CN107501245B

CN107501245B - Mitochondrion-targeted double-signal turn-on formaldehyde fluorescent nano probe and preparation and application thereof

Info

Publication number: CN107501245B
Application number: CN201710650829.XA
Authority: CN
Inventors: 朱勍; 谢振达; 方红梨; 朱伸; 赵成艳
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2017-08-02
Filing date: 2017-08-02
Publication date: 2020-02-21
Anticipated expiration: 2037-08-02
Also published as: CN107501245A

Abstract

The invention discloses a preparation method and application of a mitochondrion-targeted double-signal turn-on formaldehyde fluorescent nano probe, and relates to a formaldehyde fluorescent nano probe based on the combined action of fluorescence energy resonance transfer and self-assembly. The nano probe has almost no fluorescence in water, and two free fluorescent substances are obtained after the nano probe reacts with formaldehyde, so that the effect of double turn-on is realized, and the accuracy of a detection result is improved. The nano probe has good specificity to formaldehyde. Confocal fluorescence microscope imaging experiments well prove that the nanoprobes can penetrate cell membranes to enter mitochondria of cells and can detect the change of formaldehyde concentration in the cells, so that a new tool is provided for researching the metabolic mechanism of formaldehyde in the mitochondria of the cells, and the nanoprobes have good prospects in the biological field.

Description

Mitochondrion-targeted double-signal turn-on formaldehyde fluorescent nano probe and preparation and application thereof

Technical Field

The invention relates to a mitochondrion-targeted double-signal turn-on formaldehyde fluorescent nano probe and a preparation method and application thereof.

Background

1. Activated carbon clusters are a class of compounds with one or more carbonyl groups that play a key role in human physiological processes. Formaldehyde, a recognized carcinogen, is the smallest molecule in the activated carbon cluster. On the one hand, formaldehyde can originate from industrial production, building materials, leather, fuels, even food. On the other hand, 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. Most of the existing formaldehyde detection methods cannot monitor the concentration of formaldehyde in cells in real time, so that it is necessary to develop a new method capable of detecting formaldehyde in cells.

2. Mitochondria is an organelle of two membranes present in most cells and is the energizing center of the cell. It has been shown that mitochondria have aldehyde dehydrogenase 2, an enzyme that degrades formaldehyde to formate. Meanwhile, excessive formaldehyde may lower mitochondrial membrane potential to affect mitochondrial function. At present, no relevant literature exists for studying the mitochondrial formaldehyde concentration, so that the development of a formaldehyde probe targeting mitochondria in cells is urgently needed.

3. Fluorescent probes are concerned due to the advantages of high sensitivity, real-time detection and the like, but most of the existing probes only have the characteristic of single signal turn-on output when detecting analytes, but the single signal is easily influenced by the external environment and cannot accurately detect results. The invention aims to develop a formaldehyde fluorescent nano probe with double-signal turn-on output, namely, when the probe is used for detecting formaldehyde, two different exciting lights can be selected for excitation so as to obtain two corresponding turn-on emission signals.

Disclosure of Invention

The invention aims to provide a mitochondrion-targeted double-signal turn-on formaldehyde fluorescent nano probe.

The second aim of the invention is to provide a preparation method of the mitochondrion-targeted double-signal turn-on formaldehyde fluorescent nano probe.

The third purpose of the invention is to provide the application of the mitochondrion-targeted double-signal turn-on formaldehyde fluorescent nano probe.

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

a fluorescent nanoprobe, characterized in that it consists of a compound (I)

Self-assembly in water to produce:

the structural formula of the compound (I) is as follows:

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

carrying out nucleophilic substitution reaction on p-hydroxybenzaldehyde and 3-bromopropyne under an alkaline condition to generate a compound (II); the compound (II) and ammonia methanol solution generate Schiff base compound, and then the Schiff base compound and propenyl boronic acid ortho-di-tertiary alcohol ester generate substitution reaction to generate a compound (III); under an acidic condition, generating a Schiff base intermediate product by using the compound (III) and the compound (IV), and reducing a carbon-nitrogen double bond of the Schiff base intermediate product by using a reducing agent sodium triacetoxyborohydride to generate a compound (V); the compound (VI) and 3-azidopropylamine are subjected to amide reaction to generate a compound (VII); finally, the compound (V) and the compound (VII) are catalyzed by monovalent copper to generate a compound (I); dissolving the compound (I) in DMSO (dimethyl sulfoxide) to serve as mother liquor, diluting the mother liquor with ultrapure water or PBS (phosphate buffer solution) or cell culture solution, performing ultrasonic treatment for several minutes, then violently shaking, and performing self-assembly on the compound (I) to obtain the fluorescent nano probe;

the reaction route of the fluorescent nano probe is as follows:

furthermore, the preparation method specifically comprises the following steps:

(1) reacting p-hydroxybenzaldehyde and 3-bromopropyne according to the mass ratio of 1:2 under alkaline conditions, and performing post-treatment A to prepare a compound (II); the reaction temperature is 60-70 ℃, the reaction time is 3 hours, and the reaction solvent is acetone;

(2) mixing the compound (II) with an ammonia methanol solution and propenyl boronic acid ortho-di-tertiary alcohol ester at 0 ℃, then transferring to 20-35 ℃ for reaction, and performing post-treatment B to prepare a compound (III); the reaction time is 12 hours, and the reaction solvent is methanol; the mass ratio of the compound (II) to ammonia and the propenyl boronic acid ortho-di-tertiary alcohol ester in the ammonia methanol solution is 1:10: 1.5;

(3) mixing the compound (IV), the compound (III) and sodium triacetoxyborohydride according to the mass ratio of theoretical substances of 1:1.2:4 at the temperature of 0 ℃ under an acidic condition, then transferring to 20-30 ℃ for reaction, and preparing a compound (V) through post-treatment C; the reaction time is 12 hours, and the reaction solvent is tetrahydrofuran;

(4) reacting the compound (VI) with 3-azidopropylamine according to the mass ratio of 1:1.2, and carrying out post-treatment D to prepare a compound (VII); the reaction temperature is room temperature, the reaction time is 10 hours, and the reaction solvent is dichloromethane;

(5) reacting a compound (V) with a compound (VII) according to the quantitative ratio of theoretical substances of 1:1 under the catalysis of monovalent copper, evaporating the solvent to dryness, and purifying the crude product by using a high performance liquid chromatography to obtain a compound (I); the reaction temperature is room temperature, the reaction time is 6 hours, and the reaction solvent is tetrahydrofuran and water;

(6) dissolving a compound (I) in DMSO to prepare a probe mother solution with the concentration of 0.1-2 mM, diluting the probe mother solution to 99 times of the original mother solution volume by using ultrapure water or PBS buffer solution or DMEM culture medium, performing ultrasonic treatment for several minutes, then violently shaking, and performing self-assembly on the compound (I) to obtain the fluorescent nano probe.

The compound (IV) of the present invention is a compound disclosed in the following references [ H.park, S. -K.Chang, signalling of water content in organic solvents by solvents, a-based meritorium, DyesPigm.122(2015) 324-330 ].

Further, the alkaline condition in the step (1) is realized by adopting an acid-binding agent potassium carbonate, and the amount of the acid-binding agent is 1.5 times of the equivalent of the p-hydroxybenzaldehyde.

Further, the ammonia concentration in the ammonia methanol solution in the step (2) is 7 mol/L.

Further, the acidic condition in the step (3) is realized by adopting acetic acid, and the mass ratio of the acetic acid to the compound (IV) is 8-20: 1, preferably 10:1.

Further, 1-hydroxybenzotriazole and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride can be added in the step (4) of the invention to serve as an amide reaction condensing agent, and triethylamine is used as an acid-binding agent, wherein the recommended ratio of the three substances is 3:3: 4.

The post-treatment A of the invention is as follows: removing the solvent from the reaction liquid by rotary evaporation under reduced pressure, adding water, extracting with ethyl acetate, combining organic phases, washing the organic phases with water and saturated saline solution for a plurality of times, drying with anhydrous sodium sulfate, filtering, drying the solvent by rotary evaporation, and carrying out column chromatography separation to obtain a target product, wherein an eluant is ethyl acetate and petroleum ether with the volume ratio of 1: 10;

the post-treatment B of the invention is as follows: carrying out reduced pressure rotary evaporation on the reaction liquid to remove the solvent, carrying out chromatographic column separation on the crude product to obtain a target product, wherein an eluent is dichloromethane and methanol with the volume ratio of 40: 1;

the post-treatment C of the invention is as follows: carrying out reduced pressure rotary evaporation on the reaction liquid to remove the solvent, carrying out chromatographic column separation on the crude product to obtain a target product, wherein an eluent is dichloromethane and methanol with the volume ratio of 20: 1;

the post-treatment D of the invention is as follows: separating the reaction solution by thin layer chromatography, wherein the developing agent is dichloromethane and methanol with the volume ratio of 20:1 to obtain the target compound.

The invention provides an application of the fluorescent nano probe in detecting the concentration of formaldehyde.

Furthermore, the formaldehyde exists in the form of an aqueous solution, the concentration of the formaldehyde is 0-5 mmol/L, and the meaning of the concentration of 0 is infinitely close to 0 but not equal to 0.

Furthermore, the formaldehyde is preferably intracellular formaldehyde, and the concentration of the intracellular formaldehyde is 0-1 mmol/L.

Still further, the cell of the invention is a human breast cancer cell MCF-7.

The fluorescence nanoprobe is the mitochondrion-targeted double-signal turn-on formaldehyde fluorescence nanoprobe nano-MTDF.

The formaldehyde fluorescent nano probe which is self-assembled by the compound (I) and can be used as a mitochondrion-targeted double-signal turn-on can be applied to the fluorescent quantitative detection of formaldehyde. The fluorescence detection principle of the quantitative formaldehyde concentration is as follows: the method comprises the steps of taking a nano probe nano-MTDF as a fluorescent probe, reacting with formaldehyde to generate an intermediate product, then carrying out 2-aza-coppu rearrangement and hydrolysis to generate a fluorescent substance compound VIII and a fluorescent substance compound IX, and measuring the fluorescence intensity change under the excitation of 440nm and 535nm, thereby obtaining the formaldehyde concentration.

The principle of detecting the concentration of formaldehyde in water by using the novel mitochondrion-targeted double-signal turn-on formaldehyde fluorescent probe is as follows:

the mitochondrion-targeted double-signal turn-on formaldehyde fluorescent nano probe has almost no fluorescence in water, namely the fluorescence of two fluorophores 1, 8-naphthalimide and rhodamine B in the probe is quenched, wherein the principle is that the fluorescence of the 1, 8-naphthalimide is quenched due to fluorescence resonance energy transfer, meanwhile, the hydrophobicity of the 1, 8-naphthalimide causes the compound (I) to be assembled into nano particles in water, and the fluorescence of the rhodamine B is quenched due to aggregation-induced quenching. When the nano-probe nano-MTDF reacts with formaldehyde, two separated fluorophores are released, the nano-probe is self-assembled and removed, and the fluorescence of the 1, 8-naphthalimide and the rhodamine B is recovered simultaneously, so that the effect of a double-signal turn-on is realized. Wherein N in the rhodamine B structure⁺It also has the effect of targeting mitochondria.

Compared with the prior art, the invention has the following beneficial effects: the 1, 8-naphthalimide and rhodamine B fluorophore selected in the invention are self-assembled into the nano-scale fluorescent probe in water, so that the effect of double signal turn-on can be realized, and the double signal turn-on nano probe has the advantage that the double signal turn-on nano probe can be self-contrasted by comparing two different turn-on signals after reacting with formaldehyde, so that the accuracy of probe detection is improved. The nano-probe nano-MTDF has good specificity to formaldehyde aqueous solution, the lower limit of detection at the excitation wavelength of 440nm is 3 mu M, and the lower limit of detection at the excitation wavelength of 535nm is 8 mu M. Meanwhile, the nanoprobe nano-MTDF realizes the imaging experiment of the formaldehyde in the mitochondria in the cells, and the research is not reported in related documents. In conclusion, the novel mitochondrion-targeted double-signal turn-on formaldehyde fluorescent nanoprobe nano-MTDF provides an effective research tool for researching the physiological action of formaldehyde 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 shows the particle size of the nanoprobe nano-MTDF (1 μ M) prepared in example 1 of the present invention measured by dynamic light diffraction under DMSO/water (v/v: 1/99) and the nanoparticle imaging by transmission electron microscopy (standard size 1 μ M).

FIG. 4 is a fluorescence emission spectrum of the nano-probe nano-MTDF (1 μ M) prepared in example 1 in different DMSO/water ratios. The graph a shows the fluorescence emission spectrum with an excitation wavelength of 440 mm. Panel b shows fluorescence emission spectra with excitation wavelength of 535 nm.

FIG. 5 shows fluorescence emission spectra of the nanoprobe nano-MTDF (1 μ M) prepared in example 1 of the present invention under DMSO/PBS buffer (pH 7.4, v/v 1/99) with different equivalents of formaldehyde added. The graph a shows the fluorescence emission spectrum with an excitation wavelength of 440 mm. Panel b shows fluorescence emission spectra with excitation wavelength of 535 mm.

FIG. 6 is a fluorescence diagram showing the change with time of the nanoprobe nano-MTDF (1. mu.M) prepared in example 1 of the present invention in the presence of formaldehyde (1mM) in DMSO/PBS buffer (pH 7.4, v/v 1/99). Graph a excitation wavelength 440nm and emission wavelength 540 nm. Panel b excitation wavelength 535nm and emission wavelength 585 nm.

FIG. 7 is a fluorescence diagram showing the selective results of the nanoprobe nano-MTDF (1 μ M) prepared in example 1 of the present invention under the condition of DMSO/PBS buffer (pH 7.4, v/v 1/99). 1-17 are PBS, formaldehyde, acetaldehyde, methylglyoxal, benzaldehyde, p-nitrobenzaldehyde, p-hydroxybenzaldehyde, acetone, formic acid, sodium pyruvate, glucose, glutathione, homocysteine, cysteine, sodium hydrogen sulfate, hydrogen peroxide and tert-butyl hydroperoxide respectively. Graph a excitation wavelength 440nm and emission wavelength 540 nm. Panel b excitation wavelength 535nm and emission wavelength 585 nm.

FIG. 8 is a fluorescence image of the nanoprobe nano-MTDF (1. mu.M) prepared in example 1 of the present invention before and after reacting with formaldehyde under the condition of DMSO/different pH buffer (v/v. 1/99). Graph a excitation wavelength 440nm and emission wavelength 540 nm. Panel b excitation wavelength 535nm and emission wavelength 585 nm.

Fig. 9 shows a high performance liquid chromatogram and a mass spectrum of the nanoprobe nano-MTDF prepared in example 1 of the present invention before and after adding formaldehyde under the condition of DMSO/PBS buffer (pH 7.4, v/v 1/99).

FIG. 10 is the fluorescence imaging of formaldehyde in cells by the nanoprobe nano-MTDF prepared in example 1 of the present invention.

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 (II)

1.22g of p-hydroxybenzaldehyde (10mmol) was dissolved in 50mL of acetone solution, followed by addition of 2.07g of potassium carbonate (15mmol), reaction at 60-70 ℃ for half an hour, addition of 2.37g of 3-bromopropyne (20mmol), reaction at 60-70 ℃ for 2 hours, and then termination, and the solvent was distilled off under reduced pressure. To the mixture was added water, extracted with ethyl acetate, the organic phases were combined, washed several times with water and saturated brine, dried over anhydrous sodium sulfate, filtered, the solvent was dried by spinning, and separated by column chromatography (ethyl acetate: petroleum ether ═ 1:10 as eluent) to give compound (II) as a white solid (1.53g, 95% yield).¹H NMR(500MHz,CDCl₃)δ9.88(s,1H),7.95–7.75(m,2H),7.18–6.98(m,2H),4.77(d,J＝2.4Hz,2H),2.58(t,J＝2.4Hz,1H).¹³CNMR(126MHz,CDCl₃)δ190.69,162.30,131.81,130.51,115.11,77.51,76.35,55.87.ESIcalcd.for C₁₀H₈O₂[M+H]⁺161.05,found 161.18。

(2) Preparation of Compound (III)

0.64g of compound (II) (4mmol) was dissolved in 40mL of methanol, ice-cooled to 0 ℃, 6mL of aminomethanol solution (7mol/L, 42mmol) was added, and the reaction was carried out at 0 ℃ for half an hour, followed by addition of 1g of vicinal ditertiary propenyl borate (6mmol), and the reaction was switched to 25-35 ℃ and allowed to react overnight. Removing solvent by rotary evaporation under reduced pressure, and separating the crude product with chromatography column (dichloromethane: methanol 40:1 for elution)Reagent) to give compound (III) as a colorless oily liquid (0.613g, 76% yield).¹H NMR(500MHz,CDCl₃)δ7.32–7.25(m,2H),6.99–6.91(m,2H),5.79–5.71(m,1H),5.16–5.05(m,2H),4.69(d,J＝2.4Hz,2H),3.98–3.95(m,1H),2.53(t,J＝2.4Hz,1H),2.49–2.41(m,1H),2.40–2.29(m,1H).¹³C NMR(126MHz,CDCl₃)δ156.51,138.87,135.46,127.35,117.56,114.75,78.65,75.42,55.82,54.71,44.17.ESI calcd.for C₁₃H₁₅NO[M+H]⁺202.12,found 202.29。

(3) Preparation of Compound (V)

0.15g of compound (IV) (0.5mmol) was added to 15mL of anhydrous tetrahydrofuran, cooled to 0 ℃ and then 0.18g of compound (III) (0.6mmol), 0.3g of acetic acid (5mmol) and 0.42g of sodium triacetoxyborohydride (2mmol) were sequentially added. The reaction was switched to 20-30 ℃ and reacted overnight. The solvent was removed by rotary evaporation under reduced pressure, and the crude product was subjected to column chromatography (dichloromethane: methanol ═ 20:1) to give compound (V) as an orange solid (0.14g, 59% yield).¹H NMR(500MHz,CDCl₃)δ8.38(t,J＝7.2Hz,2H),7.93(s,1H),7.53(t,J＝7.8Hz,1H),7.30(d,J＝8.6Hz,2H),7.03(d,J＝8.6Hz,2H),5.81-5.73(m,1H),5.22–5.13(m,2H),4.71(d,J＝2.4Hz,2H),4.13–4.05(m,2H),4.02(d,J＝14.2Hz,1H),3.91(d,J＝14.2Hz,1H),3.81(t,J＝7.0Hz,1H),2.70–2.59(m,2H),2.56(t,J＝2.3Hz,1H),1.70–1.64(m,2H),1.49–1.37(m,2H),0.97(q,J＝7.8Hz,3H).¹³CNMR(126MHz,CDCl₃)δ164.60,163.76,162.42,157.39,133.86,132.93,132.48,131.08,128.81,128.29,124.93,123.02,121.68,118.77,115.34,111.71,78.39,75.74,61.41,55.85,49.83,41.43,39.98,30.21,24.81,20.39,13.85.ESI calcd.For C₃₀H₃₀N₂O₄[M–H]^-481.22,found 481.27。

(4) Preparation of Compound (VII)

0.06g (0.1mmol) of the compound (VI) was dissolved in 5mL of methylene chloride, followed by addition of 1-hydroxybenzotriazole (0.02g, 0.15mmol), 0.03g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (0.15mmol), 0.02g of triethylamine (0.2mmol) and 0.01g of 3-azido-propylamine (0.12mmol) in this order, reaction was carried out overnight at room temperature, after completion of the reaction,the crude product was isolated by preparative thin layer chromatography (dichloromethane: methanol ═ 20:1) to give compound (VII) (0.057g, 82% yield) as a dark red solid.¹H NMR(400MHz,DMSO)δ7.89(s,1H),7.83–7.66(m,3H),7.54(s,1H),7.13(dt,J＝9.7,5.6Hz,4H),6.95(d,J＝1.7Hz,2H),3.66(dd,J＝13.6,6.5Hz,8H),3.56–3.16(m,10H),3.08(dd,J＝12.2,6.2Hz,2H),2.50(d,J＝1.5Hz,3H),2.29(s,2H),1.73–1.49(m,2H),1.21(t,J＝6.9Hz,12H).¹³C NMR(75MHz,DMSO)δ171.70,170.45,166.94,159.23,158.77,158.30,157.83,157.44,155.95,155.51,135.64,132.13,131.05,130.76,130.14,127.87,118.34,114.63,113.41,96.29,48.73,47.13,45.75,44.63,41.44,36.12,30.59,28.80,28.07,12.76.ESI calcd.for C₃₉H₄₉N₈O₄[M]⁺693.39,found693.48。

(5) Preparation of Compound (I)

0.027g of Compound (VII) (0.04mmol) and Compound (V) (0.04mmol) were dissolved in 3mL of tetrahydrofuran, and an aqueous solution (3mL) of 0.004g of vitamin C (0.02mmol) and 0.003g of copper sulfate pentahydrate (0.02mmol) were added to the above mixture, followed by reaction at room temperature for 6 hours. The solvent was evaporated to dryness and the crude product was purified by preparative high performance liquid chromatography to give dark red old compound (I) (0.03g, 65% yield). Where vitamin C acts to reduce the divalent copper to monovalent copper.

¹H NMR(300MHz,DMSO)δ8.43(d,J＝7.9Hz,1H),8.26(t,J＝3.5Hz,2H),7.98(s,2H),7.72(ddd,J＝13.2,7.3,4.6Hz,3H),7.46(ddd,J＝28.1,12.0,6.5Hz,4H),7.18–7.01(m,6H),6.91(d,J＝11.8Hz,2H),5.56(td,J＝17.0,6.8Hz,1H),5.16(s,2H),5.04(t,J＝12.8Hz,2H),4.38(t,J＝6.8Hz,2H),4.24(dd,J＝9.2,5.5Hz,2H),3.96(dd,J＝20.2,13.1Hz,6H),3.63(d,J＝6.9Hz,18H),3.25(dd,J＝33.1,17.3Hz,10H),3.04(d,J＝5.9Hz,3H),2.94–2.58(m,3H),2.30(s,2H),1.93(dd,J＝11.2,4.5Hz,2H),1.63–1.43(m,2H),1.30(dd,J＝14.8,7.4Hz,2H),1.19(dd,J＝12.5,5.8Hz,12H),0.90(t,J＝7.3Hz,3H).¹³C NMR(75MHz,DMSO)δ175.28,171.87,170.47,166.91,164.51,163.03,158.72,157.38,155.91,155.46,142.82,135.74,135.61,133.43,132.07,131.86,131.05,130.88,130.77,130.67,130.18,130.08,130.03,128.28,127.86,126.90,125.00,122.23,121.37,119.09,115.27,114.60,114.28,113.37,100.35,96.26,61.61,60.13,48.00,47.57,47.05,45.75,41.46,38.96,37.95,36.03,30.59,30.35,30.29,29.33,28.79,28.10,20.20,14.13,12.78.HRMS(ESI)calcd.for C₆₉H₇₉N₁₀O₈[M]⁺1175.6082,found1175.6067。

(6) Preparation of nano probe nano-MTDF

Dissolving the compound (I) in DMSO as a mother solution, adding the mother solution into ultrapure water, PBS buffer solution or cell culture solution, performing ultrasonic treatment for several minutes, and then performing vigorous shaking to obtain the nano-probe nano-MTDF.

Example 2 nanoprobe nano-MTDF particles were tested for particle size by dynamic light diffraction in DMSO/water buffer (pH 7.4, v/v 1/99) and nanoparticles imaged by transmission electron microscopy.

Accurately weighing a certain amount of the compound (I) prepared in example 1, preparing a probe mother solution with the concentration of 0.1mM by DMSO, sucking 0.02mL by a pipette gun, adding into 1.98mL of water, carrying out ultrasonic treatment for several minutes, then violently shaking to obtain a nano probe nano-MTDF, then measuring the particle size of the nano-MTDF in the water by using a nano-zs90particle analyzer, simultaneously dripping the mixed solution on a copper net, drying at 37 ℃ and carrying out projection electron microscope imaging, wherein the result is shown in figure 3.

Referring to FIG. 3(a), it can be seen that the dynamic light diffraction test results in that the average particle diameter of the particles was 161.9nm and the polydispersity PDI index was 0.262. Referring to FIG. 3(b), the nanoparticle size obtained from the transmission electron microscope (standard bar: 1 μm) was substantially identical to the data obtained from dynamic light diffraction, thus confirming that Compound (I) formed a nanomaterial in water.

Example 3 fluorescence spectroscopy detection of nanoprobe nano-MTDF (1. mu.M) at different DMSO/water ratios.

An amount of compound (I) prepared in example 1 was weighed out accurately, prepared with DMSO at 0.1mM concentration as a probe stock solution, pipetted 0.02mL into 1.98mL of different DMSO/water ratios (DMSO 1%, 5%, 10%, 20%, 40%, 60%, 70%, 80%, 90%), sonicated for several minutes, then shaken vigorously, and then the fluorescence spectrum of compound (I) was determined.

The experimental result shows that the fluorescence of the compound (I) is enhanced along with the increase of the proportion of DMSO, thereby proving that the aggregation effect of the compound (I) is enhanced along with the decrease of the proportion of DMSO, the fluorescence at different excitation wavelengths is weakened, and the fluorescence of rhodamine B is quenched due to aggregation. Meanwhile, the probe has only one emission peak under the excitation wavelength of 440nm no matter the proportion of DMSO, which indicates that the fluorescence of naphthalimide is quenched by rhodamine B through fluorescence resonance energy transfer. The fluorescence spectrum is shown in FIG. 4.

Example 4 the nanoprobe nano-MTDF (1 μ M) of the present invention was subjected to fluorescence spectroscopy under DMSO/PBS buffer (pH 7.4, v/v 1/99) with different equivalents of formaldehyde added.

Accurately weighing a certain amount of the probe (I) prepared in example 1, preparing a mother solution with the concentration of 0.1mM by using dimethyl sulfoxide, sucking 0.02mL by using a pipette gun, adding the mother solution into 1.96mL of PBS buffer solution, carrying out ultrasonic treatment for several minutes, then carrying out vigorous shaking to obtain the nano-probe nano-MTDF, sucking 396 mu L of nano-probe nano-MTDF solution each time, adding 4 mu L of formaldehyde solutions with different equivalent weights (the final concentrations of formaldehyde in water are respectively 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, 2and 5mM), reacting at 37 ℃ for 3 hours, and then determining the fluorescence value. The excitation wavelength was 440nm or 535nm, respectively, and the fluorescence spectrum is shown in FIG. 5.

The experimental result shows that with the increase of formaldehyde equivalent, two free fluorophores generated by the nano-probe nano-MTDF are increased, and the fluorescence intensity of the two fluorophores is respectively increased.

Example 5 fluorescence profiles of nanoprobes nano-MTDF (1 μ M) of the present invention with time during the action with formaldehyde (1mM) in DMSO/PBS buffer (pH 7.4, v/v 1/99).

Accurately weighing a certain amount of probe (I), preparing a mother solution with the concentration of 0.1mM by using dimethyl sulfoxide, sucking 0.02mL by a pipette gun, adding the mother solution into 1.96mL of PBS buffer solution, carrying out ultrasonic treatment for several minutes, then violently shaking to obtain the nano-probe nano-MTDF, sucking 396 mu L of nano-probe nano-MTDF solution each time, adding 4 mu L of formaldehyde aqueous solution (finally, the concentration of formaldehyde in water is 1mM), reacting at 37 ℃, and measuring the fluorescence value of the nano-probe at different time points (0, 0.5, 1, 1.5, 2, 2.5, 3 and 4 hours respectively). Fluorescence spectrum 6 (a): excitation wavelength was 440nm, emission wavelength was 540nm, fluorescence spectrum is shown in FIG. 6 (b): the excitation wavelength was 535nm and the emission wavelength was 585 nm.

Experiments prove that the fluorescence intensity of the two fluorophores can be enhanced along with the increase of time, and the effect of detecting formaldehyde by the probe is met.

Example 6 fluorescence spectroscopy detection of the selectivity results of nanoprobes nano-MTDF (1 μ M) in the present invention under DMSO/PBS buffer (pH 7.4, v/v 1/99).

Accurately weighing a certain amount of probe (I), preparing mother liquor with the concentration of 0.1mM by using dimethyl sulfoxide, sucking 0.02mL by a pipette, adding the mother liquor into 1.96mL of PBS buffer solution, carrying out ultrasonic treatment for several minutes, then violently oscillating to obtain the nano-probe nano-MTDF, sucking 396 mu L of nano-probe nano-MTDF solution each time, 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, methylglyoxal, acetone, formic acid, 4-hydroxybenzaldehyde, 4-nitrobenzaldehyde, benzaldehyde, hydrogen peroxide, tert-butyl hydroperoxide, sodium hydrosulfide, glutathione, cysteine, homocysteine, sodium pyruvate and glucose, the final concentration is 1mM), reacting for 3 hours at 37 ℃, and determining the fluorescence value. Fluorescence spectrum 7 (a): excitation wavelength is 440nm, emission wavelength is 540nm, fluorescence spectrum is shown in FIG. 7 (b): the excitation wavelength was 535nm and the emission wavelength was 585 nm.

Experimental results show that the fluorescence intensity of the nano-probe nano-MTDF is basically unchanged in the presence of other related bioactive molecules except formaldehyde, and the anti-interference capability of the nano-probe nano-MTDF is very good, namely the specificity of the nano-probe nano-MTDF to formaldehyde is good.

Example 7 fluorescence spectra of the nanoprobe nano-MTDF (1 μ M) of the present invention before and after reaction with formaldehyde in DMSO/different pH buffer (v/v-1/99) were detected.

Accurately weighing a certain amount of probe (I), preparing a mother solution with the concentration of 0.1mM by using dimethyl sulfoxide, sucking 0.02mL by a pipette gun, adding the 0.02mL into 1.96mL of buffer solutions with different pH values (the pH values are respectively 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 and 10.5), carrying out ultrasonic treatment for a plurality of minutes, then violently shaking to obtain the nano-probe nano-MTDF, sucking 396 mu L of nano-probe nano-MTDF solution each time, adding 4 mu L of formaldehyde aqueous solution (the final concentration of formaldehyde in water is 0 and 1mM), reacting at 37 ℃ for 3 hours, and determining the fluorescence value. Fluorescence spectrum 8 (a): excitation wavelength was 440nm, emission wavelength was 540nm, fluorescence spectrum is shown in FIG. 8 (b): the excitation wavelength was 535nm and the emission wavelength was 585 nm.

Experiments prove that the change of the pH has little influence on the nano-MTDF in the pH range from 5 to 7.5, namely the nano-MTDF is suitable for detecting the concentration of formaldehyde in organisms.

Example 8 the mechanism of the nanoprobe nano-MTDF (1 μ M) of the present invention after the reaction with formaldehyde under the condition of DMSO/PBS buffer (pH 7.4, v/v 1/99) was demonstrated.

Accurately weighing a certain amount of probe (I), preparing a mother solution with the concentration of 2mM by using dimethyl sulfoxide, sucking 0.02mL by a pipette gun, adding the solution into 1.96mL of PBS buffer solution, carrying out ultrasonic treatment for a plurality of minutes, then carrying out violent oscillation to obtain a nano probe nano-MTDF, sucking 0.99mL of nano probe nano-MTDF solution, respectively adding 10 mu L of formaldehyde aqueous solution, reacting overnight, and then analyzing by using high performance liquid chromatography. The HPLC chromatogram is shown in FIG. 9.

Experiments prove that the mechanism of the reaction of the nanoprobe nano-MTDF and formaldehyde is correct. The nano-MTDF and formaldehyde generated two free fluorescent substances, compound VIII and compound IX, to test the effect of the dual signal turn-on.

Example 9 Formaldehyde imaging analysis of the Nanoprobe nano-MTDF of the present invention in cancer cells

Accurately weighing a certain amount of probe (I), preparing mother liquor with the concentration of 0.5mM by using dimethyl sulfoxide, sucking 0.02mL by a liquid transfer gun, adding the mother liquor into 1.98mL of DMEM culture medium, carrying out ultrasonic treatment for several minutes, and then violently shaking to obtain the nano-probe nano-MTDF. Adding 1mL of culture solution containing nanoprobe nano-MTDF into MCF-7 cells, incubating at 37 ℃ for 0.5h, washing twice with fresh DMEM medium, incubating for 3h with different formaldehyde concentrations (final formaldehyde concentrations are 0 and 1 respectively), washing twice with fresh DMEM medium, addingCommercialized

Deep Red FM, in which, incubation was 20min at 37 ℃, PBS washed twice, and finally imaged with Perkinelmer UltraView Vox Spinning Disk confocal fluorescence. FIG. 10 is a diagram showing the effect of confocal fluorescence imaging of cells. (a1, a2, a3): formaldehyde (0mM), (a1, a2, a3) formaldehyde (1 mM). Na-channel (a1, b1): lambda_ex＝440nm,λ_em＝455–515nm；Rho-channel(a2,b2):λ_ex＝514nm,λ_em＝524.5–649.5nm；Deep red-channel(a3,b3):λ_ex＝640nm,

λ

_em660 and 750 nm. c1 coincidence of b1 and b 3; c2 is the superposition coefficient of Na-channel and Deep red-channel; c3 superposition of b2and b 3; c4 coincidence coefficient of Rho-channel and Deep red-channel, scale bar, 20 μm.

The experimental result shows that under the condition of increasing the concentration of formaldehyde, the fluorescence signal in the cells is also enhanced, which indicates that the substance can detect the formaldehyde in the cells. At the same time, by commercial and commercial

Image comparison of deep FM, the Pearson correlation coefficients are 0.81 (lambda)_ex440nm) and 0.85(λ)_ex514nm), which demonstrates that nano-MTDF is able to detect formaldehyde in mitochondria within cells.

Claims

1. A fluorescent nanoprobe, characterized in that it is prepared by self-assembly of compound (I) in water:

the structural formula of the compound (I) is as follows:

2. the method for preparing the fluorescent nanoprobe as claimed in claim 1, characterized in that the preparation method comprises:

3. the method for preparing a fluorescent nanoprobe according to claim 2, characterized in that the method specifically comprises:

(2) mixing the compound (II) with an ammonia methanol solution and propenyl boronic acid ortho-di-tertiary alcohol ester at 0 ℃, then transferring to 25-35 ℃ for reaction, and performing post-treatment B to prepare a compound (III); the reaction time is 12 hours, and the reaction solvent is methanol; the mass ratio of the compound (II) to ammonia and the propenyl boronic acid ortho-di-tertiary alcohol ester in the ammonia methanol solution is 1:10: 1.5;

(3) mixing the compound (IV), the compound (III) and sodium triacetoxyborohydride according to the mass ratio of 1:1.2:4 under an acidic condition at 0 ℃, then transferring to 20-30 ℃ for reaction, and preparing a compound (V) through aftertreatment C; the reaction time is 12 hours, and the reaction solvent is tetrahydrofuran;

(5) reacting a compound (V) with a compound (VII) according to the mass ratio of 1:1 under the catalysis of cuprous, evaporating the solvent to dryness, and purifying the crude product by using a high performance liquid chromatography to obtain a compound (I); the reaction temperature is room temperature, the reaction time is 6 hours, and the reaction solvent is tetrahydrofuran and water;

4. The method for preparing a fluorescent nanoprobe according to claim 3, characterized in that: the alkaline condition in the step (1) is realized by adopting potassium carbonate.

5. The method for preparing a fluorescent nanoprobe according to claim 3, characterized in that: the acidic condition in step (3) is realized by adopting acetic acid.

6. The method for preparing a fluorescent nanoprobe according to claim 3, characterized in that:

the post-treatment A comprises the following steps: removing the solvent from the reaction liquid by rotary evaporation under reduced pressure, adding water, extracting with ethyl acetate, combining organic phases, washing the organic phases with water and saturated saline solution for a plurality of times, drying with anhydrous sodium sulfate, filtering, drying the solvent by rotary evaporation, and carrying out column chromatography separation to obtain a target product, wherein an eluant is ethyl acetate and petroleum ether with the volume ratio of 1: 10;

the post-treatment B comprises the following steps: carrying out reduced pressure rotary evaporation on the reaction liquid to remove the solvent, carrying out chromatographic column separation on the crude product to obtain a target product, wherein an eluent is dichloromethane and methanol with the volume ratio of 40: 1;

the post-treatment C comprises the following steps: carrying out reduced pressure rotary evaporation on the reaction liquid to remove the solvent, carrying out chromatographic column separation on the crude product to obtain a target product, wherein an eluent is dichloromethane and methanol with the volume ratio of 20: 1;

the post-treatment D is as follows: separating the reaction solution by thin layer chromatography, wherein the developing agent is dichloromethane and methanol with the volume ratio of 20:1 to obtain the target compound.

7. Use of the fluorescent nanoprobe of claim 1 in detecting formaldehyde concentration.

8. The use of claim 7, wherein: the formaldehyde exists in the form of aqueous solution, and the concentration of the formaldehyde is 0-5 mmol/L.

9. The use of claim 8, wherein: the formaldehyde is intracellular formaldehyde, and the concentration of the formaldehyde is 0-1 mmol/L.

10. The use of claim 9, wherein: the cell is human breast cancer cell MCF-7.