Narrow emission peak organic fluorescent nanoprobe with adjustable luminescent wavelength and preparation method and application thereof

Abstract

The present invention provides an organic fluorescent nanoprobe with adjustable luminescent wavelength and narrow emission peak, and a preparation method and application thereof, to solve the common problems of existing probes such as low brightness, serious influence of aggregation quenching, small Stokes shift, poor photostability, strong signal crosstalk between multiple channels, etc. The technical solution adopted by the present invention to solve the technical problem is: using molecules with aggregation-induced luminescence properties as energy transfer entities, dye molecules with narrow emission peaks as energy acceptors, and luminescent nanoparticles formed by loading or self-assembly of water-oil amphiphilic polymers.

Description

Technical Field

[0001] The invention relates to the technical field of nano-biomaterials, and in particular to an organic fluorescent probe with adjustable luminescent wavelength and narrow emission peak that can be used for fluorescent imaging, and a preparation method and application thereof. Background Art

[0002] In recent years, fluorescent probes have been widely used in biomedicine, 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, which is attributed to their high quantum yield, tunable emission spectra, small size, easy-to-modify structure, and consistent single-molecule optical properties. Although promising, the brightness of a single dye is much lower than that of single-luminescent nanomaterials such as QDs (quantum dots), lanthanide nanocrystals, and polymer dots, due to the limited ability of the dye to absorb photons, which in turn leads to optical imaging with a lower signal-to-noise ratio. Therefore, the development of organic dye-doped nanoparticles (ODNPs) has been proven to be an effective strategy for designing fluorescent probes with higher brightness. This strategy can both maintain the excellent fluorescence properties of the dye and improve the photostability of the dye, but there are still limitations in brightness enhancement.

[0003] AIE molecules are organic fluorescent molecules with aggregation-induced emission properties, which show strong fluorescence in the aggregated state or solid state. This unique property makes AIE molecules a promising candidate for constructing high-brightness nanofluorophores 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 photostability. Despite their obvious advantages and remarkable development, their wide emission peaks limit their research in biomedicine, especially in multiple fluorescence analysis. Therefore, there is an urgent need in the art to develop a fluorescent nanoprobe with high quantum efficiency, good photostability and narrow emission peak. Summary of the invention

[0004] In view of the above-mentioned problems and current status of fluorescent probes, the present invention provides a narrow emission organic fluorescent nanoprobe with adjustable emission wavelength based on the aggregation-induced emission mechanism, a preparation method thereof, and an application in multi-channel fluorescence imaging. The series of narrow emission fluorescent nanoprobes based on the aggregation-induced emission mechanism synthesized by the present invention have excellent characteristics such as excellent photostability, large Stokes shift, high brightness and tunable emission peak, and can realize multi-channel fluorescence imaging of living mouse lymph nodes, providing important support for future applications in multiple in vivo labeling and clinical surgery.

[0005] Technical solution:

[0006] The purpose of the present invention is to provide a narrow emission peak organic fluorescent nanoprobe with adjustable luminescence wavelength, so as to solve the common problems of existing probes such as low brightness, serious influence of aggregation quenching, small Stokes shift, poor photostability, and strong signal crosstalk between multiple channels.

[0007] The technical solution adopted by the present invention to solve the technical problem is: using molecules with aggregation-induced luminescence properties as energy transfer entities, dye molecules with narrow emission peaks as energy acceptors, and luminescent nanoparticles formed by loading or self-assembling water-oil amphiphilic polymers.

[0008] Molecules with aggregation-induced emission properties are characterized in that they have the following structure:

[0009]

[0010] Wherein, the electron withdrawing group QA is selected from any of the following structural formulas:

[0011]

[0012] The electron donating groups Ar1 and Ar2 are independently selected from the following groups:

[0013]

[0014] in,

[0015] R1 is any one of sulfur, selenium, oxygen, nitrogen, and carbon atoms;

[0016] R2 and R3 are each independently selected from hydrogen, trifluoromethyl, cyano, nitro, halogen, hydroxyl, amine, optionally substituted alkyl, alkylamine, alkoxy, alkylthio, alkenyl, alkynyl, cycloalkyl, cycloalkyloxy, cycloalkyloxythio, acyl, aromatic, heterocyclic, heteroaryl, heterocycloalkyl, monosubstituted amine or disubstituted amine.

[0017] Molecules with narrow emission peaks, characterized in that they belong to any of the following categories:

[0018] 1) This type of molecule has any of the following structures:

[0019]

[0020] Wherein, R1 and R2 are independently selected from the following groups:

[0021]

[0022] 2) This type of molecule has the following structure:

[0023] Wherein, R is selected from any one of cyano, trifluoromethyl, and pentafluorobenzene;

[0024] R1, R2, R3, and R4 are each independently selected from a group of any of the following structural formulas:

[0025]

[0026] 3) This type of molecule has the following structure:

[0027]

[0028] Wherein, M is a hydrogen atom, a silicon atom or other transition metal atom, and the Ar group is an aromatic group.

[0029]

[0030] 4. The water-oil amphiphilic polymer is selected from one or more of polystyrene and its derivatives, polyethylene glycol and its derivatives, polyacrylic acid and its derivatives, polypeptides, and proteins.

[0031] The method for preparing the fluorescent nanoprobe of the present invention comprises the following steps:

[0032] The organic phase mixture of the molecule with aggregation-induced emission properties, the luminescent molecule with a narrow emission peak, and the amphiphilic unit was sonicated to obtain a clear solution. The mixture was then quickly injected into water, stirred in a fume hood, and the organic solvent was 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°C for further use.

[0033] The present invention also provides a single particle brightness comparison between the prepared fluorescent nanoprobe and commercial quantum dots. As a single nanofluorophore, in the same wavelength band, the fluorescent nanoprobe of the present invention has higher brightness than commercial quantum dots, proving the excellent performance of the fluorescent nanoprobe of the present invention.

[0034] Specifically, the present invention also provides the use of the prepared fluorescent nanoprobe in multi-channel bioluminescence imaging for imaging cancer cells in vitro and lymph nodes in vivo.

[0035] Beneficial effects: The series of fluorescent nanoprobes with narrow peak emission provided by the present invention are simple to prepare and easy to synthesize. A small-sized organic fluorescent nanoprobe is synthesized using molecules with aggregation-induced emission properties as energy transfer agents and narrow-peak emission fluorophores as emitters. The fluorescent probe of the present invention has the characteristics of narrow emission peak, adjustable emission wavelength, high brightness and large Stokes shift. The emission peak half-width of the fluorescent probe of the present invention ranges from 18nm to 36nm, which is 3-6.3 times narrower than the emission peak of traditional AIEdots. The single-particle brightness test shows that in the same wavelength band, the fluorescent nanoprobe in the present invention is brighter than commercial quantum dots, which proves the excellent performance of the fluorescent nanoprobe of the present invention. Multi-channel fluorescence imaging shows that the high-brightness narrow emission fluorescent nanoprobe provided by the present invention is of considerable significance and value for multiplexed cell labeling and multiplexed fluorescence imaging of living mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is the normalized absorption spectrum of the fluorescent nanoprobe AIEdots-1 of the present invention in the aqueous phase;

[0037] Figure 2 is the normalized absorption spectrum of the fluorescent nanoprobe AIEdots-2 of the present invention in the aqueous phase;

[0038] Figure 3 is the normalized absorption spectrum of the fluorescent nanoprobe AIEdots-3 of the present invention in the aqueous phase;

[0039] Figure 4 is the normalized emission spectrum of the fluorescent nanoprobe AIEdots-1 of the present invention in the aqueous phase;

[0040] Figure 5 is the normalized emission spectrum of the fluorescent nanoprobe AIEdots-2 of the present invention in the aqueous phase;

[0041] Figure 6 is the normalized emission spectrum of the fluorescent nanoprobe AIEdots-3 of the present invention in the aqueous phase;

[0042] Figure 7 is the change in particle size of the fluorescent nanoprobe AIEdots of the present invention at different times;

[0043] Figure 8 This is a bright field image of human cervical cancer cells (HeLa) incubated with the fluorescent nanoprobe AIEdots-1 of the present invention;

[0044] Fig. 9 It is a fluorescence imaging diagram of human cervical cancer cells (HeLa) incubated with the fluorescent nanoprobe AIEdots-1 of the present invention in the range of 520-580nm of the luminescent signal wavelength;

[0045] Fig.10 This is a bright field image of human cervical cancer cells (HeLa) incubated with the fluorescent nanoprobe AIEdots-2 of the present invention;

[0046] Fig.11 It is a fluorescence imaging diagram of human cervical cancer cells (HeLa) incubated with the fluorescent nanoprobe AIEdots-2 of the present invention in the range of 700-765nm of the luminescent signal wavelength;

[0047] Fig.12 This is a bright field image of human cervical cancer cells (HeLa) incubated with the fluorescent nanoprobe AIEdots-3 of the present invention;

[0048] Fig.13 It is a fluorescence imaging diagram of human cervical cancer cells (HeLa) incubated with the fluorescent nanoprobe AIEdots-3 of the present invention in the range of 760-824nm of the luminescent signal wavelength;

[0049] Fig.14 The fluorescent imaging diagrams are taken before and after the nude mouse foot pads are injected with the fluorescent nanoprobes of the present invention and within the range of a luminescent signal wavelength greater than 495 nm. DETAILED DESCRIPTION

[0050] The present invention will be further explained below in conjunction with the embodiments.

[0051] Aiming at the existing problems of nano bioluminescent probes in the prior art, such as cumbersome preparation process, high cost, poor spectral tunability, etc., the present invention provides a high-performance bioluminescent nanoprobe that can be easily prepared, and constructs a spectrally tunable narrow emission fluorescent nanoprobe by using molecules with high light-harvesting ability and aggregation-induced emission properties as energy donors and narrow emission fluorophores as energy acceptors.

[0052] The following is a further description of the organic fluorescent nanoprobe with adjustable luminescent wavelength and narrow emission peak and its application in conjunction with the accompanying drawings and embodiments:

[0053] Example 1: Preparation of product AIEdots-1

[0054] This embodiment provides a narrow emission peak organic fluorescent nanoprobe with adjustable luminescent wavelength. The chemical structural formulas of the AIE molecule, narrow emission dye molecule and amphiphilic molecule used in the present invention are as follows:

[0055]

[0056] A mixture of 0.4 mg AIE-1, 4 μg Dye-1, 0.1 mg PS-PEGCOOH and 0.5 mL tetrahydrofuran was sonicated to obtain a clear solution. The mixture was then quickly injected into 5 mL deionized water, stirred in a fume hood for 2 hours and concentrated under vacuum. The solution was then filtered through a membrane filter (diameter = 0.22 μm) to obtain the corresponding fluorescent nanoprobe AIEdots-1, and the prepared fluorescent nanoprobe AIEdots was stored at 4 ° C for further use.

[0057] Example 2: Preparation of product AIEdots-2

[0058] This embodiment provides a narrow emission peak organic fluorescent nanoprobe with adjustable luminescent wavelength. The chemical structural formulas of the AIE molecule, narrow emission dye molecule and amphiphilic molecule used in the present invention are as follows:

[0059]

[0060] A mixture of 0.4 mg AIE-2, 4 μg Dye-2, 0.1 mg PS-PEGCOOH and 0.5 mL tetrahydrofuran was sonicated to obtain a clear solution. The mixture was then quickly injected into 5 mL deionized water, stirred in a fume hood for 2 hours and concentrated under vacuum. The solution was then filtered through a membrane filter (diameter = 0.22 μm) to obtain the corresponding fluorescent nanoprobe AIEdots-2, and the prepared fluorescent nanoprobe AIEdots was stored at 4 ° C for further use.

[0061] Example 3: Preparation of product AIEdots-3

[0062] This embodiment provides a narrow emission peak organic fluorescent nanoprobe with adjustable luminescent wavelength. The chemical structural formulas of the AIE molecule, narrow emission dye molecule and amphiphilic molecule used in the present invention are as follows:

[0063]

[0064] A mixture of 0.4 mg AIE-2, 4 μg Dye-3, 0.1 mg PS-PEGCOOH and 0.5 mL tetrahydrofuran was sonicated to obtain a clear solution. The mixture was then quickly injected into 5 mL deionized water, stirred in a fume hood for 2 hours and concentrated under vacuum. The solution was then filtered through a membrane filter (diameter = 0.22 μm) to obtain the corresponding fluorescent nanoprobe AIEdots-3, and the prepared fluorescent nanoprobe AIEdots was stored at 4 ° C for further use.

[0065] Example 4: Study on the optical properties of the product

[0066] Absorption and emission spectra Figures 1 to 6 As shown, the fluorescent nanoprobe AIEdots of the present invention has the characteristics of large Stokes shift, high quantum efficiency, and narrow emission peak. As the probe required for biological fluorescence imaging technology, it is beneficial to reduce the interference of background signals, improve the signal-to-noise ratio of imaging, and better realize multiplexed fluorescence imaging.

[0067] Example 5: Study on product stability

[0068] In the stability test, 20 μg / ml of the fluorescent nanoprobe AIEdots of the present invention was added to 2 ml of the matrix, and the particle size was tested at different time points. Figure 7 As shown, the product prepared by the present invention exhibits high stability over time in a medium such as water, and has considerable potential as a multiplexed fluorescent nanoprobe in a complex system.

[0069] Example 6: Study on the brightness of a single particle of the product

[0070] The fluorescent nanoprobe AIEdots and commercial quantum dots prepared by the present invention are diluted in glycerol and oleic acid, respectively. The fluorescence imaging process is carried out on a customized wide-field fluorescence microscope (Nikon-Ti2, Japan). A high-power xenon lamp is used as an excitation light source under different specifications of bandpass or short-pass filters, and different long-pass filters and bandpass filters are used to collect fluorescence signals from different channels (520-580nm, 600-660nm, 700-765nm, 760-824nm), and the fluorescence intensity emitted by a given particle is estimated by integrating the sCMOS signal on the fluorescent point. Compared with commercial quantum dots, the product AIEdots-1 of the present invention is 5.2 times brighter than QD525 with a similar wavelength, and the brightness of AIEdots-2 emitted in the near-infrared region is comparable to QD625.

[0071] Example 7: Cell culture

[0072] Human cervical cancer cells HeLa were provided by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (SIBS). HeLa cells were cultured in DMEM medium containing 10% fetal bovine serum and incubated in a humidified incubator at 37°C containing 5% CO2. Before the experiment, the cells were grown on 14 mm coverslips for 24 h and remained attached.

[0073] Example 8: Fluorescent probe staining of cells

[0074] The cultured cells were incubated with 20 μg / mL fluorescent nanoprobes at 37°C in the presence of 5% CO2 for 12 h, and then the cells were washed three times with phosphate buffered saline (PBS) before imaging.

[0075] Example 9: Application of the product in in vitro cancer cell and in vivo imaging

[0076] In vivo and in vitro fluorescence imaging was performed using a homemade imaging system. Animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Experimental Animal Center of Nantong University. An EMCCD camera was used to collect the luminescent signals from the fluorescent nanoprobes of the present invention. The homemade fluorescence imaging system was equipped with different long-pass filters (495LP, 600LP) and band-pass filters (550BP, 630BP, 732BP, and 792BP) to collect the fluorescence signals of different AIEdots in different acquisition channels. The light source used for in vitro fluorescence imaging excitation was a xenon lamp light source filtered by a 500SP (<500nm) filter, and the excitation light source for in vivo fluorescence imaging was a 450nm LED lamp. See Figures 8 to 13 In in vitro fluorescence imaging, the naked fluorescent nanoprobes mainly accumulated around the nucleus and existed as bright spots. In addition, strong fluorescence signals from the probes could only be observed in the corresponding channels, and the narrow emission peaks avoided the crosstalk between the probes in different channels. For in vivo fluorescence imaging, nude mice (n=3) were subcutaneously injected with AIEdots-1, AIEdots-2, and AIEdots-3 into different foot pads, with a total dose of 0.5 mg / mL (25 μL). The average signals of the solution and tissue were analyzed by Bruker imaging software and Origin 8.0 software. See Fig.14 It can be clearly observed from the results of in vivo fluorescence imaging that the injected fluorescent nanoprobes are enriched in the corresponding legs and lymph nodes, and clearly distinguishable fluorescence signals can be observed through 550BP, 732BP and 792BP filters.

[0077] In view of this, the present invention can be used as a promising barcoding strategy to design fluorescent probes for multiplex labeling and imaging.

[0078] The adjustable narrow emission fluorescent nanoprobe product based on the aggregation-induced emission mechanism of the present invention can successfully realize multi-channel labeling and imaging in vivo or in vitro. In addition, since the nanoprobe prepared by the present invention mainly uses a luminophore with aggregation-induced emission properties as an antenna group, and efficiently transfers energy to a series of narrow-peak emission dyes doped at extremely low ratios, the invention of high-brightness narrow-emission fluorescent nanoprobes is realized, which has considerable significance and value for multiplexed cell labeling and barcode applications. In addition, the present invention has the advantage of being simple and easy to obtain in material preparation, which can further highlight the competitiveness of the high-brightness narrow-peak emission fluorescent probe of the present invention that cannot be ignored.

[0079] The present invention only takes the above two AIE molecules as energy donors, three narrow-peak emission dyes as energy acceptors, and PS-PEGCOOH as amphiphilic units to construct narrow-peak emission fluorescent nanoprobes as embodiments. For professionals in related fields, improvements or changes can be made according to the description in the present invention, including the use of other aggregation-induced luminophores as energy donors, or other narrow-peak emission units as energy acceptors, or other amphiphilic materials to coat or self-assemble the prepared nanoprobes and their applications in biology.

[0080] It is obvious to those skilled in the art that the present invention is not limited to the details demonstrated in the above embodiments. Without departing from the main features of the present invention, technicians in chemistry, biology or related fields can implement or modify the present invention in other forms. However, it should be noted that the present invention is not limited to the procedures, properties and details in the above exemplary embodiments defined above. Therefore, the claims attached to the present invention are not limited by the above description, but are intended to include the present invention and all modifications in the form of various modifications of the present invention and equivalent meanings and scopes within the present invention, and the modifications and adjustments made should fall within the scope of protection of the claims attached to the present invention.

[0081] In addition, it should be understood that each example of the present invention only includes one or a class of independent technical solutions. Researchers in related fields should take the specification as a whole and may make corresponding improvements or changes to the description of the present invention, but all changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. An organic fluorescent nanoprobe with adjustable luminescence wavelength and narrow emission peak, characterized in that: Molecules with aggregation-induced emission properties are used as energy transfer entities, dye molecules with narrow emission peaks are used as energy acceptors, and luminescent nanoparticles are formed through water-oil amphiphilic polymer loading or self-assembly; The chemical structural formulas of molecules with aggregation-induced emission properties, dye molecules with narrow emission peaks, and water-oil amphiphilic polymers are as follows:

2. The method for preparing the nanoprobe according to claim 1, characterized in that: The method comprises the following steps: subjecting an organic phase mixture of a molecule having aggregation-induced luminescence properties, a luminescent molecule having a narrow emission peak, and an amphiphilic unit to ultrasonic treatment to obtain a clear solution; injecting the mixture into water, stirring, and removing the organic solvent; Then, the solution was filtered through a membrane filter to obtain the corresponding fluorescent nanoprobes.

3. Use of the nanoprobe according to claim 1 in multi-channel bioluminescence imaging in vitro or in vivo.

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