2. They have the following structural formula:

the invention provides a method for synthesizing two near-infrared fluorescent probes based on basic dyes, which comprises the following steps:

(1) dissolving p-hydroxybenzyl alcohol in dichloromethane, stirring for 5min in an ice-water bath, then adding triethylamine, stirring for 30min, then dropwise adding 1-chloro-2, 4-dinitrobenzene in dichloromethane, stirring for reacting overnight, detecting the reaction progress by TLC, extracting and washing with saturated sodium chloride (3X 50mL) and dichloromethane (3X 50mL), drying the organic phase with anhydrous sodium sulfate, and using petroleum ether/ethyl acetate ═ 3: 1, purifying by a column to obtain light yellow powder, namely a product (4- (2,4-dinitrophenoxy) phenyl) methanol, wherein the mass ratio of the raw material p-hydroxybenzyl alcohol: triethylamine: the mol ratio of 1-chlorine-2, 4-dinitrobenzene is 1: 3: 1.

(2.1) dissolving (4- (2,4-dinitrophenoxy) phenyl) methanol, 3,7-bis (diethylamino) -10H-phenoxazine-10-carbonyl chloride, sodium carbonate and 4-dimethylaminopyridine in dichloromethane, and stirring for reacting for 8 hours under the conditions of nitrogen protection and ice bath. The solvent was removed and the crude product was purified by silica gel column chromatography to give 4- (2,4-dinitrophenoxy) benzyl 3,7-bis (diethylamino) -10H-benzoxazine-10-carboxylate (named L1) as a brown solid. Wherein (4- (2,4-dinitrophenoxy) phenyl) methanol: 3,7-bis (diethylamino) -10H-phenoxazine-10-carbonyl chloride: sodium carbonate: the feeding molar ratio of the 4-dimethylaminopyridine is 1.2: 1: 3: 1.

(2.2) dissolving (4- (2,4-dinitrophenoxy) phenyl) methanol, 3,7-bis (dimethylamino) -10H-phenothiazine-10-carbonyl chloride and sodium carbonate 4-dimethylaminopyridine in dichloromethane, and stirring for reacting for 8 hours under the conditions of nitrogen protection and ice bath. The solvent was removed and the crude product was purified by silica gel column chromatography to give 4- (2,4-dinitrophenoxy) benzyl 3,7-bis (dimethylamino) -10H-phenothiazine-10-carboxylate (named L2) as a brown solid. (4- (2,4-dinitrophenoxy) phenyl) methanol: 3,7-bis (dimethylamino) -10H-phenothiazine-10-carbonyl chloride: sodium carbonate: the feeding molar ratio of the 4-dimethylaminopyridine is 1.2: 1: 3: 1.

the near-infrared fluorescent probes L1 and L2 synthesized based on basic dye fluorophores can be used for detecting GSTs.

The invention provides a method for detecting GSTs, which comprises the following steps:

(1) a phosphate buffer solution (10mM, 10% absolute ethanol) having a pH of 7 was prepared as a reaction system, and L1 and L2 were dissolved in DMSO to prepare a 2mM solution, and a 20mM Glutathione (GSH) solution and a GSTs solution of 1mg/mL were prepared.

(2) 900 μ L of the reaction solution, 1 μ L L1(L2) in DMSO, 50 μ L of the GSH solution, and 50 μ L of the GSTs solution were added to a fluorescence cuvette, and fluorescence scanning was performed in a water bath at 37 ℃ with increasing time, and the fluorescence intensity at 676nm (L1)686nm (L2) was gradually increased.

(3) 944. mu.L, 943. mu.L, 942. mu.L, 941. mu.L and 940. mu.L of the system solution were added to 5 EP tubes of 2mL, respectively; 1 μ L L1 in DMSO; 50 mu L of GSH solution and different amounts of GSTs solution, the concentration of GSTs in the system is respectively 6 mu g/mL, 7 mu g/mL, 8 mu g/mL, 9 mu g/mL, 10 mu g/mL, after shaking and placing in a water bath at 37 ℃ for 3h, the fluorescence intensity of each concentration is scanned at 676 nm. Plotting and drawing the concentration of the GSTs as an abscissa and the 676nm fluorescence intensity as an ordinate to obtain a working curve of the GSTs; the linear regression equation is: 6.507x +48.218, R²0.99445. The unit of x is μ g/mL.

(4) Adding 950 mu L of system solution into 6 EP tubes with 2 mL; 1 μ L L2 in DMSO; 50 mu L of GSH solution and different amounts of GSTs solution to make the concentration of GSTs in the system respectively be 50ng/mL, 100ng/mL, 200ng/mL, 300ng/mL, 400ng/mL and 500ng/mL, shaking and placing in 37 ℃ water bath for 3h, then scanning at 686nmFluorescence intensity at each concentration. Plotting and drawing the GSTs concentration as an abscissa and the 686nm fluorescence intensity as an ordinate to obtain a GSTs working curve; the linear regression equation is: 0.08197x +11.1103, R²0.99145. The unit of x is ng/mL.

Compared with the prior art, the invention has the following advantages and effects:

1. the basic dye near-infrared fluorescent derivative is simple to synthesize and low in cost;

2. the detection method is simple, and only an ultraviolet spectrophotometer and a fluorescence detector are needed;

3. the near-infrared fluorescent probes L1 and L2 can realize dynamic detection of GSTs, and have the advantages of high selectivity, good sensitivity and low detection limit;

4. the detection signal is obvious and is a near-infrared fluorescence signal, and the color change of the reaction solution is visible by naked eyes and changes from colorless to light blue.

Drawings

FIG. 1 example 1 preparation of fluorescent Probe L1 Hydrogen Spectrum

FIG. 2 carbon spectrum of fluorescent probe L1 prepared in example 1

FIG. 3 Mass spectrum of fluorescent probe L1 prepared in example 1

FIG. 4 hydrogen spectrum of fluorescent probe L2 prepared in example 1

FIG. 5 carbon spectrum of fluorescent probe L2 prepared in example 1

FIG. 6 Mass spectrum of fluorescent probe L2 prepared in example 1

FIG. 7 fluorescence spectra of the effect of example 2L1 on GSH and GSTs

FIG. 8 fluorescence spectra of the effect of example 2L2 on GSH and GSTs

FIG. 9 GSTs working curve of example 3L1

FIG. 10 GSTs working curve of example 4L2

FIG. 11 Selective fluorescence histogram of example 5L1

FIG. 12 histogram of selective fluorescence of example 5L2

FIG. 13 fluorescence imaging of examples 6L1, L2 in HepG-2

FIG. 14 fluorescence images of examples 7L1, L2 incorporating EA and NEM, respectively, in HepG-2

Detailed Description

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

Example 1

Synthesis and characterization of L1, L2

(1) P-hydroxybenzyl alcohol (5mmol, 620.6mg) was dissolved in 15mL dichloromethane, stirred in an ice-water bath for 5min, followed by addition of triethylamine (15mmol, 2mL), stirred for 30min, then 1-chloro-2, 4-dinitrobenzene (5mmol, 1005mg) dissolved in 10mL dichloromethane was added dropwise, the reaction was stirred overnight, the progress of the reaction was checked by TLC, extracted and washed with saturated NaCl (3 × 50mL) and dichloromethane (3 × 50mL), the organic phase was dried over anhydrous sodium sulfate, and the ratio of petroleum ether/ethyl acetate ═ 3: purification on column 1 gave the pure product (4- (2,4-dinitrophenoxy) phenyl) methanol (3.87mmol, 1121.2mg) in 77.3% yield.

(2) (4- (2,4-dinitrophenoxy) phenyl) methanol (173.8mg, 0.6mmol), 3,7-bis (diethylamino) -10H-phenoxazine-10-carbonyl chloride (194.1mg, 0.5mmol), sodium carbonate (Na)₂CO₃160mg, 1.5mmol) and 4-dimethylaminopyridine (DMAP, 61.1mg, 0.5mmol) were dissolved in 20mL of dichloromethane and the reaction was stirred under nitrogen in an ice bath for 8 h. The solvent was removed and the crude product was purified by silica gel column chromatography to give 4- (2,4-dinitrophenoxy) benzyl 3,7-bis (diethylamino) -10H-benzoxazine-10-carboxylate (L1, 51.3mg, 0.08mmol) as a brown solid; the yield was 16%. 1H NMR (600MHz, DMSO) δ 8.90(s,1H),8.43(d, J ═ 9.2Hz,1H),7.55(d, J ═ 8.1Hz,2H),7.30(dd, J ═ 12.9,8.8Hz,5H),7.18(d, J ═ 9.2Hz,1H),6.40(d, J ═ 8.8Hz,2H),6.33(s,2H),5.25(s,2H),3.31(d, J ═ 6.8Hz,8H),1.06(t, J ═ 6.7Hz,12H).¹³C NMR(151MHz,DMSO)δ154.81,153.36,153.01,150.78,146.09,141.47,139.46,134.34,130.33,129.67,125.21,121.94,120.38,119.40,116.37,106.26,98.68,66.59,43.83,12.36.

(3) (4- (2,4-dinitrophenoxy) phenyl) methanol (173.8mg, 0.6mmol), 3,7-bis (dimethylamino) -10H-phenothiazine-10-carbonyl chloride (174.5mg, 0.5mmol), sodium carbonate (Na)₂CO₃124.4mg, 1.17mmol) and 4-bisMethylaminopyridine (DMAP, 61.1mg, 0.5mmol) was dissolved in 20mL dichloromethane and the reaction stirred under nitrogen for 8h under ice bath conditions. The solvent was removed and the crude product was purified by column chromatography on silica gel to give 4- (2,4-dinitrophenoxy) benzyl 3,7-bis (dimethylamino) -10H-phenothiazine-10-carboxylate (L2, 90.15mg, 0.15mmol) as a brown solid in 30% yield. 1H NMR (600MHz, DMSO) δ 8.90(d, J ═ 2.8Hz,1H),8.43(dd, J ═ 9.3,2.8Hz,1H),7.48(d, J ═ 8.5Hz,2H),7.35(d, J ═ 8.8Hz,2H),7.27(d, J ═ 8.5Hz,2H),7.18(d, J ═ 9.3Hz,1H),6.69(d, J ═ 2.6Hz,2H),6.67(dd, J ═ 8.9,2.7Hz,2H),5.21(s,2H),2.89(s,12H).¹³C NMR(151MHz,DMSO)δ154.78,153.53,153.27,148.64,141.47,139.46,134.46,131.99,129.87,129.64,127.47,127.01,121.90,120.29,119.41,110.88,109.73,66.36,40.19.

Example 2

Preparing a phosphate buffer solution (10mM, 10% absolute ethanol) system solution with pH 7, dissolving L1(L2) in DMSO to prepare a 2mM preparation solution, and preparing a 20mM Glutathione (GSH) solution and a 1mg/mL GSTs solution; 900 μ L of the reaction solution, 1 μ L L1(L2) in DMSO, 50 μ L of the GSH solution, and 50 μ L of the GSTs solution were added to a fluorescence cuvette, and fluorescence scanning was performed in a water bath at 37 ℃ with increasing time, and the fluorescence intensity at 676nm (L1)686nm (L2) was gradually increased. The fluorescence emission patterns are shown in FIGS. 7 and 8.

Example 3

Preparing a phosphate buffer solution (10mM, 10% absolute ethanol) system solution with pH 7, dissolving L1 in DMSO to prepare a 2mM preparation solution, and preparing a 20mM Glutathione (GSH) solution and a 1mg/mL GSTs solution; 944. mu.L, 943. mu.L, 942. mu.L, 941. mu.L and 940. mu.L of the system solution were added to 5 EP tubes of 2mL, respectively; 1 μ L L1 in DMSO; 50 mu L of GSH solution and different amounts of GSTs solution, the concentration of GSTs in the system is respectively 6 mu g/mL, 7 mu g/mL, 8 mu g/mL, 9 mu g/mL, 10 mu g/mL, after shaking and placing in a water bath at 37 ℃ for 3h, the fluorescence intensity of each concentration is scanned at 676 nm. Plotting and drawing the concentration of the GSTs as an abscissa and the 676nm fluorescence intensity as an ordinate to obtain a GSTs working curve as shown in FIG. 9; the linear regression equation is: 6.507x +48.218, R²0.99445. The unit of x is mu g/mL;

example 4

Preparing a phosphate buffer solution (10mM, 10% absolute ethanol) system solution with pH 7, dissolving L1 and L2 in DMSO to prepare a 2mM preparation solution, and preparing a 20mM Glutathione (GSH) solution and a 1mg/mL GSTs solution; in 6 EP tubes with 2mL, 950 mu L of system solution is firstly and respectively added; 1 μ L L2 in DMSO; 50 mu L of GSH solution and different amounts of GSTs solution, the concentration of GSTs in the system is respectively 50ng/mL, 100ng/mL, 200ng/mL, 300ng/mL, 400ng/mL and 500ng/mL, after shaking and placing in a water bath at 37 ℃ for 3h, the fluorescence intensity of each concentration is scanned at 686 nm. Plotting and drawing the GSTs concentration as abscissa and the 686nm fluorescence intensity as ordinate to obtain the GSTs working curve as shown in FIG. 10; the linear regression equation is: 0.08197x +11.1103, R²0.99145. The unit of x is ng/mL.

Example 5

A phosphate buffer solution (10mM, 10% absolute ethanol) having a pH of 7 was prepared as a system solution. Dissolving L1 or L2 in DMSO to prepare a 2mM preparation solution, and preparing a 20mM Glutathione (GSH) solution and a 1mg/mL GSTs solution; the system solution, 1. mu. L L1 or L2 in DMSO, was added to 9 EP tubes (2 mL), followed by blank, GSH (1mM), Cys (1mM), Hcy (1mM), H, respectively₂S (1mM), GST (50. mu.g/mL), GSH (1mM) + Lysozyme (50. mu.g/mL), GSH (1mM) + human albumin (50. mu.g/mL), GSH (1mM) + GST (50. mu.g/mL), shaking uniformly, placing in a water bath at 37 ℃ for 3h, pouring into a fluorescent cup, and scanning on a fluorescence photometer to obtain the fluorescence intensity of each analyte 676nm (L1) and 686nm (L2). The histogram is plotted, and as shown in FIGS. 11 and 12, only the fluorescence intensity of GSH (1mM) + GSTs (50. mu.g/mL) is significantly increased, and the fluorescence intensity of other analytes is not substantially changed.

Example 6

Preparing a PBS buffer solution with the pH of 7.4 and the concentration of 10mM, and preparing DMSO solutions of 2mM L1 and L2; 10 u L L1, L2 DMSO solution into 2mL PBS solution, so that its concentration is 10M; adding the solution into HepG-2 cells, incubating for 30min at 37 ℃, and imaging in confocal manner to show strong red fluorescence under a fluorescence imager; as shown in fig. 13.

Example 7

Preparing a PBS buffer solution with the pH of 7.4 and the concentration of 10mM, and preparing DMSO solutions of 2mM L1 and L2; incubation of HepG-230 min with 2mL of 500. mu.M solution of EA (inhibitors of GSTs) in PBS, washing twice with 2mL of PBS, and addition of 10. mu. L L1, L2 solution in DMSO to 2mL of PBS to give a concentration of 10. mu.M; adding the solution into HepG-2 cells, incubating for 30min at 37 ℃, and imaging in confocal manner to show that red fluorescence is weakened under a fluorescence imager; see fig. 14.

Incubation of HepG-230 min with 2mL of 50. mu.M NEM (inhibitor of GSH) in PBS followed by two washes with 2mL PBS and addition of 10. mu. L L1, L2 in DMSO to 2mL of PBS to give a concentration of 10. mu.M; adding the solution into HepG-2 cells, incubating for 30min at 37 ℃, washing with 2mL of PBS twice, adding 2mL of PBS solution for confocal imaging, and displaying red fluorescence reduction under a fluorescence imager; see fig. 14.

Claims

1. A near-infrared fluorescent probe capable of detecting the content of GSTs is characterized in that the structural formula is L1 or L2:

2. the method for synthesizing a near-infrared fluorescent probe capable of detecting the content of GSTs according to claim 1, which comprises the following steps:

dissolving p-hydroxybenzyl alcohol in dichloromethane, stirring in an ice-water bath for 5min, then adding triethylamine, stirring for 30min, then dropwise adding 1-chloro-2, 4-dinitrobenzene dissolved in dichloromethane, stirring for reaction overnight, detecting the reaction progress by TLC, extracting and washing with saturated sodium chloride and dichloromethane, drying an organic phase by using anhydrous sodium sulfate, and performing separation and separation on the organic phase by using petroleum ether/ethyl acetate ═ 3: 1, purifying by a column to obtain light yellow powder, namely a product (4- (2,4-dinitrophenoxy) phenyl) methanol; the molar ratio of the p-hydroxybenzyl alcohol to the triethylamine to the 1-chloro-2, 4-dinitrobenzene is 1: 3: 1;

dissolving (4- (2,4-dinitrophenoxy) phenyl) methanol, 3,7-bis (diethylamino) -10H-phenoxazine-10-carbonyl chloride, sodium carbonate and 4-dimethylaminopyridine in dichloromethane, and stirring for reacting for 8 hours under the conditions of nitrogen protection and ice bath; removing the solvent and purifying the crude product by silica gel column chromatography to obtain brown solid 4- (2,4-dinitrophenoxy) benzyl 3,7-bis (diethylamino) -10H-benzoxazine-10-carboxylate L1; the molar ratio of the (4- (2,4-dinitrophenoxy) phenyl) methanol to the 3,7-bis (diethylamino) -10H-phenoxazine-10-carbonyl chloride to the sodium carbonate to the 4-dimethylaminopyridine is 1.2: 1: 3: 1;

or,

dissolving (4- (2,4-dinitrophenoxy) phenyl) methanol, 3,7-bis (dimethylamino) -10H-phenothiazine-10-carbonyl chloride, sodium carbonate and 4-dimethylamino pyridine in dichloromethane, and stirring and reacting for 8 hours under the conditions of nitrogen protection and ice bath. Removing solvent and purifying the crude product by silica gel column chromatography to obtain brown solid 4- (2,4-dinitrophenoxy) benzyl 3,7-bis (dimethylamino) -10H-phenothiazine-10-carboxylate L2; the molar ratio of the (4- (2,4-dinitrophenoxy) phenyl) methanol to the 3,7-bis (dimethylamino) -10H-phenothiazine-10-carbonyl chloride to the sodium carbonate to the 4-dimethylaminopyridine is 1.2: 1: 3: 1.

3. use of the near-infrared fluorescent probe according to claim 1 for detecting GSTs.

4. A method of detecting GSTs, comprising the steps of:

(1) preparing a phosphate buffer solution with pH 7 and 10mM and containing 10% absolute ethyl alcohol as a reaction system, dissolving L1 or L2 in DMSO to prepare a 2mM preparation solution, and preparing a 20mM Glutathione (GSH) solution and a 1mg/mL GSTs solution;

(2) adding 900 mu L of reaction system solution, 1 mu L L1 or L2 DMSO solution and 50 mu L of GSH solution into a fluorescence cuvette, carrying out fluorescence scanning at 37 ℃ in a water bath, and gradually increasing the fluorescence intensity at 676nm (L1) or 686nm (L2) along with the increase of time;

(3) 944. mu.L, 943. mu.L, 942. mu.L, 941. mu.L and 940. mu.L of the system solution were added to 5 EP tubes of 2mL, respectively; 1 μ L L1 in DMSO; 50 μ L of GSH solution, GSTs solution with different amount, the concentration of GSTs in the system is respectively 6 mug/mL, 7 mug/mL, 8 mug/mL, 9 mug/mL, 10 mug/mL, after shaking up and placing in water bath with 37 ℃ for 3h, the fluorescence intensity with different concentration obtained by scanning at 676 nm; plotting and drawing the concentration of the GSTs as an abscissa and the 676nm fluorescence intensity as an ordinate to obtain a working curve of the GSTs; the linear regression equation is: 6.507x +48.218, R²0.99445. The unit of x is mu g/mL;

(4) adding 950 mu L of system solution and 1 mu L L2 of DMSO solution into 6 EP tubes with the volume of 2 mL; 50 mu L of GSH solution and different amounts of GSTs solution, the concentration of GSTs in the system is respectively 50ng/mL, 100ng/mL, 200ng/mL, 300ng/mL, 400ng/mL and 500ng/mL, after shaking up and placing in a water bath at 37 ℃ for 3h, the fluorescence intensity of each concentration is scanned at 686 nm; plotting and drawing the GSTs concentration as an abscissa and the 686nm fluorescence intensity as an ordinate to obtain a GSTs working curve; the linear regression equation is: 0.08197x +11.1103, R²0.99145. The unit of x is ng/mL.

5. Use of the probe of claim 1 for the preparation of a reagent for cell imaging.