CN114672302A - Preparation and application of a near-infrared MOF fluorescent probe based on silicon rhodamine - Google Patents

Abstract

The invention relates to the preparation and application of a near-infrared MOF fluorescent probe for the detection of adenosine triphosphate (ATP) based on silicon rhodamine. The structure of the fluorescent probe is composed of a nanoscale metal-organic framework (ZIF-90) and its Consists of a silicon-rhodamine-based near-infrared fluorophore encapsulated inside. The present invention provides synthesis by using 3-Br-N,N-dimethylaniline, n-butyllithium, dichlorodisilane, 2-formylbenzoic acid, imidazole-2-formaldehyde and zinc acetate dihydrate as raw materials. The preparation method of the fluorescent probe; the fluorescent probe is a near-infrared MOF fluorescent probe of adenosine triphosphate based on silicon rhodamine; firstly, the fluorescent probe has a simple synthesis method, which can produce 8.5 times of fluorescence enhancement to ATP; secondly, The fluorescent probe has relatively ideal selectivity for ATP, and is not interfered by other nucleotides and common ions in organisms; thirdly, the fluorescent probe interacts with ATP rapidly, and the response time is within 500s; in addition, the fluorescent probe The needle has a longer near-infrared emission wavelength and can be used to detect the content of ATP in living cells.

Description

Preparation and application of a near-infrared MOF fluorescent probe based on silicon rhodamine

technical field

The invention belongs to the technical field of fluorescent probes, in particular to the preparation and application of a silicon-rhodamine-based adenosine triphosphate near-infrared MOF fluorescent probe.

Background technique

Adenosine triphosphate (ATP) is mainly produced in mitochondria, which is the source of energy required for life activities in organisms (Schulz G E.Binding of nucleotides by proteins[J].Curr.Opin.in Struc.Biol.,1992,2,61 -67; Roberts J. Control of the supply line [J]. Science, 1997, 278, 2073-2074.). It is involved in various physiological processes such as protein synthesis, energy transfer, cellular respiration, enzyme catalysis, and signal transduction (Szewcyk A, Pikula S. Adenosine 5'-triphosphate: an intracelluar metabolic messenger[J].Biochim.Biophys.Acta Rev .Cancer, 1998, 1365, 333-353; Zhou Y, Tozzi F, Chen J, Fan F, Xia L, Wang J, Gao G, Zhang A, Xia X, Brasher H, Widger W, Ellis L M, Zhang W. Intracellular ATP levels are pivotal determinant of chemoresistance in colon cancer cells[J]. Cancer Res., 2011, 72.). Moreover, the concentration of ATP in cells is closely related to many diseases, such as ischemic injury, platelet aggregation, inflammation and malignant tumors, etc. Therefore, ATP can be used as a marker for certain diseases to be studied (Morciano G, Sarti A C, Marchi S, Missiroli S, Falzoni S, Raffaghello L, Pistoia V, Giorgi C, Di Virgilio F, Pinton P. Use of luciferaseprobes to measure ATP in living cells and animals[J].Nature Protoc.,2017,12:1542;Abraham E H, Okunieff P, Scala S, Vos P, Oosterveld M J S, Chen A Y, Shrivastav B, Guidotti G. Cystic fibrosis transmembrane conductance regulator and adenosine triphosphate [J]. Science, 1997, 275, 1324-1326.). It is necessary to develop a simple and effective ATP detection method for our further understanding and study of ATP-related physiological and pathological processes.

In recent years, various methods have been developed for ATP detection, such as colorimetry, chemiluminescence, electrochemical analysis, fluorescence detection, etc. (Li S, Zhao X, Yu X, Wan Y, Yin M, Zhang W, Cao B, Wang H. Fe ₃ O ₄ nanozymes with aptamer-tuned catalysis for selective colorimetric analysis of ATP in blood[J].Anal.Chem.,2019,91,14737-14742;Yao W,Wang L , WangH, Zhang X, Li L. An aptamer-based electrochemiluminescent biosensor for ATP detection[J]. Biosens. Bioelectron., 2009, 24, 3269-3274; Xie H, Chai Y, Yuan Y, Yuan R. Highly effective molecule converting strategy based on enzyme-free dualrecycling amplification for ultrasensitive electrochemical detection of ATP[J].Chem.Commun.,2017,53,8368-8371.). Compared with other detection methods, fluorescence method has the advantages of high sensitivity, good selectivity, fast response and strong imaging resolution, and is widely used in biological detection and analysis (Zhou Y, Xu Z, Yoon J. Fluorescent and colorimetric chemosensors for detection of nucleotides, FAD and NADH: highlighted research during 2004-2010 [J]. Chem. Soc. Rev., 2011, 40, 2222-2235.). However, most of the ATP fluorescent probes reported so far have shortcomings such as insufficient hydrophilicity, poor biocompatibility, and complex synthesis, making it difficult to meet the requirements of in vivo imaging. Therefore, it is necessary to design a near-infrared-emitting fluorescent probe that can be used to detect ATP levels in vivo.

Zeolitic imidazole frameworks (ZIFs), a subclass of metal-organic frameworks (MOFs), are formed by the self-assembly of metal ions and imidazole linkers. Due to its tunable pores, controllable structure, high loading rate, good biocompatibility, easy synthesis and functionalization, etc., it has been widely studied and used in the fields of catalysis, gas storage and separation, drug delivery, bioimaging and sensing. Application (Khan N A, Hasan Z, Jhung S H. Beyond pristine metal-organic frameworks: Preparation and application of nanostructured, nanosized, and analogous MOFs [J]. Coordin. Chem. Rev., 2018, 376, 20-45; Xu C, Fang R, Luque R, Chen L, LiY.Functional metal–organic frameworks for catalytic applications[J].Coordin.Chem.Rev.,2019,388,268-292; Jiao L, Wang Y, Jiang H L, Xu Q. Metal-organic frameworks as platforms for catalytic applications[J].Adv.Mater.,2018,30,1703663.). In recent years, a large number of MOF materials have been reported for the encapsulation of drugs, proteins, and small molecule dyes. However, there are few reports using it to detect ATP in vivo. Therefore, it is of great significance to combine nanomaterials and near-infrared fluorescent dyes to develop a MOFs-based near-infrared fluorescent probe to detect ATP in vivo.

SUMMARY OF THE INVENTION

According to the proposed requirements, the inventors have conducted in-depth research on this, and after a lot of creative work, provide a near-infrared nano-fluorescent probe of adenosine triphosphate based on metal organic framework (ZIF-90).

The technical scheme of the present invention is a near-infrared MOF fluorescent probe based on silicon rhodamine, the structure of which is self-assembled by metal organic framework (ZIF-90) and silicon rhodamine near-infrared fluorescent dye (SiB).

A preparation method of a near-infrared MOF fluorescent probe based on silicon rhodamine. Proceed as follows:

1) Preparation method of silicon rhodamine near-infrared fluorescent dye (SiB): at 0 °C, under the protection of N, add 1.5 to _2.5 equivalents of 3-Br-N,N-dimethylaniline to a solution containing 60 mL of diethyl ether. Into a 200mL double-necked round-bottom flask, 1.5-2.5 equivalents of n-butyllithium were added, and the reaction was carried out for 1.5-2.5h. Dissolve 0.5 to 1.5 equivalents of dichlorodimethylsilane in 10 mL of ether, slowly add it to the above reaction, after the reactant gradually returns to room temperature, react overnight, and add 50 mL of water to quench the reaction. The reaction mixture was extracted with ether, washed with brine, and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography, the eluent was petroleum ether:ethyl acetate (80:1), and the solvent was distilled off under reduced pressure to obtain a light yellow oily intermediate product (yield 75%). 8-12 equivalents of the intermediate product, 40-60 equivalents of 2-formylbenzoic acid and 0.5-1.5 equivalents of copper bromide were added to a 15 mL sealed tube, and the reaction was stirred at 140 °C for 5 h; cooled to room temperature, the reaction was The mixture was dissolved in dichloromethane, extracted with 2M NaOH solution, the organic layer was washed with brine and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography, the eluent was petroleum ether:ethyl acetate:triethylamine (50:1:1), and the solvent was distilled off under reduced pressure to obtain colorless needle-like crystals (yield 45%) .

2) Preparation method of near-infrared MOF fluorescent probe (ZIF-90@SiB) based on silicon rhodamine: at room temperature, 2 mL of N,N-dimethylformamide was added to 90-110 equivalents of zinc acetate dihydrate Make it completely dissolved, at the same time, completely dissolve 180-220 equivalents of imidazole-2-carbaldehyde and 0.5-1.5 equivalents of near-infrared fluorescent dye SiB into another 2 mL of N,N-dimethylformamide. After the solution is completely dissolved, mix it thoroughly, put it into an ultrasonic cleaner, shake for 4-6 minutes, then add 6-8 mL of N,N-dimethylformamide, continue to ultrasonicate for 20 minutes, and make the suspension The nanoparticles were further stabilized, then, the obtained suspension was centrifuged at 10,000 rpm for 4-6 min, the upper layer liquid was discarded, and the lower layer solid was washed 2-3 times with N,N-dimethylformamide, and then anhydrous Wash with ethanol for 8-12 times, and finally, put the obtained solid in a vacuum drying box, and dry at room temperature for 24 hours to obtain an off-white solid, which is the fluorescent probe.

The beneficial effect of the present invention is that a near-infrared MOF fluorescent probe based on silicon rhodamine has good spectral response performance to adenosine triphosphate (ATP). First, the fluorescence spectral properties of the probe were studied. Before adding ATP, the probe itself has no obvious fluorescence emission; after adding ATP, there is an obvious fluorescence emission peak in the near-infrared region (670 nm). With the increase of ATP concentration, the near-infrared fluorescence intensity of the probe molecules increased continuously. When 7 mM ATP was added, the fluorescence intensity was enhanced 8.5-fold, so this probe could be used to detect ATP. The fluorescence enhancement of the probe has a linear relationship with the concentration of ATP in the detection range of 1mM to 7mM, indicating that the probe can reflect the concentration of ATP by detecting the fluorescence intensity in this range. Second, the UV absorption spectra of the probes were studied. When ATP is not added, the probe has no UV absorption; after adding ATP, the probe has an absorption peak at 640 nm. Next, the selectivity of the probe is studied, and the probe and other nucleotides (ADP, AMP, GTP, CTP, UTP), common ions in organisms (P ₃ O ₁₀ ^5- , P ₂ O ₇ ^4- , H ₂ PO ₄ ^- , HPO ₄ ^2- , Cl ^- , SO ₄ ^2- , NO ₃ ^- , CH ₃ COO ^- , CO ₃ ^2- ), and the fluorescence response of the test substance (ATP). It was found that ATP can cause a strong change in the fluorescence spectrum, and other detection substances have little effect on the fluorescence spectrum of the probe. Then, the effect of pH value on the determination of ATP by the fluorescent probe was studied, when the pH value was between 5.0 and 8.0, it did not affect the determination of ATP by the fluorescent probe. In addition, the fluorescent probe responds rapidly, with a response time of less than 500 s.

An application of adenosine triphosphate near-infrared fluorescent probe. No obvious fluorescence was observed in the cells of the control group. When the fluorescent probe was added to the cells, strong fluorescence could be observed, indicating that the probe could detect the ATP present in the cells. When cells were pretreated with Apyrase to remove intracellularly generated ATP, the fluorescence was found to be significantly reduced. These results indicate that the fluorescent probe can detect the changes of intracellular ATP content. This provides a reliable means of monitoring ATP levels in living cells.

Description of drawings

Figure 1 is a schematic diagram of the preparation route of the fluorescent probe and its interaction with ATP.

Figure 2 is the fluorescence spectrum of the fluorescent probes reacted with different concentrations of ATP.

The abscissa is the wavelength, and the ordinate is the fluorescence intensity. The concentrations of fluorescent probes were all 4 mg/mL, and the ATP concentrations were: 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 mM, respectively. The fluorescence excitation wavelength is 640nm, and the emission wavelength is 650nm-800nm.

FIG. 3 is a graph showing the linear relationship of fluorescence of fluorescent probes to different ATP concentrations.

Figure 4 shows the UV-vis absorption spectra before and after the interaction of the fluorescent probe with ATP.

The abscissa is the wavelength, and the ordinate is the absorbance. The concentration of fluorescent probe was 4 mg/mL and the concentration of ATP was 7 mM.

Figure 5 is a graph of the selectivity of fluorescent probes.

The concentration of fluorescent probe was 4 mg/mL, the concentration of ATP was 7 mM, and the other analytes were all at 7 mM.

Figure 6 is a graph showing the effect of pH on fluorescent probes.

FIG. 7 is a graph showing the relationship between the fluorescence intensity and the time change of the fluorescent probe and different concentrations of ATP. ATP concentrations were: 1.0, 2.0, 4.0, 7.0 mM, respectively.

Figure 8 is a graph of the cytotoxicity experiment. The abscissa is the concentration of fluorescent probe, and the ordinate is the cell viability.

Figure 9 (a) Cell imaging diagram of the interaction of fluorescent probes with ATP; (b) relative fluorescence intensity diagram.

Detailed ways

The present invention is described in detail below with reference to the accompanying drawings and specific embodiments, but is not limited thereto.

Example 1:

Preparation of fluorescent probes

Preparation method of silicorhodamine near-infrared fluorescent dye (SiB): at 0 °C, under the protection of N, add ₂ equivalents of 3-Br-N,N-dimethylaniline to a 200 mL double-necked circle containing 60 mL of diethyl ether In the bottom flask, 2 equivalents of n-butyllithium were added, and the reaction was continued for 3h. 1 equivalent of dichlorodimethylsilane was dissolved in 10 mL of diethyl ether, and slowly added to the above reaction. After the reactant gradually returned to room temperature, the reaction was performed overnight, and 50 mL of water was added to quench the reaction. The reaction mixture was extracted with ether, washed with brine, and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography, the eluent was petroleum ether:ethyl acetate (80:1), and the solvent was distilled off under reduced pressure to obtain a light yellow oily intermediate product (yield 75%). 10 equivalents of intermediate product, 50 equivalents of 2-formylbenzoic acid and 1 equivalent of copper bromide were added to a 15 mL sealed tube, and the reaction was stirred at 140 °C for 5 h; cooled to room temperature, and the reaction mixture was dissolved in dichloromethane , extracted with 2M NaOH solution, the organic layer was washed with brine, and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography, the eluent was petroleum ether:ethyl acetate:triethylamine (50:1:1), and the solvent was distilled off under reduced pressure to obtain colorless needle-like crystals (yield 45%) . ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.96 (d, J=7.5 Hz, 1H), 7.64 (t, J=7.4 Hz, 1H), 7.54 (t, J=7.5 Hz, 1H), 7.30 (d ,J=7.6Hz,1H),6.97(d,J=2.9Hz,2H),6.78(d,J=8.9Hz,2H),6.55(dd,J=8.9,2.9Hz,2H),2.96(s ,12H),0.64(s,3H),0.61(s,3H).

Preparation method of silicon-rhodamine-based near-infrared MOF fluorescent probe (ZIF-90@SiB): As shown in Figure 1, at room temperature, 2 mL of N,N-dimethylacetate was added to 100 equivalents of zinc acetate dihydrate at room temperature. Formamide makes it completely dissolved. At the same time, 200 equivalents of imidazole-2-carbaldehyde and 1 equivalent of near-infrared fluorescent dye SiB are completely dissolved in another 2 mL of N,N-dimethylformamide. After completely dissolving, mix it thoroughly, put it into an ultrasonic cleaner, shake it for 5 minutes, then add 7 mL of N,N-dimethylformamide, continue to ultrasonicate for 20 minutes, to further stabilize the nanoparticles in the suspension, and then , the obtained suspension was centrifuged at 10,000 rpm for 5 min, the upper layer was discarded, and the lower solid was washed 3 times with N,N-dimethylformamide, and then 10 times with absolute ethanol. Finally, the obtained The solid was placed in a vacuum drying oven, and dried at room temperature for 24 hours to obtain an off-white solid, which is the fluorescent probe.

Example 2:

Fluorescent probe and ATP solution preparation

Preparation of probe solution: Weigh a certain amount of probe and disperse it in distilled water to prepare a 4 mg/mL probe solution. Preparation of ATP solution: Weigh a certain amount of adenosine-5'-triphosphate disodium salt, dissolve it in distilled water, prepare a 20mM ATP solution, and store it in an environment of 4-8°C.

Example 3:

Determination of the fluorescence spectrum of the interaction between fluorescent probes and ATP

Figure 2 shows the fluorescence spectrum of the interaction between the fluorescent probe and ATP. The concentration of the fluorescent probe is 4 mg/mL, and the concentration of ATP is 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0 mM. The excitation wavelength was fixed at 640 nm, and the emission wavelength ranged from 650 to 800 nm. The slit width is 5.0 nm/5.0 nm, and the fluorescence measuring instrument used is Hitachi F4600 fluorescence spectrophotometer. As can be seen from Figure 2, before adding ATP, the fluorescent probe has no obvious fluorescence emission; after adding ATP, an emission peak appears in the near-infrared region (670 nm). This is because ATP competitively binds with Zn ²⁺ in the structure of the probe, leading to the collapse of the zeolitic imidazole structure of the probe, thereby releasing the fluorophore SiB encapsulated in it and emitting near-infrared fluorescence. At the same time, with the increase of ATP concentration, the near-infrared fluorescence intensity of the probe molecules increased continuously. When 7 mM ATP was added, the fluorescence intensity increased 8.5-fold, so it could be used to detect ATP. Figure 3 is a graph of the linear response of the probe to different ATP concentrations. When the ATP concentration ranged from 1.0 to 7.0 mM, the fluorescence intensity of the probe showed a linear relationship with the ATP concentration, indicating that the probe could quantitatively detect ATP within this concentration range.

Example 4:

Determination of UV-Vis Absorption Spectra of the Interaction of Fluorescent Probes with ATP

FIG. 4 is the UV-Vis absorption spectra before and after the interaction of the fluorescent probe with ATP. The concentration of the fluorescent probe is 4 mg/mL, and the amount of ATP added is 7 mM. The instrument used for UV-Vis absorption spectroscopy was an Agilent Cary60 UV-Vis spectrophotometer. It can be seen from Figure 4 that the probe has no obvious absorption when ATP is not added; after adding ATP, the probe has an absorption peak at 640 nm, which is consistent with the UV absorption peak of SiB itself, indicating that ATP causes the structure of the probe collapse, thereby releasing the fluorescent dye SiB, resulting in UV absorption.

Example 5:

Selectivity of Fluorescent Probes for ATP Determination

Figure 5 is a graph of the selectivity of fluorescent probes for ATP assay. Investigate the addition of ATP (7 mM) and other nucleotides (ADP, AMP, GTP, CTP, UTP), and common ions in organisms (P ₃ O ₁₀ ) to the fluorescent probe suspension at a concentration of 4 mg/mL. Fluorescence response of ^5- ,P ₂ O ₇ ^4- ,H ₂ PO ₄ ^- ,HPO ₄ ^2- ,Cl ^- ,SO ₄ ^2- ,NO ₃ ^- ,CH ₃ COO ^- ,CO ₃ ^2- )(7mM) . From Figure 5, it can be seen that only ATP can cause an 8.5-fold fluorescence enhancement, while other detectors have no obvious effect on the fluorescence intensity of the probe. These results indicate that the fluorescent probes have good selectivity for ATP.

Example 6:

Effect of solution pH on the fluorescence properties of ATP measured by fluorescent probes

The influence of pH value on the fluorescence spectrum of ATP measured by fluorescent probe was investigated, and the results are shown in Figure 6. The pH range of our study was 2.0–12.0, the concentration of fluorescent probe was 4 mg/mL, and the concentration of ATP was 7 mM. It can be seen from the figure that when the pH of the fluorescent probe is between 5.0 and 8.0, the fluorescence intensity is basically unchanged, indicating that the pH within this range has no effect on the probe itself and the detection of ATP by the probe, which is a more suitable pH value range. . This is very beneficial for the determination of ATP in real samples using this probe.

Example 7:

Determination of the Response Time of Fluorescent Probe and ATP

We studied the response time of the fluorescent probe to ATP, and the ATP concentration was 1.0, 2.0, 4.0, 7.0 mM, and the results are shown in Figure 7. It can be seen from the figure that the response time of the probe to various concentrations of ATP is within 500s, which can meet the requirements of real-time monitoring in actual samples. It can also be seen from Fig. 7 that after the fluorescence intensity reaches the maximum value, there is almost no change in the following time, which indicates that the fluorescent probe has good photostability.

Example 8:

Application of Fluorescent Probes in Living Cells

First, we did a cytotoxicity assay, as shown in Figure 8. When 0～100μg/mL fluorescent probe was added, the cell survival rate was above 90%. This indicates that the fluorescent probe is less toxic and can be used to detect ATP in living cells. Then, we studied the application of fluorescent probes in living cells, and selected HeLa cells for confocal microscopy imaging, and the results are shown in Figure 9. In control cells, almost no fluorescence was observed. Then the probe (4 mg/mL) was added to the cells, and strong fluorescence could be observed, indicating that the fluorescent probe interacted with ATP in the cells to generate fluorescence. When the ATP scavenger Apyrase was added to the cells for pretreatment, and then the probe was added, it was found that the intracellular fluorescence almost disappeared. These results indicate that the probe can detect changes in intracellular ATP levels, providing a reliable means for monitoring the dynamic changes of ATP levels in living cells.

Claims (3)

1. The structure of the near-infrared MOF fluorescent probe based on the silarhodamine is composed of a nano-grade metal organic framework ZIF-90 and a near-infrared fluorescent dye SiB encapsulated inside.

2. The preparation method of the near-infrared MOF fluorescent probe based on the silarhodamine of claim 1 is characterized by comprising the following steps:

adding 2mL of N, N-dimethylformamide into 90-110 equivalents of zinc acetate dihydrate at room temperature to completely dissolve the zinc acetate dihydrate, simultaneously completely dissolving 180-220 equivalents of imidazole-2-formaldehyde and 0.5-1.5 equivalents of near-infrared fluorescent dye SiB into another 2mL of N, N-dimethylformamide, fully mixing the two solutions after the two solutions are completely dissolved, then putting the mixed solutions into an ultrasonic cleaning machine, oscillating for 4-6 min, subsequently adding 6-8 mL of N, N-dimethylformamide, continuing ultrasonic treatment for 20min to further stabilize nanoparticles in the suspension, then centrifuging the obtained suspension for 4-6 min at 10000rpm, discarding the lower-layer solid for 2-3 times by using the N, N-dimethylformamide, then washing for 8-12 times by using absolute ethyl alcohol, and finally, and (3) putting the obtained solid into a vacuum drying oven, and drying for 24 hours at room temperature to obtain an off-white solid, namely the fluorescent probe.

3. The application of the near-infrared MOF fluorescent probe based on the silarhodamine of claim 1, wherein the nano fluorescent probe can perform near-infrared fluorescence imaging detection on adenosine triphosphate in living cells.

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