CN115651640A - Narrow emission peak organic fluorescent nano probe with adjustable light-emitting wavelength and preparation method and application thereof - Google Patents
Narrow emission peak organic fluorescent nano probe with adjustable light-emitting wavelength and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a narrow emission peak organic fluorescent nano probe with adjustable light-emitting wavelength, a preparation method and application thereof, and aims to solve the common problems of low brightness, serious influence of aggregation quenching, small Stokes shift, poor light stability, strong signal crosstalk among multiple channels and the like of the conventional probe. The technical scheme adopted by the invention for solving the technical problem is as follows: the luminescent nano-particle is formed by taking a molecule with aggregation-induced emission property as an energy transfer donor and a dye molecule with a narrow emission peak as an energy acceptor and carrying or self-assembling through a water-oil amphiphilic polymer.
Description
Technical Field
The invention relates to the technical field of nano biological materials, in particular to a narrow emission peak organic fluorescent probe with adjustable light-emitting wavelength for fluorescence imaging and a preparation method and application thereof.
Background
In recent years, fluorescent probes have been widely used in biomedical applications, such as fluorescence imaging, flow cytometry, surgical navigation, and immunofluorescence analysis. To date, a variety of luminescent materials have been developed and widely used as fluorescent probes. Among them, organic dyes are the most commonly used fluorescent probes due to their high quantum yield, tunable emission spectra, small size, easily modifiable structure and consistent single-molecule optical properties. Although promising, the brightness of single dyes is much lower than that of single-emitting nanomaterials such as QDs (quantum dots), lanthanide nanocrystals and polymer dots, due to the limited ability of dyes to absorb photons, which in turn leads to optical imaging with lower signal-to-noise ratio. Therefore, the development of organic dye-doped nanoparticles (ODNPs) has proven to be an effective strategy for designing fluorescent probes with higher brightness. This strategy can both maintain the excellent fluorescent properties of the dye and improve the photostability of the dye, but there is still a limitation in brightness enhancement.
An AIE molecule is an organic fluorescent molecule with aggregation-induced emission characteristics that exhibits intense fluorescence in the aggregate or solid state. This unique property makes AIE molecules promising candidates for the construction of high brightness nanophosphors with high luminescent center doping content. In recent years, AIE molecule-doped fluorescent nanodots (AIEdots) have attracted great interest due to their ultra-small size, high brightness, large stokes shift, and excellent light stability. Despite their obvious advantages and remarkable developments, their broad emission peak limits their application in biomedical research, especially in multiplex fluorescence analysis. Therefore, there is an urgent need in the art to develop a fluorescent nanoprobe having high quantum efficiency, good photostability, and a narrow emission peak.
Disclosure of Invention
Aiming at the problems and the current situation of the existing fluorescent probe, the invention provides a narrow-emission organic fluorescent nano probe with adjustable light-emitting wavelength based on an aggregation-induced light-emitting mechanism, a preparation method thereof and application thereof in multi-channel fluorescence imaging. A series of narrow-emission fluorescent nano probes synthesized by the invention based on the aggregation-induced emission mechanism have the characteristics of excellent light stability, large Stokes shift, high brightness, tunable emission peak and the like, can realize multichannel fluorescence imaging of living mouse lymph nodes, and provides important support for application in vivo multiple labeling and clinical operation in the future.
The technical scheme is as follows:
the invention aims to provide a narrow emission peak organic fluorescent nano probe with adjustable light-emitting wavelength, which solves the common problems of low brightness, serious influence by aggregation quenching, small Stokes shift, poor light stability, strong signal crosstalk among multiple channels and the like of the conventional probe.
The technical scheme adopted by the invention for solving the technical problem is as follows: the luminescent nano-particle is formed by taking a molecule with aggregation-induced emission property as an energy transfer donor and a dye molecule with a narrow emission peak as an energy acceptor and carrying or self-assembling through a water-oil amphiphilic polymer.
A molecule having aggregation-induced emission properties, wherein the molecule has the structure:
wherein the electron-withdrawing group QA is selected from a group of any one of the following structural formulas:
electron donating group Ar 1 And Ar 2 Each independently selected from the group of any of the following structural formulae:
wherein the content of the first and second substances,
R 1 is any one of sulfur, selenium, oxygen, nitrogen and carbon atoms;
R 2 、R 3 each independently selected from hydrogen, trifluoromethyl, cyano, nitro, halogen, hydroxyl, amino, optionally substituted alkyl, alkylaminoalkyl, alkoxy, alkylthio, alkenyl, alkynyl, cycloalkyl, cycloalkyloxy, cycloalkylthio, acyl, aryl, heterocyclyl, heteroaryl, heterocycloalkyl, monosubstitutedAn amine group or a disubstituted amine group.
Molecules with narrow emission peaks, characterized in that the class of molecules belongs to any of the following classes:
1) Such molecules have any of the following structures:
wherein R is 1 、R 2 Each independently selected from the group of any of the following structural formulae:
2) Such molecules have the following structure:
wherein R is selected from any one of cyano, trifluoromethyl and pentafluorobenzene;
R 1 、R 2 、R 3 、R 4 each independently selected from the group of any of the following structural formulae:
3) Such molecules have the following structure:
wherein M is a hydrogen atom, a silicon atom or other transition metal atom, and Ar group is an aromatic group.
4. The water-oil amphiphilic polymer is selected from one or more of polystyrene and derivatives thereof, polyethylene glycol and derivatives thereof, polyacrylic acid and derivatives thereof, polypeptide and protein.
The preparation method of the fluorescent nano probe comprises the following steps:
an organic phase mixture of molecules with aggregation-induced emission properties, luminescent molecules with narrow emission peaks, amphiphilic units is sonicated to obtain a clear solution. The mixture was then quickly poured into water, stirred in a fume hood and the organic solvent removed under vacuum. Then, the solution was filtered through a membrane filter to obtain the corresponding fluorescent nanoprobe, and the prepared fluorescent nanoprobe was stored at 4 ℃ for further use.
The invention also provides the contrast of the single-particle brightness of the prepared fluorescent nano probe and the commercial quantum dot, and the fluorescent nano probe is higher than the commercial quantum dot in brightness under the same wave band as a single nano fluorophore, thereby proving the excellent performance of the fluorescent nano probe.
Specifically, the invention also provides application of the prepared fluorescent nano probe in multichannel bioluminescence imaging of in vitro cancer cells and in vivo lymph nodes.
Has the advantages that: the series of fluorescent nano probes with narrow peak emission provided by the invention are simple to prepare and easy to synthesize. Molecules with aggregation-induced emission properties are used as energy transfer donors, narrow-peak emission fluorophores are used as emitters, and the small-sized organic fluorescent nano probe is synthesized. The fluorescent probe has the characteristics of narrow emission peak, adjustable emission wavelength, high brightness, large Stokes shift and the like. The half-peak width of the emission peak of the fluorescent probe is different from 18nm to 36nm, and is 3-6.3 times narrower than that of the traditional AIEdots. Single particle brightness tests show that the fluorescent nano-probe has higher brightness than commercial quantum dots under the same wave band, and the excellent performance of the fluorescent nano-probe is proved. The multichannel fluorescence imaging shows that the high-brightness narrow-emission fluorescence nanoprobe provided by the invention has important significance and value for multiplex cell marking and living mouse multichannel fluorescence imaging.
Drawings
FIG. 1 is a normalized absorption spectrum of the fluorescent nanoprobe AIEdots-1 of the present invention in an aqueous phase;
FIG. 2 is a normalized absorption spectrum of the fluorescent nanoprobe AIEdots-2 of the present invention in an aqueous phase;
FIG. 3 is a normalized absorption spectrum of the fluorescent nanoprobe AIEdots-3 of the present invention in an aqueous phase;
FIG. 4 is a normalized emission spectrum of the fluorescent nanoprobe AIEdots-1 of the present invention in an aqueous phase;
FIG. 5 is a normalized emission spectrum of the fluorescent nanoprobe AIEdots-2 of the present invention in an aqueous phase;
FIG. 6 is a normalized emission spectrum of the fluorescent nanoprobes AIEdots-3 of the present invention in an aqueous phase;
FIG. 7 shows the particle size change of the fluorescent nanoprobes AIEdots of the present invention at different times;
FIG. 8 is a bright field image of human cervical cancer cells (HeLa) incubated with the fluorescent nanoprobes AIEdots-1 of the present invention;
FIG. 9 is a fluorescence imaging graph of human cervical cancer cells (HeLa) incubated by the fluorescent nanoprobe AIEdots-1 of the present invention at a luminescence signal wavelength ranging from 520 nm to 580 nm;
FIG. 10 is a bright field image of human cervical cancer cells (HeLa) incubated with the fluorescent nanoprobes AIEdots-2 of the present invention;
FIG. 11 is a fluorescence image of human cervical cancer cells (HeLa) incubated with the fluorescent nanoprobes AIEdots-2 of the present invention at a luminescence signal wavelength in the range of 700-765 nm;
FIG. 12 is a bright field image of human cervical cancer cells (HeLa) incubated with the fluorescent nanoprobes AIEdots-3 of the present invention;
FIG. 13 is a graph showing fluorescence images of human cervical cancer cells (HeLa) incubated with the fluorescent nanoprobes AIEdots-3 of the present invention at a luminescence signal wavelength in the range of 760 to 824 nm;
FIG. 14 is a fluorescence imaging diagram of a nude mouse foot pad injected with the fluorescent nanoprobe of the present invention respectively before and after dissection within a range that the wavelength of the luminescence signal is greater than 495 nm.
Detailed Description
The present invention is further illustrated by the following examples.
Aiming at the problems of the prior art, such as complicated preparation process, high cost, poor spectrum tuning property and the like, of the nano bioluminescent probe. The invention provides a simply prepared high-performance biological fluorescent nano probe, which is constructed by taking a molecule with high light-capturing capacity and aggregation-induced emission property as an energy donor and a narrow-emission fluorophore as an energy acceptor.
The narrow emission peak organic fluorescent nanoprobe with adjustable light-emitting wavelength and the application thereof are further explained by combining the accompanying drawings and the embodiment as follows:
example 1: preparation of product AIEdots-1
The present embodiment provides a narrow emission peak organic fluorescent nanoprobe with adjustable emission wavelength, and the chemical structural formulas of the AIE molecule, the narrow peak emission dye molecule and the amphiphilic molecule used in the present invention are as follows:
a mixture of 0.4mg AIE-1, 4. Mu.g Dye-1, 0.1mg PS-PEGCOOH and 0.5mL tetrahydrofuran was sonicated to obtain a clear solution. The mixture was then quickly poured into 5mL of deionized water, stirred in a fume hood for 2 hours and concentrated under vacuum. Then, the solution was filtered through a membrane filter (diameter =0.22 μm) to obtain the corresponding fluorescent nanoprobes AIEdots-1, and the prepared fluorescent nanoprobes AIEdots were stored at 4 ℃ for further use.
Example 2: preparation of product AIEdots-2
The present embodiment provides a narrow emission peak organic fluorescent nanoprobe with adjustable light emission wavelength, and the chemical structural formulas of the AIE molecule, the narrow peak emission dye molecule and the amphiphilic molecule used in the present invention are as follows:
a mixture of 0.4mg AIE-2, 4. Mu.g Dye-2, 0.1mg PS-PEGCOOH, and 0.5mL tetrahydrofuran was sonicated to obtain a clear solution. The mixture was then quickly poured into 5mL of deionized water, stirred in a fume hood for 2 hours and concentrated under vacuum. Then, the solution was filtered through a membrane filter (diameter =0.22 μm) to obtain the corresponding fluorescent nanoprobes AIEdots-2, and the prepared fluorescent nanoprobes AIEdots were stored at 4 ℃ for further use.
Example 3: preparation of product AIEdots-3
The present embodiment provides a narrow emission peak organic fluorescent nanoprobe with adjustable emission wavelength, and the chemical structural formulas of the AIE molecule, the narrow peak emission dye molecule and the amphiphilic molecule used in the present invention are as follows:
a mixture of 0.4mg AIE-2, 4. Mu.g Dye-3, 0.1mg PS-PEGCOOH, and 0.5mL tetrahydrofuran was sonicated to obtain a clear solution. The mixture was then quickly poured into 5mL of deionized water, stirred in a fume hood for 2 hours and concentrated under vacuum. Then, the solution was filtered through a membrane filter (diameter =0.22 μm) to obtain the corresponding fluorescent nanoprobes AIEdots-3, and the prepared fluorescent nanoprobes AIEdots were stored at 4 ℃ for further use.
Example 4: investigation of optical Properties of products
Absorption and emission spectra are shown in figures 1-6, and the fluorescent nano probe AIEdots has the characteristics of large Stokes shift, high quantum efficiency and narrow emission peak, is used as a probe required by a biological fluorescence imaging technology, and has the advantages of reducing the interference of background signals, improving the signal-to-noise ratio of imaging, better realizing multiplex fluorescence imaging and the like.
Example 5: study of product stability
In the stability test, 20 μ g/ml of the inventive fluorescent nanoprobes AIEdots were added to 2ml of the matrix and the particle sizes were tested separately at different time nodes, as shown in fig. 7, the products prepared by the present invention show high stability over time in the medium using water as an example, and rather have the potential to be used as multiplexed fluorescent nanoprobes in a complex system.
Example 6: study on Single particle Brightness of the product
The fluorescent nanoprobes AIEdots and the commercial quantum dots prepared by the invention are respectively diluted in glycerol and oleic acid. The fluorescence imaging procedure was performed on a custom-made wide-field fluorescence microscope (Nikon-Ti 2, japan). High power xenon lamps are used as excitation light sources under the filtering of band pass or short pass filters of different specifications, and fluorescence signals (520-580nm, 600-660nm,700-765nm and 760-824 nm) are collected from different channels by using different long pass filters and band pass filters, and the fluorescence intensity emitted by given particles is estimated by integrating sCMOS signals on fluorescence spots. Compared to commercial quantum dots, the product of the invention, AIEdots-1, is 5.2 times brighter than QD525 with a similar band, while the near infrared region emits AIEdots-2 with a brightness comparable to QD 625.
Example 7: cell culture
HeLa, a human cervical cancer cell, was provided by the institute of biochemistry and cell biology (SIBS) of the Chinese academy of sciences. HeLa cells were cultured in DMEM medium containing 10% fetal bovine serum and contained 5% CO at 37% 2 Incubating in a humidified incubator. Cells were grown on 14mm coverslips for 24h and remained attached prior to the experiment.
Example 8: staining of cells with fluorescent probes
The cultured cells contained 5% CO at 37 ℃ with 20. Mu.g/mL of fluorescent nanoprobe 2 Was incubated for 12h, and then the cells were washed three times with Phosphate Buffered Saline (PBS) and imaged.
Example 9: application of product in-vitro cancer cell and in-vivo imaging
In vivo and in vitro fluorescence imaging was performed using a home-made imaging system. Animal experiments were performed according to the guidelines of the institutional animal care and use committee of the university of southeast university laboratory animals. The luminescence signal from the fluorescent nanoprobes of the invention was collected using an EMCCD camera. The autofluorescence imaging system was equipped with different long pass filters (495 LP, 600 LP) and band pass filters (550 BP, 630BP, 732BP, and 792 BP) for acquiring fluorescence signals of different AIEdots in different acquisition channels. The light source for in vitro fluorescence imaging excitation is a xenon lamp light source filtered by a 500SP (< 500 nm) optical filter, and the excitation light source for in vivo fluorescence imaging is a 450nm LED lamp. Referring to fig. 8 to 13, in vitro fluorescence imaging, the naked fluorescent nanoprobes are mainly gathered around the nucleus and exist in the form of bright spots. In addition, strong fluorescence signals from the probes can be observed only in the corresponding channels, and the narrow emission peaks avoid mutual crosstalk of the probes in different channels. For in vivo fluorescence imaging, nude mice (n = 3) were injected subcutaneously with AIEdots-1, AIEdots-2, AIEdots-3, respectively, to different footpads for a total dose of 0.5mg/mL (25 μ L). The mean signals of the solution and tissue were analyzed by Bruker imaging software and Origin 8.0 software. Referring to fig. 14, it is clearly observed from the results of fluorescence imaging in vivo that the injected fluorescent nanoprobes were enriched at the corresponding leg and lymph node sites, and clearly distinguishable fluorescence signals were observed through 550BP, 732BP and 792BP filters.
In view of this, fluorescent probes for multiplex labeling and imaging can be designed based on the present invention as a promising barcode strategy.
The adjustable narrow-emission fluorescence nanoprobe product based on the aggregation-induced emission mechanism can successfully realize in-vivo or in-vitro multi-channel marking and imaging. In addition, the prepared nano probe mainly takes a luminophore with aggregation-induced emission property as an antenna group, and efficiently transfers energy to a series of narrow-peak emission dyes doped at an extremely low proportion, so that the invention of the high-brightness narrow-emission fluorescent nano probe is realized, and the invention has important significance and value for multiplex cell marking and bar code application. In addition, the invention has the advantages of simplicity and easy obtainment in material preparation, and can highlight that the narrow-peak emission fluorescent probe with high brightness has non-negligible competitiveness.
The invention only takes the two AIE molecules as energy donors, three narrow-peak emission dyes as energy acceptors, and the narrow-peak emission fluorescent nano-probe respectively constructed by taking PS-PEGCOOH as amphiphilic units as an example, and for professionals in related fields, the invention can be improved or changed according to the description in the invention, and the content comprises that other aggregation-induced luminophores are used as energy donors, or other narrow-peak emission units are used as energy acceptors, or other amphiphilic materials are used for coating or self-assembling to prepare the nano-probe and the application thereof in biology.
It is obvious to the person skilled in the art that the invention is not limited to the details exemplified in the above-described embodiments. The present invention may be embodied or modified in other forms by those skilled in the chemical, biological or related arts without departing from the essential characteristics thereof. It should be noted, however, that the invention is not limited to the procedures, nature and details of the exemplary embodiments described above. Therefore, the appended claims are not to be limited by the foregoing description, but are intended to include within the invention all such modifications and changes as fall within the true spirit and scope of the invention, and all such modifications and changes are intended to be included within the scope of the appended claims.
In addition, it should be understood that each embodiment of the present invention includes only one or a few independent technical solutions, and those skilled in the relevant art will recognize that the present invention as a whole may be modified or changed accordingly, all of which are intended to be within the scope of the appended claims.
Claims (9)
1. A narrow emission peak organic fluorescent nano probe with adjustable light-emitting wavelength is characterized in that molecules with aggregation-induced emission properties are used as energy transfer donors, dye molecules with narrow emission peaks are used as energy receptors, and light-emitting nano particles are formed through loading or self-assembly of water-oil amphiphilic polymers.
2. The narrow emission peak organic fluorescent nanoprobe with adjustable luminescence wavelength of claim 1, wherein the molecule with aggregation-induced emission property has the following structure:
wherein the electron-withdrawing group QA is selected from a group of any one of the following structural formulas:
electron donating group Ar 1 And Ar 2 Each independently selected from the group of any of the following structural formulae:
3. the narrow emission peak organic fluorescent nanoprobe with adjustable luminescence wavelength of claim 2, wherein R is 1 Is any one of sulfur, selenium, oxygen, nitrogen and carbon atoms; r 2 、R 3 Each independently selected from hydrogen, trifluoromethyl, cyano, nitro, halogen, hydroxy, amino, optionally substituted alkyl, alkylaminoalkyl, alkoxy, alkylthio, alkenyl, alkynyl, cycloalkyl, cycloalkyloxy, cycloalkylthio, acyl, aryl, heterocyclyl, heteroaryl, heterocycloalkyl, mono-substituted amino, or di-substituted amino.
4. The narrow emission peak organic fluorescent nanoprobe with tunable emission wavelength of claim 1, wherein the molecule with the narrow emission peak has any one of the following structures:
wherein R is 1 、R 2 Each independently selected fromA group of any one of the formulae:
5. the narrow emission peak organic fluorescent nanoprobe with adjustable luminescence wavelength of claim 1, wherein the structure of the molecule with the narrow emission peak is as follows:
wherein R is selected from any one of cyano, trifluoromethyl and pentafluorobenzene;
R 1 、R 2 、R 3 、R 4 each independently selected from the group of any of the following structural formulae:
6. the narrow emission peak organic fluorescent nanoprobe with adjustable luminescence wavelength of claim 1, wherein the structure of the molecule with the narrow emission peak is as follows:
wherein M is a hydrogen atom, a silicon atom or other transition metal atom, and Ar group is an aromatic group;
7. the narrow emission peak organic fluorescent nanoprobe with adjustable luminescence wavelength of claim 1, wherein the water-oil amphiphilic polymer is selected from one or more of polystyrene and derivatives thereof, polyethylene glycol and derivatives thereof, polyacrylic acid and derivatives thereof, polypeptide and protein.
8. The method for preparing the nanoprobe as claimed in any one of claims 1 to 7, which comprises the steps of: subjecting an organic phase mixture of molecules with aggregation-induced emission properties, luminescent molecules with narrow emission peaks, amphiphilic units to ultrasound treatment to obtain a clear solution; then the mixture is injected into water, stirred and the organic solvent is removed; then, the solution was filtered through a membrane filter to obtain the corresponding fluorescent nanoprobe.
9. Use of the nanoprobe of any of claims 1 to 7 in multichannel bioluminescence imaging in vitro or in vivo.
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