CN108727265B

CN108727265B - Dual-functional fluorescent probe for detecting formaldehyde and pH (potential of hydrogen), and preparation method and application thereof

Info

Publication number: CN108727265B
Application number: CN201810581986.4A
Authority: CN
Inventors: 朱勍; 付曼琳; 谢振达; 尹彪
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2018-06-07
Filing date: 2018-06-07
Publication date: 2020-08-21
Anticipated expiration: 2038-06-07
Also published as: CN108727265A

Abstract

The invention discloses a bifunctional fluorescent probe for detecting formaldehyde and pH, and a preparation method and application thereof. The structure of the fluorescent probe is shown as the formula (I), and the preparation method comprises the following steps: under the action of cesium carbonate, generating a compound (IV) by using a compound (II) and a compound (III); under the alkaline condition, the compound (IV) reduces the azide into amino to finally generate the compound (I). The invention provides an application of the fluorescent probe in detecting formaldehyde and/or pH value. The invention provides a fluorescent probe capable of simultaneously detecting formaldehyde and pH in cells, and provides an effective research tool for researching the physiological action of formaldehyde and pH in cells.

Description

Dual-functional fluorescent probe for detecting formaldehyde and pH (potential of hydrogen), and preparation method and application thereof

Technical Field

The invention relates to a bifunctional fluorescent probe for detecting formaldehyde and pH, a preparation method and application thereof

Background

1. Formaldehyde belongs to an activated carbon cluster and is a recognized carcinogen. Formaldehyde is derived from endogenous metabolites produced by intracellular oxidase and demethylase, and the concentration of formaldehyde in normal cells can reach about 0.4 mM. Excessive formaldehyde in the body can cause neurodegenerative diseases, alzheimer's disease, various cancers, and the like. However, most of the current formaldehyde detection methods cannot monitor the intracellular formaldehyde concentration in real time, and therefore, it is imperative to develop a novel fluorescent probe for detecting the intracellular formaldehyde.

2. pH and formaldehyde concentration levels are interdependent in physiological environments, pH affects formaldehyde production by altering the activity of lysine-specific demethylase, while FA and acidic microenvironment can synergistically induce bone cancer pain. Thus, simultaneous imaging of pH and formaldehyde in complex organisms would likely help to further understand the relationship between each other. In recent years, the development of fluorescent probes has been used for pH or formaldehyde imaging in living cells or tissues. However, no single probe can simultaneously image two analytes in the same system.

3. Fluorescent probes are receiving attention from researchers due to their advantages of high sensitivity, real-time detection, small biological damage, etc. The invention aims to develop a novel formaldehyde and pH fluorescent probe which can accurately detect the concentration and pH value of formaldehyde in cells.

Disclosure of Invention

The invention aims to provide a bifunctional fluorescent probe capable of detecting the concentration and the pH value of formaldehyde.

The second purpose of the invention is to provide a preparation method of the fluorescent probe.

The third purpose of the invention is to provide the application of the fluorescent probe in formaldehyde and pH detection.

The invention adopts the following technical scheme for realizing the purpose:

a fluorescent probe has a structure shown in formula (I):

a preparation method of the fluorescent probe comprises the following steps:

under the action of cesium carbonate, generating a compound (IV) by using a compound (II) and a compound (III); reducing azide into amino in the compound (IV) under the alkaline condition to finally generate a compound (I);

the reaction route is as follows:

further, the preparation method specifically comprises the following steps:

(1) compound (III), Cs₂CO₃According to the mass ratio of 1: 1-1.5 (preferably 1:1), mixing the components in the mixtureCompound (II) is added dropwise to a solution in aqueous DMF and the mixture is taken up in N₂Stirring overnight at 40-50 ℃ under protection; removing the solvent by rotary evaporation, and purifying the obtained residue by silica column chromatography (preferably using ethyl acetate/petroleum ether as eluting agent at a volume ratio of 1:10) to obtain compound (IV);

(2) adding tin dichloride, triethylamine and thiophenol into an acetonitrile solvent according to the mass ratio of 1: 2-3.5 (preferably 1:3:3), stirring at 30-40 ℃ for 15-30 min, and then adding a compound (IV), wherein the mass ratio of the compound (IV) to tin dichloride dihydrate is 0.5-1: 1 (preferably 0.7: 1), and continuously stirring for 12 to 14 hours at the temperature of between 30 and 40 ℃; the reaction mixture was concentrated under reduced pressure and purified by silica column chromatography (preferably with ethyl acetate/petroleum ether in a volume ratio of 1:3 as the elution reagent) to give compound (I).

The compounds (II) of the present invention are disclosed, and their preparation is described in the literature (K.J.Bruemmer, R.R.Walvoord, T.F.Brewer, G.Burgos-Barragan, N.Wit, L.B.Pontel, K.J.Patel and C.J.Chang, Development of a General Aza-code Reaction Trigger Applied fluorescence Imaging of a generalized electrolyte in Living Cells, J.Am.chem.Soc.,2017,139,5338.)

The compound (III) of the present invention is a disclosed compound, and a preparation method thereof can be referred to in literature (2. H).

Park,S.-K.Chang,Signaling of water content in organic solvents bysolvatochromism of a hydroxynaphthalimide-based merocyanine dye.Dyes andPigments,2015,122,324.)

The invention provides application of the fluorescent probe in formaldehyde detection.

Further, the formaldehyde is preferably intracellular formaldehyde, and the concentration of the formaldehyde is not higher than 1 mmol/L.

Furthermore, the cell is human cervical carcinoma cell Hela cell.

The invention also provides application of the fluorescent probe in detecting the pH value.

Further, the pH is preferably a pH of intracellular lysosomes at a concentration of pH 4.5-5.5.

Furthermore, the cell is human cervical carcinoma cell Hela cell.

The invention further provides application of the fluorescent probe in detecting formaldehyde and pH value.

Further, the formaldehyde is intracellular formaldehyde, and the concentration of the intracellular formaldehyde is not higher than 1 mmol/L; the pH value is the pH value of lysosome in cells, and the concentration is pH 4.5-5.5.

Furthermore, the cell is human cervical carcinoma cell Hela cell.

The compound (I) provided by the invention is used as a fluorescent probe and can be applied to fluorescent quantitative detection of formaldehyde. The fluorescence detection principle of the quantitative formaldehyde concentration is as follows: after the compound (I) reacts with formaldehyde, a reactive group is removed to generate a fluorescent substance compound (III), and the change of fluorescence intensity of the probe at an emission wavelength of 555nm when the excitation is 455nm is measured, so that the formaldehyde concentration is obtained. The principle of detecting the concentration of formaldehyde by using the fluorescent probe of the invention is as follows:

the compound (I) provided by the invention is used as a fluorescent probe and can be applied to fluorescent quantitative detection of pH. The fluorescence detection principle for quantifying the pH concentration is as follows: after the compound (I) reacts with pH, an amino group is taken as a pH sensitive group to obtain a proton to form NH₃ ⁺The reaction group, the fluorescence intensity increased, and the change in fluorescence intensity of the probe at an emission wavelength of 455nm upon excitation at 365nm was measured, thereby obtaining the pH value. The principle of detecting pH concentration using the fluorescent probe of the present invention is as follows:

confocal fluorescence microscope imaging experiments well prove that the fluorescent probe disclosed by the invention can detect the change of the concentration of formaldehyde in cells and the acid-base environment in the cells, provides a new tool for researching the metabolic mechanism in the cells, and has a good prospect in the biological field.

Compared with the prior art, the invention has the beneficial effects that: the invention provides a fluorescent probe capable of simultaneously detecting formaldehyde and pH in cells, and provides an effective research tool for researching the physiological action of formaldehyde and pH in cells.

Drawings

FIG. 1 is a nuclear magnetic hydrogen spectrum of compound (I) prepared in example 1 of the present invention.

FIG. 2 shows a nuclear magnetic carbon spectrum of Compound (I) prepared in example 1 of the present invention.

Fig. 3 is a graph showing fluorescence absorption spectra of compound (I) prepared in example 1 of the present invention, when different equivalents of formaldehyde solution were added under the conditions of DMSO/PBS buffer (pH 7.4, v/v 1/199).

Fig. 4 is a graph showing fluorescence absorption spectra of compound (I) prepared in example 1 of the present invention in DMSO/PBS buffer solutions (v/v-1/199) at pH 3.5 and pH 7.5, respectively.

Fig. 5 shows fluorescence emission spectra of compound (I) prepared in example 1 of the present invention when different equivalents of formaldehyde solution were added under the conditions of DMSO/PBS buffer (pH 7.4, v/v 1/199). FIG. 5 excitation wavelength 455nm and emission wavelength 555 nm.

Fig. 6 is a fluorescence plot of compound (I) (0.5 μ M) prepared in example 1 of the present invention as a function of time during the action with formaldehyde (0.5mM) in DMSO/PBS buffer (pH 7.4, v/v 1/199). FIG. 6 excitation wavelength 455nm and emission wavelength 555 nm.

Fig. 7 is a fluorescence diagram of the selectivity results of compound (I) prepared in example 1 of the present invention in DMSO/PBS buffer (pH 7.4, v/v 1/199). 1-15 are PBS, acetaldehyde, benzaldehyde, p-nitrobenzaldehyde, p-hydroxybenzaldehyde, acetone, formic acid, glucose, glutathione, homocysteine, cysteine, sodium bisulfate, hydrogen peroxide, tert-butyl hydroperoxide and formaldehyde respectively. FIG. 7 excitation wavelength 455nm and emission wavelength 555 nm.

Fig. 8 shows a high performance liquid chromatogram and a mass spectrum of compound (I) prepared in example 1 of the present invention before and after addition of formaldehyde under DMSO/PBS buffer (pH 7.4, v/v 1/199).

FIG. 9 shows fluorescence imaging of formaldehyde in cervical cancer cells by the compound (I) prepared in example 1 of the present invention.

FIG. 10 shows fluorescence imaging of endogenous formaldehyde in cervical cancer cells by the compound (I) prepared in example 1 of the present invention.

Fig. 11 shows fluorescence emission spectra of compound (I) prepared in example 1 according to the present invention, added to DMSO/PBS buffer (v/v-1/199) at different pH values, and a linear relationship between fluorescence intensity and pH. Excitation wavelength 365nm and emission wavelength 455 nm.

Fig. 12 shows the change of fluorescence intensity of the probe with time in DMSO/PBS buffer (v/v ═ 1/199) at pH4 and pH 7.4, respectively, for compound (I) prepared in example 1 according to the present invention. Excitation wavelength 365nm and emission wavelength 455 nm.

Fig. 13 is a dot plot of the change in fluorescence intensity versus the value at different pH for compound (IV) (0.5 μ M) prepared in example 1 of the present invention in DMSO/PBS buffer (pH 7.4, v/v 1/199). Excitation wavelength 365nm and emission wavelength 455 nm.

Fig. 14 is a fluorescence diagram of the selectivity results of compound (I) prepared in example 1 of the present invention in DMSO/PBS buffer (pH 7.4, v/v 1/199). 1-15 are PBS, acetaldehyde, benzaldehyde, p-nitrobenzaldehyde, p-hydroxybenzaldehyde, acetone, formic acid, glucose, glutathione, homocysteine, cysteine, sodium bisulfate, hydrogen peroxide, tert-butyl hydroperoxide and pH respectively. FIG. 7 excitation wavelength 365nm and emission wavelength 455 nm.

FIG. 15 is a photograph of an image of a compound (I) prepared in example 1 of the present invention and a commercially available Lyso-Tracker Red.

Fig. 16 is a dot plot of the change in fluorescence intensity and the value at different pH for compound (IV) (0.5 μ M) prepared in example 1 of the present invention in DMSO/PBS buffer (pH 7.4, v/v 1/199). Excitation wavelength 365nm and emission wavelength 455 nm.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.

Example 1

(1) Preparation of Compound (IV)

The compound (III) (0.65g,2.4mmol) and Cs₂CO₃(0.78g, 2.4mmol) to a solution in 10ml of anhydrous DMF was added dropwise compound (II) (0.67g,2mmol) and the mixture was taken up in N₂Stirring at 40-50 deg.c overnight. The solvent was removed under high vacuum and the resulting residue was purified by column chromatography on silica using ethyl acetate/petroleum ether (v/v, 1:10) as eluent to give 0.57g of compound (IV) (yield 30%).¹H NMR(500MHz,CDCl₃)8.50–8.34(m,3H),7.59(dd,J＝8.3,7.4Hz,1H),6.94(d,J＝8.3Hz,1H),6.00(dd,J＝17.5,10.9Hz,1H),5.20–5.03(m,2H),4.34(t,J＝6.7Hz,2H),4.17–4.02(m,2H),2.22(dd,J＝13.8,6.9Hz,1H),2.10(dt,J＝14.2,7.0Hz,1H),1.67(tt,J＝7.7,6.6Hz,2H),1.45(s,3H),1.41(dt,J＝14.2,7.2Hz,2H),1.13(s,6H),0.95(t,J＝7.4Hz,3H).¹³C NMR(126MHz,CDCl₃)164.18,163.58,159.52,143.42,133.07,131.20,129.08,128.22,125.69,123.19,122.23,114.94,114.06,105.61,67.10,65.70,45.53,39.89,34.57,30.13,22.31,22.13,20.29,18.01,13.75.C₂₅H₃₀N₄O₃(M+H)435.2,found 435.2.

(2) Preparation of Compound (I)

Tin dichloride dihydrate (1g, 0.33mmol) was added to a round bottom flask, then thiophenol (0.11g, 1mmol) and triethylamine (0.10g, 1mmol) were added to mix in 5ml acetonitrile solvent and the mixture was stirred at 30-40 ℃ for 15min, then compound (IV) (0.1g, 0.23mmol) was added to acetonitrile solvent and stirred at 30-40 ℃ for another 12 h. The reaction was concentrated under reduced pressure and purified by column chromatography on silica gel using ethyl acetate/petroleum ether (v/v, 1: 3) as eluent to give 120mg of the final compound (I) probe DPFP.¹H NMR(500MHz,CDCl₃)8.47(s,3H),7.58(s,1H),7.09(d,J＝8.3Hz,1H),6.07(dd,J＝17.4,10.8Hz,1H),5.29–5.06(m,2H),4.56(s,1H),4.45(dd,J＝15.8,8.2Hz,1H),4.30–4.00(m,2H),2.21(s,1H),1.71(ddd,J＝12.7,8.6,6.7Hz,2H),1.47(dt,J＝15.1,7.4Hz,2H),1.35–1.16(m,9H),0.99(t,J＝7.4Hz,3H).¹³CNMR(126MHz,CDCl₃)164.38,163.89,133.31,131.35,128.30,125.69,122.35,115.06,106.09,58.47,53.43,40.09,30.28,20.43,18.45,13.88.C₂₅H₃₀N₄O₃(M+H)409.2413,found409.2492.

Example 2 fluorescence spectroscopy detection of compound (I) (5 μ M) at different formaldehyde concentrations.

An amount of the compound (I) prepared in example 1 was weighed out accurately, a probe stock solution having a concentration of 0.1mM was prepared using dimethylsulfoxide, 2 μ L of the solution was pipetted into 0.394mL of PBS buffer (pH 7.4), 4 μ L of different equivalent formaldehyde solutions (concentrations of final formaldehyde in the mixed solution were 0, 0.0025, 0.0075, 0.01, 0.025, 0.04, 0.05, 0.06, 0.075, 0.1, 0.15, 0.25, 0.4, 0.5, 0.6, 0.75, 1, 2, 5mM, respectively) were added, and after reacting at 37 ℃ for 3 hours, the mixture was added to a cuvette, and then the fluorescence absorption spectrum of the compound (I) was measured.

The experimental results show that the absorption of compound (I) at a wavelength of 455nm increases with increasing concentration of formaldehyde. As a result, compound (I) reacts with formaldehyde to cause a "turn-on" phenomenon, and the fluorescence spectrum is shown in FIG. 3.

Example 3 fluorescence absorbance detection of compound (I) (5 μ M) at different pH values.

An amount of the compound (I) prepared in example 1 was accurately weighed, prepared into a probe stock solution with a concentration of 0.1mM using dimethyl sulfoxide, and 2. mu.L of the solution was pipetted into 0.398mL of PBS buffers with different pH values (the final mixed solution was adjusted to pH values of 3.5 and 7.5); after 2. mu.L of the probe stock solution was aspirated using a pipette gun, added to 0.394mL of PBS buffer (pH 7.4), and then added with 4. mu.L of formaldehyde solution (to make the concentration of formaldehyde in the final mixed solution 2mM) to react at 37 ℃ for 3 hours, the mixture was added to a cuvette, and the fluorescence absorption spectrum of compound (I) was measured.

The experimental results show that at pH 3.5, compound (I) increases at a wavelength of 365nm, indicating that compound (I) generates a "turn on" phenomenon in an acidic environment. When the probe reacts with formaldehyde, the absorption of the compound (I) at the 455nm wavelength is enhanced, and a 'turn-on' phenomenon is generated, and the fluorescence spectrum is shown in figure 4.

Example 4 fluorescence spectroscopy of compound (I) (5 μ M) of the present invention under DMSO/PBS buffer (pH 7.4, v/v 1/199) with different equivalents of formaldehyde added.

An appropriate amount of the compound (I) prepared in example 1 was weighed out accurately, a probe stock solution having a concentration of 0.1mM was prepared using dimethyl sulfoxide, 2 μ L of the solution was pipetted into 0.394mL of PBS buffer (pH 7.4), 4 μ L of different equivalent amounts of formaldehyde solutions (to give final concentrations of 0, 0.0025, 0.0075, 0.01, 0.025, 0.04, 0.05, 0.06, 0.075, 0.1, 0.15, 0.25, 0.4, 0.5, 0.6, 0.75, 1, 2, 5mM, respectively) were added, and after reaction at 37 ℃ for 3 hours, the mixture was added to a 96-well plate, and then the fluorescence spectrum of the compound (I) was measured.

The experimental results show that the fluorescence intensity of compound (I) at 555nm increases with increasing concentration of formaldehyde when excited at a wavelength of 455 nm. It is shown that the reaction of compound (I) with formaldehyde generates more and more "turn-on" phenomena of compound (I). The lower limit of detection of the formaldehyde fluorescent probe is 10 mu M, R²The fluorescent spectrum is shown in figure 5, and meets the requirement of intracellular formaldehyde detection.

Example 5 compound (I) (5 μ M) of the present invention was added to 0.5mmol of formaldehyde in DMSO/PBS buffer (pH 7.4, v/v 1/199) to obtain a linear relationship of fluorescence intensity with time.

A certain amount of the compound (I) prepared in example 1 was accurately weighed, a probe stock solution with a concentration of 0.1mM was prepared using dimethyl sulfoxide, 2 μ L of the solution was pipetted into 0.394mL of PBS buffer (pH 7.4), 4 μ L of formaldehyde solution was added to make the final formaldehyde concentration 0.5mmol in the buffer, the buffer was added to a 96-well plate, the fluorescence intensity was measured every 5min at 37 ℃, the total reaction time was 3 hours, the data was counted, and a correlation curve of a linear relationship was plotted. The fluorescence excitation wavelength is 455nm, and the emission wavelength is 555 nm.

The data show that the fluorescence intensity of the formaldehyde fluorescent probe increases with time. The fluorescence spectrum is shown in FIG. 6.

Example 6 fluorescence spectroscopy detection of selective results of compound (I) (5 μ M) in the present invention under DMSO/PBS buffer (pH 7.4, v/v 1/199).

Accurately weighing a certain amount of probe (I), preparing a mother solution with the concentration of 0.1mM by using dimethyl sulfoxide, sucking 2 mu L of the probe by a pipette, adding the mother solution into 0.394mL of PBS buffer solution (the pH is 7.4), respectively adding 4 mu L of formaldehyde aqueous solution (the final concentration of formaldehyde in water is 1mM) and biologically-related active small molecule aqueous solution (acetaldehyde, acetone, formic acid, 4-hydroxybenzaldehyde, 4-nitrobenzaldehyde, benzaldehyde, hydrogen peroxide, tert-butyl hydroperoxide, sodium hydrosulfide, glutathione, cysteine, homocysteine and glucose, the final concentrations of which are 1mM), reacting at 37 ℃ for 3h, and measuring the fluorescence value of the probe. The fluorescence excitation wavelength is 455nm, and the emission wavelength is 555 nm.

The experimental result shows that the fluorescence intensity of the compound (I) is basically unchanged in the presence of other related bioactive molecules except formaldehyde, and the anti-interference capability of the compound (I) is very good, namely the specificity of the compound (I) to formaldehyde is relatively good. The fluorescence spectrum is shown in FIG. 7.

Example 7 the mechanism of the reaction of compound (I) (5 μ M) of the present invention with formaldehyde under the conditions of DMSO/PBS buffer (pH 7.4, v/v 1/199) was demonstrated.

An appropriate amount of probe (I) was weighed accurately, prepared into a 0.1mM mother solution using dimethyl sulfoxide, and 2 μ L of the solution was pipetted into 0.394mL of PBS buffer (pH 7.4), and 4 μ L of an aqueous formaldehyde solution (final concentration of formaldehyde in water was 1mM) was added thereto, respectively, to react overnight, and then analyzed by high performance liquid chromatography. The HPLC chromatogram is shown in FIG. 8.

The experiments demonstrate that the mechanism by which we describe the reaction of compound (I) with formaldehyde is correct. Compound (I) forms an intermediate with formaldehyde and is eventually converted to compound (III), thereby signaling the effect of turn-on.

Example 8 imaging analysis of exogenous Formaldehyde in cervical cancer cells by Compound (I) of the present invention

A certain amount of the probe (I) was accurately weighed, prepared into a 10mM stock solution with dimethyl sulfoxide, and 2. mu.L of the stock solution was pipetted into 1.998 mM DMMEM medium. 1mL of the cells were added to Hela cells, incubated at 37 ℃ for 0.5h, washed twice with fresh DMEM medium, then incubated with different formaldehyde concentrations (final formaldehyde concentrations 0, 0.3mM, 0.6mM, 1mM, 2mM, respectively) for 3h, washed twice with fresh DMEM medium, and finally imaged with Olympus Fluoview FV 1200 confocal microscope. FIG. 10 is a diagram of the effect of confocal fluorescence imaging of cells: (a) PBS (pH 7.4), (b) Formaldehyde 0.3mM, (c) Formaldehyde 0.6mM, (d) Formaldehyde 1mM, or (e) Formaldehyde 2mM, and the fluorescence spectrum is shown in FIG. 9

EXAMPLE 9 imaging analysis of endogenous Formaldehyde in cervical cancer cells by Compound (I) of the present invention

An amount of probe (I) was accurately weighed, prepared into a 0.1mM stock solution using dimethyl sulfoxide, and 2. mu.L of the solution was pipetted into 0.198mL of MEM medium. Taking 1mL of NaHSO containing inhibitor₃(Final NaHSO)₃Concentration 0.2mmol) or NAC (final NAC concentration 0.2mmol) were incubated at 37 ℃ for 0.5h, respectively. The culture broth of compound (I) was then added to Hela cells, incubated at 37 ℃ for 0.5h, washed twice with DMEM medium and finally imaged with Olympus Fluoview FV 1200 confocal microscope. FIG. 10 is a diagram of the effect of confocal fluorescence imaging of cells: (a) blank probe control without inhibitor (b) NaHSO₃The concentration is 0.2 mmol; (c) NAC concentration of 0.2 mmol; reference bar, 20 μm. The fluorescence spectrum is shown in FIG. 10.

The experimental result shows that the fluorescence intensity in the cell can be observed to be continuously enhanced along with the continuous increase of the concentration of the formaldehyde in the cell, which indicates that the compound (I) can detect the change of the concentration of the formaldehyde in the cell.

Example 10 fluorescence emission spectra of compound (I) (5 μ M) at different pH. The excitation wavelength was 365nm and the emission wavelength was 455 nm.

An amount of the compound (I) prepared in example 1 was accurately weighed, prepared into a probe stock solution with a concentration of 0.1mM using dimethyl sulfoxide, and 2. mu.L of the solution was pipetted into 0.398mL of PBS buffer solutions with different pH values (finally, pH values in water were 3.5 to 10), reacted at 37 ℃ for 3 hours, added to a 96-well plate, and then the fluorescence emission spectrum of the compound (I) was measured.

The experimental result shows that when the compound is excited by 365nm wavelength and the pH value is lower, the fluorescence intensity of the compound (I) at 455nm is stronger; when the pH is neutral and basic, the fluorescence intensity of compound (I) at 455nm is weak. Indicating that the probe is pH sensitive, the fluorescence spectrum is shown in FIG. 11.

Example 11 change of fluorescence intensity of the probe with time in DMSO/PBS buffer (v/v ═ 1/199), pH4, and pH 7.4, respectively, for compound (I) of example 11.

An amount of the compound (I) prepared in example 1 was accurately weighed, a 1mM probe stock solution was prepared using dimethyl sulfoxide, 2. mu.L of the solution was pipetted into 0.398mL of PBS buffers having different pH values (final pH values were 4 and 7.4, respectively), reacted at 37 ℃ for 0.5 hour, and then added to a 96-well plate, and the fluorescence spectrum of the compound (I) was measured.

The data show that compound (I) is fully reacted at around 25 min. And at pH4 and pH 7.4, there was a large difference in fluorescence intensity. The fluorescence spectrum is shown in FIG. 12.

Example 12 the change in fluorescence intensity of compound (IV) (5 μ M) prepared in example 1 of the present invention was plotted in the form of dots at different pH values under the conditions of DMSO/PBS buffer (pH 7.4, v/v 1/199).

An amount of the compound (IV) prepared in example 1 was accurately weighed, prepared into a probe stock solution with a concentration of 1mM using dimethyl sulfoxide, and 2. mu.L of the solution was pipetted into 0.398mL of PBS buffers with different pH values (the final pH values were adjusted from 3.5 to 9.5, respectively) and added to a 96-well plate, reacted at 37 ℃ for 3 hours, and the data was counted and plotted in a linear relationship. The fluorescence excitation wavelength is 365nm, and the emission wavelength is 455 nm.

The data show that compound (IV) is not pH sensitive, and further illustrate that the "turn on" phenomenon occurs when the amino group of compound (I) is pH sensitive. The fluorescence spectrum is shown in FIG. 13.

Example 13 fluorescence spectroscopy detection of selective results of compound (I) (5 μ M) in DMSO/PBS buffer (pH 7.4, v/v 1/199) in the present invention.

A certain amount of the compound (IV) prepared in example 1 is accurately weighed, a probe mother solution with the concentration of 1mM is prepared by dimethyl sulfoxide, 2 mu L of the probe mother solution is sucked by a pipette and added into 0.394mL, then 4 mu L of biologically relevant active small molecule aqueous solution (acetaldehyde, acetone, formic acid, 4-hydroxybenzaldehyde, 4-nitrobenzaldehyde, benzaldehyde, hydrogen peroxide, tert-butyl hydroperoxide, sodium hydrosulfide, glutathione, cysteine, homocysteine and glucose with the final concentration of 1mM) is respectively added, the reaction is carried out for 0.5h at the temperature of 37 ℃, and the fluorescence value is measured. The fluorescence excitation wavelength is 365nm, and the emission wavelength is 455 nm.

The experimental result shows that except for pH, the fluorescence intensity of the compound (I) basically has no obvious change under the existence of other related bioactive molecules, and the anti-interference capability of the compound (I) is very good, namely the specificity to the pH is relatively good. The fluorescence spectrum is shown in FIG. 14.

Example 14 cellular imaging of Compound (I) of the present invention with a commercial Lyso-Tracker Red.

A certain amount of the probe (I) was accurately weighed, prepared into a 10mM stock solution with dimethyl sulfoxide, and 2. mu.L of the stock solution was pipetted into 1.998 mM DMMEM medium. 1mL of the culture containing compound (I) was added to Hela cells, incubated at 37 ℃ for 0.5h, washed twice with DMEM medium, then incubated with commercially available Lyso-Tracker Red (0.5. mu. mol) at 37 ℃ for 20min, washed twice with 1mL of PBS buffer (pH 7.4), and finally subjected to fluorescence imaging using an Olympus Fluoview FV 1200 confocal microscope. FIG. 7 is a diagram of the effect of confocal fluorescence imaging of cells: (a) the compound (I) has an excitation wavelength of 405nm and a receiving wavelength range of 430-480 nm, (b) a Lyso-Tracker Red, an excitation wavelength of 543nm and a receiving wavelength range of 590-640 nm; (c) a mixing channel; reference bar, 20 μm.

The experimental result shows that the compound (I) can detect the pH of lysosomes in cells, and the Fiji software is used for comparing and analyzing the fluorescence intensity in two groups of cells to obtain the Pearson correlation coefficient of 0.88, which indicates that the compound (I) can more accurately detect the pH of lysosomes. The fluorescence spectrum is shown in FIG. 15.

Comparative example 1

The following compounds (1) are disclosed in the references (x.f.wu, l.h.li, w.shi, q.y.gong, x.h.li, and h.m.ma, Sensitive and selective ratio strategies for Probes for Detection of intracellular genes Monoamine oxide a, anal.chem.,2016,88, 1440):

the compound (1) is used as a fluorescent probe, can only realize single monoamine oxidase detection in cells, and cannot realize dual-function detection of pH and formaldehyde.

Comparative example 2: the change in fluorescence intensity of compound (IV) (5 μ M) prepared in example 1 of the present invention was plotted in the form of a dot plot of the change in pH and the change in fluorescence intensity in DMSO/PBS buffer (pH 7.4, v/v 1/199).

The data show that compound (IV) is not pH sensitive, and further illustrate that the "turn on" phenomenon occurs when the amino group of compound (I) is pH sensitive. The fluorescence spectrum is shown in FIG. 16.

Comparative example 3

To examine the uniqueness of the probe of the present invention, the present invention also examined whether the compound (2) having a structure similar to the probe can be used for pH detection:

the replacement of compound (IV) in comparative example 2 with compound (2) showed that compound (2) was not sensitive to pH under the same assay conditions.

Claims

1. The application of the fluorescent probe with the structure shown as the formula (I) in the preparation of a reagent for detecting formaldehyde and pH value is as follows:

2. the use of claim 1, wherein: the formaldehyde is intracellular formaldehyde, and the concentration of the formaldehyde is not higher than 1 mmol/L; the pH value is the pH value of lysosome in cells, and the concentration is pH 4.5-5.5.

3. Use according to claim 2, characterized in that: the cell is human cervical carcinoma cell Hela cell.