CN115975140A - Two-dimensional covalent organic framework quantum dot, preparation method thereof and application thereof in two-photon fluorescence imaging - Google Patents

Abstract

The invention discloses a two-dimensional covalent organic framework quantum dot, a preparation method thereof and application thereof in two-photon fluorescence imaging.

Description

A two-dimensional covalent organic framework quantum dot and its preparation method and application in two-photon fluorescence imaging

technical field

The invention relates to the technical field of porous organic crystalline materials and the field of two-photon fluorescence imaging, in particular to a new type of two-dimensional covalent organic framework quantum dot material and its preparation method and application in cell imaging.

Background technique

Fluorescence imaging is of great significance for biomedical therapeutics due to its simplicity, visibility, and high selectivity. However, single-photon fluorescence imaging is generally limited to biological applications due to its unsatisfactory deep tissue imaging due to poor tissue penetration at short excitation wavelengths; in addition, autofluorescence of the intrinsic biological environment can also interfere. True fluorescence signal, resulting in low signal-to-noise ratio for single-photon fluorescence imaging. Two-photon fluorescence imaging itself has the characteristics of high resolution, high throughput, non-invasiveness, and large imaging depth. Combined with the localization and expression technology of markers such as fluorescent proteins and fluorescent dyes in cells, it can be used in living organisms and cells. The dynamic observation of its function in the active state allows people to study the neuronal activity in the animal brain in a physiological state.

Two-dimensional (2D) fluorescent covalent organic frameworks (FCOFs), as a new class of emerging crystalline porous polymers with long-range π-conjugated structures, have attracted much attention in the fields of biosensing and bioimaging. The 2D highly ordered π framework endows it with a large two-photon absorption cross-section, but the strong π-π stacking between layers of 2D FCOFs leads to its weakened two-photon fluorescence. The preparation of highly dispersed FCOFs nanosheets or nanodots by 2D FCOFs by ultrasonic stripping and dispersion can avoid the fluorescence attenuation or quenching caused by the stacking and aggregation of π-π bonds in aqueous solution. So far, there have been few reports on two-photon 2D FCOFs, and their application in in vivo and in vitro bioimaging is limited by their large size and unsatisfactory fluorescence emission. Therefore, it is of great significance to study a facile, highly dispersed, and high quantum yield two-photon covalent organic framework quantum dots (FCOF QDs).

Pluronic F-127 is a non-ionic surfactant (average molecular weight about 12.6kDa), which can promote the dissolution and dispersion of water-insoluble dyes or other materials in a physiological culture environment. Usually used with dyes containing acetoxymethoxy ester (AM) form to enhance the dispersibility of dyes in aqueous solution. Pluronic F-127 is suitable for the separation of membrane proteins, and its UV absorbance is very low, which is valuable in some experimental procedures using UV methods to detect dissolved proteins.

Contents of the invention

The purpose of the present invention is to provide a novel triphenylamine two-dimensional covalent organic framework quantum dot and its preparation method and application. The triphenylamine two-dimensional covalent organic framework in the invention has high crystallinity, high quantum yield, large specific surface area, sensitive light response, and two-photon absorption characteristics, and has good application prospects in two-photon cell fluorescence imaging.

Technical scheme of the present invention is as follows:

A two-dimensional covalent organic framework quantum dot material formed by interconnecting three-connected nodes A and three-connected nodes B in two-dimensional space;

In at least a part of the two-dimensional covalent organic framework quantum dot material, each three-connected node A is respectively connected to three adjacent three-connected nodes B to form a covalent organic framework structure with a uniform hexagonal channel ; The molar ratio of three-connected node A to three-connected node B can be arbitrary, and different ratios of connected points will produce different shapes, preferably 1:1;

The three-connection node A is as shown in formula (1):

The three-connection node B is shown in formula (2a), (2b) or (2c):

In formula (2a), (2b) or (2c),

In formula (1), (2a), (2b) or (2c),

X indicates the linking site.

The BET specific surface area of the two-dimensional covalent organic framework quantum dot material of the present invention is 40-2000m ² /g, and the pore diameter is 1.0-5.0nm.

The two-dimensional covalent organic framework quantum dot material has regular hexagonal channels.

The linking group of the two-dimensional covalent organic framework quantum dot material contains dynamic covalent bonds, and the linking mode is selected from -C=N-, -C=N-N=C-, -C=N-NH-, -C One of =C-, -C=C(CN)-, preferably -C=N-.

Preferably, when the connection mode is -C=N-, the basic framework of the two-dimensional covalent organic framework quantum dot material is as follows:

A method for preparing a two-dimensional covalent organic framework quantum dot material, comprising the steps of:

(1) Preparation of two-dimensional covalent organic framework materials

Mix three-connected node molecules (I), three-connected node molecules (II), organic solvents and catalysts, freeze in liquid nitrogen, freeze-thaw and degas, seal and place at 80-180°C (preferably 120°C) for 72-168 hours , the solid product was separated by filtration, soaked, washed and dried to obtain a two-dimensional covalent organic framework material;

Described organic solvent is the mixed solvent of anisole and ethanol volume ratio 1:1;

The catalyst is an aqueous solution of 6M acetic acid; the volume ratio of the catalyst to the organic solvent is preferably 0.1:1;

Specifically, the method of soaking and washing the solid product is as follows: first soak in N,N-dimethylformamide for 6h, repeat twice, then soak in acetone for 6h, repeat twice, and then use THF and acetone to Soxhlet extract respectively 12h; the drying conditions are: in a vacuum drying oven, vacuumize to 20mTorr at 80°C, and dry for 24h;

(2) Preparation of two-dimensional covalent organic framework quantum dot materials

Add the two-dimensional covalent organic framework material obtained in step (1) into the PF-127 aqueous solution, then use a cell pulverizer to sonicate for more than 25 h, and then centrifuge at 2000 rpm/min for 10 min (to remove large-sized residues), and take The supernatant was centrifuged at 21000rpm/min for 15min, and the lower layer of solid was washed with deionized water to obtain a two-dimensional covalent organic framework quantum dot material (referred to as COF@PF-127QDs, dispersed in deionized water for subsequent use);

The concentration of the PF-127 aqueous solution is 4.95 mg/mL; under the premise that PF-127 is used as a template protecting agent and modifier, after a series of ultrasonic and centrifugation treatments, a covalent organic compound with an average size of about 5 nm is obtained. frame quantum dots;

           

In formula (I), (II),

One of R ₁ and R ₂ is an amino group (-NH ₂ ), and the other is an aldehyde group (-CHO).

The two-dimensional covalent organic framework quantum dot material described in the present invention can be applied to two-photon cell fluorescence imaging.

Compared with the prior art, the present invention has the following beneficial technical effects:

The present invention combines building unit design and morphology control to prepare two-dimensional covalent organic frameworks (COFs) with specific structures, and uses PF-127 as template protection and modification to convert COFs into amphiphilic quantum dots with an average diameter of about 5nm. For the first time, a COF quantum dot material that can be used for two-photon fluorescence imaging has been prepared.

Significant advantage of the present invention is:

(1) Covalent organic frameworks are a class of ordered porous materials with the advantages of adjustable structure and functionalization, as well as high porosity, nanoscale pore size, high temperature stability, and chemical stability. The present invention designs several new COFs for the first time, and prepares these COFs into quantum dot materials with an average diameter of about 5nm, which can be used for two-photon fluorescence imaging, which is the first in COF materials.

(2) The covalent organic framework quantum dot material used for two-photon fluorescence imaging of the present invention has mild and simple preparation conditions, is universal and easy to implement, is conducive to large-scale promotion, and has broad application prospects.

(3) In the preferred condition, the as-prepared COF quantum dots exhibit outstanding two-photon fluorescence with a two-photon absorption cross-section of 49 GM, and have good dispersion and photostability under physiological conditions.

Description of drawings

Figure 1 is a flow chart of the preparation of two-dimensional covalent organic framework material (TAPBA-TFBPA-COF).

Fig. 2 is a powder X-ray test spectrum and a simulated spectrum of a two-dimensional covalent organic framework material (TAPBA-TFBPA-COF).

Fig. 3 is the infrared spectrum of the two-dimensional covalent organic framework material (TAPBA-TFBPA-COF).

Fig. 4 is a transmission electron microscope image of a two-dimensional covalent organic framework material (TAPBA-TFBPA-COF).

Figure 5 shows the transmission electron microscope image and particle size distribution of two-dimensional covalent organic framework quantum dots (TAPBA-TFBPA-COF QDs).

Figure 6 shows the fluorescence emission spectra of TAPBA-TFPBA-COF QDs at different excitation wavelengths.

Figure 7 shows the two-photon absorption cross-sections of TAPBA-TFPBA-COF QDs at different excitation wavelengths.

Figure 8 shows the two-photon fluorescence emission spectra of TAPBA-TFPBA-COF QDs at different excitation wavelengths.

Detailed ways

The present invention is further described below through specific examples, and the following examples help those in the relevant fields to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from Under the premise of the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention.

Example 1

A preparation of a two-dimensional covalent organic framework quantum dot and its application in two-photon fluorescence imaging, comprising the following steps:

(1) Preparation of two-dimensional covalent organic framework (TAPBA-TFBPA-COF) materials

Referring to Fig. 1, tris (4-aminobiphenyl) amine (TAPBA, 28mg, 0.055mmol) and tris (4-formyl biphenyl) amine (TFPBA, 31mg, 0.055mmol) were added to the solution containing anisole (0.9mL) and ethanol (0.9mL) in a Schlenk tube. The solution was sonicated for 5 minutes to disperse evenly to obtain a yellow cloudy solution. Aqueous 6M acetic acid (0.18 mL) was added to the Schlenk tube as a catalyst. The Schlenk tube was quickly frozen at 77K using a liquid nitrogen bath, and degassed three times by freeze-thaw degassing. After returning to room temperature, the Schlenk tube was placed in an oven at 120°C for 3 days. After the reaction, the green solid product was separated by filtration, soaked in N,N-dimethylformamide for 6 hours, repeated twice, and then soaked in acetone for 6 hours, repeated twice. After the sample is naturally air-dried, it is then extracted with tetrahydrofuran and acetone for 12 hours to further activate the sample. After the Soxhlet extraction, the sample is transferred to a vacuum oven and vacuumed at 80°C to 20mTorr for 24 hours to obtain green powder TAPBA - TFPBA-COF material (yield: 50.1 mg, 85%).

(2) Preparation of covalent organic framework quantum dots (TAPBA-TFBPA-COF QDs)

Add TABPA-TFBPA (13.5mg) into 3mL PF-127 (Sigma-Aldrich, CAS: 9003-11-6, Mn～12600g/mol) aqueous solution (4.95mg mL ^-1 ), and then use a cell disruptor (P = 350W) ultrasonic treatment over 25h. Afterwards, the reaction solution was centrifuged at 2000 rpm for 10 min to remove large-sized residues. The resulting supernatant was further centrifuged at 21,000 rpm for 15 min, and then the lower layer of solid was washed twice with deionized water to obtain TAPBA-TFPBA-COF QDs, which were dispersed in deionized water for subsequent use.

(3) Product characterization and performance testing

Referring to Figure 2, TAPBA-TFBPA-COF has diffraction peaks at 3.1, 5.2, and 7.8 degrees as measured by powder X-ray diffraction, which proves the successful synthesis of TAPBA-TFPBA-COF. The structure simulation was carried out by Materials Studio software, and the crystal structure of TAPBA-TFPBA-COF was analyzed. The simulated PXRD pattern generated by the corresponding simulated structure matched well with the experimental PXRD pattern, which proved the correctness of the structure.

Referring to Figure 3, Fourier transform infrared (FT-IR) spectrum test, by comparing the infrared spectrum of the relevant monomers required for synthesis with the corresponding product TAPBA-TFPBA-COF, the product produced C=N at 1623cm ^-1 The characteristic stretching vibration of the bond, demonstrates the successful synthesis of TAPBA-TFPBA-COF.

Referring to FIG. 4 , the transmission electron microscope image shows that the prepared TAPBA-TFPBA-COF has a uniform layered morphology.

Referring to Figure 5, the transmission electron microscope image shows that the prepared TAPBA-TFPBA-COF QDs are uniformly dispersed nanoparticles with a diameter of about 5 nm.

Referring to Figure 6, through fluorescence tests excited by light sources of different wavelengths, it is shown that TAPBA-TFBPA-COF QDs have typical two-photon fluorescence characteristics.

Referring to Figure 7, through the two-photon absorption cross-section test under the excitation of different wavelength light sources, it shows that the two-photon fluorescence absorption cross-section of TAPBA-TFBPA-COF QDs reaches 49GM at the excitation wavelength of 800nm.

The above-mentioned embodiments only express the preferred implementation modes of the present invention, and the description thereof is more specific and detailed, but should not be construed as limiting the protection scope of the present invention. The technical features of the embodiments can be combined arbitrarily. For the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, but as long as there is no contradiction in the combinations involved, they should be considered as the scope described in this manual. For those skilled in the art, without departing from the concept of the present invention, several modifications and changes can be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims (9)

1. A two-dimensional covalent organic framework quantum dot material, characterized in that it is formed by connecting three connection nodes A and three connection nodes B in two-dimensional space; In at least a part of the two-dimensional covalent organic framework quantum dot material, each three-connected node A is respectively connected to three adjacent three-connected nodes B to form a covalent organic framework structure with a uniform hexagonal channel ; The three-connection node A is as shown in formula (1):

The three-connection node B is shown in formula (2a), (2b) or (2c):

In formula (2a), (2b) or (2c),

In formula (1), (2a), (2b) or (2c), X indicates the linking site. 2. The two-dimensional covalent organic framework quantum dot material according to claim 1, wherein the molar ratio of the three-connected node A to the three-connected node B is 1:1. 3. two-dimensional covalent organic framework quantum dot material as claimed in claim 1, is characterized in that, the linking group of described two-dimensional covalent organic framework quantum dot material contains dynamic covalent bond, and connection mode is selected from- One of C=N-, -C=N-N=C-, -C=N-NH-, -C=C-, -C=C(CN)-. 4. The two-dimensional covalent organic framework quantum dot material as claimed in claim 1, wherein the connection mode of the two-dimensional covalent organic framework quantum dot material is -C=N-, and the two-dimensional covalent organic framework quantum dot material Framework The basic framework of quantum dot materials is as follows:

5. A method for preparing a two-dimensional covalent organic framework quantum dot material, comprising the steps of: (1) Preparation of two-dimensional covalent organic framework materials Mix three-connection node molecule (I), three-connection node molecule (II), organic solvent and catalyst, freeze in liquid nitrogen, freeze-thaw and degas, seal and place at 80-180°C for 72-168 hours, and filter to separate the solid The product is soaked, washed and dried to obtain a two-dimensional covalent organic framework material; Described organic solvent is the mixed solvent of anisole and ethanol volume ratio 1:1; Described catalyst is the aqueous solution of 6M acetic acid; (2) Preparation of two-dimensional covalent organic framework quantum dot materials Add the two-dimensional covalent organic framework material obtained in step (1) into the PF-127 aqueous solution, then use a cell pulverizer to sonicate for more than 25 hours, then centrifuge at 2000rpm/min for 10min, and take the supernatant and centrifuge at 21000rpm/min After 15 minutes, remove the lower layer of solid and wash it with deionized water to obtain a two-dimensional covalent organic framework quantum dot material;

In formula (I), (II), One of R ₁ and R ₂ is an amino group, and the other is an aldehyde group. 6. The preparation method of two-dimensional covalent organic framework quantum dot material as claimed in claim 5, characterized in that, in step (1), the volume ratio of the catalyst to the organic solvent is 0.1:1. 7. The preparation method of the two-dimensional covalent organic framework quantum dot material as claimed in claim 5, is characterized in that, in step (1), the method for soaking and washing the solid product is: first in N,N-dimethyl formazan Soak in amide for 6h, repeat twice, then soak in acetone for 6h, repeat twice, then Soxhlet extract with tetrahydrofuran and acetone for 12h respectively; drying conditions are: in a vacuum drying oven, vacuum at 80°C to 20mTorr, Dry for 24h. 8. The preparation method of two-dimensional covalent organic framework quantum dot material as claimed in claim 5, is characterized in that, in step (2), the concentration of described PF-127 aqueous solution is 4.95mg/mL. 9. The application of the two-dimensional covalent organic framework quantum dot material as claimed in claim 1 in two-photon cell fluorescence imaging.

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